Continental J. Biomedical Sciences - Volume 4 (2010)

Continental J. Biomedical Sciences 4: 1 - 5, 2010 ISSN: 2141 – 419X

©Wilolud Journals, 2010 http://www.wiloludjournal.com

PATIENTS’ AWARENESS AND THE ISSUE OF MALARIA CHEMOPROPHYLAXIS

Anyanwu, E. B1., Mabiku, T. O.1, and Okperi, B.2

1Department of Family Medicine, Faculty of Clinical Medicine, College of Health Science, Delta State University, Abraka, 2Department of Paediatrics, Faculty of Clinical Medicine, College of Health Science, Delta State University,

ABSTRACT

Malaria causes an acute febrile illness which maybe characterized by periods of febrile paroxysm. Mankind is infested by four species of the genus plasmodium, but Plasmodium falciparum predominates in the Sub-Saharan Africa. There is therefore a risk of non-immune travellers to malaria-endemic countries acquiring malaria. Exposure prophylaxis and chemoprophylaxis are established methods of preventing malaria during travel in malarial-endemic countries. Persons from non-endemic countries are considered non-immune and stand a chance of acquiring malaria with possible fatal outcome. This case report emphasizes the need for chemoprophylaxis for all returnees from non-endemic areas.

KEYWORDS: Patient awareness, counseling, chemoprophylaxis, diaspora

INTRODUCTION

Malaria infection is an endemic illness in the Sub-Saharan Africa in which Nigeria is located. Malaria infection has been reported to be responsible for the death of over one million people each year. These deaths are mainly in children under five years. Malaria infection of children during the first five years of life can cause severe and potentially fatal illness, which usually is rapidly progressive particularly in highly endemic region (WHO).

The risk of severe and potentially fatal infection is greater in person with little or no acquired immunity such as young children or immigrants from malaria free zones (Breman 1988).

Severe malaria is usually confined to the first five years of life and becomes progressive less common with increasing age. Passive transfer of antibody across the placenta helps protect the neonate for the first six months of life, until he stars to acquire his own antibody by the age of six months. Following this period of relative protection, children becomes increasingly susceptible to the more severe clinical manifestation of malaria. But from around the fourth year of life, the severity of clinical attacks begins to decline. Deaths from malaria is uncommon after the fifth year of life and thereafter clinical attacks becomes less frequent and less severe until in adulthood when significant disease is uncommon (White and Strickland 1991).

Repeated malaria infection leads to the acquisition of a specific partial immunity by members of the indigenous population.

Our index case has lived all her life in a western country and never had malaria fever. Also she was aged only eighteen months and therefore her immunity against malaria was expectedly low or none at all.

Non-immune persons and other highs risk people such as pregnant women, infants and young children should therefore be protected if they have to be in malaria zones and the aim of this study is to re-emphasis the need for chemoprophylaxis for such groups of persons and all non-immune travellers going to malaria zones.

Anyanwu, E. B et al.,: Continental J. Biomedical Sciences 4: 1 - 5, 2010

CASE HISTORY

A young girl aged about 1½ years was brought into a busy group medicine practice by her mother and aunty with a history of a high grade fever for two days. Apparently, she has been managed at home with analgesics/antipyretic mixture and tepid sponging to no avail. There was an associated non-pruritic rash on her face and upper anterior chest wall. Appetite was poor, but she was not vomiting nor stooling.

Further questioning revealed that the family had just come back from their residence abroad and the young girl had never traveled outside the western country where they reside. Her parents have lived in that country for three uninterrupted years and this was the first girl’s trip to Nigeria.

Apparently, the girl and even her parents were not placed on any kind of prophylaxis against malaria before they left the American continent where they live, and only started taking proguanil tablets on arrival here in Nigeria. She soon broke down with a high grade fever and a temperature maximum of 39.30c and was subsequently brought to the clinic.

Examination revealed a febrile child, who was not pale, not dehydrated and not cyanosed. Systemic examination was essentially normal.

A working diagnosis of malaria fever was made and a malaria parasite smear that was ordered for came back as positive (2+). She was subsequently started on an appropriate anti-malaria medication and an injectable antipyretic was administered to help lower the temperature.

She was reported to have been febrile overnight and her attendants spent the night tepid sponging her.

By the following morning, one of the baby’s attendants who happened to be a trained health worker (based and work in Europe), came with the entourage that brought the baby girl back to the clinic. She “wondered aloud” why we did not “properly” educate them on what to expect following our line of management.

They claimed that all we told them was that the baby will be “alright”, but that we did not explain to them what drugs we gave and what the drugs were expected to do. They also wondered why the baby still had a high temperature despite the medication that we gave to her.

She was essentially accusing us of not giving them a proper health talk which explains our diagnosis, the laboratory finding and then our line of management and choice of anti-malaria medication, and of any adverse reactions if any. They felt that we ought to have explained the therapeutic effect of all drugs given and our reason for choosing the medicines that we gave.

At this point, we had a group talk explaining the rationale for the choice of medication, what to expect and possible outcome. We also explained to them the expected rate of decline of temperature as we then encouraged them to give the medication as prescribed.

Finally, we again re-emphasized the need for a proper prophylaxis against malaria whenever they are to come home to Africa again.

DISCUSSION

Malaria infection is found commonly in the Sub-Saharan Africa in which Nigeria is found. It is responsible for about one million deaths each year, mainly in children under five years in Africa. An average child has about six bouts of malaria a year. Over a third of the world population live in zones where there is a high risk of infection (Chinnock, 1997).

Malaria is the most important parasitic disease of man. This human disease is a protozoan infection of red blood cells transmitted by the bite of a blood-feeding female Anopheles mosquito (White, 1996).

Anyanwu, E. B et al.,: Continental J. Biomedical Sciences 4: 1 - 5, 2010

Malaria transmission occurs in more than 100 countries throughout Africa, Asia and Latin America. More than one billion inhabitants of these areas are exposed to risk of malaria infection. The estimated annual global incidence of malaria subsequently is over 200 million cases (White 1996, & Strickland 1991).

Malaria infection in children during the first five years of life can cause severe and potentially fatal illness, which usually is rapidly progressive particularly in highly endemic regions (WHO 1986).

The causative agent of this parasitic disease is Plasmodium. This protozoan is transmitted to man by the bite of the female Anopheles mosquito (Bouree, et al 1993).

Mankind is infected by four species of the genus plasmodium. These are plasmodium falciparum, Plasmodium ovale, Plasmodium vivax and Plasmodium malariae.

Only Plasmodium falciparum, the most widespread can lead to death through cerebral involvement in the form of cerebral malaria (Russel, 1995, Philips, 1990). Plasmodium falciparum predominates in the Sub-Sharan Africa, Papua New Guinea and Haiti.

Plasmodium falciparumis associated with the heaviest degree of parasitemia, and the severity of malaria that it causes is accentuated by the increasing resistance to commonly used drugs, requiring the use of more and more potent prophylactic and therapeutic agents (Gilbert, et al., 2003 & Prince ,. 1994).

Control measures are directed at reducing the population of anopheles mosquito, chemotherapy for infected persons and chemoprophylaxis for person traveling to endemic areas. The most important is the prevention of mosquito bites through the use of bed nets impregnated with insecticide, mosquito repellants and protective clothing (Warrel, 1993)

Infection by any of the human malaria species produces symptoms which, in non-immune subjects are temporarily debilitating and, in the case of Plasmodium falciparum are potentially fatal. Malaria should therefore be prevented if possible.

Those at most risk of contracting debilitating or life threatening malaria are:
 * Children with sickle cell disease
 * Pregnant mothers in endemic areas
 * Child or adult who returns to an endemic area after an absence of over one year even if he/she is originally from that region.
 * Non-immune individuals: people from non-endemic areas such as military personnel and other task forces working in malarious area.
 * Indigenous populations in malarious areas before they have acquire protective immunity (i.e. infants and children up to the age of about five years) (Warrel, 1993 & Molyneux,. 2002).

The state of immunity has a bearing on the use of drugs since people who have, by a prolonged exposure to the infection, acquired a degree of immunity can be cures or protected much more easily than those who have not.

The clinical manifestation of malaria is dependent on the previous status of the host. In areas of stable endemicity which includes Nigeria, repeated malaria infection leads to the acquisition of a specific partial immunity by members of the indigenous population. In these areas, people are infected repeatedly throughout their lives. Thus, a form of immunity develops which is sufficient to control, but not prevents infection (Marsh,. 1993).

The risk of severe and potentially fatal infection is greater in persons with little or no acquired immunity such as young children or immigrants from malaria free areas (Breman, et al 1988 & Warrel et al., 1990).

All travelers to malarious area of the world are advised to use an appropriate drug regimen and personal protection measures to prevent malaria. However, travelers are to be informed that regardless of methods employed, malaria

Anyanwu, E. B et al.,: Continental J. Biomedical Sciences 4: 1 - 5, 2010

may be contracted. Malaria symptoms can develop as early as eight days after initial exposure in a malaria-endemic area and as late as several months after departure from a malarious area, after chemoprophylaxis has been stopped. Travelers should understand that malaria can be treated effectively early in the course of the disease, but that delay of appropriate therapy can result in a serious or even fatal outcome (CDC, 1990 & CDC, 1993).

Because of the nocturnal feeding habits of Anopheles mosquitoes, malaria transmission occurs primarily at night. Travels should take protective measures to reduce contact with mosquitoes especially during these times. Such measures include remaining in well-screened areas, using mosquito nets, and wearing clothes that cover most of the body.

Malaria chemoprophylaxis should begin 1 -2 weeks before travel to malarious area. This allows for any potential side effects to be evaluated and treated by the travellers’ doctor before departure, and also assures for adequate blood levels of the drug before departure. Chemoprophylaxis should continue during travel in the malaria area and for 4 weeks after persons leaves the malarious areas (CDC, 1990 & CDC, 1993).

Children of any age can contract malaria; young children should avoid travel to areas with chloroquine-resistant Plasmodium falciparum, unless they can take a highly effective drug.

In a study to determine the effect of chemoprophylaxis on the case fatality rate of malaria, it was demonstrated that chemoprophylaxis significantly reduces fatality rates among non-immune malaria patients. This supports all efforts towards chemoprophylaxis (Kruase, et al 2006). Our index client was not given any chemoprophylaxis before she left the foreign country where she was delivered in and had lived her life before now.

She apparently contacted malaria while here in the country and promptly reported for proper evaluation. She did well eventually on management but then the risk taken could have been better avoided if proper prophylactic measures had been taken.

This report is essentially to call to our remembrance the need for prophylaxis wherever our kinsmen/women in diaspora are coming home for holidays or for any other ceremonies.

REFEREES

Bouree, P., Taugourdeau Ph., Ng – Anh Van. Malaria. In: Bouree, P., Taugourdeau, Ph. (Eds). Malaria, Smithkline

Beacham Pharmaceuticals Bibliography; 1993 pp. 1 – 40.

Breman, J. G. and Campbell, C. C. Combating severe malaria in African children. Bull WHO, 1988; 66(5): 611 620.

Center for Disease Control and Prevention Recommendations for the Prevention of Malaria Among Travelers.

March 09, 1990/39 (RR – 3); 1 – 10.

Center for Disease Control and Prevention Malaria: Health Information for International Travel http;//

wonder.CDC.gov/wonder/ prevguid/p0000375/p0000375.asp.

Chinnock, P. Malaria: Action at last? African Health, 1997 September; Vol. 19, No. 6: pp 26 – 27.

Gilbert, D. N., Moellering, R. C., and Sande, M. A. Treatment of Parasitic Infections. In: The Sanford Guide to

Anti-microbial Therapy. 33rd edition; 2003. Jeb. C. Sanford (Publisher) USA, pp. 90 – 97.

Kruase, G., Schoneberg, I., Altmann, D., and Stark, K. Chemoprophylaxis and malaria death rates. Emerg infect Dis

(serial on the inernet). 2006. http:// www.CDC.gov/ncidou/E1D/vol12no03/05-0736.htm.

Marsh, K. Immunology of human malaria. In Gilles, H. M and Warrel, D. A. (Eds). Bruce – Chewatt’s Essential

<p style="margin-left: 36pt;">Malarialogy. 3rd edition. Edward Arnold (Publ). 1993. pp. 60 – 78.

Anyanwu, E. B et al.,: Continental J. Biomedical Sciences 4: 1 - 5, 2010

<p style="margin-left: 36pt;">Molyneux, E. Malaria. In: Southall, D., Coulter, B., Ronald, C., Nicholson, S., and Parke, S (Eds). International

<p style="margin-left: 36pt;">Child Health Care: A Practical Manual for Hospitals Worldwide, Child Advocacy International. BMJ Books, 2002,

<p style="margin-left: 36pt;">pp. 473 – 476.

<p style="margin-left: 36pt;">Philips, R. E., and Solomon, T. Cerebral malaria in Children. The Lancet 1990; 336: 1355 – 1360.

<p style="margin-left: 36pt;">Prince, A. Infectious disease systemic protozoual infection-Malaria. In: Behraman, R. E., and Kliegman, R. M.

<p style="margin-left: 36pt;">(Eds). Nelson’s Essential Pediatrics. 2nd edition. Philadelphia: WB Saunders Company; 1994, pp. 389 – 390.

<p style="margin-left: 36pt;">Russel, W., Streel and Baffoe – Bonnie B. Cerebral Malaria in Children. ''Pediatr Infect Dis. J''. 1995; 14 (4) 281 –

<p style="margin-left: 36pt;">285.

<p style="margin-left: 36pt;">Strickland, G. T. Infections of the blood and reticuloendothelial system-Malaria. In: Strickland, G.T (Ed). Hunters

<p style="margin-left: 36pt;">Tropical Medicine. 7th edition. Philadelphia; WB Saunders Company: 1991 pp 586 – 614.

<p style="margin-left: 36pt;">Warrel, D. A. Treatment and Prevention of Malaria. In: Gilles, H. M., and Warrel, D. A (Eds). Bruce Chwatts

<p style="margin-left: 36pt;">Essential Malarialogy. 3rd edition. Edward Annold (Publ) 1993; pp. 163 – 195.

<p style="margin-left: 36pt;">Warrel, D. A., Molyneux, M. E. and Beales, P. F. (Eds) World Health Organisation Division of Control of Tropical

<p style="margin-left: 36pt;">Disease. Severe and complicated malaria. 2nd edition. Trans Roy Soc Trop Med Hyg, 1990; 84 (Supplement 2) 1 –

<p style="margin-left: 36pt;">65.

<p style="margin-left: 36pt;">White, N. J. Malaria. In: Gordon C. Cook(Ed); Manson’s Tropical Diseases. 20th edition. London; WB. Saunders

<p style="margin-left: 36pt;">Company; 1996. pp 1087 – 1164.

<p style="margin-left: 36pt;">WHO Expert Committee on Malaria.Technical Report Series 18th Report, 735, 1986

Received for Publication: 02/04/2010

Accepted for Publication: 12/05/2010

Corresponding Author:

<p style="margin-left: 180pt;">E. B. Anyanwu,

<p style="margin-left: 180pt;">Department of Family Medicine, Faculty of Clinical Medicine, Delta State University, Abraka, Nigeria

<p style="margin-left: 180pt;">E-mail: ebirian@yayoo.com

Continental J. Biomedical Sciences 4: 6 - 9, 2010 ISSN: 2141 – 419X

©Wilolud Journals, 2010 http://www.wiloludjournal.com

UNDERSTANDING AND PREVENTION OF CHILDREN’S ACCIDENTS

1Anyanwu, E. B.,1Mabiaku, T. O., 2Akathor, A. and 3Okperi, B.

<p style="margin-left: 18pt;">1Department of Family Medicine, 2Department of Surgery, 3Department of Paediatrics, Faculty of Clinical Medicine, Delta State University, Abraka, Nigeria

<p style="margin-left: 43.2pt;">ABSTRACT

<p style="margin-left: 43.2pt;">Accidents are preventable but some say that they are bound to happen. With some extra care or caution, most accidents can be prevented, and even when they do occur, the consequences can be minimal. Children are very vulnerable and do not have the ability to judge between what may or may not be dangerous. They are therefore, more at risk to various forms of accidents both at home and outside. This short review reports two cases and highlights some factors that can be helpful in preventing accidents especially among children.

<p style="margin-left: 187.2pt;">KEYWORDS: Children, accident, prevention, inequality between the rich and the poor, parental role, storage of medicines

INTRODUCTION

Most accidents can be prevented. Accidents are the commonest cause of death among children aged and results in many children being permanently disabled annually.

Childhood accidents are intimately related to the children’s development. An older child can climb a tree and fall, or fall in a play ground. While a crawling child can climb upstairs and fall back.

Accidents are responsible for about eighty percent of deaths in 15 – 19 years olds and sixty percent of death in 10 – 14 years old. On any given day in the United States of America, about 23 teenagers die in motor vehicle accidents while about six are victims of homicide (Ditmar, 1997).

This article is essentially to bring to our attention the urgent need for parents to be more alert to their children’s safety and seek improvement in their preventive knowledge.

CASE REPORT

A young family of five children were gently driving around in one of the towns in Delta State, when a close family friend of theirs saw them and pulled over for routine greetings. The family in this report also pulled over across the road and their father ran across the road to great their friend.

After a while, the two year old son of the family opened their car’s door, came down and without anyone knowing it ran as fast as his legs could carry him across the tarmac to his father on the other side of the road.

Without any doubt, the boy did not look to check if there were any vehicles coming from either direction. His young mind cannot understand nor comprehend that accident do occur and that running across any road carelessly can result in an accident occurring, which may be fatal.

Fortunately, there were no cars coming from either direction. Except for an “Okada” (commercial motorcyclist) who carefully avoided the on-rushing boy and then verbally abused the parent thoroughly for being so careless and irresponsible. The adults absorbed the insult without saying a word and even thanked the “Okada man” for being careful.

The boy’s father quietly carried the boy and explained to him that what he just did was bad and wrong. The boy was made to promise that he will never do it again.

A second family reported that because of the incessant biting of mosquitoes at night in their home, that they bought some locally prepared anti-mosquito agents.

Anyanwu, E. B et al.,: Continental J. Biomedical Sciences 4: 6 - 9, 2010

They have to drop this chemical at the corners of their rooms. To guide against wastage, the father of the house got a syringe which they fill with the chemical agent and then carefully spray out this agent at the desired locations within the house.

Little did he know that he was courting problem by using a syringe to spray the anti-mosquito agent. The family’s mother usually administers prescribed medicinal syrups to her children with the help of syringes, since this help her to measure the quantity of medicines to be given very accurately.

A young son of the family apparently has been observing his father withdrawing the anti-mosquito agents and probably thought that the agent was another medicine. When no adult was around, the boy possibly withdrew some of the agent into the syringe and possibly drank some before he was noticed to be coughing aggressively, and was salivating copiously, was restless and then becoming breathless. On a closer observation, the odour of the anti-mosquito agent was perceived from the boy’s mouth and clothings and they found the abandoned syringe containing some of the agent on the floor of their parent’s room.

It was total chaos from this moment as the whole household rushed the boy to a nearby clinic where he was admitted and treated. Of course, the whole family stayed with the boy throughout the duration of his treatment. He did well and was discharged without any complications. As a unit, the family promptly discarded the anti-mosquito agent on arrival from the hospital.

DISCUSSION

Accidents are the commonest cause of death among children aged between 1 – 4 years of life, causing about half of all deaths of children aged between 10 -14 years of life. Accident also results in many children having permanent disability (Southhall, 2002).

Children’s accidents are closely related to stage of their development. For instance, a new born baby can only fall if dropped by its caretaker, or if the caretaker falls while holding the baby. And older child can climb a tree or fall in the playground. Also, children under five years of age experience accident mostly at home, while school-aged children experience accident at school, on sports playgrounds, and are of particular danger of accidental deaths as pedestrians (Southhall, 2002).

Accidents are also linked to inequalities in environment. Children from poor homes are twice as likely to die from an accident as compared with children from wealthy families. This does not mean that poor parents care less about their children than rich parents, or that they do not know about accident risks. It may mean that there are many other pressures – over crowding, poor housing and lack of money and therefore being unable to afford safety equipment to make changes for safety. Accidents are more common in homes where there is stress from mental illness or marital discord (Southhall, 2002).

Most accidents can be prevented. The three levels of operation at accident prevention:
 * 1) Accidents can be prevented completely/totally. This is known as primary precaution.
 * 2) The consequences of an accident can be minimized. This is known as secondary prevention. Example is the wearing of a seatbelt which can reduce injury even if a car accident occurs.
 * 3) Then lastly, quick attention by professionals can reduce mortality and morbidity. This is known as tertiary prevention which is seen in the pouring of cold water on burns and scalds.

Accidents are prevented therefore through three main approaches which are:
 * 1) Education, whereby increases in knowledge and changes in attitude and behaviour are achieved.
 * 2) Engineering, with safer designs of building and making drug bottles difficult for children to open.
 * 3) Enforcement, which is the role of legislations, regulations and standards in accident preventions (Southhall, 2002).

Anyanwu, E. B et al.,: Continental J. Biomedical Sciences 4: 6 - 9, 2010

The Federal Road Safety Commission was set up essentially to help reduce carnage and loss of lives on highways. They have encouraged the use of seatbelts, advised all drivers to drive carefully past schools and now are campaigning for the use of crash helmets by all cyclists (both motorcycle and bicycle).

The Dean Health System have since 1990 been working to reduce the risk of head injuries by encouraging everyone to wear a crash helmet when riding even a bicycle (Dean Health System)

They have shown that crash helmet when worn correctly acts as a second skull and prevent up to 88% of brain injuries during an accident. They advised that the habit of wearing crash helmets should start early, and also, that adults should set good examples by putting on helmet whenever riding a motorcycle.

A fall from as little as two feet can result in a traumatic brain injury. That is why wearing a helmet while riding a cycle is so important for people of all ages (Dean Health System). Helmets appear to be similarly effective for all age groups, including young children and older adults.

The history of the prevention of accidents to children in England started with the measures taken to improve the safety and health of children who were employed in factories and farms in the 18th – 19th centuries. Before the industrial revolution, children were the victims of accidents of ordinary everyday life.

It was the industrial revolution that changed matters for the best. Large numbers of children were employed into industries with no consideration to their safety, but overtime several legislative acts were passed which focused on the children’s safety (Jackson, 1995).

Accidental injuries to infants and children are often serious, but are largely prevented with appropriate information and safe practices. Young children are particularly vulnerable to accidents due to their innate desire to explore their world and the inability to perceive the dangers of their actions. As children learn through experience, minor injuries are therefore inevitable but the provision of a safe environment can reduce the risks, with close supervision and setting limits of safety by guardians and parents (Shendurnikar, N., Internet Journal).

Therefore, the following advice can be helpful to both parents and all guardians everywhere.
 * 1) Always read labels carefully before administering any medicine to a child. All medicines should be kept away from children’s reach. Discard all old and partially used medicines.
 * 2) Young children should not play in the road. Teach the “stop, look, listen” code at the roadside whenever you are with the child. Teach your child to look left and right before crossing roads.
 * 3) Keep your cupboards securely locked, as these are one of the favourite places for children to hide. Accidental closure can result in choking.
 * 4) Small objects like beans, buttons, beads and even safety pins must be kept out of reach of children particularly below the age of four years.
 * 5) Young children should be made to sit in the backseat of the car. If they sit in front, the use of seatbelt should be mandatory.
 * 6) Do not allow children to play with plastic bags covering chairs heads and faces, as these can cause asphyxiation.
 * 7) Do not allow children to perform new skills without giving them proper demonstration and training.
 * 8) Do not allow children to play and run with sharp objects in their mouths. Accidental falls can cause severe laceration in the mouth and throat.
 * 9) Do not hold your baby in the lap while drinking anything hot or while cooking.
 * 10) Keep household chemicals and kerosene out of reach preferably in a locked cupboard.
 * 11) Never leave young children alone in the house.
 * 12) Keep matches out of reach of young children.
 * 13) Be particularly careful of pots and kettles containing hot fluids. They can easily be pulled on to children, resulting in severe burns (Southhall, 2002 & Shendurnikar, N., Internet Journal)..

Anyanwu, E. B et al.,: Continental J. Biomedical Sciences 4: 6 - 9, 2010

Accidents do occur, but conscious effort must be made to prevent them from occurring. Our children must be protected at all times and all parents are hereby called upon to improve their household and possibly support media campaigns.

REFERENCES

<p style="margin-left: 36pt;">Dean Health System. Crash Helmet Safety Program. http://www.deancare.com/dhs/crash/

<p style="margin-left: 36pt;">Ditmar, M. F., 1997: Adolescent Medicien. In: Pediatric Secrets. Polin, R. A., and Ditmar, M. F. (Eds.) 2nd Edition.

