Effect Of Ethanolic Extract Of Ocimum gratissimum (scent leave) Leaves On Sodium Nitrite Induced Changes In The Liver Of Adult Wistar Rats

Ibegbu A.O
Department of Human Anatomy,Faculty of Medicine, Ahmadu Bello University Zaria, Kaduna State-Nigeria. 81006.
Iduh M.U, Livinus P.P.; Eze S.M;

Department of Pathology Ahmadu Bello University Zaria, Kaduna State-Nigeria
Idoko J; Akpulu S.P; Adamu Sadeeq A.

Department of Medical Microbiology Usmanu Danfodiyo University Sokoto, Nigeria

All correspondence to: aoibegbu@yahoo.com.

ABTSRACT

The Effects of ethanolic extract of Ocimum gratissimum leaves on the liver of Adult Wistar rats were studied. Twenty four adult Wistar rats weighing between 150-250g were randomly divided into six groups of four rats each. Group 1 received 2mlv/v of distilled water, Group 2 received 54mg/kg of sodium Nitrite (NaNO2), Group 3 received 750mg/kg of extract +54mg/kg NaNO2, Group 4 received 375mg/kg of extract+54gm/kg NaNO2, Group 5 received 54mg/kg NaNO2 + 2ml/V of Olive oil while Group 6 received 2ml/V of Olive oil orally. At the end of the experiment which lasted for 21 days, the animals were sacrificed. Blood samples were collected for biochemical analysis and the liver was harvested and fixed in Bouin’s fluid for histological studies. The result showed decreased physical activities and increase rate of respiration in Group 2 and a decrease in the body weight of Groups 3 and 4 animals. The result of biochemical analyses of Liver parameters, showed a statistical significant increase in ALT, AST and a decrease in ALP in Group 2 when compared to the Control (p=0.001). The result of the histological studies of the liver showed that there was some damages in the Liver of the animals while the administration of O.gratissimum showed a mild effect in a dose dependent manner. The results from the present study suggest that O.gratissimum has the ability to ameliorate liver injury in Wistar rats.

Key Words: Osimum Gratissimum, Scent Leaves, Sodium Nitrite, Liver, Wistar Rats.

INTRODUCTION

Sodium nitrite (NaNO2) is an inorganic salt used in the manufacture of dyes and considered as one of the important food additives for fishes and meat that has been used in vivo and in vitro experiments for decades to prevent growth of Clostridium botulinum (Luca, et al., 1987). It is also used in drug industries and in medicine as antidote for cyanide poisoning (Filvo, et al., 1993). Most synthetic food additives causes health hazard such as hepatotoxicity, nephrotoxicity, endocrinal disturbances, methemoglobenemia and growth retardation (Hassan, 2007, Hassan, et al.,2008; Hassan, et al., 2009). Reactive nitrogen species produced by exposure to nitrate is considered one of the most important causes of carcinogenesis through its reaction with body tissues and triggering lipid peroxidation, DNA lesions, enzyme
inactivation and damage of different organs (Abdeen, et al., 2008; El-Wakf, et al., 2009). The high oxidative stress indicator; lipid peroxidation could be attributed to the oxidative cytotoxicity of nitrite (Patsoukis and Georgiou, 2007). These observations have been reinforced by the successful use of several endogenous and exogenous antioxidants against nitric oxide induced cellular damage.
Sidney, 1986, reported that trends in controlling and treating diseases tend to prefer natural compounds use rather than synthetic ones in scavenging produced free radicals species. Nitrite reacts with amines to produce nitrosamines and with amides to produce nitrosamides. Nitrosamines and nitrosomides constitute the N-nitroso compounds (NNC) and the reaction with nitrite is called nitrosation. Nitrosamines readily induced tumors of the liver, oesophagus, kidney, nasal cavity and pancreas and notrosamine chiefly induce tumors of glandular stomach, small intestine and nervous as well as lymphoid systems. The tissue effects depend on the species of the NNC and the system involved (Sidney, 1986). Excessive quantities of nitrate and nitrite consumption through food can be harmful to health (Leszczy-nska, et al., 2009; Chan, 2011). The ability of animals to resist the toxic effects of environmental agents is dependent on the detoxification and antioxidant systems. Several nutrients and other chemicals have been shown to be effective antioxidants, such as vitamins, trace elements, amino acids and their derivatives, fatty acids and plant phenolics (Bhattacharya, et al., 1987; Son, et al., 2004; Ayo, et al., 2006; Suteu, et al., 2007). The use of herbal products for medicinal benefits have important roles in nearly every culture on earth and herbal Medicine was practiced by the ancient people of Asia, Europe and the Americas (Wargovish, et al., 2001).
Many natural and artificial Agents possessing anti-oxidative properties have been Proposed to prevent and treat liver and kidney damages induced by Oxidative stress (Lieber, 1997; Cervinkova and Drahota,1998). There is increasing evidence for the protective role of hydroxyl and polyhydroxy-organic compounds particularly from vegetables, fruits and some herbs (Bass, 1999).
Ocimum gratissimum known as Scent leaf is a plant with root, stem and leaf systems (Iwu, 1993). O.gratissimum belongs to the family leguminocaeae, widely used local plant in Nigeria for both nutritional and Therapeutic purposes. It is naturally used in the treatment of different diseases which includes: upper respiratory tract infections, diarrhea, headache, conjunctivitis, skin disease, pneumonia, tooth and gum disorder, fever and as a mosquito repellant (Onajobi, 1986; Ilori, et al., 1996; Okigbo and Mneka, 2006). This study is aimed at evaluating the effect of ethanolic extract of Ocimum gratissimum leaves against sodium nitrite induced changes in the liver of adult Wistar rats.