<p style="margin-left: 36pt;">Hanley and Belfus, Inc Philadephia (Publ.) pg. 1 – 24.

<p style="margin-left: 36pt;">Jackson, R. H., (1995): The history of childhood accident and injury prevention in England: background to the

<p style="margin-left: 36pt;">foundation of the Children Accident Prevention Trust. Inj Prev. 1 (1): 4 – 6.

<p style="margin-left: 36pt;">Shendurnikar, N. Accident Prevention in childhood. Available from: http://www.indiaparenting.com/ raising

<p style="margin-left: 36pt;">child/data/raising child027.shtml.

<p style="margin-left: 36pt;">Southhall, D., (2002): Childhood accidents and their prevention. In: Southhall, D., Coulter, B., Ronald, C.,

<p style="margin-left: 36pt;">Nicholson, S., and Parke, S. (eds). International Child Health Care. A Practical Manual for Hospitals Worldwide.

<p style="margin-left: 36pt;">Child Advocacy International. BMJ Books. United Kingdom, pp 493 – 494.

Received for Publication: 13/12/2009

Accepted for Publication: 12/05/2010

Corresponding Author:

<p style="margin-left: 180pt;">E. B. Anyanwu,

<p style="margin-left: 180pt;">Department of Family Medicine, Faculty of Clinical Medicine, Delta State University, Abraka, Nigeria

<p style="margin-left: 180pt;">E-mail: ebirian@yayoo.com

Continental J. Biomedical Sciences 4: 10 - 15, 2010 ISSN:2141 – 419X

©Wilolud Journals, 2010 http://www.wiloludjournal.com

ETHANOLIC EXTRACT OF P'hoenix dactylifera L. PREVENTS LEAD INDUCED HEMATOTOXICITY IN RATS

1Alhassan Abdul Wahab, 2Mohammed Abdel-Aziz Mabrouk, 1Joshua Mathew Joro, 3Salawu Emmanuel Oluwatobi, 4Zainab Mahmood Bauchi and 5Ajibade Adeshina John

Department of Human Physiology, Faculty of Medicine,Ahmadu Bello University, Zaria, Nigeria, 2Department of Human Physiology, Faculty of Medicine, Bayero University, Kano, Nigeria,3Department of Physiology,Faculty of Basic Medical Sciences,College of Medicine, Ladoke Akintola University of Technology, Ogbomoso, Nigeria,4Department of Human Anatomy, Faculty of Medicine, Ahmadu Bello University, Zaria, Nigeria,5Department of Anatomy,Faculty of Basic Medical Sciences,College of Medicine,Ladoke Akintola University of Technology, Ogbomoso, Nigeria

<p style="margin-left: 43.2pt;">ABSTRACT

<p style="margin-left: 43.2pt;">Lead is a heavy metal that has been known for its adverse effects on many body organs and systems and thus their functions. In this study, the toxic effect of lead on blood was investigated, and ethanolic extract of Phoenixdactylifera L. (EPD) (a well known nutritious, antioxidant, and medicinal fruit) was administered orally to prevent lead’s toxicity. Forty adult male Wister rats, randomly divided into four groups (n = 10), were used for this study Group B and Group D were given 200 g of EPD/Kg Body Weight/Day (orally) and 1% sodium acetate and Lead acetate respectively, while group A (control) and group C were given sodium acetate and lead acetate respectively. All treatments were for eight weeks. The animals were weighed and then sacrificed twenty-four hours after the last treatment. Hematocrit, red and white blood cell counts (RBC and WBC), differential count, hemoglobin concentration and other hematological parameters were determined. The data obtained were compared using t-test. The results showed that lead caused a significant decrease in hematocrit, RBC, WBC, hemoglobin concentration, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration,andlymphocyte and monocyte count; and significant increase in neutrophil count. These were,however, prevented in the EPD treated groups. It is therefore concluded that oral admistration of EPD promotes blood’s healthand annuls leadinduced hematotoxicity.'''

<p style="margin-left: 43.2pt;">KEY WORDS: Phoenix dactylifera, lead, blood, anemia, date palm

INTRODUCTION

Lead is one of the first metals to be smelted and used (Aicheson, 1960). It probably was first mined in Turkey around 3500 BC (Alan, 2010). Its density, workability and corrosion resistance were among the metals attractions (Flora, 2008). Reasonably, lead poisoning [plumbism or painter’s Colic (Kosnett, 2006)] is one of the oldest known environmental hazards (Flora, 2008). Also, no level of lead in the body below which no harm can occur has been discovered (Merrill et al., 2007). Worst still, the levels of lead found in most people today are of magnitudes greater than those of pre-industrial times (Merrill et al., 2007; Patrick 2006). In fact, humans get exposed to lead through various media: air, water, soil, food and consumer products (Gärtner, 1997; Gidlow, 2004; Thompson, 2006).

Lead interferes with a variety of body processes and is toxic to the body systems including the cardiovascular, reproductive, haematopoietic, gastrointestinal, and nervous systems (Kosnett, 2006), renal functions (Patocka and Cerný, 2003), release of glutamate and learning (Xu et al., 2006). It affects the hematological system [even at concentrations below 10µg/dl (ATSDR, 2005)] by inhibiting the activities of several enzymes [particularly Amino Levulinic Acid Dehydratase (ALAD)] involved in heme biosynthesis and shortening of erythrocyte life span (Sakai et al., 1999) as well as inducing inappropriate production of erythropoietin leading to inadequate maturation of red cell progenitors and affecting the introduction of Fe2+ into protoporphyrin IX (Taketani et al., 2001). Even though manifestations of Pb poisoning in humans are non-specific, they are always accompanied by oxidation (Hande et al., 2004).

On the other hand date palm (Phoenix dactylifera) fruits, an important component of diet in the arid and semiarid regions of the world (Biglari et al., 2008), are widely used in traditional medicine for the treatment of various

Alhassan Abdul Wahab et al.,: Continental J. Biomedical Sciences 4: 10 - 15, 2010

disorders e.g. memory disturbances, fever, inflammation, paralysis, loss of consciousness, nervous disorders (Nadkarni, 2006), and as a detersive and astringent in intestinal troubles. It is also used in the treatment for sore throat, colds, bronchial asthma, to relieve fever, cystitis, gonorrhea, edema, liver and abdominal troubles and to counteract alcohol intoxication (Barh and Mazumdar, 2008). It possesses anticancer, antimutagenic, antihyperlipidemic, nephroprotective, and in vivo antiviral activities, and the ability to increase the concentration of testosterone, follicle stimulating hormone and luteininzing hormone (Bahmanpour et al., 2006). Many researchers have also documented the antioxidant property of Phoenix dactylifera (Mohamed and Al-Okbi,2004; Allaith and Abdul, 2005; Al-Qarawi et al., 2008).

Since humans are inseparable from their environment and almost entirely not free from exposure to lead (most especially that lead has almost always found an important position in many industries), it is very necessary to figure out ways by which our body can be made to still maintain homeostasis (basis of good health) even on exposure to relatively high level of lead exposure. This research work, therefore, aims at knowing whether or not oral administration of extract of Phoenix dactylifera (EPD) prevents lead induced hemato-toxicity base on the knowledge that EPDhas nutritious, medicinal, and antioxidant components.

MATERIALS AND METHODS

Forty adult male Wister rats [average Body Weight (BW) 186.70 ± 0.511 g] obtained from the animal house section of Faculty of Pharmaceutical Science, Ahmadu Bello University, Zaria, Nigeria, were used for this study. The animals were kept in the animal house section of Human Physiology Department, Ahmadu Bello University, Zaria, Nigeria, and allowed to acclimatize over a period of ten days.

PLANT MATERIALS

The fruits of Phoenix dactylifera were bought from a local market in Zaria, Kaduna State, Nigeriaand authenticated at the Department of Biological Science, Ahmadu Bello University, Zaria, Nigeria.

PREPARATION OF ETHANOLIC EXTRACT OF PHOENIX DACTYLIFERA (EPD)

The extract of the fruits was prepared in the Department of Pharmacognosy and Drug Development, Ahmadu Bello University, Zaria. The extract was prepared by removing the shells of the fruit and the fruit grounded into fine powder. The fine powder was left to dry under shade, it was soaked in ethanol (inside a separating funnel plugged with cotton wool) for 24 hours. It was drained and rewashed with more ethanol. The filtrate was poured in an evaporating dish and placed on a water bath at 80 oC, and reduced pressure (70% atmospheric pressure) to remove the solvent. After all the solvent had evaporated, the extract was scrapped and stored in a dry glass container at a temperature of 0 – 4 oC. Fresh solution of the extract was prepared in distilled water just before use to maintain its potency.

ANIMAL TREATMENT

The forty rats were randomly grouped into four (Group A, B, C and D, n = 10). Rats in group A served as the control and were neither exposed to lead nor treated withEPD, but they were allowed to drink 1% sodium acetate ad libitum. Group B rats were allowed to drink 1% sodium acetate ad libitum and also administered 200 mg of EPD/Kg BW/Day. Group C and D were allowed to drink 1 % lead acetate ad libitum, while group D rats were in addition administered 200 mg of EPD/Kg BW/Day. All treatments were for eight weeks.

ANIMAL SACRIFICE AND COLLECTION OF SAMPLES

Twenty-four hours after the last treatment, each animal was weighed and then sacrificed by cervical dislocation and blood samples were collected via cardiac puncture. Blood sample obtained from each rat was immediately transferred into EDTA bottle and mixed thoroughly although gently.

COLLECTION OF DATA AND STATISTICAL ANALYSIS

PCV was determined using plain capillary tubes filled with anti-coagulated blood and spinning at 5.1 x g for 20 min. RBC and WBC were determined using Improved Neubauer counting chamber and following the procedure documented by Chessbrough (1976). Field’s stain A and Field’s stain B were used for the Differential WBC.

The control and “Test groups” were compared using t-test. The significant level was set to P value < 0.05.

Alhassan Abdul Wahab et al.,: Continental J. Biomedical Sciences 4: 10 - 15, 2010

RESULTS

The following results were obtained and are presented as mean ± SEM and level of significance is taken at “p value < 0.05” (*), “p value < 0.001” (**) and/or “p value < 0.0001” (***).

WEIGHT INCREASE (G)

Comparing their final and initial weight showed that there was significant weight gain (P-value < 0.05) is in all the groups over the 10 weeks of the research. There was, however, no significant difference (P-value > 0.05) in weight gain of NA+EPDG, LA+EPDG and Control, while weight increase in LAGhad a significantly lower value when compared to the Control (Table 1).

PACKED CELL VOLUME (PCV, %), RED BLOOD CELL COUNT (RBC, CELLS/mm3), AND WHITE BLOOD CELL COUNT (WBC, CELLS/mm3), HEMOGLOBIN CONCENTRATION (Hb, G/DL), MEAN CORPUSCULAR VOLUME (MCV, FL), MEAN CORPUSCULAR HEMOGLOBIN (MCH, PG), AND MEAN CORPUSCULAR HEMOGLOBIN CONCENTRATION (MCHC, G/DL)

The PCV and RBC of NA+EPDG and LA+EPDG were significantly (P-value < 0.05, and P-value < 0.001 respectively) higher than that of the control. However, PCV and RBC of LAG was found to be significantly (P-value < 0.05) lower than that of the control.Similar trends were noted for Hb, MCH, and MCHC. While WBC and MCHC of LAG were found to be significantly (P-value < 0.01) lower than that of the control; but LA+EPDG and NA+EPDG showed no significant (P-value > 0.05) difference from the Control (Table 2).

DIFFERENTIAL COUNT

Differential count showed that LAG, unlike NA+EPDG and LA+EPDG, has significantly (P-value < 0.05) lower percentage of Lymphocyte and Monocyte, but significantly (P-value < 0.05) higher percentage of Neutrophil. Percentages of Eosinophil and Basophil of NA+EPD and LA+EPD were however not different from that of the Control (Table 3).

DISCUSSION

The results of this study show thatchronic exposure to Pb significantly (P-value < 0.05) reduces weight gain (Table 1), this is in support of the findings of Suzan and Ghayasuddin (1999) and can be linked to the less efficient metabolic processes associated with Pb toxicity (Struzyńska et al., 1997), that is the anti-metabolic effect of Lead (Biswas and Ghosh, 2004). Administration of 200 mg of EPD/Kg BW/Day, however, annuls this Pb’s adverse effect on weight gain; it in fact had a significant (P-value < 0.05)enhancing effect on the final BW of group B and group D. This may be partly due to the fatty acid composition of EPD (National Institute for Health and Welfare, 2009);and mainly due to its relatively high caloric value (284 Kcal/100g) (National Institute for Health and Welfare, 2009) and the presence of health-protective antioxidants such as melatonin, vitamin E, and ascorbic acidin EPD (Al-Qarawi et al., 2008)despite its relatively low protein content (6% by weight) (National Institute for Health and Welfare, 2009). These can similarly explain the significant (P-value < 0.05)decrease in PCV noticed in animals exposed to Pb (LAG) and the non significant (P-value > 0.05)decrease in PCV noticed in animals treated with EPD alongside Pb exposure (LA+EPDG) (Table 2). This finding that Pb reduces PCV is in support of the findings of Anetor et al., (2002)that some indices of erythropoietic activity such as Hb, PCV and MCHC are significantly decreased in workers exposed to lead. It however opposes the findings of Franson et al., (1983) that PCV is not affected in birds by 50 ppm of Pb in their feed; and that of Arvind and Chopra, (2003) that PCV is not affected in calves by 100 ppm Pb in the diet. It is, however, possible that the findings of Franson et al., (1983) and Arvind and Chopra, (2003) are due to the low concentration of Pb (</= 100 ppm) administered as well as the relatively short period of administration.

The significantly (P-value < 0.05) high RBC in both NA+EPDG and LA+EPDG, and the significantly (P-value < 0.05) low RBC in LAG compared to the control [Table 2] support the findings of James, (2007) and Salawu et al., (2010)that lead induces anemia especially when the exposure is chronic; and the findings of Abuharfeil et al., (1999) that Phoenix dactylifera promotes RBC by preventing hemolysis.These observations could be accounted for by the ability of Pb to interfere with energy metabolism of erythrocytes (Irena and Alina, 2003) which is one of the determinants of ATP concentration in erythrocytes and thus erythrocytes’ life span and RBC. This reduction in RBC could still be linked to the findings of Bernard et al., (1972) that acute lead (Pb) toxicity in mice produced transient

Alhassan Abdul Wahab et al.,: Continental J. Biomedical Sciences 4: 10 - 15, 2010

erythroidhypoplasia and impaired utilization of RBC 59Fe for heme synthesis. The administered EPD would therefore be responsible for the prevention of these lowering effects of Pb on RBC (and even the enhancement of RBC) in LA+EPDG\ probably by preventing its adverse effects on energy metabolism of erythrocytes and on the utilization of RBC 59Fe for heme synthesis.This becomes more evident by the significantly high (P-value < 0.05) RBC found in NA+EPDG as well (Table 2).

The significantly (P-value < 0.0001) lower WBC in LAG, and the non-significant (P-value > 0.05) difference in the WBC of both NA+EPDG and LA+EPDG with respect to the control, further establishes that lead adversely affects blood and blood cells. This observation is in line with the previous publications of Nedjet et al., (2009) and Karmaus et al., (2005) that lead exposure is associated with a reduction in WBC. It also lends supports to scientific claims that lead exposure may lower body’s immunity (Grasman and Scanlon, 1995; Rabinowitz et al., 1990), more so that white blood cells are important part of the immune system (Karmaus et al., 2005). Therefore, EPD must have somehow prevented Pb’s adverse effects on WBC, such that there was no significant difference in WBC of control and that of LA+EPDG. This could most reasonably be linked to the detoxification as well as hepatoprotective effect of EPD (Al-Quarawi et al., 2005).

In the present study, just as documented by Sembulingam and Sembuligam (2006)that Pb (like some other chemicals and drugs) causes netrophilia, Pb caused a significant (P-value < 0.05) increase in Neutrophil count but significantly (P-value < 0.05) decreased Lymphocyte and Monocyte counts (Table 3). This is as well in support of the findings of Di Lorenzoet al., (2006)that mean absolute neutrophil count was significantly higher in workers exposed to Pb with respect to workers not exposed to Pb.In addition Neutrophils and lymphocytes were noticed to vary in opposite direction, which is as well in support of the documentation of Sembulingam and Sembuligam, (2006). However, EPD was able to prevent Pb from affecting not only WBC but differential count as well.

Conclusively, lead causes significant decrease in hematocrit, RBC, WBC, hemoglobin concentration, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration,andlymphocyte and monocyte count; and significant increase in neutrophil count. However, oral administration of EPD prevents these lead’s adverse effects (in rats). It is therefore reasonable to conclud that oral admistration of EPD promotes blood’s healthand annuls leadinduced hematotoxicity.

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<p style="margin-left: 4.5pt;">Aicheson L. A History of Metals; Interscience, New York. 1960: 54-61.

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<p style="margin-left: 4.5pt;">Arvind K, Chopra RC. Lead concentration in feeds and fodders and it's effect on growth, nutrient utilization and certain blood parameters in crossbred calves. Indian J Anim Nutr. 2003; 20(4):412-20.

<p style="margin-left: 4.5pt;">Bernard SM, Gerald JG, Dennis GG. Abnormal Erythroid Maturation Following Acute Lead Toxicity in Mice. Blood. 1972; 39(5):713-720.

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<p style="margin-left: 4.5pt;">Biswas NM and Ghosh P. Effect of Lead on gonadal activity in albino rats. Kathmandu University Medical Journal 2004;2: 43-46.

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<p style="margin-left: 4.5pt;">Di Lorenzo L, Silvestroni A, Martino MG, Gagliardi T, Corfiati M, Soleo L. Evaluation of peripheral blood neutrophil leucocytes in lead-exposed workers. International archives of occupational and environmental health. 2006; 79(6):491-498.

<p style="margin-left: 4.5pt;">Flora SJ, Mittal M and Mehta A. Heavy metal induced Ox idative stress and its possible reversal by Chelation therapy. The Indian Journal of Medical Research 2008;128(4): 501–23.

<p style="margin-left: 4.5pt;">Franson JC, Sileo L, Pattee OH, and Moore JF. Effects of chronic dietary lead in American kestrels (Falco sparverius). Journal of Wildlife Diseases. 1983;19(2):110-113

<p style="margin-left: 4.5pt;">Gärtner C, Stahl W, Sies H. Lycopene is more bioavailable from tomato paste than from fresh tomatoes. ASCN Annual Meeting. 1997; 66(1):199-222.

<p style="margin-left: 4.5pt;">Gidlow DA. Lead toxicity. Occup Med (Lond) 2004;54:76-81.

<p style="margin-left: 4.5pt;">Grasman KA, Scanlon PF. Effects of acute lead ingestion and diet on antibody and T-cell mediated immunity in Japanese quail. Archives of Environmental Contamination and Toxicology 1995;28(2):161-7.

<p style="margin-left: 4.5pt;">Hande GO, Handan US and Hilal Ö. Correlation between clinical indicators of lead poisoning and oxidative stress parameters in controls and lead-exposed workers. Toxicology. 2004; 195(2-3):147-154.

<p style="margin-left: 4.5pt;">Irena BB, Alina JH. The effect of lead ions on the energy metabolism of human erythrocytes in vitro Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology. 2003; 134(3):403-416.

<p style="margin-left: 4.5pt;">Karmaus W, Brooks KR, Nebe T, Witten J, Obi-Osius N, Krus H. Immune function biomarkers in children exposed to lead and organochlorine compounds: a cross-sectional study. Environmental Health 2005;4(5):1-10.

<p style="margin-left: 4.5pt;">Kosnett, (2006). Global approach to reducing Lead exposure and poisoning.Mutation research, 659(1-2): 166-175.

<p style="margin-left: 4.5pt;">Merrill D nad Morton, Soileau R. Mechanisms of Lead induced hypertension and cardiovascular diseases.American Journal of physiology 2007;295(2):454–465.

<p style="margin-left: 4.5pt;">Mohamed, D. A. and Al-Okbi, S. Y. (2004). In vivo evaluation of antioxidant and anti-inflammatory activity of different extracts of date fruits in adjuvant arthritis. Polish journal of food and nutrition sciences, 13: 397-402.

<p style="margin-left: 4.5pt;">National Institute for Health and Welfare. (2009). Fineli: Walnut. Available from: http://www.fineli.fi/food.php?foodid=1203&lang=en. Accessed on: 16/11/2009, 11:12 GMT.

<p style="margin-left: 4.5pt;">NedjetS, Ali K, Yildiray K, Osman NK, Bunyami Unal. Spirulina platensis feeding inhibited the anemia- and leucopenia-induced lead and cadmium in rats. Journal of Hazadous Materials 2009;164(2-3):1304-9.

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<p style="margin-left: 4.5pt;">Sakai, T., Yanagihara S. and Ushio, K. (1999). Erythrocyte factors concerned in the inhibition of ALAD by lead. British'Journal of Industrial Med'icine, 38, 268-274

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<p style="margin-left: 4.5pt;">Sembulingam K, Sembulingam P. Essentials of medical physiology. 4th ed. Jaypee Brothers Medical Publisher (P) LTD. New Delhi. 2006; 72-73.

<p style="margin-left: 4.5pt;">Struzyńska L, Dabrowska-Bouta B, Rafałowska U. Acute lead toxicity and energy metabolism in rat brain synaptosomes. Acta Neurobiol Exp (Wars). 1997; 57(4):275-81.

<p style="margin-left: 4.5pt;">Suzan AW, Ghayasuddin A. Effects of Lead on the Male Reproductive System in Mice. Journal of Toxicology and Environmental Health, 1999; Part A, 56(7): 513 – 521.

<p style="margin-left: 4.5pt;">Thompson KA, Marshall MR, Sims CA, Wei CI, Sargent SA, Scott JW. Cultivar, Maturity, and Heat Treatment on Lycopene Content in Tomatoes. Journal of Food Science. 2006; 65(5):791-795.

<p style="margin-left: 4.5pt;">Xu HH, Chen ZP and Shen Y. Meta analysis for effect of lead on male reproductive function. Journal of Industrial hygiene and occupational disease 2006; 24(10): 634-36.

Table 1: Weight Increase (g) after the 10 Weeks of Research *“P-value < 0.05”

<p style="margin-left: 72pt;">Table 2: Packed Cell Volume (PCV, %), Red Blood Cell Count (RBC, cells/mm3), and White Blood Cell Count (WBC, cells/mm3), Hemoglobin concentration (Hb, g/dl), Mean Corpuscular Volume (MCV, fl), Mean Corpuscular Hemoglobin (MCH, pg), and Mean Corpuscular Hemoglobin Concentration (MCHC, g/dl) *“P-value < 0.05”, **“P-value < 0.001”, ***“P-value < 0.0001”

<p style="margin-left: 72pt;">Table 3: Comparison of Percentage of White Blood Cells that is Neutrophil, Lymphocyte, Monocyte, Eosinophil and Basophil Sperm Motility across the three Groups *“P-value < 0.05”

Received for Publication: 29/05/2010

Accepted for Publication: 23/06/2010

Corresponding Author

Salawu Emmanuel Oluwatobi

Box 233, Ejigbo, Osun State, Nigeria

Email: [mailto:seocatholic@gmail.com seocatholic@gmail.com]

Continental J. Biomedical Sciences 4: 16 - 20, 2010 ISSN:2141 – 419X

©Wilolud Journals, 2010 http://www.wiloludjournal.com

STRESS REVERSIBLY AFFECTS IMMUNITY IN RATS

1SALAWU Emmanuel Oluwatobi, 2ALHASSAN Abdul Wahab, 3MABAYOJE Victor Olatunji, 4ADEEYO Olusola Atilade, 1SAKA Waidi Adeoye, 1ISHOLA Olufunto Olayinka

1Department of Physiology, and 4Department of Anatomy, Faculty of Basic Medical Sciences, Ladoke AkintolaUniversity of Technology, Ogbomoso, Nigeria, 2Department of Human Physiology, Faculty of Medicine, Ahmadu Bello University, Zaria, Nigeria, 3Department of Heamatology, Faculty of Basic Medical Sciences, College of Medicine, Ladoke Akintola University of Technology, Osogbo, Nigeria

<p style="margin-left: 43.2pt;">ABSTRACT

<p style="margin-left: 43.2pt;">This study investigated the effects of warm water swim stress and its withdrawal on the immunity of rats. Thirty-two adult male Wistar rats, average body weight (BW) 191.84 ± 1.61g grouped into four groups (n=8) were used for this study. Group A was the control and was not exposed to any particular stress; Group B rats were made to swim in warm (34 ± 0.5 oC) water for 3 minutes/day; and Group C rats in warm (37 ± 0.5 oC) water for 6 min/day; while Group D rats were made to swim in warm (40 ± 0.5 oC) water for 12 min/day. These continued for three weeks, after which each group was divided into two Sub-Groups (nSG=4). Rats in one subgroup from each of the four groups were sacrificed 24 h after the last day of the 3 weeks of swim-stress, while the other sub-group from each of the four groups were left for 3 more weeks (to recover from the stress) before they were sacrificed. The WBC, CD4, and Differential WBC counts in the test and control groups were compared using independent-sample t-test. The results showed that the stress in groups B and C was moderate and significantly boosted the rats’ immune components, while the stress in group D was severe and significantly reduce the rats’ immunity. However, these changes were reversible (although not completely) upon stress withdrawal for three weeks.

<p style="margin-left: 43.2pt;">KEYWORDS: Stress, immunity, blood, WBC, CD4, rat, differential WBC

INTRODUCTION

Various quantity and/or types of pressure (large or small; physical or psychological) could impose stress of different degrees on different individuals (Selye, 1950; Viner, 1999; Keil, 2004). Mild to moderate stress can be motivational and improve performance. In fact, some stress may help the body to prepare for certain challenges (Selye, 1950; Viner, 1999). However, a considerable amount of literature have documented it that too much of stress lead to physical, mental, and emotional (i.e. health) problems (Coren, 2008). In other words, sever and prolonged (unlike moderate) stress could breakdown mind and body systems.

Since stress is able to affect the state of health, it is reasonable and straight forward to believe that stress is capable of affecting or influencing the protective functions of the immune system. If stress would affect the immune system (Segerstrom and Miller, 2004; Herbert and Cohen, 1993), then: “What becomes of stress’s effects on immunity after stress withdrawal?” “Will the effects be permanent or reversible?” If reversible, “how long will the required unaided recovery period be?” “Will the recovery be complete?” All of these make important research questions that had neither been answered nor previously thoroughly explored.

This research, therefore, evaluated the effects of graded amounts of stress and different durations of stress, as well as their withdrawal on immune system of adult male Wistar rats, with the aim of providing answers to the above research questions.

MATERIALS AND METHODS

Thirty-two adult male Wistar rats, average body weight (BW) 191.84 ± 1.61g, obtained from the animal house section of the Faculty of Pharmaceutical Sciences, Ahmadu Bello University, Zaria, Nigeria were used for this study. The animals were allowed to acclimatize over a period of ten days.

SALAWU Emmanuel Oluwatobi et al.,: Continental J. Biomedical Sciences 4: 16 - 20, 2010

Experimental Design and Animal Treatment

The thirty-two rats were randomly grouped into four (Group A, B, C and D, n = 8). Rats in group A were the control and were not exposed to any particular stress; Group B rats were made to swim in warm (34 ± 0.5 oC) water for 3 min/day; and Group C rats in warm (37 ± 0.5 oC) water for 6 min/day; while Group D rats were made to swim in warm (40 ± 0.5 oC) water for 12 min/day. These continued for three weeks, after which each group was divided into two Sub-Groups (nSG=4). Rats in one subgroup from each of the four groups were sacrificed 24 hours after the last day of the 3 weeks of swim-stress, while the other sub-group from each of the four groups were left for 3 more weeks (to recover from the stress) before they were sacrificed.

Collection of Samples

Each rat was weighed before sacrificing by cervical dislocation, and blood samples were collected via cardiac puncture. Blood sample obtained from each rat was immediately transferred into EDTA bottle and mixed gently and thoroughly.

Collection of Data and Statistical Analysis

WBC, CD4 counts were determined using Improved Neubauer counting chamber and following the procedure documented by Chessbrough (1976). Field’s stain A and Field’s stain B were used for the Differential WBC.

The control and “Test groups” were compared using independent-samples t-test. The significant level was set to P value < 0.05.

RESULTS

The following results were obtained and are presented as mean ± SEM and level of significance is taken at “p value < 0.05” (*).

Weight Increase (g) 

Comparing their final and initial weight showed that there was significant weight gain (P-value < 0.05) is in all the groups over the three or six weeks of the research. There was, however, no significant difference in weight gain of groups B C, and DR compared to the control, while weight increase in DNR had a significantly lower value when compared to the Control (Table 1).

White Blood Cell Count (WBC), CD4, and Differential Count

The WBC was significantly reduced in both DNR and DR, while it was significantly higher in the BNR and CNR compared to the control. However, the WBC for BR and CR were not significantly different from that of the respective control (Table 2).

The CD4 count was significantly higher for BNR, BR, CNR and CR, while it was significantly lower in DNR and DR compared to the respective controls.

For the differential count, the percentages of WBC made up by neutrophils were significantly higher for DNR and CNR, but significantly lower for BNR, while all the recovery groups showed no significant different in the percentages of neutrophils from those of the control (Table 2). An opposing trend was noted for the percentages of WBC made up by lymphocytes, such that significantly lower for DNR and CNR, but significantly higher for BNR. The percentages for monocytes and basophils were, however, not significantly different throughout all the groups (Table 2).

The percentages of WBC made up by Eosinophils were found to be significantly (P-value < 0.01) lower in CNR, but not significantly affected in other groups compared to the control (Table 2).

DISCUSSION

The significantly lower weight gain in DNR, and the non-significant difference in the weight gain of all the subgroups of group B and C, and DR (Table 1) is a telescope to the adverse effects of considerable amount of stress (i.e. sever and chronic stress) on living systems. Although, significant growth impairment cannot be used solely for a specific diagnosis, still it is a non-specific indicator of health problem, as it at least tells us that something is wrong somewhere in one of the body systems, perhaps in the immunological system as the case may be. Previous

SALAWU Emmanuel Oluwatobi et al.,: Continental J. Biomedical Sciences 4: 16 - 20, 2010

publications of Wallace et al. (1995), Powera et al. (2000), Markowitz and Daum (2008), etc have earlier documented similar association between the different disease states and body weight loss/growth impairment.

The speculations from the gain in body weight/growth impairment (above) becomes more evident and better based by the significantly low WBC in both DNR and DR, the significantly high WBC in BNRand CNR; and the non-significant difference in the WBC of BR and CR compared to the control (Table 2). In other words, the significantly increased WBC in BNR and CNR is an indication that moderate stress could raise immune level, while the highly significantly lower WBC in DNR indicates that severe stress has potential to adversely affect body immune level. The (considerably) significantly high CD4 count in BNR and BR, and the less significantly high CD4 count in CNR and CR (Table 2) can be linked to the activities of adrenergic stress hormones: cortisol, epinephrine, and norepinephrine. It is believed that when undergoing stress, the adrenal cortex releases the adrenergic stress hormones which increase the synthesis and release of cytokins, which consequently brings about the production of more white blood cells, more helper T-cells (CD4 cells), and thus boosts the immune system (Rassnick et al., 1994). This can explain the extremely high CD4 count in BNR and BR, since the adrenergic stress hormones (cortisol, epinephrine, and norepinephrine) released from the adrenal cortex (in response to the stress) would have augmented the production of white blood cells, more helper T-cells (CD4 cells), and thus boosts the immune system. One would normally expect even higher white blood cells and helper T-cells (CD4 cells) counts, and more boosts to the immune system in CNR and DNR, since more adrenergic stress hormones (cortisol, epinephrine, and norepinephrine) would be released from the adrenal cortex (in response to higher degree and much prolonged stress in groups C, and D). this was, however, not the case. The observed lower white blood cells and helper T-cells (CD4 cells) counts in CNR and DNR (Table 2) could be a surprise at first thought. But subsequent thoughts, and the fact that a high degree and prolonged stress leads to the build up of cortisol and other adrenergic stress hormones (not used up) in the body, would make this observation justifiable, since the build up of these unused adrenergic stress hormones is known to hinder the normal functioning of the immune system as well as reduced WBC and CD4 counts. In fact, previous works of Coren (2008) and Naliboff et al. (1991) among others had documented similar findings.

In a similar way, the withdrawal of the source of stress in the recovery groups caused the WBC and CD4 counts for each of the experimental (recovery) groups to return towards the values of the control groups (Table 2). This could be linked to the using up of the previously built up adrenergic hormones. It was however noted that these values (of WBC and CD4 counts in the recovery groups) could not totally return to the values of the control in the three weeks allowed for recovery. This observation raises an important speculation that the time it may require to completely recover (unaided) from an episode of stress upon stress withdrawal will most often be more than the actual duration of the stress, even though considerable recovery could still be accomplished within a recovery period that is of the same length as the duration of stress exposure. In other words, withdrawal of stress for three weeks might not just be enough to completely (and naturally) recover from a three-week episode of considerable stress, but could bring about the accomplishment of a considerable amount of recovery.

The significantly increased lymphocyte count in BNR,and the significantly reduced lymphocyte count in CNR and DNR (Table 2) is parallel to other findings of this work and further establishes that moderate stress augments immunity while, severe stress brake it down, since the lymphocytes (and plasma cells) function mainly in connection with the immune system (Guyton and Hall, 2006). Dorian et al. (1982) and Kenji et al. (2000) have also documented similar trend for lymphocyte count with respect to exposure to stress. Dorian et al. (1982) found that there was stress-induced transiently elevated numbers of lymphocytes in eight psychiatry trainees taking their final oral fellowship examinations. For Kenji et al. (2000) surgical stress increases lymphocyte subsets and decreases the subsets that promote cellular immunity leading to cellular immunosuppression. This is parallel to many findings of this research.

However, neutrophils and lymphocytes were noticed to vary in opposite direction [Table 2], which is as well in support of the documentations of Sembulingam and Sembuligam (2006) as well as the findings of Kenji et al. (2000). In other words, neutrophils count was significantly reduced in BNR,but significantly increased in CNR and DNR. This increase in neutrophils (a typical granulocytes) in the groups exposed to considerably severe stress presents another important (otherwise hidden) fact that even though considerably severe stress may significantly hinder normal immune functions, it, however, may not completely have adverse affects on all the body’s defence lines/levels, since a typical granulocyte (neutrophils, in this case) which protects the body against invading

SALAWU Emmanuel Oluwatobi et al.,: Continental J. Biomedical Sciences 4: 16 - 20, 2010

organisms mainly by ingesting them (that is, by phagocytosis), was significantly elevated in the rats exposed to considerably severe stress. However, this point would have being more solidified if other granulocytes (eosinophils, basophils) and monocytes in addition to neutrophils were as well significantly increased in CNR and DNR rather than just being comparable (P > 0.05) to those of the control. Perhaps a longer or more sever episode of stress would bring about such trend.

CONCLUSION

Stress, depending on the degree and duration, has both beneficial and suppressive effects on immunity; however, its either effects are often not permanent: they are reversible after its withdrawal. But the period it would take to completely and naturally recover (if at all possible) from the effects of an episode of stress is often more than the actual period of the stress. Even though, this research work establishes that stress could have either beneficial or detrimental effects (depending on the amount of the stress, and the duration of exposure to the stress), and that either of the effects is considerably reversible, we are at present not able to determine the specific amount of stress that is beneficial, and that which is detrimental. A better understanding of the amount of stress that is beneficial, and that which is detrimental could be determined if successive research could focus on estimation of stress and the derivation of a standard unit that could be used universally for the amount of stress in both humans and experimental animals.

REFERENCES

Chessbrough M. (1976). A laboratory manual for rural tropical hospitals. 35-77.

Coren A. Stress - Introduction. [Online]. Accessed from: http://www.nhs.uk/conditions/stress/pages/introduction.aspx Accessed on June, 2010.

Dorian B, Garfinkel P, Brown G, Shore A, Gladman D, Keystone E. (1982). Aberrations in lymphocyte subpopulations and function during psychological stress. Clin Exp Immunol. 50(1):132–8.

Guyton AC, Hall JE. (2006). Textbook of medical physiology. 11th ed. China: Elsevier. p. 429-50.

Herbert TB, Cohen S. (1993). Stress and immunity in humans: a meta-analytic review. Psychosom Med.55:364-379.

Keil RMK. (2004). Coping and stress: a conceptual analysis. J. Adv. Nursing. 45(6):659-65.

Kenji O, Masanori H, Takao K, Minoru M, Kanako H, Takeshi S, Yoshihiko N, Toshihiko H, Tetsuro K. (2000). Suppression of cellular immunity by surgical stress. Surgery.127(3):329-36.

Markowitz J, Daum F. (2008). Growth Impairment in Pediatric Inflammatory Bowel Disease. The American Journal of Gastroenterology. 89(3):319-26.

Naliboff BD, Benton D, Solomon GF, Morley JE, Fahey JL, Bloom ET, Makinodan T, Glimore SL. (1991). Immunological changes in young and old. 65(7):234-56.

Powera C, Lia L, Manorb O. (2000). A prospective study of limiting longstanding illness in early adulthood. International Journal of Epidemiology. 29:131-9.

Rassnick S, Sved AF, Rabin BS. (1994). Locus coeruleus stimulation by corticortropin releasing hormone suppresses in vitro cellular immune responses. Journal of Neuroscience. 14:6033-40.

Sembulingam K, Sembulingam P. (2006). Essentials of medical physiology. 4th ed. Jaypee Brothers Medical Publisher (P) LTD. New Delhi. p. 72-73.

Selye H. (1950). Stress and the general adaptive syndrome. Br. Med. J. 4667:1383-92.

SALAWU Emmanuel Oluwatobi et al.,: Continental J. Biomedical Sciences 4: 16 - 20, 2010

Segerstrom SC, Miller GE. (2004). Psychological stress and the human immune system: A meta-analytic study of 30 years of inquiry. Psychological Bulletin. 130:601–30.

Viner R. (1999). Putting stress in life: Hans Selye and the making of stress theory. Social Studies of Science. 29(3):391-410.

Wallace JI, Schwartz RS, LaCroix AZ, Uhlmann RF, Pearlman RA. (1995). Involuntary weight loss in older outpatients: incidence and clinical significance. J Am Geriatr Soc. 43(4):329-37.

Table 1: Weight Increase (g) after the 10 Weeks of Research Data are expressed in mean ± SEM.


 * =“P-value < 0.01” indicated significant difference when compared with the control group by independent samples t test

Table 2: Comparison of White Blood Cell Count (WBC), CD4, Percentage of White Blood Cells that is Neutrophils (N), Lymphocytes (L), Monocyte (M), Eosinophil (E) and Basophil (B) Data are expressed in mean ± SEM.


 * =“P-value < 0.05”, ** =“P-value < 0.01”, *** =“P-value < 0.001” indicated significant difference when compared with the control group by independent samples t test.

Received for Publication: 29/05/2010

Accepted for Publication: 23/06/2010

Corresponding Author

SALAWU Emmanuel Oluwatobi

Box 233, Ejigbo, Osun State, Nigeria

Email: seocatholic@gmail.com

Continental J. Biomedical Sciences 4: 21 - 36, 2010 ISSN:2141 – 419X

©Wilolud Journals, 2010 http://www.wiloludjournal.com

ENZYMATIC TRANSESTERIFICATION OF LIPASES OBTAINED FROM BACTERIAL AND PLANT SOURCES

1Gayatri Nahak, 2Chandra Kanti Mohanty, 2N.K. Mohapatra and 1R.K. Sahu

1B.J.B. Autonomous College, Bhubaneswar, Orissa, 2Academy of Management and Information Technology, Khorda, Orissa

<p style="margin-left: 43.2pt;">ABSTRACT

<p style="margin-left: 43.2pt;">The use of vegetable oil as a fuel source in diesel engine is as old as the diesel engine itself. However, the demand to develop and utilize plant oils and animal fats as biodiesel fuels has been limited recently. The technical definition of biodiesel is “The mono alkyl ester of long fatty acids derived from renewable lipid feedstock such as vegetable oils or animal fats, for use in compression ignition (diesel) engines” (National Biodiesel Board, 1996). It is one of the many alternative fuel options that can help in oil dependence. Non-edible oils likeJatropha curcas, Ricinus communis, Calophyllum inophyllum etc can be used for the production of biodiesel. With regard to protein/lipase content of the different sources as has been found out, highest protein content was found to be in Callophylum seed followed by J. curcas per gm. of source material. However gross estimate represents the soluble protein of which the activity screening further justifies the use of enzyme extract from Xanthomonas Spp as lipase source for transesterification in a cost effective way. The protocol followed is simple without involvement of much technical complication may be further looked into to standardize a general acceptable method. The bacterial lipase and lipase from Jatropha curcas appear to have highest efficiency in transesterifying Jatropha oil as enzyme used is partially pure. The ester yield is as high 80-85%, can be considered for further research to increase the production keeping the cost of process at lowest minimum. There was no scope to go for kinetic study of enzyme from individual sources along with assessment of quality of ester, therefore it is premature to comment on standardized protocol and experimentally designed to highlight the scale up.

<p style="margin-left: 43.2pt;">KEYWORDS: Biodiesel, Enzymatic transesterification, Lipase, Immobilized enzyme, Ester.

INTRODUCTION

Biodiesel is a renewable fuel that can be synthesized from edible, non-edible and waste oils. Due to diminishing petroleum reserves, vegetable oils have attracted attention as a potential renewable source for the production of alternatives to petroleum based fuel. A number of processes have been developed for biodiesel production involving chemical or enzyme catalysis or supercritical alcohol treatment (Fukuda et al., 2001; Warabi et al., 2004). Enzymatic transesterification of triglycerides is a good alternative to chemical process due to its eco-friendly, selective nature and low temperature requirements (Kaieda et al., 1999; Du et al., 2005). Many starting materials such as soybean oil (Schwab et al., 1988; Samukawa et al., 2000), sunflower oil (Belafi Bako et al., 2002; Dossat et al., 2002), cotton seed oil (Oznur et al., 2002), rapeseed oil (Nelson et al., 1996), palm oil (Abigor et al., 2000; Crabbe et al., 2001) and restaurant kitchen wastes (Hsu et al., 2002) have been evaluated for preparation of biodiesel by the enzymatic route. In many countries, like India, where edible oils are not in surplus supply, there is a need to search for alternative starting materials, such as from non-edible oils. Oil of Jatropha curcas (Euphobiaceae), non-edible oil, has been chosen for the present investigation. The oil content of Jatropha seed ranges from 30 to 50% by weight, whereas in kernel the oil content ranges from 45 to 60%. The fatty acid composition of Jatropha oil consists of oleic acid 43.1%, linoleic acid 34.3%, stearic acid 6.9%, palmitic acid 4.2% and other acids 1.4%. Jatropha curcas is a low-growing tree, generally planted as a hedge for protecting crops from animals. It can be grown on barren land under harsh conditions and can be cultivated as a part of the strategy for reclaiming degraded lands. Keeping all this in view, the Indian Government has announced a “National Mission on Biodiesel” for Jatropha plantation in wasteland regions is to be implemented on an area of 400,000 ha over the next five years (Francis et al., 2005).

Gayatri Nahaket al.,; Continental J. Biomedical Sciences 4: 21 - 36, 2010

There are many reports on biodiesel production using enzyme catalysis by free or immobilized lipases (Kamini et al., 2001; Du et al., 2004; Schwab et al., 1988; Nelson et al.,1996; Hsu et al., 2002 and Shimada et al., 1999; Watanabe et al., 2000). Immobilized lipase in particular recovery from the reaction mixture. There are two major limitations of lipase-catalyzed biodiesel synthesis. One is higher cost (which can be reduced up to a certain extent by immobilization) and another is its inactivation by methanol and glycerol. It has been reported that as methanol is insoluble in vegetable oils, it inhibits the immobilized lipases and thereby decreases the catalytic activity of the transesterification reaction. Further, the hydrophilic by-product glycerol is also insoluble in oil, so it is easily adsorbed onto the surface of the immobilized lipase leading to a negative effect on lipase activity and operational stability. (Shimada et al., 2002). Use of several solvents such as n-hexane and petroleum persisted since the reaction medium has been reported (Ghamuia et al., 2004) but the problem persisted since the inhibition of lipase still occurred due to poor solubility of methanol and glycerol in hydrophobic solvents (Dossat et al., 1999). There are some reports on enhanced biodiesel synthesis in the presence of t-butanol as a solvent (Li et al., 2006; Shah et al., 2004). As both methanol and glycerol are soluble in t-butanol, the inhibitory effect of methanol and glycerol on lipase activity is reduced. Moreover, t-butanol is not a substrate for the lipase because it does not act on tertiary alcohols.

Several micro-organisms that produce solvent-tolerant lipases, either 1,3-specific from Fusarium spp. (Shimada et al., 1993) or non-specific from Pseudomonas and Bacillus spp. (Sugihara et al., 1992; Ogino et al., 1999), have been reported. Shimada et al., 1999 reported continuous conversion of vegetable oils to methyl esters using lipase in organic. Solvent-free environment. They reported that immobilized Candida antarctica lipase was found to be the most effective for the methanolysis among lipases tested. Using same lipase as described above Watanabe et al., developed also continuous production of methyl esters from vegetable oils in three step methanolysis. A mixture of vegetable oil and 1:3 molar equivalent of methanol were fed into the first column that immobilized Candida antarctica lipase. Abigor et al., 2000 reported that lipase catalyzed production of alkyl esters by transesterification of palm kernel and coconut oil with different alcohols using PS30 (Psudomonas cepacia) lipase as a catalyst. Biodiesel synthesis from Jatropha oil has been reported by Chromobacterium viscosum and Pseudomonas cepacia lipases. In both the cases the ethanolysis of Jatropha oil for biodiesel synthesis has been carried out (Shah and Gupta, 2007; Shah et al., 2004). Immobilized P. cepacia lipase was used for the transesterification of soybean oil with methanol and ethanol (Noureddini et al., 2005). Fattyacid ethyl esters have also been prepared from castor oil using-hexane as solvent and two commercial lipases, Novozyme 43 and Liposome IM, as catalysts (De Oliveira et al., 2004). Novozyme 435 have also been used to catalyze the transesterification of crude soybeanoils for biodiesel production in a solvent-free medium (Du et al., 2004 ).Simple alkyl ester derivatives of restaurant grease were prepared using immobilized lipases from Thermomyces lanuginose and C. antarctica, as biocatalysts (Hsu et al.,2002). Fatty acids esters we reproduced from two Nigerian lauric oils, palm kernel oil and coconut oil, by transesterification of the oils with different alcohols using PS30 lipase as a catalyst. In the conversion of palm kernel oil to alkyl esters (biodiesel), ethanol gave the highest conversion of 72%. Some of the fuel properties compared favorably with international biodiesel specifications (Abigor et al.,2000).Despite its importance, studies on the mechanisms of production of microbial lipases and the role of lipidic substances used as inducers in lipase production are scarce(Shimada et al.,1992).Lipases represent an extremely versatile group of bacterial extracellular enzymes that are capable of performing a variety of important reactions, thereby presenting a fascinating field for future research(Jaeger et al., 1994). The understanding of structure–function relationships will enables researchers to tailor new lipases for biotechnological applications (Jaeger et al., 1999).

At the drop of the works already undertaken in said direction the present work aims at screening of different sources of lipase preferably from plants like Jatropha curcus, Ricinus, and Callophylum and then compared with one species of bacteria like Xanthomonas species in three different aspects such as lipase content in terms of soluble protein, enzyme activity and efficiency of transesterification ofJatropha oil. Germinated seeds for optimal lipase activity/the screening of the sources is essential to augment further studies on enzymatic transesterificaion to evolve a suitable strategy with a view to economizing a standard protocol. Moreover the transesterification of Jatropha oil with the enzyme extract from the above sources both in free and immobilized form in an ideal reaction environment reported earlier.

Gayatri Nahaket al.,; Continental J. Biomedical Sciences 4: 21 - 36, 2010

MATERIALS AND METHODS

Plant materials

The seeds of Jatropha curcas, Ricinus communis, Calophyllum inophyllum, were collected from Department of Forestry, Orissa University of Agriculture and Technology (OUAT), Bhubaneswar, Orissa

Bacterial organisms

The bacterial cultureXanthomonas species was collected from the Department of Microbiology, OUAT, Bhubaneswar, Orissa and then they were grown for pure culture in the basal medium for further use.

Chemical and Reagents

Petroleum ether(40-600C), 1% phenolphthalein, 95% ethanol, 0.1N potassium hydroxide,0.5N HCl, KOH, Silica gel, CaCl2, Benzene, Phosphate buffer (pH 7.3)50mM, Sodium taurocholate( Bile salt), Acetone, Ammonium sulphate, Sodium alginate, Calcium chloride, 50% H2SO4, Nutrient broth, glucose, Sterilize sand and double distilled water.