MATERIALS AND METHODS
Experimental protocol:
Twenty-four apparently healthy adult Wistar rats of both sexes weighing between 150g to 250g were purchase from the Department of Human Anatomy, Faculty of Medicine, Ahmadu Bello University Zaria Kaduna State. The animals were acclimatized for three weeks in the Department Animal house. The animals were fed with standard pellet and water ad libitum throughout the experimental period. The rats were divided into six Groups of four rats each.
Fresh leaves of O.gratissimum were purchased from Sabon Gari Market Zaria Kaduna State-Nigeria. Identification and authentication was done in the herbarium of the Department of Biological Sciences, Ahmadu Bello University Zaria with voucher number 285.
The leaves were washed with distilled water and air dried for the period of one week and was extracted in the Department of Pharmacognosy, Faculty of Pharmaceutical Sciences, Ahmadu Bello University Zaria. The dried fresh leaves were grounded into coarse powder of 500g. The powder was subjected to absolute ethanol extraction using Soxhlet apparatus for 10 hours. The extract was concentrated by using evaporating dish to dryness in a Water bath regulated at 600C and 10%w/w dark green
extract was obtained.

PHYTOCHEMICAL SCREENING
The Ethanolic extracts obtained were subjected to preliminary phytochemical screening to identify the chemical constituents. The methods of analysis employed were those described by Brain and Turner (1975).

Chemicals and Reagents
12gm of sodium Nitrite manufactured by May and Baker limited Dagenhan England was purchased from Steve Moore Chemicals Limited Samaru Zaria-Nigeria. Goya Olive oil was purchased from Beautiful Gate Pharmaceutical Limited Samaru Zaria-Kaduna State, Nigeria. Growers feed from Vital feed was obtained from Samaru Market Zaria, Kaduna, Nigeria and was used to feed the animals throughout the experimental period.

Experimental Procedure
The Dose of the extract was determined using LD50 of 2500mg/kg body weight following the method of Rabelo et al., 2003. The stock solution was prepared by dissolving 12gm of the extract in 160 ml of Olive oil to form the stock solution, 30% (750mg/kg body weight) and 15% (375mg/kg body weight) of the LD50 were used in this study for the high and low dose respectively. The animals were randomly divided into six groups of four animals per group. Group 1 received 2ml/kg body weight of distilled water, Group 2 received 54mg/kg body weight of NaNO2, Group 3 received 750mg/kg body weight of the extract+54mg/kg body weight of NaNO2, Group 4 received 375mg/kg body weight of the extract+ 54gm/kg body weight of NaNO2, Group 5 received 54mg/kg body weight of NaNO2+2ml/kg body weight of Olive oil, Group 6 received 2ml/kg body weight of Olive oil.

Animal Sacrifice
After the last day of administration, the animals were left for 48 hours and were fasted overnight before sacrificed. The animals were humanely sacrificed by cervical dislocation and the blood collected through cardiac puncture for hematological and biochemical analyses.
Incisions were made through the abdomen and the liver was removed and fixed in Bouin`s fluid. The tissues were processed, sectioned and stained with hematoxylin and eosin and Cresyl fast violet methods.

STATISTICAL ANALYSIS
Data obtained from the haematological and biochemical analysis was reported as Mean ± Standard Error of Mean (SEM). One way Analysis of Variance (ANOVA) was used to compare the means w;ith values of p<0.05 was considered to be statistically significant. Sigmastat 2.0 (Systat Inc, Point Richmond, CA) was used for the statistical analysis.