Instrumentations

Soxhlet apparatus, Rotary shaker, Cooling Centrifuge, UV-Vis spectrophotometer, Incubator, Hot-air oven, Laminar-air flow, Separating funnel and mortar and pestle.

Seed germination

The seeds of J. curcas and Ricinus, Callophylum were thoroughly washed with tap water followed by distilled water. Then they were taken in Petri dishes and kept for germination for 1-2 days for further use.

Bacterial culture

All glass wares were cleaned by soaking in dichromate and sulfuric acid for not less than 24 hours, washing in tap water, and rinsing in distilled water. Then the basal medium was prepared. In practice, the inorganic components were dissolved in about half the necessary volume of water, supplemented by test nutrients if any, made up to volume, distributed in flasks, and autoclaved. Sterile glucose was then added aseptically in the form of a 25 or 50 per cent solution prepared by treating glucose solutions twice at pH 3.0 with norit charcoal, filtering each time with the aid of super-cel, and autoclaving the resulting solution (Hutner, 1944). Series of these media were inoculated under the laminar-air flow chamber uniformly with Xanthomonas isolates, which are able to grow in the basal medium, and, after incubation at 37 0C in an incubator it gets its turbidities.

Free enzyme Preparation

Preparation of Acetone powder

The germinated seeds were homogenized in mortar and pastel with cold petroleum ether. Then it was centrifuged successively with ether mixture and then finally ground with cold acetone to fine powder and air dried and preserved at -40C. The enzyme was extracted from acetone powder by phosphate buffer (pH 7.3). Then they were centrifuged at 15000g for 10min at -40C. The supernatant was preserved for further analysis and the residue was discarded.

The Nutrient broth containing bacterial species was centrifuged and the bacterial pellet was taken in a mortar and pestle. Then the cells were disrupted using sterile sand as abrasive. Again it was centrifuged and the pellet containing the enzyme was collected and kept at -4oC in a refrigerator for further use during the course of study.

Extraction and Purification of Enzymes

The enzyme was extracted from Acetone powder by phosphate buffer (pH 7.3) and in case of bacterial source the extraction was done by mascarating the bacterial cells or pellets in phosphate buffer with sterilized sand or abrasive. The materials were then centrifuged at 1500g for 10 minutes at -4oC. The supernatant was preserved for further analysis and the residue was discarded.

The crude enzyme extract was partially purified by salting out method using 70% ammonium sulfate. The precipitated proteins from all samples were recovered after discarding the supernatant. The precipitate was redissolved in phosphate buffer (50mM) pH 7.3 and dialyzed to make it free from ammonium sulfate. The dialyzed samples were taken as enzyme source.

Gayatri Nahaket al.,; Continental J. Biomedical Sciences 4: 21 - 36, 2010

Assay of Lipase from Acetone Powder

The prepared acetone powder (2gm.) was slightly ground with mortar and pestle with buffer solution (30% KOH+ 50% KH2PO4+ 20ml ice cold H2O). Then it was centrifuged at 15000 rpm for 15 minutes. The pellet was discarded and the supernatant was used as enzyme source (Sadasivam and Manickam., 2008). Same procedure was followed for Xanthomonas species.

To 20ml. of substrate 5ml. of phosphate buffer was added and the contents were stirred slowly by keeping the beaker on top of a magnetic stirrer-hot plate maintaining the temperature at 35oC and pH 7.0. 0.5ml. of enzyme was added and the pH was recorded immediately at zero time with timer on (pH at zero time). At regular intervals (10 min) or as the pH drops by about 0.2 units 0.1N NaOH was added to bring the pHback to the original level. This titration was repeated for 30-60 minutes and the volume of NaOH consumed was noted to estimate the protein content in the enzyme sample.

Calculation

The enzyme activity as the amount of enzyme required to release one milliequivalent of free fatty acid/ min./gm. sample and specific activity as milliequivalent/ min./ mg. protein.

Screening of Efficiency of Lipase from Different Sources in Transesterification

Immobilization of Enzymes

3g of Sodium alginate was taken in 100ml distilled water and was properly mixed to make a 3% solution. Approximately 0.015g of lipase enzyme was mixed with 10ml of 3% sodium alginate solution. An excess (100ml) of CaCl2 (0.2M) was stirred by placing the beaker on a magnetic stirrer. Then lipase alginate mixture was added drop wise manner through a pipette into the stirred calcium chloride solution. This resulted in formation of spherical beads of get immobilized enzyme. Then these beads were stored at 5oC for further use.

Enzymatic Transesterification

The enzymatic transesterification was done followed by the methodJatropha oil (10gm) and ethanols (2gm) were taken in the ratio of 1:4 (mole mle-1) in a 100 ml conical flask. To this mixture different concentrations of enzymes showing different activity of enzymes in terms of milliequivalent/min/mg protein were taken in solution form were added and stirred. Then heated to 400C with constant shaking at 200 rpm. Then the samples were withdrawn and analyzed for maximum conversion.

RESULTS AND DISCUSSION

Transesterification by using Free Enzymes from different sources

From Table-1 the lipase content of Callophylum per gm of material is highest with 0.353gms. Followed by germinated seed of Jatropha with 0.181gms. the enzyme activity was determined in millimequivalent/min/mg of protein. Assay of the enzyme was conducted with 15 min. time interval of fat hydrolysis and it is clear from the data from Table-2 is that the highest activity was observed in case of Xanthomonas sp. Followed by seeds ofCallophylum with activities of 0.008 milliequivalent and 0.003 milliequivalent.

Comparing the Table-1 and Table-2 it is interesting to observe althoughCallophylum has higher lipase content, activity wise the bacterial sources top the list followed byJatropha seeds having low lipase content keeping aside the Xanthomonas, the three other plant sources have varied degree of their efficiencies with high efficiency to be found is Jatropha seeds probably due to compatibility of the enzyme with the Jatropha oil being originated from same source. The efficiency of enzymes from all the given sources in transesterifying J. curcas was too arrived at a comparative assessment in terms of gms. of biodiesel produced using equal concentration of ethanol with varying enzymatic activity level. The optimum level of ethanol concentration for the maximum synthesis of biodiesel was investigated. As was expected, an increase in the number of moles of alcohol with respect to the triglycerides resulted in an increase in the production of esters. Ultimately, the formation of esters reached a maximum level with 1:4 Jatropha oil:methanol molar ratio, and a further increase in alcohol concentration resulted in a decrease in the

Gayatri Nahaket al.,; Continental J. Biomedical Sciences 4: 21 - 36, 2010

formation of esters. The maximum biodiesel synthesis rates from rapeseed oil and cotton oil were reported at 1:3:6 and 1:4 oil: methanol ratio, respectively (Li et al., 2006; Royon et al., 2007). The other constituents produced as byproducts in gms. has also been taken into consideration. The reaction environment was maintained at 40oC with reaction time which was happened to be optimum reaction condition as reported earlier. The data of different sources are showing in Table-3, 4, 5, and 6. The effect of enzyme concentration on the transesterification was also investigated. The enzyme concentration was varied in both the case i.e. in free and immobilized forms from 2.5 to 4.5 gm equivalent of material as shown in figure-3, 4, 5, and 6 in free enzyme and Figure-8, 9, 10 and 11. The synthesis of fatty acid initially increased within the enzyme content with maximum conversion rate 90% in case of free enzyme and 90.2% in case of Immobilized form as shown in Table-11 and Figure-7. A previous study has demonstrated that the yield of 1-butyl oleate increased when the amount of Rhizopus oryzae was increased from 30 to 60U and remained almost constant with increase in lipase amount beyond 60U (Ghamuia et al., 2004). Similar results were reported for the methanolysis of rice brain oil catalyzed by Cryptococcus Spp. S-2 lipase (Kamini et al.,2001). This may be due to the fact that in the presence of a high amount of lipase, the active site can not be exposed to the substrates and many molecules of the enzyme aggregate together. Agglomeration using immobilized lipase in a solvent-free system has been previously reported (Liou et al., 1998; Foresti and Ferreira, 2005).

A common thread in all studies of enzymes in organic media is that the amount of water associated with the enzyme is a key determinant of the properties (for e.g. activity, stability and specificity) that the enzyme exhibits. It was observed that the percentage conversion was a maximum when no additional water in the reaction mixture. Since the enzyme taken for study already contain water at minimum level, the effect of solvent in the reaction mixture has not been studied and the result does not show any remarkable difference in both free and immobilized system. Although water is necessary for acquisition in essentially anhydrous organic solvents, water is also involved in many enzyme inactivation processes (Volkin et al., 1991). As long as a minimal amount of water is associated with the enzymes, its activity in the organic media is retained. However, too much water facilitates enzyme aggregation, which leads to a decrease in enzyme activity (Iso et al.,2001; Nelson et al., 1996).

Transesterification by using Immobilized Enzymes from different sources

The repeated use of immobilized enzyme may help to bring down the product cost and make the enzymatic process economically viable (Ye et al., 2006). Operational stability or reusability is of importance in determining immobilized enzyme efficiency. However, it was observed that reaction behavior changes when an immobilized enzyme is used repeatedly. This may be due to loss of enzyme during filtration and drying, prolonged interaction of the organic solvent with the immobilized enzyme resulting in the denaturation of the enzyme, and production substantial quantities of co-product water after each cycle (Dave et al., 2006). From the data found in Table-11 and Figure-7 showed that there is not any remarkable difference (in percentage of conversion) in both free and immobilized system but the results obtained from Immobilized system show better in comparison to enzyme in free form.

From this study we found that although the immobilized system does not play a significant role in the process as the comparative result of percentage of conversion in both the cases, but it has been proven to be useful techniques for improving enzyme activity, which is in accordance with the paper (Persson et al., 2002).

CONCLUSION

The present study shows that Xanthomonas Spp. shows the best performance followed by the seed of Jatropha curcas. From these findings we can try further enhancement of lipase production may be achieved by genetic engineering. High levels of expression of lipases from several micro-organisms have been successfully achieved using Saccharomyces cerevisiae as the host. Accordingly further investigation with these two sources can be taken up for in depth kinetic study before standardizing the protocol without searching for different sources in heat and trial methods. The steps had already been taken by government of India to improveJatropha plants both in increasing seed production and lipase content. It is imperative that investigation must confine with production of bio-diesel from Jatropha curcas oil which is non-edible using enzyme from same sources. Preliminary results show a positive energy balance and impact on global warming potential. However more research is needed to get a good insight in the environment sustainability of this production system. It is premature to conclude for sure, the protocol used in this study to be standard as further modification to reduce various steps maximizing ester yield in future

Gayatri Nahaket al.,; Continental J. Biomedical Sciences 4: 21 - 36, 2010

cannot be denied. The high operational stability of immobilized lipase also indicates the efficiency of the process. From the present work, it has been demonstrated that esterification of jatropha oil could be effectively carried out in this novel system with a good operational stability of the lipases. However, further research and development on additional fuel property measures, long-term run and wear analysis of biodioesel-fulled engines is necessary.

ACKNOWLEDGMENT

The authors are thankful to (N.K.M) one of the author for providing proper facilities and constant supervision for research work. We are also thankful to Chandrakanti Mohanty for constant encouragement till the completion of research work. Sabitri Nahak is also acknowledging for her timely help to the author (G.N).

Table 1 : Lipase contents of different sources in terms of soluble proteins

Table 2: Measurement of Enzyme activity

Table-3: Transesterification by using free enzyme extracted from xanthomonas Sp.

Gayatri Nahaket al.,; Continental J. Biomedical Sciences 4: 21 - 36, 2010

Table-4: Transesterification by using free enzyme extracted fromRicinus

Table 5: Transesterification by using free enzyme extracted from Jatropha curcus

Table 6: Transesterification by using free enzyme extracted from Callophylum

Table-7: Effect of immobilized lipase extracted from seeds of J. curcus Gayatri Nahaket al.,; Continental J. Biomedical Sciences 4: 21 - 36, 2010

Table-8: Effect of immobilized lipase extracted from seeds of Ricinus

Table 9: Effect of immobilized lipase extracted from seeds of Callophylum

Table 10:Effect of immobilized lipase extracted from seeds of bacterial source

Table 11: Summery of the ester yield in Free and Immobilized enzyme system

Gayatri Nahaket al.,; Continental J. Biomedical Sciences 4: 21 - 36, 2010

<p style="margin-left: 36pt;">J.curcus Ricinus Callophylum Xanthomonas Sp.

<p style="margin-left: 36pt;">Figure-1: Lipase content of different sources in terms of soluble protein

  J.curcus                Ricinus         Callophylum         Xanthomonas Sp.

<p style="margin-left: 36pt;">Figure-2: Measurement of Enzymatic activity

<p style="margin-left: 36pt;">Figure-3: Enzyme in gm equivalents of material

Gayatri Nahaket al.,; Continental J. Biomedical Sciences 4: 21 - 36, 2010

<p style="margin-left: 36pt;">Figure-4: Enzyme in equivalent of material

<p style="margin-left: 36pt;">Figure-5: Enzyme in gm equivalent of material

<p style="margin-left: 36pt;">Figure-6: Enzyme in gm equivalents of material

Gayatri Nahaket al.,; Continental J. Biomedical Sciences 4: 21 - 36, 2010

Figure-7: Ester yield in Free Enzyme and Immobilized Enzyme

<p style="margin-left: 36pt;">Figure-8: Enzyme in gm equivalents of material

<p style="margin-left: 36pt;">Figure-9: Enzyme in gm equivalents of material

Gayatri Nahaket al.,; Continental J. Biomedical Sciences 4: 21 - 36, 2010

Figure10: Enzyme in gm equivalents of material

<p style="margin-left: 72pt;">Figure-11: Enzyme in gm equivalents of material

<p style="margin-left: 72pt;">Figure-12: Sources of Enzymes

Gayatri Nahaket al.,; Continental J. Biomedical Sciences 4: 21 - 36, 2010

<p style="margin-left: 72pt;">Figure-13: Sources of Enzymes

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Gayatri Nahaket al.,; Continental J. Biomedical Sciences 4: 21 - 36, 2010

Received for Publication: 19/07/2010

Accepted for Publication: 18/08/2010

Correspondence Author

R. K. Sahu

B.J.B Autonomous College, Bhubaneswar, Orissa

[mailto:sahurajani@yahoo.co.in sahurajani@yahoo.co.in]

Continental J. Biomedical Sciences 4: 37 - 42, 2010 ISSN:2141 – 419X

©Wilolud Journals, 2010 http://www.wiloludjournal.com

ANTIDIABETIC ACTIVITY OF DRIED LEAVES OF  Aginanthus brunneus  ON ALLOXAN-INDUCED DIABETIC RATS

V.O Aina, H.M Inuwa, D.A Ameh

Department of Biochemistry, Ahmadu Bello University, Zaria - Nigeria.

<p style="margin-left: 43.2pt;">ABSTRACT

<p style="margin-left: 43.2pt;">The present study was aimed at evaluating the antidiabetic potential of Aginanthus brunneus (a specie of Africa mistletoe) leaves on alloxan induced experimental diabetes in wister Albino rats. Oral administration of ethanolic extracts of the leaves (200mg/kg body weight/rat/day) for 30 days significantly reduced the levels of blood glucose and glycosylated hemoglobin in diabetic rats. Determination of plasma insulin levels revealed the insulin stimulating action of the leaf extracts. Furthermore, the changes observed in the activities of carbohydrate and glycogen metabolizing enzymes were reverted back to normal after 30 days of treatment with the extracts. The efficacy of the leaf extracts was comparable with Gliben clamide (a sulphony urea), a well known hypoglyceamic drug.

<p style="margin-left: 43.2pt;">KEYWORDS: Aginanthus brunneus, Eucalyptus, Ethanolic extract, diabetes carbohydrate metabolism, Glibenclamide.

INTRODUCTION

Diabetes mellitus (DM) is considered as one of the leading causes of death in the world about 160 million people are suffering from diabetes mellitus worldwide. Diabetes is a complex multisystemic disorder characterized by a relative or absolute insufficiency of insulin secretion insulin dependent diabetes mellitus (IDDM) or concomitant resistance of metabolic action of insulin on target tissues non insulin dependent diabetes mellitus (NIDDM) (Garber. A 1998). There is need for search for drugs with low cost, more potentials, and without adverse side effects which is being searched for throughout the world.

Many traditional plants have been found successful for ant diabetic activity. It is necessary to provide scientific proof as to whether it is justified to use a plant or its active principles for treatment (Singh et al 2000). This parasitic plant Aginanthus brunneus parasitizing on Eucalyptus is native to Nigeria and belongs to the Lorantheceae family of Africa mistletoe.

<p style="margin-left: 36pt;">FIGURE 1: Aginanthus brunneus parasitizingon Eucalyptus

Being a parasite, it is unable to acquire all the nutrient it needs by its own independent processes and hence takes what it needs from it’s living host through special sucker-like outgrowth which penetrate the tissues of the host plant. No systematic work on it’s antidiabetogenic activity has been reported in literature. Hence the present study was aimed to evaluate the pharmacologic effect of ethanolic extract of Aginanthus brunneus on carbohydrate and

V.O Aina et al.,: Continental J. Biomedical Sciences 4: 37 - 42, 2010

glycogen metabolism in both normal and alloxan induced diabetic rats. The effects of Aginanthus brunneus were compared to glibenclamide that is often used as a standard drug.

MATERIALS AND METHODS

CHEMICAL

Alloxan was purchased from sigma chemical co U.S.A All other chemicals used were of analytical grade.

PLANT MATERIAL

Fresh mature Globimetulla brownii (Lorantheceae) leaves were collected from Eucalyptus tree in botanical garden of Ahmadu Bello University, Zaria Nigeria. The plant was identified and authenticated by Mr. U.S. Galla of biological Sciences Department with a voucher number 1968

Preparation of Aginanthus brunneus Extracts

Dried leaves were powdered in an electrical grinder and stored at 5oC until further use. 100g of the powder was extracted with petroleum ether (60-80oC) to remove lipids it was then filtered and the filtrate discarded the residue was extracted with 95% ethanol by Soxhlet extraction. The ethanol was evaporated in a rotary evaporator at 40-50oC under reduced pressure. The yield of the extract was 8.5g/100g.

Animals

Adults male albino rat of Wistar strain weighing approximately 150 – 180g were procured from NITOR (Nigerian Institute for Trypanosomiasis and Oncocerchiasis Research) Kaduna and were acclimatized to Animal house conditions in the department of Applied Science, Kaduna Polytechnic, Kaduna, Nigeria and fed with standard rat feeds supplied by Pfizer feeds.

Toxicity studies

To study any possible toxic effects and/or changes in behavioral pattern, rats were treated with graded doses of Aginanthus brunneus extract (100 – 500 mg 1kg body weight/rat/day) and kept under close observation for 8 hr daily for 30 days.

Induction of Experimental diabetes

The animals were fasted overnight and diabetes was induced by a single intrapezitoneal injection of a freshly prepared Alloxan solution (50mg/1kg body weight) in 0.1m citrate buffer (pH 4.5) (Sekar et al (1990). The animals were allowed to drink 5% glucose solution overnight to overcome the drug induced hypoglycemia. Control rats were infected with citrate buffer alone. After a week time for the development of diabetes, the rats with moderate diabetes having glycosuria and hypoglycemia (blood glucose range above 250mg/dl) were considered diabetic and used for drug treatment. The leaf treatment was administered orally at a concentration of 200 mg/1kg body weight/rat/day for 30 day.

Experimental design

The animals were divided into two sets, one for the evaluation of a glucose tolerance test and a second one for the analysis of biochemical parameters. Each set was further divided into four groups, each comprising a minimum of six animals in each group shown below: Group i: Normal control rats

Group ii: Diabetic control rats

Group iii: Diabetic rats administered Aginanthus brunneus

leaf extracts (200mg 1kg body weight)

Day/ rat) in aqueous solution orally for 30days (Pari et al 2000)

Group iv: Diabetic rats administered with glibenclamide (600 mg 1kg body weight/rat/day)

In aqueous solution orally for 30days (Pau et al 2000).

The body weight gain and fasting blood glucose levels of all the rats were recorded at regular intervals during the experimental period

V.O Aina et al.,: Continental J. Biomedical Sciences 4: 37 - 42, 2010

Glucose Tolerance Test

After 30days of treatment, a fasting blood sample was collected from all the groups in heparinized tubes blood samples were also collected at the time intervals of 30, 60, 90 and 120 min after administration of the glucose at a concentration of 2g/kg of body weight (Joy et al 1999)

Biochemical Assays

After 30 days of treatment, a fasting blood sample was collected from all the groups, and were sacrificed by cervical decapitation, fasting blood glucose was estimated by 0-toluidine method of (Sasaki et al 1972). The levels of hemoglobin and glycosylated hemoglobin were estimated according to the method of Drabkin et al (1972) Nayak et al (1981) respectively the hexokinase activity was assayed by the method of Brandstrup et al (1967).

The activities of glucose – 6- phosphatase and fructose 1, 6 – bis phosphatase were assayed according to the method of Koide and Oda (1999) and Gancedo and Gancedo (1971) respectively, Lactate dehydrogenase by Kind (1999), Glycogen synthesis (Leloir etal (1982) and phosphorylase by Cornblath (1973).

RESULTS

Fig 1 changes in Body weight of control and Experimental Groups of Rats

V.O Aina et al.,: Continental J. Biomedical Sciences 4: 37 - 42, 2010

Fig 1 shows the change in weight gain of control and experimental groups of rats. There was significant decrease in the body weight of diabetic rats compared with control rats. Upon treatment with Globimetulla brownii and glibenclamide the body weight gain was improved but the effect was more pronounced in Aginanthus brunneus treated rats than glibenclamide.

Fig 2 Glucose Tolerance Test Graph of Control and Experimental Groups of Rats.

The levels of blood glucose in control and experimental groups of rats after oral administration of glucose is shown in figure 2 the blood glucose value in the control rats rose to a peak value 60min after glucose load and decreased to near normal level at 120min. in diabetic control rats, the peak increase in blood glucose concentration was observed after 60min and remained high over the next 60min. Aginanthus brunneus and glibenclamide treated diabetic rats showed significant decrease in blood glucose concentration at 60 and 120min compared with diabetic groups of rats.

V.O Aina et al.,: Continental J. Biomedical Sciences 4: 37 - 42, 2010

Table 1 changes in the level of blood glucose, plasma insulin, Hemoglobin, Glycosylated Hemoglobin and Urine Sugar in Control and Experimental Groups of Rats

Table 1 shows the level of blood glucose, plasma insulin, total hemoglobin, glycosylated hemoglobin and urine sugar in normal and experiment rats.

There was a significant elevation in blood glucose, urine sugar and glycosylated hemoglobin, while the level of plasma insulin and total hemoglobin decreased during diabetes when compared to control group. Administration of Aginanthus brunneus brought back to rear normal values as that of the standard drug glibenclamide treatment.

Table 2: changes in the activities of hepatic hexokinase, lactate dehydrogenase, Glucose 6 phosphatase and fructose 1,6 Bisphosphatase of control and Experimental Groups of Rats

Table 2 shows a significant decrease in the activity of hepatic hexokinase, a significant increase in the activities of lactate dedydrogenase, glucose-6-phosphatase and fructose 1, 6 bis phosphatse, in Alloxan induced diabetic rats when compared to control rats.

Treatment with Aginanthus brunneus extracts (group iii) and glibenclamide (Group iv) significantly controlled the alterations and restored the altered levels to near normalcy. Aginanthus brunneus treatment exerted more effect than glibenclamide in diabetic rats.

DISCUSSION

Glibenclamide is often used as a standard antidiabetic drug in Alloxan induced moderate diabetes to compare the efficacy of a variety of hypoglycemic compounds. (Paredes et al 2000). The present study was conducted to assess the hypoglycemic activity of Aginanthus brunneus leaves on alloxan induced diabetic rats the ability of Aginanthus brunneus leaf extract in significantly increasing the body weight and effectively controlling the increase in blood glucose levels in diabetic group of rats may be attributed to its antihyperglycemic effects. Further, the antihyperglycemic activity of Aginanthus brunneus was associated with an increase in plasma insulin level suggesting an insulinogenic activity of the leaf extract. The observed increase in the level of plasma indicates that Aginanthus brunneus leaf extracts stimulates insulin secretion from the remnant bcells or from regenerated bcells

V.O Aina et al.,: Continental J. Biomedical Sciences 4: 37 - 42, 2010

the observed increase in the levels of glycosylated hemoglobin in diabetic control group of rats is due to the presence of excessive amounts of blood glucose.

The hepatic gluconeogenic enzymes, glucose 6 phosphatase and fructose 1, 6 bisphosphatase were increased significantly in diabetic rats. The increased activities of these two gluconeogenic enzymes may be due to the activation or increased synthesis of the enzymes contributing to the increased glucose production during diabetes by the liver (Baque et al 1998). In the present study, the experimental diabetic rats treated with Aginanthus brunneus leaf extract and glibenclamide treated groups restored the level of hepatic glycogen by means of decreasing the activity of glycogen phosphorylase and increasing the activity of glycogensynthase.

In conclusion, the present work/study show that the ethanolic extract of Aginanthus brunneus leaves has potential hypoglycemic action in alloxan induced diabetic rats and the effect was found to be more effective than glibenclamide.

REFERENCES

Baque, N.Z, Gupta, D and Raju, J (1998) Regulation of metabolic pathway in liver and kidney during experimental diabetes. Effect of antidiabetic compound. Ind.J. Clin biochem, 13, 63-80.

Brandstrup, N, Kir, J.E and Bruni, C. (1977) Determination of hexokinase in tissue. J. Gerantol 12, 166-171

Drabkin, D.C and Austin, J.M (1972) Spectrophotometric constants for common haemoglobin derivatives in human, dog and rabbit blood. J. Biol. Chem., 98 719-733

Garber. A. (1998) Diabetes mellitus. In international Medicine (stein, J. H.Ed) Mosby, st louis, pp. 1850-1854.

Joy, K.L and Kuttan, R. (1999). Antidiabetic activity of Picrorrhiza Kurroa extract J. Ethnopharmacol, 67, 143-148.

Nayak, SS and Pathabiraman, T.N (1981). A new colorimetric method for the estimation of glycosylated hemoglobin. Clin chim Acta, 109, 267-274

Paredes, A, Hasegawa, M, Prieto F, Mendez, J. Rodriguez, M and Rodriquez Ortega, M. (2001) Biological activity of Guatteria cardoniana fractions. J. Ethno pharmacol, 78, 129-132.

Pari, L, and Ummamaheswari, J(2000) Antihyperglycemic activity of Musa Sapientum flowers: effect on lipid peroxidation in alloxan diabetic rats. Phy tother. Res, 14, 136-138.

Sasaki, T, Matsy, S and Soroe, A(1972). Effect of acetic acid concentration on the colour reaction in the O-toluidine boric acid method for blood Glucose estimation. Riush. Vagakur, 1, 346-353

Sekar, N, kanthasamy, S., William, S., Subramanian, S., and Gorindasamy, S. (1990). Insulinic acitions of Vanadate in diabetic rats pharmacol Res., 14, 136-138

Singh, R.P, Padmavathi, B and Raa, A.R (2000) Modulatory influence of  Adhatoda veisca  ( justice  adhatoda ) leaf extract on the enzyme of Xeno biotic metabolism, antioxidant status and lipid peroxidation in mice. Mal. Cell Biochem 213, 99-109.

Received for Publication: 19/07/2010

Accepted for Publication: 18/08/2010

Correspondence Author

V.O Aina

Department of Biochemistry, Ahmadu Bello University, Zaria - Nigeria.

E-mail address: vocwummi2006@yahoo.com

Continental J. Biomedical Sciences 4: 43 - 49, 2010 ISSN:2141 – 419X

©Wilolud Journals, 2010 http://www.wiloludjournal.com

COMPARATIVE STUDY OF MICROSCOPY WITH ELISA ANTIBODY BASED AMOEBIASIS DIAGNOSIS IN PATIENTS PRESENTING WITH DYSENTERY AT GOVERNMENT HOSPITALS IN KADUNA METROPOLIS

<p style="margin-left: 72pt;">Dawah, I. S, Inabo, H. I.,Jatau, E.D

Department of Microbiology, Ahmadu Bello University, Zaria, Nigeria.

<p style="margin-left: 43.2pt;">ABSTRACT

<p style="margin-left: 43.2pt;">This study investigated the prevalence and risk factors of Entamoeba histolytica infection among dysentery patients presenting at government hospitals in Kaduna metropolis. Three hundred and seventy eight (378) samples were collected, comprising 189 stool and 189 serum specimens. The stool and serum specimens were analysed using Microscopy and Enzyme Linked Immunosorbent Assay (ELISA) respectively. Of the 189 patients sampled, 27 (14.3%) were positive for Entamoeba histolytica, 13 (6.9%) Entamoeba coli and 4 (2.1%) Giardia lamblia. The prevalence of E. histolytica infection (14.3%) was higher than the 10% recorded by WHO (1995) in the developing world. Individuals in the age group 1-10 years had the highest prevalence (29.40%) and it decreased with age except for age group 41 years and above. Males were more infected (14.6%) than females (14.0%) but the difference was not statistically significant (p>0.05). The infection rate was higher in the rainy season (15.5%) than the dry season (12.9%). The study also revealed that E. histolytica infection was not statistically associated with educational status and type of toilet facility (p>0.05), but it was however, statistically associated with the occupational status and source of drinking water of the patients (p<0.05).

<p style="margin-left: 43.2pt;">Key words: Entamoeba histolytica, dysentery, Microscopy, Enzyme Linked Immunosorbent Assay

INTRODUCTION

The World Health Organization (WHO) estimates that the protozoan Entamoeba histolytica is a major cause of morbidity worldwide, causing approximately 50 million cases of dysentery and 100,000 deaths annually (WHO, 1997; Ravdin et al., 2005).

Intestinal amoebiasis due to the infection of E. histolytica is ranked third on the list of parasitic protozoan infections leading to death behind malaria and schistosomiasis (Farthing et al., 1996).

Amoebiasis is second only to malaria as a cause of death resulting from a protozoan parasite (WHO, 1997).

Amoebiasis is a rare occurrence in developed countries of the world, but only found in travelers, immigrants,homosexuals and institutionalized persons. E. histolytica-associated dysentery is a common occurrence in the less developed and developing countries of the world., but is more common in areas of low socio-economic status, poor sanitation and nutrition especially in the tropics (Ravdin et al., 2005). Thus the majority of E. histolytica infections, morbidity and mortality occur in Africa, Central and South America and the Indian Sub-continent (Haque et al., 2006). However, the rate varies geographically, For instance, 39% was recorded in Bangladesh, 21% in Vietnam and 33% in Columbia (Blessmann and Le van Tannieth, 2006).

Studies in parts of Africa reported prevalence rates of 22% and 21% in South Africa and Egypt respectively (Stauffer et al., 2006). In Nigeria, prevalence rates of 22.3% in Calabar (Meremiku et al., 1997), 21.6% in Enugu (Ozumba, 1997) and 13.7% in Ilesa have been reported (Ogunlesi et al., 2005).

In Kaduna metropolis however, such records do not exist. In addition sewage disposal is mainly by pit latrines and water cistern methods. Indiscriminate disposal of human waste in the poorly developed outskirts where farming is also done as well as inconsistent supply of pipe borne water have resulted in the use of other sources of water which are exposed to faecal contamination.

<p style="margin-left: 72pt;">Dawah, I. S et al.,: Continental J. Biomedical Sciences 4: 43 - 49, 2010

There is a need to use a rapid method such as the ELISA antigen detection technique instead of the conventional microscopy which may not detect all positive cases. It is therefore our aim to use microscopy and ELISA antibody detection technique to determine the prevalence of Entamoeba histolytica infections in patients presenting at government hospitals in Kaduna .The appropriate health authorities would therefore, need baseline data to enable them take decisions with regards to diagnosis of E. histolytica infection.

MATERIALS AND METHODS

Ethical Consideration

Informed consent was obtained from each of the patient or their parents. The work was also approved by the Ethical Committee of the government hospitals used in this study.

Enrolment of patients

All patients presenting to the selected government hospitals in Kaduna with acute and persistent diarrhoea or dysentery within the 18 month period of study were enlisted having consented to participate and fulfilled the inclusion criteria which included acute or persistent diarrhoea and dysenteric syndrome.

Patients with diarrhoea or dysentery on antimicrobial agents were excluded.

Patients visiting the hospital for reasons other than diarrhoea and had no diarrhoeal illness within the last 2 weeks were used as control.

Specimen collection and processing

Three hundred and seventy eight samples (378) comprising 189 stool and 189 blood specimens were aseptically collected from dysentery patients presenting at Barau Dikko, Gwamna Awan and Yusuf Dantsoho general hospitals in Kaduna metropolis.

Formol- Ether Concentration Method

The stool samples were analysed using the Formol-Ether concentration method of Cheesbrough (2005). The emulsified faecal samples were filtered in a two-layered gauze and the filtrate transferred to a conical centrifuge tube containing equal volume of ether and centrifuged for 1 minute at 3,000rpm. After discarding the faecal debris and ether, the sediment was transferred to a clean glass slide and a drop of iodine was added. The entire preparation covered with a cover slip and examined microscopically under x40 objective to identify the trophozoites.

ELISA Antibody Detection Technique

The sera from the blood samples were analysed using the Enyzme Linked Immunosorbent Assay (ELISA) made by TechLab (USA). The first well of the microplate was left blank (control) while 100ul of negative and positive controls were added to the second and third wells respectively. Two drops of the diluted test samples were added to the remaining wells and incubated at room temperature (15-20oC) for 10 min. The contents were then shaken and washed 3 times with diluted buffer. After washing, 2 drops of enzyme conjugate were added to each well and again incubated at room temperature for 5 minutes.

This was followed by another shaking and washing again with buffer after which 2 drops of chromagen was added to each well and again incubated at room temperature. Finally, 2 drops of the stop solution were added to each well and mixed by tapping the strip holder. The results were read with a ELISA machine set for biochromatic readings at 450/650-620nm. The prevalence of E. histolytica was determined by the percentage of patients who tested positive for microscopy and ELISA, while the Chi-square was used to determine the association of between E histolytica and the selected variables. All statistical analyses were carried out using the SPSS statistical software.

RESULTS

Of the 189 dysentery patients sampled, 27 (14.3%) were positive for E. histolytica, 13 (6.9%) E. coli and 4 (2.1%) G. lamblia respectively (Table 1). The results in Table 2 below show that E. histolytica was statistically associated with age (p=0.05), but was not associated with gender and season (p>0.05). However, males were more infected (14.6%) than females (14.0%). The rate of infection decreased with age except for age 41 and above (18.2%). The infection rate was higher in the rainy season (15.5%) than dry season (12.9%). E. histolytica infection was not

<p style="margin-left: 72pt;">Dawah, I. S et al.,: Continental J. Biomedical Sciences 4: 43 - 49, 2010

statistically associated with the educational status and type of toilet facility used (p>0.05) Table 3. However the results in Table 4 show that the infection was associated with the occupational status and source of drinking water of the patients (p<0.05).

Table 1. Prevalence of pathogenic protozoa among diarrhoiec patients

Table 2: Prevalence of E. histolytica infection based on gender, age and season. <p style="margin-left: 36pt;">Key: NE = Number Examined, M = Microscopy, E=Enzyme Linked Immunosorbent Assay (ELISA), ME= Microscopy and ELISA.

Table 3: Prevalence of E. histolytica infection based on educational status and type of toilet facility. <p style="margin-left: 36pt;">Key: NE = Number Examined, M = Microscopy, E=Enzyme Linked Immunosorbent Assay (ELISA), ME= Microscopy and ELISA.

<p style="margin-left: 72pt;">Dawah, I. S et al.,: Continental J. Biomedical Sciences 4: 43 - 49, 2010

Table 4: Prevalence of E. histolytica based on occupational status and source of drinking water. Key: NE = Number Examined, M = Microscopy, E=Enzyme Linked Immunosorbent Assay (ELISA)

ME = Microscopy and ELISA.

DISCUSSION

Diarrhoea and Dysentery remain major problems in the developing countries due mainly to poverty, characterized by the absence of potable drinking water, proper sanitary habits, absence of good faecal disposal system, poor hygienic practices by the impoverished citizens and overcrowding (World Health Organization. 1998). Intestinal

Amoebiasis due to the infection of E. histolytica is ranked third on the list of parasitic protozoan infection leading to death behind malaria and Schistosomiasis (Farthing et al., 1996).

The findings of this study revealed that E. histolytica had a prevalence of 14.3% in Kaduna metropolis. This was slightly higher than the 10% cited by WHO, (1995) in the developing world. It was also higher than the 6.3% reported in Calabar (Meremiku et al., 1997) and 0.67% in Ilorin (Fadeyi et al.,2009),13.7% in Ilesa (Ogunlesi et al., 2005) but much lower than 21.6% reported in Enugu (Ozumba, 1997). The reason for the disparity was unclear but it could be attributable to differences in study design, patient selection and environmental conditions in the various study centres.

The higher prevalence observed in males (14.6%) than in females (14.0%) agreed with the findings of Erko et al (1996), Agi (1997) and Chambers et al (1997). The decrease in the infection rate with age agreed with the work of Kobayashi et al (1995) and Rai (1997) while the higher prevalence observed in the wet season (15.1%) than dry season (13.5%) agreed with the findings of Ahmed et al (1996); Park(2002) and Mawashi (2003).

Subjects using pipe borne water were less infected with E.histolytica when compared with people using well water. The association of E. histolytica with the source of drinking water of the patients agreed with the findings of Cox (2001), Olsen et al (2001), Ogunlesi et al (2005) and Rinne et al (2005).Most of the wells in the study area were manually dug, uncemented with no casing or covering. Sometimes, the well is contaminated with surface run-off which may be faecally contaminated.

Entamoeba histolyticais often missed in routine stool microscopy as can be seen. The use of single stool specimen per patient for microscopic study may however be contributory to this result. Antigen and Antibody detection tests

<p style="margin-left: 72pt;">Dawah, I. S et al.,: Continental J. Biomedical Sciences 4: 43 - 49, 2010

are more sensitive than microscopy and results are reproducible as shown by various workers.(Gonin and Trudel,2003) They can also distinguish between the invasive and the non-invasive E. dispar. There are several factors responsible for the disparity in the prevalence rate of Entamoeba histolytica  in any locality. These may include the socio-economic status and population size, expertise of the researchers, the number of subjects enrolled in the study and the duration of the study.

ELISA diagnostic method is a technique which does not require collection of multiple stool specimens per patient and is specific for the diagnosis of Entamoeba histolytica infection against microscopy.

Correlation was observed between seropositivity and stool colonization with E.dispar and/or ''E. histolytica. Data in this study showed that 77.8% of seropositive individuals as detected by ELISA antibody technique to be colonized with E. histolytica-E. dispar ''complex and 21.8% as detected by microcopy. This is in agreement with the work of Haque et al (2000) who found an association between seropositivity and stool colonization with E. histolytica. In their study, 52% of the children that were colonized with E. histolytica were seropositive and 13% of children whose stools were E. histolytica negative were seropositive

Based on the findings of this study; efforts must be made towards the control of E. histolytica infection in Kaduna Metropolis. It is recommend that routine screening of dysentery patients for amoebic dysentery should be emphasised as well as the empirical treatement with metronidazole (the anti-parasitic drug recommended by WHO (1995) pending the availability of bacteriological reports.

The poor water supply in the metropolis be improved and additional boiling of water for drinking be emphasized; since some chemical methods of water purification (chlorination) are reportedly ineffective against the cyst of the parasite (Park, 2002).

The general public health enlightenment should be intensified. Poor hand washing practices, for instance, create large pool of carriers of the parasite (Cuttinget al, 2006).

Other aspects of epidemiology of E. histolytica infection like the sanitary conditions of the patients’ homes should be studied because such findings would be of great help in reduction and control of the amoebic dysentery.

REFERENCES

Agi, P. I. (1997). Comparative helminth infections of man in two rural community of the Niger Delta, Nigeria. West African Journal of Medicine. 1694: 2:232-238.

Ahmed, E. S. Danfalla, A. Chiratensen, N. O., Madsen, H. (1996). Pattern of infection and transmission of human Schistsoma mansoni and Schistosoma haematobium in White Nile province, Sudan. Annals of Tropical Medicine and Parasitology 90:173-80.

Blessmann, J. and Le Van Tanniech, E. (2006) Epidemiology and Treatment of Amoebiasis in Hue, Vietnam. ''Arch. Medical Research'' 37:270-272.

Chambers, R., Longhurst, R. W., Bradley, D. J. and Feacham, R. G. (1997). Seasonal dimensions to rural poverty: analysis and practical implications''. Journal of Tropical Medicine and Hygiene 82:'' 156-172.

Cheesbrough, M. (2005). District Laboratory practice in Tropical Countries. ''Cambridge University Press. ''

Cox, F. E. (2001) Concomitant infections, parasites and immune responses. Parasitology. 122 supp. 23-28.

Cutting, W. A. M., Hawkins, P. (2006). The role of water in relation to diarrhoeal diseases. Tropical Medicine and Hygiene. 85:31-9.

Erko, B., Birrie, H., Tedla, S. (1995). Amoebiasis in Ethiopia ''Tropical and Geo. Medicine.'' 47: (1): 30-32.

<p style="margin-left: 72pt;">Dawah, I. S et al.,: Continental J. Biomedical Sciences 4: 43 - 49, 2010

Fadeyi A; Nwabuisi C; Adegboro B; Akanbi II AA; Fowotade A and Odimayo M.S(2009)Apparent Rarity of Entamoeba histolytica and other Intestinal Parasites in Acute and Persistent Diarrhoeic Patients Attending Ilorin Hospitals: Time for ELISA Antigen Based Amoebiasis Diagnosis.European Journal of Scientific Research.31(3):388-397

Farthing, M S; Cavellos A M; Kelly P; Cook G C (1996). Intestinal Protozoa; In Manson’s Tropical Disease. 20th Edition, London W.B. Saunder Company. 1255-1267.

Gonin,P. and Louise Trudel, L.(2003). Detection and Differentiation of Entamoeba histolytica and Entamoeba dispar Isolates in Clinical Samples by PCR and Enzyme-Linked Immunosorbent Assay. Journal of Clinical Microbiology, 41 (1):. 237–241

Haque,R; Mollah, Ali,I.K.M. Alam,K Eubanks, Lyerly,A.D and William A. Petri, JR, W.A (2000). Diagnosis of Amebic Liver Abscess and Intestinal Infection with the TechLab Entamoeba histolytica II Antigen Detection and Antibody Tests. Journal of Clinical Microbiology. 38( 9 ):3235–3239

Haque, D., Duggal, P., Kabir M., Roy, S., Fair, B. M. (2006). Entamoeba histolytica infection in children and prevention from subsequent Amoebiasis. Infect Immun. 74: 904-909.

Kobayashi, J., Hasegawa, H., Forli, A. A., Vashimura, N. F., Yamanaka, A., Sato, Y. (1995). Prevalence of Intestinal parasitic infections in five farms in Holambra, Sau Paulo, Brazil, Rev. Tropical Medicine Sau-Paulo. 37 (1): 13-18.

Mawashi, K. Y. (2003). The prevalence of intestinal parasites in some parts of Katsina State Unpublished ''Masters’ Thesis.Department of Microbiology, Ahmadu Bello University, Zaria. ''

Merimiku, M.M.,sindi A.A.and Anitam Obong, O.E.(1997). The influence of breastfeeding on the occurrence of dysentery, persistent diarrhea and malnutrition among Nigerian children with diarrheoa. ''West Afri. J. Med''. 16:20

Ogunlesi, T. A., Okeniyi, J. A. O., Oyedeji, O. A., Oseni, S. B. A., Oyelami, O. A., Njokonma, O. F. (2005) Childhood Dysentery in Ilesa, Nigeria: the unusual role of E. histolytica. The Internet Journal of Tropical Medicine. 2. (2).

Olsen, A., Samuelson, H., Onyango-Ouma (2001). Study of risk factors for intestinal helminths and protozoan infection using epidemiological approaches. ?? Journal Biosoc-Society. 33: (4) 56 9-584.

Ozumba, U. C. (1997). Antimicrobial susceptibility pattern and serogroup distribution of Shigella species at Enugu, Nigeria. Post-Graduate Medicine Journal. 4: 1-3.

Rai, S. K., Hirai, K., Ohno, Y. and Matsunmuro, T. (1997). Village health and sanitary profile from eastern hill region, Napal Kobe. Journal of Medical Science 43 (3-4): 121-133.

Stauffer, W.,Abd-Alla,M.,Ravdin,J.I.(2006).Prevalence and incidence of Entamoeba histolytica in South Afric and Egypt. Arch. Med. Res.7:266 -299.

Park, K. (2002). Amoebiasis. In Park K. (ed). Park’s Textbook of preventive and Soc. Med. 17th Edition. M/S Barnasidas Bhanor Publishers, Jabalpur, India. 184-185.

Ravdin, J. I., Stauffer, M. M. (2005). Entamoeba histolytica (Amoebiasis). In Mendell, G. L., Benneth, J. E. Dolin, R. (ed) Mendell, Doglas and Benneth) Principles and Practice of Infectious Diseases. 6th ed. Philadelphia, P. A. Churchill Livingstone.

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Rinne, S., Rodas, E. J., Galer-until, R., Glickman, L.T (2005). prevalence and Risk factors for protozoan and nematodes infections among children in an Ecuadorian highland community. Transaction of the Royal Society of Tropical Medicine. 99: 585-92.

World Health Organization, (1995). The treatment of diarrhoea: A manual for Physicians and other Senior Health Workers. WHO/CDC/95:33.

World Health Organization, (1997). Weekly Epidemiological Records. 72: 97-100.

World Health Organization. (1998).The state of World Health. In: The World Health Reports. Life in the 21st century: a vision for all. WHO Geneva. 57-58.

Received for Publication: 19/07/2010

Accepted for Publication: 18/08/2010

Correspondence Author

Inabo, H. I.

Department of Microbiology, Ahmadu Bello University, Zaria, Nigeria.

Email address:heleninabo@yahoo.co.uk

Continental J. Biomedical Sciences 4: 50 - 54, 2010 ISSN:2141 – 419X

©Wilolud Journals, 2010 http://www.wiloludjournal.com

EFFECTS OF SAND BOX (Hura crepitans) SEED MEALS ON RATS’ LIVER AND SERUM ENZYME LEVELS

Fowomola, M. A and Akindahunsi ,A. A.

School of Science, Department of Biochemistry, Federal University of Technology Akure, Ondo State, Nigeria.

<p style="margin-left: 43.2pt;">ABSTRACT

<p style="margin-left: 43.2pt;">The effect of Hura crepitans seed flour, incorporated at 10% (w/w) protein on liver and serum acid phosphatase (E.C. 3.1.3.2), alkaline phosphatase (E.C.3.1.3.1), aspartate – amino transferase activity (AST) (E.C.2.6.1.1) and alanine amino transferase activity (ALT) (E.C.2.6.1.2) activities were investigated. Generally, results of the toxicological investigation showed that acid phosphatase (ACP), alkaline phosphatase (ALP), aspartate amino transferase (AST) and alanine amino transferase (ALT) activities of liver and serum of rats fed on defatted cooked diet were not significantly different (p>0.05) from those of control fed casein diet. However, their activities were significantly reduced (p≤0.05) in liver of rats fed with undefatted cooked diet, raw defatted diet and raw undefatted diet, while those of serum were significantly increased (p≤0.05).The results of this study indicated that combination of defatting and cooking processes reduced the toxicity of Hura crepitans seeds.

<p style="margin-left: 43.2pt;">KEYWORDS; Sandbox, Hura crepitans, toxicity, liver and serum.

INTRODUCTION

Hura crepitansLinn is a tropical plant belonging to the family Euphorbiacea. In Nigeria, it is known as ‘Odan Mecca’ by the Kabba people of Kogi State and “Aroyin” by the Ijesha People of Osun State. Hura crepitans is often planted in towns and villages as a cover tree. It has short, densely crowned spines on the trunk and branches, the long-stalked leaves with prominent closely parallel pinnate nerves, the purple flower spikes and the large fluted flattened, fruits are highly distinctive. This tree flowers usually at the beginning of and again at the end of rainy season. One nut is a flattened and fluted disc with 5 – 10 lobes about 2.5cm deep and 7.5cm wide on a stout stalk. The capsule splits explosively releasing one flattened circular seed about 18mm across from each chamber (Tropilab Inc. and Hear Organization ).