PHYSICAL OBSERVATION AND WEIGHT CHANGES
Several physical observations were made during the period of administration ,except the animals in group one that received 2ml/kg body weight of distilled water and group six that received 2ml/kg body weight of Olive oil which their activities including locomotion and feeding habit were observed to be normal; The animals in group two which received 54mg/kg body weight of NaNO2 were observed to be weak and breathe faster when compared to the animals in group one and six that received distilled water and olive oil respectively. However, there was no detectable change in the animals feeding habit. Meanwhile, the animals in group five which received 54kg/body weight of NaNO2 and 2ml/kg body weight of Olive oil showed physical observations similar to the animals in group two.
The animal in group three which received 54mg/kg body weight of NaNO2 and 75mg/kg body weight of the extract were observe to be feeding very well and drink a lot of water when compared with the animals in the control group. However there was no significant difference in the physical observation of the animals in group four which received 54mg/kg body weight of NaNO2 and 375mg/kg body weight of extract when compared with the animals in group three.

The mean body weight of the animals in group One, Two, Five and Six were observed to increase during the period of administration with the animals in group six showing a more
rapid increase in weight than the others. However, there was no statistical significant different in the mean body weight of the animals across the group. In the control group, apparent increase in the final mean body weight was noticed when compared with the initial mean body weight. Meanwhile the animals in group three showed a significant decrease in the final mean body weight (213.00±30.89) when compared with the initial mean body weight (218.75±36.50). However, the animals in group four were also observed to show a significant decrease in the final mean body weight (207.25±27.09) when compared with the initial mean body weight. The decrease in the final mean body weight of the animals in group Three and four could be due to the hypoglycemic and diuretic potentials of the extract. However, there was significant increase in the final mean body weight of the animal in group five (216.00±28.44) when compared with the initial mean body weight (193.75±25.03). Meanwhile, the final mean body weight of the animals in group six showed increase in their mean body weight when compared with the animal in group five. However the increase in the mean body weight of the animals in group five and six could be due to fat accumulation as a result of the administration of Olive oil. However, there was no statistical significant difference in the mean kidney and liver weight of the animals across the group when compared with the control group.

DISCUSSION

Sodium Nitrite may react with amines of food in the stomach to produce nitrosamine and free radicals may increase lipid peroxidation which is harmful to different organs including the liver. These damages can be reduced by dietary natural antioxidant and the present study was undertaken to determine if Ocimum gratissimum could prevent or reduce NaNO2 induced liver damage by examining the different biochemical parameters in the serum. The liver play important roles in toxicity by virtue of its function both qualitatively and quantitatively. Results from the present investigation show that O. gratissimum is rich in phytochemicals and specific biologically active components have been identified in extracts from the plant by previous workers (Koche,et al., 2012; Dubey, et al., 2000; Holets, et al., 2003).

The decrease in physical activities and increase in the rate of respiration observed could be due to reduced energy generation as a result of hypoxia due to methemoglobin formation. These results of the present study are in agreement with Grant and Butler (1989) and Porter, et al. (1993).The apparent decrease in the mean body weight observed could be due to the hypoglycaemic and diuretic effect of the extract. This result is in agreement with Effriam et al. (2003) and Oguanobi et al. (2012), which showed that aqueous and ethanol extracts of Ocimum gratissimum leaves possess hypoglycaemic effects in normoglycaemic and neonatal streptozocin-induced diabetes model.

The rats treated with sodium nitrite showed significant decrease in mean body weight than the Control Group throughout the experiment periods, and this reduction may be due to the reduction of food intake (Grant and Butler, 1989) or Vitamin C deficiency (Uchida, et al.., 1990). In general, the reduction in body weight may be attributed to the decrease in food intake, the disturbance in hormonal balance and direct cytotoxic effect of sodium nitrite treatment.
Normal liver functions are characterized by balanced activities of serum enzyme markers AST, ALT and ALP. Hepatocellular necrosis leads to a very high level of AST, ALT released from the liver into the blood. ALT is the best indicator of liver injury, as liver ALT represents 90% of total enzymes presents in the body (Moss, et al., 1974). ALP activities on the other hand are related to the functioning of the hepatocytes, increase in its activity is due to increased synthesis in the presence of increased biliary pressure (Pradhu et al., 2007). Ethanolic extract of Ocimum gratissimum decreases the elevated level of AST and ALP, which suggest the protection of the structural integrity of hepatocytes cell membrane or regeneration of damaged liver cells by extract. This effect is in agreement with the view that serum level of aminotransferases returns to normal with the extract while there was a decrease in the level of ALT in Group 3.