It has been reported that a person who ate a seed of Hura crepitans complained of burning in the throat; vomiting and purging; suffocating and headache[Allen (2000);Clarke (2000)]. They further stressed that people who had eaten the shells with the seeds were seized with violent vomiting and headache while those who ate the seeds without the shell, suffered from nausea and violent pain in stomach, vomited once and violent diarrhea. A milky juice that is present in all parts of the Hura plant, can cause blindness, if applied (Allen ,2000). He also reported that oil of Hura crepitans is used as purgative. Despite all these negative findings againsthura crepitans seed, the works of Fagbemi and Adebowale (2000), Adedire and Ajayi (2003) and Fowomola and Akindahunsi (2005)

revealed that Hura crepitans seed is a nutritionally promising seed and more so, there is paucity of information on its toxicity on blood and liver of rats. The present study is aimed at providing this information.

MATERIALS AND METHODS

Collection of Samples

Hura crepitansseed pods were collected from Ogidiri African Primary School, Offa, Kwara State, Nigeria. They were authenticated in the Department of Biology, School of Science, Federal University of Technology Akure, Ondo State, Nigeria. The samples were air dried in the laboratory for two weeks, Cotyledons were removed from the pods and ground to a mesh size of 1mm.

Methods

Chemical Analysis

Crude protein and lipid contents of each diet were determined using the methods described by (Horwitz,1980), ash content was determined by incineration at 5000C to a constant weight as described by (Pearson,1973) and crude

Fowomola, M. A and Akindahunsi ,A. A: Continental J. Biomedical Sciences 4: 50 - 54, 2010

fibre was measured by using methods described by (Foster and Leslie,1971). Carbohydrate content of each diet was determined by using Clegg – Anthrone Method (Norman and Waddington,1989).

Bioassay

Experimental Animal and Diets

A total of twenty – four male albino rats of Wistar strain were used. They were made up of four rats in each group and housed in individual steel cages. Animal grouping was performed on the bases of their body weights which were homogenously distributed within all groups. Six groups of rats were maintained on different protein diets as follows: Group I – control diet (Casein), Group II – basal diet (no protein), Group III – deffated cooked hura seed diet, Group IV – undafatted cooked hura seed diet, Group V – raw defatted hura seed diet and Group VI – raw underfatted hura seed diet. Quantities of protein sources incorporated were such as would keep the protein of each diet at a maximum of 10 percent as described by(A.O.A.C. ,1980). Water and feed were supplied adlibitum throughout the experimental period. Each of the experiment was run for seventeen days, three days for preliminary feeding (i.e acclimatization) and fourteen days for actual feeding.

Preparation of Samples for ALP, ACP, AST, and ALT Activities

At the end of the experiment, all rats were anaesthetized using chloroform. Blood samples were collected from dying rats by cardiac puncture using needle and syringe. Each blood sample collected was centrifuged for 15 min at 2,000 g in a centrifuge, serum was collected in a sterile bottle and kept inside a refrigerator at -40C. The stomach and thoracic regions of each rat were cut opened, liver was removed, kept inside sterile bottles and refrigerated at -40C for further studies. Each liver (1gm) was weighed and homogenized by using pestle and mortal already placed in ice. 1ml of triton detergent was added to achieve total disruption of the cell membrane. 4ml of (0.25M sucrose solution containing 10mM Tris buffer, pH 7.4) was added to the homogenate and used for analysis.

Determination of Enzyme Activities

Alkaline phosphatase (ALP) and acid phosphatase (ACP) activities were determined using the colorimetric methods described by(Wright et.al.,1972a; Wright et.al.,1972b ).Aspartate – amino transferase (AST) and Alanine – amino transferase (ALT) activities were determined using the methods described by Reitman and Frankel ( 1957).

Statistical Analysis

All data were analyzed by one – way ANOVA (P<0.05) and least significant differences between treatment means were determined by Duncan’s multiple-range test (P<0.05) (Steel and Torrie,1980).

RESULTS AND DISCUSSION

Figures 1-2 depict the effects of Hura crepitans seed meals on the ACP, ALP, AST and ALT activities of liver and serum of rats. In general, the activities of ACP, ALP, AST and ALT in liver of rats fed with deffated raw (diet V) and undefatted raw diets (diet VI) were significantly reduced (P≤0.05) compared with those of control (casein diet) (diet I ). Conversely, defatted and undefatted raw diets caused a statistically significant increase in the serum of ACP, ALP, AST and ALT. However, there was no significant (P≥0.05) difference between those of control (casein diet) and defatted cooked diet (diet III).

Only increase in serum ALP measurements are clinically important as it suggests hepatobilliary diseases or increased osteoblastic activity (Baron et.al., 1994). In addition, reduction in tissue levels of ACP and ALP activities with increase in their activities in serum could be associated with cell damage (Yakubu and Akanji ,2003; Buratai et.al., 2003). High level of ACP in the blood may indicate prostate cancer, Guacher’s diseases (which is a lipid metabolism disorder), hyper parathroidsim or Paget’s disease (Nordenson, 2002).

Increase in AST and ALT activities in the blood plasma has been attributed to cell damages (Ottah et.al.,2003). ALT is thought to be a more specific indicator of liver inflammation, since AST may be elevated in diseases of other organs such as heart or muscle disease ( David, and Johnson,1999).

The reduction of enzyme activities in liver was more pronounced in diets containing deffated raw (diet V) and undefatted raw (diet VI) hura crepitans seeds than those of defatted cooked (diet III) and undefatted cooked (diet

Fowomola, M. A and Akindahunsi ,A. A: Continental J. Biomedical Sciences 4: 50 - 54, 2010

IV). This could be as a result of presence of antinutrients present in the former diets. The toxic effect of hura crepitans seed attributed to the presence of tannins and hemagglutions in it(Adedire and Ajayi, 2003). Possible carcinogenesis is also evident by the production of liver cancer when tannins are applied to burns or administered subcutaneously repeatedly as reported by Bichel and bach (1968). Furthermore, Hura crepitans being a member of Euphorbiacaea family has been reported to contain cyanide(Anosike and Eqwuatu, 1980). Cyanide is known for its inhibitory effect on Cytochrome oxidase in cell respiration (Oke, 1997).

CONCLUSION

The present research work has shown that defatting and cooking processes improved the nutritional quality of Hura crepitans seed. Therefore, they can be used to detoxify hura crepitans seed before its incorporation into feeds.

REFERENCES

Adedire and Ajayi (2003); Potential of Sand Box Hura crepitans Seed oil for Protection of Cowpea Seeds from Bruchidae Infestation. Journal of Plant Diseases and Protection. 110(6). 1-9.

.Allen, T. F (2000); Hura crepitans.L- the Encyclopedia of Pure Material Medical Pp. 1-2.

Anosike, E. O. and Eqwuatu, C. K. (1980); Changes During the Fermentation of Castor Oil (Rinus Communis) Seeds for use as a Seasoning Agent. Qualities Plant. P1. ''Foods. Human. Nutria''.: 30, 181 – 185.

A.O.A.C. (1980) – Official Methods of Analysis of Analytical Chemists.12th Edition. Washington, D.C.Pp.1-60.

Baron, D. N., Wicher, J. T and Lee, K. E (1994). A New Short Textbook of Chemical Pathology. ELBS 5th Edition. Pp. 151 – 156.

Bichel, J. and bach, A. (1968); Investigation on the Toxicity of Small Chronic Doses of Tannic Acid with Special Reference to Possible Carcinogenicity. Acta Pharmacol Toxicol 36;Pp.41-45.

Buratai L. B., Umar, I. A, Bello, H. and Gadaka, M. A. (2003); Biochemical and Haematological Changes Following Administration of Sub – Tethal does of Dimethoate in rates. Book of Abstracts of 23rd Annual Conference of the Nigerian Society of Biochemistry and Molecular Biology, Ilorin, Nigerian.Pp. 35-36.

Clarke, J. H (2000); Hura crepitans. A Dictionary of Practical Material Medical. P 1-2.

David, E. and Johnson, M. D (1999); Special Consideration in Interpreting Liver Function Tests; American Family Physician, Published by the American Academy of Family Physicians, http: www afp. Org/afp/990415/2223.htm/

Fagbemi, T. N and Adebowale, Y. A (2000) Food Potential of Hura crepitans. Proceeding of the 24th Annual Conference of Nigeria Institute of Food Science & Technology (NIFST)Bauchi, Nigeria.

Fowomola M. A and Akindahunsi A. A (2005) Protein Quality Indices of Sandbox (Hura crepitans) seeds''. J. of Food, Agric and Environment'' Vol. 3(2).

Foster, D. S and Leslie, S. E (1971); In Crude Fibre. Encyclopedia of Industrial Chemical Analysis. Vol. II.Pp. 49 – 51.

Horwitz, W. (1980): Official Methods of Analysis (13th Edition.Association of Official Analytical Chemical, Washington D.C U.S.A).

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Nordenson, N. J (2002) Acid Phosphatase Text. http://www. Health atozconal heathatoz/atoz/ency/acid-phosphatase-test.html.

Norman, R.O.C and Waddington, D. J (1989) .In Carbohydrates. Modern Organic Chemistry 4th Edition un win hyman Ltd. Pp.289-297.

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Oke, D. B (1997). Mechanism of Action, Toxicity and Nutritional Significance of Heat – Stable Anti-nutritional Factors in some Legumes. A ''Review Nig. J. Nutria. Sci.'' Vol. 18(2) 1-5.

Ottah, G. M., Wegwu M. O and Sule O. J (2003); The Effects of Water Soluble Fraction (WSF) of Crude Oil on some Biochemical Indices of Clarias gariepinus Book of Abstracts of 23rd Annual Conference of the Nigerian Society of Biochemistry and Molecular Biology. Ilorin, Nigeria.Pp. 37.

Pearson D. (1973) Laboratory Techniques in Food Analysis Butter Worth London.

Reitman, S.and Frankel, S. A. (1957). Colorimetric Method for the Determination of Serum Glutamic Oxaloacetic and Glutamic Pyruvic Transaminases. Am J. Clin. Pathol., 28.Pp. 56-60.

Steel, R. G and Torrie, J. H (1980); Principles and Procedures of Statistics.A Biometrical Approach. 2nd Edition McGraw Hill Book Co. New –York.

Wright P. J. Leathward P. D and Plunner, D. T (1972a) Enzyme in rat urine:Alkaline Phosphatase. Enzymologia. 42.317 – 327.

Wright P. J. Leathward P. D and Plunner, D. T (1972b) Enzyme in rat urine: Alkaline Phosphatase. Enzymologia. 42.327 – 337.

Yakubu M. T and Akanji M. A (2003). Some Biochemical Changes in Selected rat tissues following Repeated Administration of Cimetidine. Book of Abstracts of 23rd Annual Conference of the Nigerian Society of Biochemistry and Molecular Biology, Ilorin, Nigeria. Pp.38-39.

Fowomola, M. A and Akindahunsi ,A. A: Continental J. Biomedical Sciences 4: 50 - 54, 2010

Received for Publication: 19/07/2010

Accepted for Publication: 18/08/2010

Correspondence Author

Fowomola, M. A

Science Technology Department, Federal Polytechnic, Offa, P.M.B 420 Offa, Kwara State Nigeria.

E-mail: [mailto:moshoodfowomola@yahoo.com moshoodfowomola@yahoo.com]

Continental J. Biomedical Sciences 4: 55 - 62, 2010 ISSN:2141 – 419X

©Wilolud Journals, 2010 http://www.wiloludjournal.com

RESPONSE PATTERN OF MIXED BACTERIAL POPULATION TO ANTIMICROBIAL AGENTS.

Okoko, F.J. and Uwafili, N.P.

Department of Microbiology, Delta State University, Abraka.

<p style="margin-left: 17pt;">ABSTRACT

<p style="margin-left: 17pt;">The effect of four antimicrobial agents, Chloramphenicol, Tetracycline, Amoxicillin and Ciprofloxacin on the growth of Staphylococcus aureus and Salmonella species was studied. Tetracycline produced the highest inhibition for Staphylococcus aureus at a concentration of 0.0625mg/ml while Ciprofloxacin produced the highest inhibition for Salmonella species at a concentration of 0.2500mg/ml. Both bacteria isolates were susceptible to amoxicillin at the same minimum inhibitory concentration (MIC) of 1.0000mg/ml. For combination thereby against individual isolates ciprofloxacin + Amoxicillin was inhibitory to Staphylococcus aureus at a concentration of 0.2500mg/ml which was the highest MIC. A combination of Tetracycline + Chloramphenicol was not inhibitory to Salmonella species. Combination therapy against mixed bacteria showed a MIC of 0.5000mg/ml for ciprofloxacin + Amoxicillin while Tetracycline + Chloramphenicol was not inhibitory. The result showed that Tetracycline and Ciprofloxacin are good antimicrobial agents for infections caused by Staphylococcus aureus and Salmonella respectively. For mixed bacterial infections cause by Staphylococcus and Salmonella species, ciprofloxacin and combination of Amoxicillin + Ciprofloxacin are effective.

<p style="margin-left: 17pt;">KEY WORDS: Antimicrobial, inhibition, isolates, concentration, combination, population.

INTRODUCTION

Drugs have been used for the treatment of infectious diseases since the 17th century. However, chemotherapy as a science began with Paul Ehrlich in the first decades of the 20th century. Paul Ehrilich developed salvarsan which was the first documented example of a chemical used successfully as antimicrobial medication. He also formulated the principle of selective toxicity and recognized the specific chemical relationships between microbial pathogens and drugs; the development of drug resistance and the role of combined therapy.

The current era of antimicrobial chemotherapy began in 1935 with the discovery of the sulfanamides. In 1940, it was demonstrated that penicillin, which was discovered in 1929, could be an effective therapeutic drug (Hoht et al (1990). During the next 25years, research on chemotherapeutic agents largely centered on substances of microbial origin called antibiotics. The isolation, concentration, purification and mass production of penicillin were followed by the development of Streptomycin, Tetracycline, Chloramphenicol and other agents. The above agents were produced by mould species, Streptomyces griseus, soil Actinomycetes and Streptomyces venezuelae (Brook et al 2002).

ANTIMICROBIAL SUSCEPTIBILITY

Treatment of uncomplicated gastroenteritis caused by non typhoidal Salmonella is a matter of some contention. People with milder infection do not seek treatment and those who seek attention of physicians are usually quite III and in great discomfort and expect antibiotic to relief their condition. However, Amoxicillin should be administered only to patients at risk for a poor outcome (Farthing et al, 1996).

Chloramphenicol is a substance produced originally from cultures of Streptomyces venezuelae but now manufactured synthetically. Chloramphenicol is rapidly absorbed from the gastrointestinal tract and widely distributed into those tissues and body fluids, excretion is mainly in the urine, 90% in inactive form. It is a potent inhibitor of protein synthesis in microorganisms. Chloramphenicol resistance is due to the distribution of the enzyme chloramphenicol acetyl transferase which is under plasmid control Moellering et al 2000.

Tetracyclines are a group of drugs that differ physically and in pharmacologic characteristics but have virtually identical antimicrobial properties and gives complete cross-resistance. All tetracyclines are readily absorbed from intestinal tracks and distributed widely in tissues but penetrate into the cerebro spinal fluid poorly. They inhibit the growth of all gram-positive and gram-negative bacteria but they do not inhibit fungi Brooks et al (2002).

Okoko, F.J. and Uwafili, N.P.: Continental J. Biomedical Sciences 4: 55 - 62, 2010

Amoxicillin are members of the family penicillin which their side chains have been modified in the laboratory to create penicillin derivatives. They are broad spectrum penicillin which retains their activities against penicillin sensitive gram positive bacteria, yet they are also active against gram negative organisms. They can be inactivated by β-lactamases. (Nester et al, 2001).

Ciprofloxacin belong to the fluoroquinolones which are synthetic drugs which inhibit one or more of a group enzyme to poisomers which maintain the supercoiling of a closed circular DNA with the bacterial cell.

The fluoroquinolones are bactericidal against a wide variety of bacteria including both gram-positive and gram-negative organisms.

TEST ORGANISMS

Staphylococcus aureus is a gram-positive spherical cell, usually arranged in grape –like irregular clusters. It grows rapidly on many types of media and is active metabolically, fermenting carbohydrates and producing pigments that vary from white to deep yellow Brooks et al, 2002. it is a normal flora of the skin and mucous membrane of humans. It is non-motile and does not cause a wide range of major and minor pathogenic infections. They can cause infections such as superficial inflammation, lesions with pus formation such as skin pustules, boils, carbuncles, phlebitis, styles, impetigo and pemphingus neonatonum.

Staphylococcusaureus rapidly develops antimicrobial resistance to many antimicrobial agents and present difficult therapeutic problems. Multiple drug resistance is explained by the acquisition of plasmid encoding for such resistance by Staphylococcus aureus strains. Certain strains of S. aureus are resistant and this occur more in hospital environments than in the community. (Baily and Scott, 1990).'''

SALMONELLA

Salmonella species are rod-shaped, motile, non-spore forming, gram-negative bacteria. They never ferment lactose or sucrose an form acid and sometimes gas from glucose and manose.

Environmental sources of salmonella include water, soil, animal feaces, raw meat, raw poultry, raw sea foods etc. they are transmitted from animals and animal products to humans where they cause enteritis, systemic infections and enteric fever. Acute symptom include nausea, vomiting, abdominal cramps, diarrhea, fever and headache. Salmonella carried by animals have been found to develop resistance to drugs such as tetracyclines incorporated into animal feeds while many species have been found to be sensitive to most antibiotics such as ceftriaxone, ciprofloxacin, afloxacin, levofloxacin, sparfloxacin, troxacin, ampillin, chloramphenicol etc, used in therapy (Brooks et al, 2002).

AIMS AND OBJECTIVES

This study is therefore designed to study the response pattern of mixed bacteria population to some selected antimicrobial agents and possible synergistic effects of these agents on the study organisms.

MATERIALS AND METHODS

Pure isolates of Staphylococcus aureus and Salmonella species used for this study were obtained from Delta State University Health Center and were identified and characterized using cultural, morphological staining and biochemical properties as described by Buchanan and Gibbons (1974), Monica (1984) and Cowan and Steel (1974).

The identified microorganisms were transferred unto agar slants in sterilized bijou bottles and saved in the refrigerator for subsequent use.

The antimicrobial agents used for this study are Tetracycline hydrochloride B.P 250mg, produced by

Yangahou No. 3 Pharmaceutical Company Limited, China. Chloramphenicol capsule, B.P. 250mg produced by Mendel Pharmaceutics PVI Limited, India, Ciprofloxacin tablets USP 500mg, produced by U.S International PVT Limited, India and Amoxicillin capsule B.P 500mg produced by Elder Pharmaceuticals Limited, Mumbere, India.

Okoko, F.J. and Uwafili, N.P.: Continental J. Biomedical Sciences 4: 55 - 62, 2010

Five hundred milligrams of the different antimicrobial agents were dispensed into four different sterilized conical flasks. Five hundred milliliter of distilled water was added and the content of each flask was swirled gently to allow for the even distribution of the antimicrobial agents.

SUSCEPTIBILITY TEST

Ten milliliter of sterilized nutrient brought was dispensed into two sterilized test tubes, inoculated with 1ml Salmonella and Staphylococcus aureus respectively and were incubated at 2370C for 24h and observed for growth. The content of each tube was washed three times making use of a bench-top centrifuge (Gallenkamp Centrifuge, EEC) at 1400mg for 30min to remove all the media. Serial dilution to 103 number of cells was made for each isolate.

Test on the susceptibility of the test microorganisms to the four antimicrobial agents was carried out using pour plate method.

One milliliter of each of the test organisms already diluted to 103 was pipetted into sterile Petri dishes using 5ml sterile pipettes after which 1ml each from the stock solutions of the antimicrobial agents was added and mixed thoroughly. The plates for each antibiotics were prepared in duplicate. One and a half milliliter of sterile nutrient agar was dispensed into each Petri dish and their content swirled gently for proper mixing after which they were incubated at 370C for 24h. The susceptibility of the test organisms was confirmed by the absence of growth on the inoculated plates. Sterile nutrient agar plates containing 1ml each of the test antibiotics were used as control.

DETERMINATION OF MINIMUM INHIBITORY CONCENTRATION (MIC)

Serial dilution was carried out on each of the antimicrobial agents making use of distilled water. Five sterile test tubes were used for each of the antimicrobial; agent and were placed in a test tube racks. Nine milliliter of distilled water was added into the first tube of each antimicrobial agent using well sterilized pipettes and labeled accordingly. One milliliter of the different antimicrobial agent was dispensed into each of the labeled test tubes and mixed thoroughly.

Serial dilution was made in decreasing concentration. One milliliter of the 103 of each serially diluted test organisms was dispensed into separate sterile petri dishes for each antibiotic and labeled. One milliliter each of the antibiotic was added to the appropriate petri dish and mixed thoroughly after which nutrient agar was poured and plates were swirled for proper mixing. They were immediately incubate at 370C for 24h and examined for visible growth. All experiments were in replicate. The least dilution for the antibiotic that yielded no growth is the minimum inhibitory concentration (MIC).

For the determination of MIC using two antimicrobial agents, 0.5ml each of the two antimicrobial agent was added to each of the petri dishes containing 1ml of the test organisms (Salmonella and S. aureus) was put in separate petri dish for the different test antibiotics and nutrient agar were poured. Each plate was mixed thoroughly and incubated at 370C for 24h.

In determining the MIC using single therapy and mixed bacteria isolates, 0.5ml each of the test organisms (Salmonella and S. aureus) was put in separate petri dish for the different test antibiotics and nutrient agar were poured. Each plate was mixed thoroughly and incubated at 370C for 24h.

The following were set-up as experimental control.

The Negative Controls are:

Nutrient broth only.

Nutrient + Sterile distilled water.

All test organisms + nutrient agar.

All test antibiotics + washed test organisms

Nutrient broth + sterile distilled water

Positive Control is: Nutrient agar + organisms

Okoko, F.J. and Uwafili, N.P.: Continental J. Biomedical Sciences 4: 55 - 62, 2010

RESULTS

Table 1 shows the various concentration of the antibiotics used for this study. The effect of tetracycline, chloramphenicol, ciprofloxacin and amoxicillin on the growth of Salmonella species is shown in Table 2. ciprofloxacin and amoxicillin showed a minimum inhibitory concentration (MIC) of 0.5000mg/ml and 1.000mg/ml respectively on the test organisms while the organisms are resistant to both tetracycline and chloramphenicol.

The effects of the antibiotics on Staphylococcus aureus was shown in Table 3. tetracycline had MIC of 0.0625mg/ml for the test organisms while ciprofloxacin and amoxicillin had MIC of 0.5000mg/ml and 1.0000mg respectively for the same organism, Staphylococcus aureus was totally resistant to chloramphenicol.

The effects of combined therapy on the test organisms was shown in Table 4 and 5. The MIC for the combination of Tetracycline and chloramphenicol on Staphylococcus aureus was 1.000mg/ml while that of ciprofloxacin and amoxicillin with a MIC of 0.5000mg/ml as shown in Table 5.

The effects of single therapy on the mixed bacteria specimen is shown in Table 6. A mixture of Staphylococcus aureus and Salmonella species were resistant to both tetracycline and chloramphenicol but inhibited by ciprofloxacin and amoxicillin with MIC of 0.5000mg/ml and 1.000mg/ml respectively.

The effects of combined therapy on the growth of mixed bacteria specimen is shown on Table 7. The test organisms (Staphylococcus aureus Salmonella species) were resistant to the combination of tetracycline and chloramphenicol while they were inhibited by the combination of ciprofloxacin and amoxicillin at MIC 0.5000mg/ml.

Table 1:Serial Dilution of Antimicrobial Agents

Table 2:Effect of the four Antimicrobial Agents on the growth of Salmonella Species

KEY

++ Heavy growth, + Light growth, - No growth, Minimum Inhibitory Concentration (MIC)

TET: Tetracycline, CHL: Chloramphenicol, CIP: Ciprofloxacin, AMO: Amoxicillin

Okoko, F.J. and Uwafili, N.P.: Continental J. Biomedical Sciences 4: 55 - 62, 2010

Table 3:Effect of the four Antimicrobial Agents on the growth of Staphylococcus aureus 

KEY

++ Heavy growth, + Light growth, - No growth, Minimum Inhibitory Concentration (MIC)

TET: Tetracycline, CHL: Chloramphenicol, CIP: Ciprofloxacin, AMO: Amoxicillin

Table 4:Effect of Combined therapy on the growth of Staphylococcus aureus

KEY

++ Heavy growth, + Light growth, - No growth, Minimum Inhibitory Concentration (MIC)

TET: Tetracycline, CHL: Chloramphenicol, CIP: Ciprofloxacin, AMO: Amoxicillin

Table 5:Effect of Combined therapy on the growth of Salmonella species

KEY

++ Heavy growth, + Light growth, - No growth, Minimum Inhibitory Concentration (MIC)

TET: Tetracycline, CHL: Chloramphenicol, CIP: Ciprofloxacin, AMO: Amoxicillin

Okoko, F.J. and Uwafili, N.P.: Continental J. Biomedical Sciences 4: 55 - 62, 2010

<p style="margin-left: 36pt;">Table 6:Effect of Single therapy on the growth of mixed bacteria (Salmonella species + Staphyloccus aureus)

KEY

++ Heavy growth, + Light growth, - No growth, Minimum Inhibitory Concentration (MIC)

TET: Tetracycline, CHL: Chloramphenicol, CIP: Ciprofloxacin, AMO: Amoxicillin

<p style="margin-left: 72pt;">Table 7: Effect of Combine therapy on the growth of mixed bacteria (Salmonella species + Staphyloccus aureus)

KEY

++ Heavy growth, + Light growth, - No growth, Minimum Inhibitory Concentration (MIC)

TET: Tetracycline, CHL: Chloramphenicol, CIP: Ciprofloxacin, AMO: Amoxicillin

DISCUSSION

Gram-positive bacteria are usually more sensitive to antibiotics than gram-negative bacteria, although some antibiotics act only on gram-negative bacteria (Thomas, 1979)

From the results obtained in this study, it could be seen that Staphylococcus aureus a gram-negative organism, was sensitive to three of the four test antimicrobial agents used for this study: tetracycline, ciprofloxacin and amoxicillin. Salmonella species which is a gram-negative organisms was sensitive to only two of the four antimicrobial agents namely ciprofloxacin and amoxicillin. Both organisms were found to be resistant to chloramphenicol. The development of resistance to antimicrobial agents by organisms may be due to misuse of drugs, use of drugs in life stock fees; the veterinary use of microbial agents may select resistant organisms and facilitate their spread.

Resistance of microorganisms to antibiotics may be mediated by chromosomally located genes by horizontal transfer through plasmids and transposons. Many of the resistant genes present as plasmids and transposons of gram-negative bacteria are integrated in integrons. Class 1 integrons are wide spread among Salmonella species.

The rates of resistance of salmonella species to tetracycline, chloramphenicol, ampicillin and trimethopraim-sulfamethoxazole increased drastically between 1984 to 2001. the susceptibility of the two bacteria isolates to ciprofloxacin is because ciprofloxacin blocks the action of bacteria enzyme catalyzing the transpeptidation reaction during cell wall synthesis in prokaryotes.  Staphylococcus aureus is susceptible to tetracycline because tetracycline inhibits synthesis in the organisms by combining with the 30S subunits of the ribosome and inhibiting of tRNA molecule to the ribosome.

Okoko, F.J. and Uwafili, N.P.: Continental J. Biomedical Sciences 4: 55 - 62, 2010

Treatment with antimicrobial combinations may be necessary in certain cases. The administration of two or more agents may be beneficial in the following situations:

To treat mixed bacteria infections in which the organisms are not susceptible to a common agent.

To achieve a synergistic antimicrobial activity against particularly resistant strains.

To overcome bacterial tolerance

To prevent the emergence of drug resistance

To minimize toxicity and

To prevent inactivation of an antibiotic by enzymes produced by other bacteria that are present.

Ideally, antimicrobial selection should be based on different mechanisms of actions and on spectra of activity that are complementary. β–lactams are often selected because their actions is unique and not only complement other drugs but also facilitates their movement through the damaged cell wall into the microbe. Examples of combination therapy for mixed infections include the use of clindamycin metronidazole or the semi synthetic penicillin in combination with amino glyxcosides for their efficacy on gram-negative organisms. Preventing of development of resistance with combination of antimicrobial therapy is best exemplified by the use of carbonicillin or amikacin together with gentamycin or tobramcyin for the treatment of Pseudomonas and other related infections.

CONCLUSION

Since many infectious microorganism have apparently developed resistance to many of the existing antimicrobial agents, it is important that more effort be directed towards research on the development of more efficacious antimicrobial agents which working singly or in combination would help check the threat of these microorganisms to human existence.

ACKNOWLEDGEMENT

I thank the Almighty for the wisdom, knowledge and good health to carry out this study and to my family I say Thank YOU for your understanding and support. May the almighty bless you all.

REFERENCES

<p style="margin-left: 54pt;">Bailey, W. R. and Scott, E. E. (1990), Diagnostic Microbiology (3rd edn). The C.V Mesby Cost Louis. 84p

<p style="margin-left: 54pt;">Bachanan, B. E. and Gibbons bacteriology (5th edn) Williams and Wilkins Co. Baltimore, MD. 128p.

<p style="margin-left: 54pt;">Brooks, G.F., Butel, J.S. and Morse, S.A. (2002). Jawetz, Melmick and Adelborg’s Medical Microbiology. (22ndEd.) McGraw-Hill, New York. 720p.

<p style="margin-left: 54pt;">Cowan, S. T., and Steel, K. J. (1974). Manual for the identification of Medical bacteria (2nd edn). Cambridge University Press, England 237p.

<p style="margin-left: 54pt;">Farthing, M., Feldman, R, and Finch, r. (2996). The management of infective gastro enteritis in adults: a consensus statement of an expert panel concerned by the British society for the study of infections. Journal of Infectious Diseases (5th edn) 33:143-152.

<p style="margin-left: 54pt;">Hohl, P., Luthy-Hottenstein, J., Zollinger-Hen, J., and Altwegg, M. (1990). In vitro activities of fleroxacin, cefetamet, ciprofloxacin, celtriaxone, trimethroprin – selfamethaxadole, amoxicillin-clavulanic acid and ampecillin against rare members of the family enterobacteriaceae primarily of human origin. Antimicrobial Agents Chemotherapy 34:1605-1608.

<p style="margin-left: 54pt;">Moellering, B.C., Mandel, G.I., Bemett, J.E. and Dolin, R. (2000). Principles and practice of infectious disease (5th Ed.) Churchill Livingstone, Edingburg 320p.

<p style="margin-left: 54pt;">Monica, C. (1984). Microbiology: Manual for tropical countries (Vol.11) Cambridge University Press, London 480p.

Okoko, F.J. and Uwafili, N.P.: Continental J. Biomedical Sciences 4: 55 - 62, 2010

<p style="margin-left: 54pt;">Nester, E.W. Roberts, C.E., Pearsall, N.N., Anderson, D.G. and Nester, M.T. (2001). Microbiology: A human perspective. (3rd Ed.) McGraw-Hill, New York 320p.

<p style="margin-left: 54pt;">Thomas, D.B. (1979). Biology of microorganisms (3rd Ed.) Prentice-Hall Inc, Eaglewood Cliff, New Jersey 802p.

Received for Publication: 19/07/2010

Accepted for Publication: 18/08/2010

Correspondence Author

Okoko, F.J.

Department of Microbiology, Delta State University, Abraka.

Continental J. Biomedical Sciences 4: 63 - 66, 2010 ISSN:2141 – 419X

©Wilolud Journals, 2010 http://www.wiloludjournal.com

FUNGAL INFECTION OF THE NAILS DUE TO PROBABLE HANDLING OF WET CURRENCY NOTES

Anyanwu, E. B1., Mabiaku, T. O1., and Okperi, B2

1Department of Family Medicine, 2Department of Paediatrics, Faculty of Clinical Medicine. College of Health Science, Delta State University, Abraka.

<p style="margin-left: 17pt;">ABSTRACT

<p style="margin-left: 17pt;">Onychomycosis is a fungal infection of the nail beds and it is caused by dermotophytes, yeast or nondermotophytes. More and more, Onychomycosis is viewed as being more than a cosmetic problem. Onychomycosis in immuncompormised clients can pose a more serious health problem. Demotophytes are the most frequently implicated causative agents. Despite improved personal hygiene and living environment, onychomycosis continues to spread and persist. This case report is that of a young Nigeria male who probably got infected by prolonged unprotected handling of wet, soggy currency notes in his place of work.

<p style="margin-left: 17pt;">KEYWORDS: Dermotophytes, onychomycosis, wet currency notes, protection.

<p style="margin-left: 108pt;">INTRODUCTION

Fungal infection is a relatively common disease worldwide. Such fungal or mycotic infection could involve the nails, hair and skin. Different fungal species are known to infect different parts of the body and many non-fungal disorders may clinically simulate a mycotic infection. This makes it necessary for proper diagnosis with basic laboratory testing such as a potassium hydroxide preparation (KOH prep) and fungal culture.

Incidentally, our index case probably contracted the infection from prolonged contact with possibly infected wet currency notes. Unfortunately, we were unable to test any of the suspected currency notes for fungal elements neither do we see other colleagues of our client under study with similar problems. We did not do fungal culture because we do not have the facility for that.

<p style="margin-left: 108pt;">Case Report

A young male Nigerian was seen at a private clinic in Warri, Delta State, complaining of prolonged deformation of all of his fingernails and some of his toenails. He said that the problem began some months ago after he was involved in sorting out old and wet currency notes in his workplace.

He works in one of the new generation banks in the country. He reported that soon after the completion of the work, which took about three weeks and which was done without wearing hand gloves, that he noticed some rashes on two of his fingers of the left hand.

This was followed by a gradual involvement of other fingernails until all the ten fingernails were involved.

Initially, what he had was a darkening around the proximal part of the nail bed but gradually over time, the entire fingernails got darkened. They eventually got heaped up and thickened, breaking easily and then became deformed.

Apparently, after one month, he noticed the same process as above on the toenails. This started on the two big toes and eventually spread to the other toenails.

It was at this point that he presented at the hospital for treatment.

Examination revealed darkened, heaped-up fingernails but normal fingers and toes. There were no signs of bacteria infection noted. The toes also revealed similar findings as above but only on four toes.

A working diagnosis of onychomycosis was made probably due to prolonged handling of infected currency notes. Retroviral test after counseling was negative and full blood count was normal and random blood sugar was normal.

Anyanwu, E. B et al.,: Continental J. Biomedical Sciences 4: 63 - 66, 2010

The problem was explained to him and treatment modality was explained to him. He was subsequently put on an oral anti-fungal medication and was to be reviewed monthly during drug refilling.

Unfortunately, he defaulted from treatment because he felt that there was no noticeable change in the texture of the fingernails after one month of treatment. He then went for traditional treatment, and applied some native herbal concoctions on the nails which led to burns and infection in the nail beds with formation of frank pus under all the fingernails.

He then reappeared at the clinic and was advised one more time on the need for compliance to drug management. Antibiotics were prescribed for him and follow-up visit showed marked improvement while adequate anti-fungal therapy was re-introduced.

DISCUSSION

Fungal infection of the nail bed, nail plate or both, otherwise known as tinea ungium or onychomycosis is mostly caused by trichophyton infection of one or more, but rarely all fingernails or toenails. The species most commonly found is Trichophyton rubrum. Saprophytic fungi may rarely cause (<5%) of onychomycosis (Berger, T. G. 1998).

Onychomycosis is a fungal infection of nails caused by dermatophytes, yeast or non-dermatophytes molds and represents about 30% of mycotic cutaneous infections. In spite of improved personal hygiene and living environment, onychomycosis continues to spread and persists. Dermatophytes are the most frequently implicated causative agents in onychomycosis (Kaur, R. et al 2007).

The prevalence rate of onychomycosis is determined by age, predisposing factors, social class, occupation, climate, living environment and frequency of travel. The prevalence is higher (25%) in patients with immunodeficiency viral infection (Kaur, R. et al 2007).

Studies have shown that prevalence of onychomycosis increases with age, reason for which may include poor peripheral circulation, diabetes, repeated nail trauma, and longer exposure to pathogenic fungi (Aditya, G. K, 1997).

Studies of patients with either psoriasis or diabetes indicate that both types of patients have a much higher chance of contracting onychomycosis than normal persons. If a diabetic is a male, older and has either peripheral vascular diseases or has a family history of onychomycosis his chances of contracting the condition at some point are increased (Aditya, G. K, 1997).

In patient with psoriasis who presents with a nail abnormality, it may be difficult to decide whether this is due to psoriasis, onychomycosis, a combination of the two or another cause (Aditya, G. K, 1997).

Also, because of the high prevalence of onychomycosis in diabetics, the availability of oral antifungal agents that treats effectively may play a role in optimal long-term management of diabetes and diseases related to it.

About 10% of the population has onychomycosis. Risk factors include tinea pedis, pre-existing nail diseases, older age, male sex and circulatory diseases.

Toenails are ten times more commonly infected than fingernails. About 60 – 80% of cases are caused by dermatophytes (Trichophyton rubrum). Many of the remaining cases are caused by non-dermatophyte molds such as Aspergillus, Scopulariopsis and Fasarium. Immuno-compromised patients may have candidial onychomycosis (Beers, M. H 2006)

Onychomycosis secondary to non-dermatophytes moulds is seen most frequently in the elderly, in patients with skin diseases that affect the nail and in immuno-compromised patients (Kaur, R. et al 2007).

There are usually no symptoms the affected nails are often brittle and friable, breaking easily. They are lusterless and hypertrophic.

Anyanwu, E. B et al.,: Continental J. Biomedical Sciences 4: 63 - 66, 2010

Onychomycosis is difficult to treat because of the long duration of therapy required and the frequency of recurrences.

Griseofulvin was the first orally effective anti-fungal agent and the only one available for over twenty years. Although it helped many patients with dermatophytic skin infection, it was ineffective against infection caused by candida, as well as systemic and subcutaneous fungal infection (Zeid S. A 1994).

The continued search for an anti-fungal drug with a broad spectrum of activity led to the introduction of new antimycotic drugs, which have opened a new era in the treatment of fungal infections (Elewski, B. 1997). They are safer and the risk for toxicity is rare.

Physicians and patient now benefit from significant advances in the treatment of superficial fungal infection of the skin, hair, nails and mucosa.

Despite these advances, treatment failure still occurs for a variety of reasons, many of which can be preventable. Whenever available, physicians are encouraged to use recommended regimens that have been substantiated as being optimal. Under-dosage or improper treatment duration commonly leads to treatment failure due to sub-therapeutic tissue concentration (Rosso Del et al 1998).

Overall, approximately 20 – 30% of onychomycosis patients exhibit only partial clearance or fail treatment with any of the newer oral agent. Occasionally, patient may present with combination of onychomycosis and “onychobateriosis” just like our client did after the application of herbal concoction on the nail beds. This too, could affect the management especially if not properly diagnosed and treated appropriately.

Our index case got infected while sorting out possibly infected bank notes. He was involved in the job without wearing protective hand gloves nor even face mask and this probably explains why his fingernails were infected first and then more severely than his toenails. Such protective devices such as wearing hand gloves would have prevented the prolonged contact between the possibly infected currency notes and the patients; and wearing face masks would have removed the risk of inhaling fungal spores in the environment. Also, the vault should have ventilators that helps clear the environment of air-borne droplets of fungal spores.

This case report is to highlight the possibility of our currency notes been able to transmit fungal infection and then to serve as a call for proper handling of the currency notes. Proper handling of currency notes must be encouraged and these includes the stoppage of the act of spraying notes during parties and the celebrants dancing on the sprayed notes; not squeezing the notes; not the writing on the currency notes and not washing the notes in our laundry. It is recommended that the currency notes be handled gently and arranged stretched out in our dry wallets.

We also recommend that central laboratories with facilities to do fungal cultures be established in the national geographical regions.

REFERENCES

<p style="margin-left: 36pt;">Aditya. G. K. 1997: Psoriatic and diabetic patients have high risks for onychomycosis. Mycol Observ, 6 (5): 6 

<p style="margin-left: 36pt;">Beers, M. H., and Porter, R. S. (Eds). 2006:The Merck Manual of Diagnosis and Therapy, 18th edition. Nail

<p style="margin-left: 36pt;">Disorder. In: Dermatologic Disorders. Merck Research Laboratories USA. pg 932 – 1027.

<p style="margin-left: 36pt;">Berger, T. G. Skin and Appendages 1998: In: Current Medical Diagnosis and Treatment. 37th edition. Tierney, L.

<p style="margin-left: 36pt;">M., McPhee, S. J., and Papadakis, M. A. (Eds). Appleton and Lange, USA. pp. 111 – 179.

<p style="margin-left: 36pt;">Elewski, B. 1997: Newer anti-fungals are treatment choice against onychomycosis. ''Mycol Observ 6, No. (5): 2. ''

<p style="margin-left: 36pt;">Kaur, R., Kashyap, B., and Bhalla, P. 2007: Onychomycosis – Epidemiology, diagnosis and management. Nig.

<p style="margin-left: 36pt;">Biomed Sci J. 4 (2) 7 – 15.

<p style="margin-left: 36pt;">Rosso Del, J. Q., and Gupta, A. K. 1998: Optimising Treatment with Oral Anti-fungal Agents. Based on lecture

<p style="margin-left: 36pt;">presentation at the 56th Annual American Academy of Dermatology Meeting, Orlando, USA.

<p style="margin-left: 36pt;">Zeid, S. A., and Halim, S. 1994: Short term Oral Lamisil (terbinafine) in the treatment of moccasin tinea pedis.

<p style="margin-left: 36pt;">African Clinician 18.Medicine Group (Journal) Ltd.

Received for Publication: 19/07/2010

Accepted for Publication: 18/08/2010

Correspondence Author

<p style="margin-left: 144pt;">Department of Family Medicine, Faculty of Clinical Science, College of Health Science, Delta State University,

<p style="margin-left: 144pt;">Abraka, Nigeria.

<p style="margin-left: 108pt;">E-Mail: ebirian@yahoo.com

Continental J. Biomedical Sciences 4: 67 - 74, 2010 ISSN: 2141 – 419X

©Wilolud Journals, 2010 http://www.wiloludjournal.com

EFFECT OF HEPATITIS C VIRUS ON HAEMOGLOBIN AND HAEMATOCRIT LEVELS IN ABUTH AND AKTH HAEMODIALYSIS PATIENTS

¹Rogo, L.D., ¹Akogwu, S., ²Muhammad, S., ²Aliyu, A., ³Owolabi, A. A., 4Malgwi, I. S. and 5Aliyu, U. D.

¹Department of Haematology and Blood Group Serology, School of Medical Laboratory Sciences, Ahmadu Bello University Teaching Hospital, Zaria.2Dialysis Unit, Department of Medicine Aminu Kano Teaching Hospital, Kano, 3Department of Haematology and Blood Group Serology, Ahmadu Bello University Teaching

Hospital, Zaria, 4Department of Human Physiology, Ahmadu Bello University Zaria, Kaduna State Nigeria, 5Department of Medical Laboratory Sciences Tudun Wada General Hospital, Kano State

<p style="margin-left: 17pt;">ABSTRACT

<p style="margin-left: 17pt;">Persistent infection with Hepatitis c virus (HCV) has emerged as one of the primary causes of chronic liver disease with an estimated 170 million people infected by HCV, more than 4 times the number of people living with HIV throughout the world. Infection with Hepatitis c virus is common among patients undergoing haemodialysis, and haemodialysis patients are at high risk for infection with such virus. The aim of this study was to assess the seroprevalence and the effect of HCV on PCV and HB in haemodialysis patients who were consented. Hepatitis c virus antibody testing was carried out using CLINOTECH DIAGNOSTIC AND PHARMACEUTICAL INC. B.C. V7A 5H5, CANADA via antibody testing kit. Information about the patient demographic factors and other variables were obtained from the patients or caregivers using a designed questionnaire. A total of 88 blood samples were analysed. The overall seroprevalence rate for HCV was 7.9%. Prevalence of HCV antibody was 6.8% in males and 1.1% in females. The age group of 61-70 years has the lowest prevalence of 1.1% while those of 51-60 years had the highest value of 4.5%. In view of the prevalence rate of HCV infection in this study, it is suggested that further epidemiological studies should be conducted to establish the exact role of HCV in liver disease among haemodialysis patients in Nigeria.

<p style="margin-left: 17pt;">KEYWORDS: Effect, Hepatitis c virus, Haemoglobin, Haematocrit, Haemodialysis, Patients.

INTRODUCTION

Despite rapid scientific progress in understanding the biology of viral illness, viral liver disease remains a common and challenging problem for physicians and their patients (Alter and Seeff, 2000). Persistent infection with Hepatitis c virus (HCV) has emerged as one of the primary causes of chronic liver disease with an estimated 170 million people infected by HCV, more than 4 times the number of people living with HIV throughout the world (WHO,2000). Of the typical hepatitis viruses, chronic infection with Hepatitis c virus remains one of the most important clinical and public health problems (El-Zayadi et al., 2004). In the western world, chronic damage from Hepatitis c is the primary cause of the end stage liver disease requiring liver transplantation (Niederan et al., 1998). The discovery of HCV in 1989 was a major breakthrough. Before that point, it was clear that a major cause of acute hepatitis after a blood transfusion was neither related to Hepatitis A nor to Hepatitis B- hence the early names for this disease, non-A, non-B hepatitis (Simmonds et al., 2005).

The virus is a small (55-65nm in size), included in Group IV, enveloped, positive sense, single stranded RNA virus, the family Flaviviridae, genus Hepacivirus, and Hepatitis c virus type species (Simmonds et al., 2005). Based on genetic differences between HCV isolates, the virus species is classified into six genotypes (1-6) with several subtypes within each genotype (represented by letters). Subtypes are further broken down into quasispecies based on their genetic diversity (Kato, 2000).

The open reading frame of the virus encodes the structural proteins core (C), enveloped (E1, E2), and the non-structural proteins (NS2, NS3, NS4a, NS4b, NS5a and NS5b) (Djebbi et al., 2004).

The most efficient transmission of HCV is through large or repeated direct percutaneous exposure to blood; example transfusion or transplantation from infectious donors, injecting drugs use (William et al., 2004).there is also evidence that the environment can serve as reservoir for infectious virus (Kamili et al.,2007). The virus has no ethnic, socio-economic, gender, age or geographic boundaries ( Perz and Alter, 2006).

Rogo, L.D et al.,: Continental J. Biomedical Sciences 4: 67 - 74, 2010

The virus binds to receptors on the surfaces of hepatocytes and subsequently enters the cell. Two putative HCV receptors are CD81 and human scavenger receptor class B1. However, these receptors are found throughout the body. The identification of hepatocyte-specific cofactors that determine observed HCV tropisms are currently under investigation (Sagnelli et al., 2005). Prolonged survival of HCV on blood contaminated skin, cloth, and other object emphasizes the importance of fomites in nosocomial spread (Kamili et al., 2007).

The main pathological features are damage to liver parenchyma which is mediated by inflammatory cytokines leading to varying degrees of hepatic fibrosis and eventually cirrhosis (Bellentani and Tribelli, 2001). Acute HCV infection is uncommonly recognised, because it is usually accompanied by mild flulike symptoms. Weight loss, fatigue, muscle or joint pain, irritability, nausea, malaise, anorexia, and jaundice have been reported in 2-26 weeks incubation period. At chronic stage of infection fatigue, depression, nausea, anorexia, abdominal discomfort, and difficulty with concentration are the predominant symptoms (Perez et al., 2005). The risk factors for HCV transmission include transfusion of blood or blood products and transplantation of solid organs from infected donors, injecting drug use, unsafe therapeutic injections and occupational exposure to blood (primarily contaminated needle sticks), birth to an infected mother, and sex with an infected partner, and sex with multiple partners (Alter, 2002).

It has been well documented that dialysis patients have a higher rate of HCV infection in the 90’s much of the world reported anti-HCV prevalence rates of 10-50% among haemodialysis patients with lower rates in such places as Ireland (1-7%) ( Niu et al.,1993; Hayashi et al.,1994). Previously, rates in Europe were as 20-30% (Touzet et al., 2000). A more recent report from Saudi Arabia showed a prevalence rate of HCV among haemodialysis patients to be 9.24% compared to 0.30% among blood donors (Qadi et al., 2004).

Currently, viral culture usually in combination with immunofluorescence is the “gold standard” for laboratory diagnosis. However, it is not a rapid diagnostic test, and therefore, its clinical value is limited. Rapid antibody / or antigen detection test are now widely used in routine laboratories for the diagnosis of viral infection (Hladik et al., 2006).

MATERIALS AND METHODS

The Study Area

The study was carried out in Ahmadu Bello Univesity Teaching Hospital (ABUTH) Zaria and Aminu Kano Teaching Hospital (AKTH) Kano haemodialysis units.

ABUTH is located in Zaria on longitude 8º and latitude 9º in Kaduna state northwestern part of Nigeria.

AKTH is located in Kano on longitude 12º 37΄N, 9º 29΄E and latitude 9º33΄S, 7º34΄W in northwestern part of Nigeria respectively (Olofin et al., 2008).

Sample Collection and Processing

All haemodialysis patients are included in the study. The samples were collected only from patients/care giver who has been given a written informed consent.

A dry sterile plastic syringe (5ml) with 21SWG (standard wire gauges) needle attached was used for blood sample collection. Blood was collected by applying soft tubing tourniquet to the arm of the patient to enable the veins seen and felt. The punctured site was cleansed using methylated spirit and allowed to air dried. The needle was inserted to the selected straight vein with the bevel of the needle directed upward in the line of the vein. Steadily the plunger of the syringe was withdrawn until 5ml of blood is obtained. The tourniquet was released and the needle was removed from the punctured vein. Pressure was applied to the punctured site to secure homeostasis. The needle was removed from the syringe and the blood was transferred to a clean anticoagulant specimen bottle (Ethelyne diamine tetracetic acid- EDTA), mixed and labelled properly. The used syringes and needles were disposed appropriately.

HB and PCV Determination

This was carried out immediately after sample collection as described by Cheesbrough (2000).

Rogo, L.D et al.,: Continental J. Biomedical Sciences 4: 67 - 74, 2010

HCV Antibody Detection

This was carried out using commercially prepared antibody testing kit based on the manufacturer’s instructions (CLINTOCH DIAGNOSTIC Ltd, USA).

Data Analysis

Data obtained from the study was analysed using correlation and the chi-square test of association with a p-value of 0.05 as statistically significant and the prevalent rate of the infection was analysed as percentage. These were achieved using computerized statistical package for social sciences (SPSS).

RESULTS

A total of 88 blood samples from haemodialysis patients were tested for the presence of HCV antibody. Among the samples tested, seven (7.9%) were seropositive.

Table 1 shows the seroprevalence of HCV antibody among the studied population with respect to age. Of the total samples tested, 7(8 %) were age group 10-20 years, 14(15.9%) were aged 21-30 years, 22(25%) were aged 31-40 years, 19(22%) were aged 41-50 years, 14(16) were aged 51-60 years, 7(8%) were aged 61-70 years, 4(6%) were aged 71-80 years, while 1(1.1%) were aged 81-90 years. The seroprevalence was highest (4.5%) in age group 51-60 years and lowest in age group 61-70 years (1.1%). The result shows increased seropositivity with age. R= 0.302, P = 0.004.

Among the 88 haemodialysis patients tested, 60(68%) were male and 28(32%) were females. This shows that there was male predominance over females, which was not statistically significant (X = 0.299, P= 0.288). As seen in Table 2.

Of the total 88 haemodialysis patients, 1 (1.1%) had PCV within 16-20%, 3(3.4%) had PCV within 21-30 and 3(3.4%) had PCV within 31-40%. Association has not been found between PCV and HCV seropositivity (Correlation co-efficient ‘r’=0.150, P value=0.162) Table 3.

Among the patients tested, 3 (3.4%) had Haemoglobin (Hb) within 1-5 g/dl but none of them was seropositive, 77 (87.5%) had Hb within 6-10 g/dl and 4(4.5%) were seropositive, 8(9%) had Hb of within 11-15 g/dl and 3(3.4%) were found to be seropositive. Although it was found not to be significant (r = 0.126, P value = 0.244) Table 4.

Table 5 shows that out of the total patients tested, 78(88.6%) within 1-100 has (3.4%) of the total 7.9% seropositivity. 3(3.4%) had dialysis within 101-200, although there was no seropositive sample found among them, 5(5.7%) had dialysis within 201-300 and 2.3% of the total 7.9% seropositivity was found in these group. 1 (1.1%) of the patients tested had dialysis within 301- 400 and 501-600 each respectively. The result shows statistical association between frequency of dialysis and HCV seropositivity (r= 0.430, P value 0.000).

Among the total population tested, 52 (59.1%) had history of blood transfusion and 36(41%) had no history of blood transfusion. 3(3.4%) seropositivity was found in those with history of blood transfusion out of the total 7.9% seroprevalence while 4.5% seropositivity was found in those without history of blood transfusion. The seropositivity was found to be more in those without history of blood transfusion, although it was not statistically significant (r= 0.043, P= 0.690) Table 6.

Rogo, L.D et al.,: Continental J. Biomedical Sciences 4: 67 - 74, 2010

Table 1: Distribution of HCV Antibody by Age. r = 0.302, P = 0.004

Table 2: Distribution of HCV Antibody by Sex X= 0.299, P = 0.288

Table 3: PCV Distribution of HCV Antibody Correlation co-efficient ‘r’= 0.150, P value=0.162

Table 4: Hb (g/dl) and Distribution of HCV Antibody r = 0.126, P value = 0.244

Table 5: Frequency of Dialysis and the HCV Antibody r= 0.430, P value = 0.000

Table 6: Blood Transfusion and Distribution of HCV Antibody r = 0.043, P= 0.690

Rogo, L.D et al.,: Continental J. Biomedical Sciences 4: 67 - 74, 2010

DISCUSSION

Despite rapid scientific progress in understanding the biology of viral illness, viral liver disease remains a common and challenging problem for physicians and their patients (Alter and Seeff, 2000). Persistent infection with Hepatitis c virus (HCV) has emerged as one of the primary causes of chronic liver disease with an estimated 170 million people infected by HCV, more than 4 times the number of people living with HIV throughout the world (WHO,2000). Of the typical hepatitis viruses, chronic infection with Hepatitis c virus remains one of the most important clinical and public health problems (El-Zayadi et al., 2004).

The present study found 7(7.9%) of the subject to be seropositive for HCV antibody. This is slightly above the finding of Nwokede et al (2007) who demonstrated 6.2% prevalence in Kano haemodialysis patients, Mendez-Sanchez et al (2004) who reported 6.7% prevalence in Mexican dialysis patients and Dede et al (2007) who demonstrated 7% prevalence in Turkey haemodialysis patients. While it contrast the finding of Kalantan-Zadeh et al (2007) who reported 12% prevalence in USA dialysis patients, Chen et al (2008) who demonstrated 20.6% prevalence in Taiwan dialysis patients, Al-Jamal et al (2009) who reported 28% prevalence in Jodanian dialysis patients, Alsaran et al (2009) who reported 33% prevalence in Saudi Arabia dialysis patients, Barril et al (2008) who demonstrated 45% prevalence in Spain dialysis patients and Sabry et al (2007) who reported 70.7% prevalence in Egyptian haemodialysis patients.

The study also reveals an increase in seropositivity with age. This finding conforms to those of Sabry et al (2007) and Barril et al (2008) who reported increase seropositivity with age. This could be due to the fact that people of older age are more susceptible to infectious micro-organism.

In assessing the sex distribution of HCV antibody seropositivity was observed to be higher in male (6.8%) than in female (1.1%).This could be as a result of behavioural exposure to the risk factor which is more in males than females. Although the result was not statistically significant (P= 0.288).

In assessing the HCV seropositivity in respect to PCV and Hb values it has been observed that HCV has no effect on PCV and Hb. This conforms to the finding of Sabry et al (2007) who has reported to have found no effect of HCV infection on PCV and Hb values. This is in contrast with the finding of Sahin et al (2003) and Chen et al (2008) who reported an increase PCV and Hb in HCV positive haemodialysis patients in their studies. The lack of consistency between the present study and that of Sahin et al (2003) and Chen et al (2008) results could be explained by: the haemodialysis duration was longer in most patients in the present study compared to the very short (12.59 months) one in the HCV-negative patients of Sahin’s study and this can be partially explain their lower haemoglobin level.

This study showed that seropositivity is higher in those with high frequency of dialysis. This conforms to the finding of with the finding of Al-Jamalet al (2009). This could be due to the fact that dialysis duration has been found to be one of the risk factor of acquiring HCV infection.

The present study shows that there is no association between HCV infection and blood transfusion. This is in contrast with the finding of Zoulin (1999), Pol et al (2002) and Dede et al (2007) that has incriminated blood transfusion as one of the risk factor for HCV infection.

CONCLUSION

The present study has revealed the importance of HCV as an etiologic agent of Liver disease in haemodialysis patients. The study emphasizes the need to rapidly diagnose viral infections for clinical management of infected dialysis patients. It also provide epidemiological data that may be useful in control effort and vaccine trials, mostly in developing countries where less information regarding viral infection in haemodialysis patients is available.

RECOMMEMDATION

In view of the prevalence rate of Hepatitis c virus seropositivity in this study, it is recommended that attention should be given to this virus for proper management of patients with liver disease especially in those with symptoms of end stage kidney malfunction.

Rogo, L.D et al.,: Continental J. Biomedical Sciences 4: 67 - 74, 2010

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Received for Publication: 19/09/2010

Accepted for Publication: 18/10/2010

Correspondence Author

Rogo, L.D

Department of Haematology and Blood Group Serology, School of Medical Laboratory Sciences, Ahmadu Bello University Teaching Hospital, Zaria.

E-Mail- [mailto:lawaldahirurogo@yahoo.com lawaldahirurogo@yahoo.com]

Continental J. Biomedical Sciences 4: 75 - 82, 2010 ISSN: 2141 – 419X

©Wilolud Journals, 2010 http://www.wiloludjournal.com

F.M. Onyije,1 V.C. Zenebo2 and Y.I. Oboma3
==1Department of Human Anatomy, 3Department of Anatomical Pathology, Faculty of Basic Medical Sciences, College of Health Sciences, Niger Delta University, Wilberforce Island, Bayelsa State, Nigeria, 2Department of Histopathology, Medical Laboratory Services, Braithwaith Memorial Specialist Hospital, Port Harcourt, Rivers State, Nigeria.==

<p style="margin-left: 17pt;">ABSTRACT

<p style="margin-left: 17pt;">Breast cancer is one of the most common cancers among females worldwide. A cross-sectional survey was conducted in Bayelsa and Rivers State between March and August 2010 to assess knowledge, attitude and practice of breast cancer self examination among female students in tertiary institutions. There was a poor knowledge on the etiology of breast cancer as Only 207 (39%) was aware that breast cancer can be inherited. 195 (37%) of the female students believe that breast cancer can be caused by evil spirit, while 129 (27%) had no idea if it was caused by evil spirit or not. Respondents had a very good knowledge of the signs and symptoms of breast cancer as 372 (72%) agreed that breast cancer usually present as a painless breast lump, 84 (16%) Disagreed while 57 (12%) were not aware of its signs and symptoms. Two hundred and thirteen (37%) of the respondents claimed to have heard about breast cancer self examination through a doctor, 153 (27%) from publications, 70 (12%) acknowledged Television, 18(3%) claimed their source was from churches/religious groups, while 33 (6%) of the respondents information from women organizations and 84(15%) from Nigerian cancer society programs. There was good of knowledge of breast cancer by the respondent but the practice of breast cancer self examination was below average which suggest that irrespective of the knowledge acquired, the practice of breast cancer self examination was still poor among female students in tertiary institutions.

<p style="margin-left: 17pt;">KEYWORDS: Breast cancer, Symptoms, Questionnaire, Women, Bayelsa and Rivers State.

INTRODUCTION

Breast cancer is one of the most common cancers among females worldwide. There is increasing evidence that breast and other cancers originate from and are maintained by a small fraction of stem/progenitor cells with self-renewal properties. Whether such cancer stem/progenitor cells originate from normal stem cells based on initiation of a de novo stem cell program, by reprogramming of a more differentiated cell type by oncogenic insults or both remains unresolved (Xiangshanet al., 2010). Global statistics show the annual incidence of breast cancer is increasing and this is occurring more rapidly in countries with a low incidence rate of breast cancer (Parkin et al., 2005; Wilson et al., 2004), from 1975–1990, Asia and Africa have experienced a more rapid rise in the annual incidence rates of breast cancer than North America and Europe (Sasco. 2001). In African breast cancer patients tend to present at a young age, with large tumors and multiple nodal involvements, and have poorer clinical and pathological prognostic factors compared with Caucasian patients. These characteristics are somewhat similar to that of African-Americans but are in contrast with those of non-Hispanic Whites in the USA, thus heightening the interest in the role of genetic factors in the etiology of breast cancer in general, and in people of African origin in particular (Adebamowo and Ajayi 2000; Rose and Royak-Schaler 2001)

Recent observations show that the frequency of breast cancer has risen over that of non-Hodgkin's lymphomas and cervical cancer in Nigeria (Thomas 2000). This trend could be attributed to several factors such as lack of knowledge about breast cancer and the usefulness of breast self-examination.

In 2008, an estimated 182 460 cases of invasive and 67 770 cases of noninvasive breast cancer were diagnosed, and 40 480 women died of breast cancer. Incidence increases with age, and the probability of a woman developing breast cancer is 1 in 69 in her 40s, 1 in 38 in her 50s, and 1 in 27 in her 60s (Heidi et al., 2009)

One of the most important risk factors for breast cancer is the occurrence of breast or ovarian cancer among family members. Women with one or more first-degree relatives with breast cancer have a 1.8-3.0-fold increased risk of

F.M. Onyije et al.,; Continental J. Biomedical Sciences 4: 75 - 82, 2010

developing the disease (Makarian et al., 2007).The prevalence of women with a family history of breast cancer has been estimated to range from 5 to 19% ( Murff et al., 2005).

The relative frequencies of breast cancer among other female cancers, from Cancer Registries in Nigeria were 35.3% in Ibadan, 28.2% in Ife-Ijesha, 44.5% in Enugu, 17% in Eruwa, 37.5% in Lagos, 20.5% in Zaria and 29.8% in Calabar (Banjo 2004). In all the centers, except Calabar and Eruwa, breast cancer rated first among other cancers. Further reports showed that majority of cases occurred in pre menopausal women, and the mean age of occurrence ranged between 43–50 years across the regions. The youngest age recorded was 16 years, from Lagos (Banjo 2004).

==There is strong evidence suggesting that older women in the developed countries are more likely to delay their presentation with breast cancer, (Ramirez et al., 1999), there is data suggesting that factors related to women's knowledge and beliefs about breast cancer and its management may contribute significantly to medical help-seeking behaviors (Ferro et al., 1992; Maxwell et al., 2001).==

==The prevalence of breast cancer in recent years has prompted women to seek medical advice randomly with minimal breast symptoms, but only a small number of women are aware of this in the society, with childbearing extending practically over the entire reproductive period of life. Due to the conservative nature of the society, many females will refrain from seeking medical advice out of shyness until their disease becomes far advanced, particularly in cases of carcinoma of the breast. Often Breast cancer engenders an exceptional level of fear among women, most probably because of its external location on the body, with all of the obvious cosmetic and psychosocial implications, coupled with proper methods of conducting breast self examina­tion (BSE) or are aware of the importance of radio­logical screening for breast cancer (Alam et al., 2006).==

==The three screening methods recommended for breast cancer includes breast self-examination (BSE), clinical breast examination (CBE), and mammography. Unlike CBE and mammography, which require hospital visit and specialized equipments and expertise, BSE is inexpensive and is carried out by women themselves. Several studies, based on breast cancer patient's retrospective self-report on their practices of the exam, have established that a positive association exists between performance of the exam and early detection of breast cancer (Philip et al., 1986).==

==Breast cancer presents most commonly as a painless breast lump and a smaller proportion with non-lump symptoms. For women to present early to hospital they need to be "breast aware"; they must be able to recognize symptoms of breast cancer through routine practice of practicable screening. At the present time, routine mammography cannot be recommended in developing countries due to financial constraints and the lack of accurate data on the burden of breast cancer in these countries (Okobia et al., 2006). There is also evidence that most of the early breast tumors are self-discovered (Smith et al 1980) and that the majority of early self-discoveries are by BSE performers (Smith et al 1980). Despite the advent of modern screening methods, more than 90% of cases of cancers of the breast are detected by women themselves, stressing the importance of breast self – examination (Parkin et al., 1992). The purpose of a BSE is to learn the topography of the breasts; which in turn will allow for one to notice changes in the future in order to detect breast masses or lumps. Breast self –examination, carried out once monthly, between the 7th and 10th day of the menstrual cycle, goes a long way in detecting breast cancer at the early stages of growth when there is low risk of spread, ensuring a better prognosis when treated (Schecter et al., 1990). The aim of this study was to evaluate the knowledge and attitude of breast cancer self examination among female students in tertiary institutions in Nigeria==

All women over the age of 20 should practice regular monthly self breast examinations. The importance of this cannot be over emphasized since an early cancer can be discovered by this method when mammography is normal. The examination should be done when the breasts are least tender, usually 7 days after the start of menstrual period. If a woman detects any changes or lumps, she should seek medical attention. Remember that 9 out of 10 women will not develop breast cancer and most breast changes are not cancerous.

F.M. Onyije et al.,; Continental J. Biomedical Sciences 4: 75 - 82, 2010

MATERIALS AND METHODS

A cross-sectional survey was conducted in Bayelsa and Rivers State between March and August 2010 to assess knowledge, attitude and practice of breast cancer self examination among female students in tertiary institutions. Bayelsa and Rivers State have the largest crude oil and natural gas deposits in Nigeria. They are mainly rural dwellers due to its peculiar terrain; Bayelsa population is estimated to be about 1,998,349, while Rivers State is estimated to be 6,689,087.

The present study was based on data from five hundred and thirty four (534) female students with age ranging from 16 to 45 with a mean ± standard deviation 30.5± 20.5. Data collection was with the aid of questionnaires designed to obtain relevant knowledge, attitude and practice towards breast cancer. Questions on knowledge of study participants on risk factors, common symptoms, methods of early detection, Breast self examination (BSE). Most of the questions were designed to elicit "yes", "no" or "don't know" answers. Socio-demographic information relating to age, educational status, religion, and marital status were collected in the first section of the questionnaire. In the second part, respondents were asked specific questions to elicit their knowledge of the common symptoms and signs of breast cancer, etiological factors and diagnostic procedures. The third section examined participant's action and attitude towards practice of breast self examination (BSE).

Informed consent was granted by individual subjects. Data analysis was by the use of PAST version 2.01: Paleontology Statistical Software Package for Education and Data Analysis.

RESULTS

The ages of the respondents ranged from 16–45 years, 441 (83%), where from age group 16-25, 81 (15%) from age group 26- 35 and 12 (2%) from age group 36-45. Only 54 (10%) of the respondents were married, while 480 (90%) were single at the time of this research. Almost all the respondents were Christians (99.4%), there was no Muslim, while less than one percent (0.6) were from other religions. 357 ( 75%) of subjects reside in an urban area, 72 (15%) in suburban and 48 (10%) in rural area. Table 1

Breast cancer is the most common cancer in women

The female students in tertiary institutions showed good knowledge of breast cancer. A greater number of the respondents 477 (89%) agreed the breast cancer is most common in women, 42 (8%) disagreed and 12 (3%) did not know if breast cancer is the most common cancer in women or not.

Breast cancer occur more commonly in old people

On group of individual in which breast cancer occur more commonly respondents had a poor knowledge as 285 (53%) said it is not common in old people, 168 (31%) agreed that it occur more in older people. While 81 (16%) had no idea.

Breast can be inherited

There was a poor knowledge on the etiology of breast cancer as Only 207 (39%) was aware that breast cancer can be inherited. 195 (37%) of the female students believe that breast cancer can be caused by evil spirit, while 129 (27%) had no idea if it was caused by evil spirit or not.

Breast cancer usually present as a painless breast lump

Respondents had a very good knowledge of the signs and symptoms of breast cancer as 372 (72%) agreed that breast cancer usually present as a painless breast lump, 84 (16%), Disagreed while 57 (12%) were not aware of its signs and symptoms.

Breast cancer is curable when detected early

A large population of the respondents 441 (84%) said “Yes” while 48 (9%) said “No” and 39 (7%) said “I don’t know” of greed with the fact that early. Table 2

F.M. Onyije et al.,; Continental J. Biomedical Sciences 4: 75 - 82, 2010

Table 1:

''* The respondent could choose more than one response category. The percentages are based on total responses and not total number of the respondent''

Distribution of respondents according to practice of breast self examination (BSE)

The respondents performed best on the question if “breast self examination is useful in early diagnosis” as 525 (98.5%) said “Yes”, 3 (0.5%) said “No” and 6 (1%) did not know. The performance was dashed when a total of 159(30%) had not practiced breast cancer self examination, 255 (49%) practice once in a month, 39(7%) practice once in two months and three to five times a year, 15(3%) practice twice a year and 18(4%)once a year.

Out of 159 (30%) that had not practiced breast cancer self examination, 75 (48%) said they don’t have breast cancer as their reason, 9 (6%) said they don’t think they should practice it, 45 (29%) said they don’t just feel like doing it, 15 (10%) said they don’t think they will find anything, 9 (6%) said they leave it for the doctors and nurses to check and 3 (2%) said they are afraid of practicing it.

F.M. Onyije et al.,; Continental J. Biomedical Sciences 4: 75 - 82, 2010

Source of knowledge of breast self examination

Two hundred and thirteen (37%) of the respondents claimed to have heard about breast cancer self examination through a doctor, 153 (27%) from publications, 70 (12%) acknowledged Television, 18(3%) claimed their source was from churches/religious groups, while 33 (6%) of the respondents information from women organizations and 84(15%) from Nigerian cancer society programs. Table 3

DISCUSSION

The reveals that Nigerian female students in tertiary institution [477 (89%)] have a good knowledge of breast cancer been the most common in women. This study agrees with an earlier study among women in Nigeria were 72.6% (Okobia et al., 2006) and in Iran were 61% (Ali et al., 2008) of the respondents agreed that breast cancer is the commonest in women. On the etiology of breast cancer there was poor knowledge as only 207 (39%) agreed that breast cancer can be inherited, while 195 (37%) disagreed that breast cancer can not be inherited and 129 (27%) claimed not to know if breast cancer can be inherited or not. This indicates that the etiology of breast cancer is not properly known among female students in tertiary schools in Nigeria. This findings is also in line with the research carried out in semi-urban area of Edo State Nigeria (Okobia et al., 2006) where only 244(26%) of that population agreed that breast cancer be inherited. Though 455 (85%) agreed that breast cancer is not caused by evil spirit, 54 (10%) still believe it is coursed by evil spirit and 24 (5%) did not know if it causes it or not, this totally not in conformity with the research carried out in the above mentioned location, where 400 (40%) of that population believed that evil spirits causes breast cancer and a lower population of 259 (25.9%) indicated that breast cancer results from an infection. These findings affirm the educational disparity between female students and semi-urban women dwellers.

There was a good knowledge of the sign / symptom of breast cancer as 372(72%) agreed that breast cancer usually presents as a painless lump. This is line with the study carried out in Lagos among teachers (Odusanya, 2001) and a study from U.K indicated that 70% of women were well aware of 'painless lump' and able to identify these symptoms in their breast self examination (Grunfeld et al., 2002) but contrary to the studies carried out in Ibadan 1.9% acknowledged a painless lump as an early warning sign (Oluwatosin and Oladepo, 2006). A large population of the respondents 441(84%) correctly noted that breast cancer is curable when detected early; this findings did not correspond with the findings in Benin where 41% agreed (Okobia et al., 2006).

Breast Self Examination was known by almost all of the respondents to useful in early diagnosis, as 525(98.5%) said yes. This is findings agreed with the study carried out in Ilorin, where 95% heard about it (Kayode, 2005), the findings also agreed with findings reported from Enugu and Lagos both in Nigeria where 92% of the respondents were aware of the procedure (Nwagbo and Akpala, 1996; Odeyemi and Oyediran, 2002). The figure reported from a study in Port-Harcourt, Nigeria was less, where 89.4% of those studied had heard about it (Uche, 1998). Irrespective of the good knowledge in the usefulness of breast cancer self examination, less than average [255(49%)] of the respondents practice breast cancer self examination once in a month. This was higher (71.8%) in the study carried out in Ilorin, Nigeria (Kayode, 2005). It was interesting to observe that 159 (30%) had never practiced breast cancer self examination.

The highest proportion of the respondents obtained their information from a doctor 213 (37%), though poor, but has not been reported in any study as been the highest source for breast cancer self examination.153 (27%) claimed they got the information from publication, Nigerian Cancer Society programs 85 (15%), television70 (12%) the radio. Women organizations 33 (6%) and Churches/religious groups 18 (3%).

This study indicates doctors as the major source of information of breast cancer self examination. There was good of knowledge of breast cancer by the respondent but the practice of breast cancer self examination was below average which suggest that irrespective of the knowledge acquired, the practice of breast cancer self examination was still poor among female students in tertiary institutions.

F.M. Onyije et al.,; Continental J. Biomedical Sciences 4: 75 - 82, 2010

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Received for Publication: 19/09/2010

Accepted for Publication: 18/10/2010

Correspondence Author