However, there was increase in the ALT in Groups 4 and 6 which is in agreement with the view that the serum level of aminotransferases returns to normal with healing of hepatic parenchyma and regeneration of hepatocytes (Surana and Jain, 2010).
Information available showed that nitrites and nitrates are both oxidation products and ready sources of nitric oxide (NO) which reacts rapidly with superoxide to form highly reactive peroxynitrite (ONOO?) and such products may increase lipid peroxidation (LPO) which can be harmful to different organs including liver (Chow and Hong, 2002; Hassan et al., 2009; Rocha et al.; 2012). The liver is liable to injury by a variety of causes and the injury may lead to profound metabolic disorders. Liver injury induced by chemicals has been recognized as one of the most toxicological problems (Cohen, 1982).
In the histological examination of the liver, there was no lesion observed in the Control Group (one) and Groups six (Olive oil only). The characteristics of the liver lesion observed in the rats given concurrent doses of sodium nitrite and Ocimum gratissimum were congestion of central vein, necrosis of the hepatocytes, dilation of sinusoid.
The administration of sodium nitrite alone caused prominent histological damage in the liver compared with the Control and these results are in accordance with Hussein et al. (2007), Klastskin and Oconn (1993) who attributed the dilatation of the sinusoids to the direct toxic effect of the toxin leading to their dilatation. When comparing Groups 3 and 4 that were given O.gratissinum to Group 2, it showed that O. gratissimum had the ability to ameliorate the effect of NaNO2 on the liver but in a dose dependent manner of which the low dose showed a better effect of O.gratissimum on the liver histology than high dose when compared to the Control.

CONCLUSION
The present study has demonstrated the NaNO2 (sodium nitrite) induced changes in the liver and that the ethanolic extract of Ocimum gratissimum has significant beneficial role in overcoming the NaNO2 induced adverse effects which may be through its antioxidant properties.

 




Breast Cancer In Men: A Review of Epidemiology, Risk Factors, Diagnosis And Prevention In Africa.

Isah, R.T.; Mohammed, I.
Department of Histopathology, Faculty of Medical Laboratory Science, Usmanu Danfodiyo University, Sokoto, Nigeria.
Avwioro, O.G.
Faculty of Science, Delta State University, Nigeria.
Abdullahi, K.
Department of Histopathology, Usmanu Danfodiyo University Teaching Hospital, Sokoto, Nigeria.
Bello, M.B.
Department of Surgery, Usmanu Danfodiyo University Teaching Hospital, Sokoto, Nigeria.

All correspondence to: Isah R.T. (tsamiyarilly@gmail.com)

ABSTRACT

The male breast in spite of being rudimentary is subject to the full spectrum of disease that affects the female breast. Male breast cancer is a rare condition globally, accounting for only 1% of all breast cancer. However, there is a wider increase in prevalence of this cancer in Africa approximately 5% – 15% as compared to developed nations. In Nigeria, several studies have shown prevalence in the range of 3.7% to 9.0% of all breast cancer cases. Mutation of BRCA 2 gene accounted for most of the cases of male breast cancer occurrence; other genetic conditions are BRCA 1 mutation, Klinefelter’s syndrome and Cowden’s disease. In addition, other risk factors associated with the disease are radiation exposure, hyperestrogenism, occupational and environmental exposure. Diagnostic methods include fairly specific techniques like routine light microscopic examination of haematoxylin and eosin stained sections of formalin fixed paraffin embedded biopsies, immunohistochemistry, fluorescent in situ hybridization and gene expression profiling. There is need for more public awareness program which focuses on behavioral modulation of diet, regular self-breast examination especially for those at risk and avoidance of exposure to environmental carcinogens.

KEY WORDS: Male breast, gynaecomastia, carcinoma, risk factors, diagnostic methods and prevention.

INTRODUCTION

Cancer can develop in any part of the body including breast of both male and females. Male breast cancer is rare compared to that of the female but they have similarities with few differences, as a result of which their diagnosis and treatment are almost similar [1, 2]. Several risk factors has been associated with development of male breast cancer which include family disposition, hyperestrogenism which may be caused by Klinefelter’s syndrome and liver cirrhosis, radiation exposure most especially at the chest region, testicular disorders, dietary source, aging and obesity [3].

Some of the signs and symptoms of breast cancer in males comprise of firm, non-painful mass located just below the nipple, skin dimpling around the breast region, nipple retraction, redness of the nipple or breast skin, bloody
discharge from the nipple and ulceration of the skin in advanced cases [4]. Pathogenesis of breast cancer in both sexes is essentially similar; much of this is linked to exposure to sex steroids (estrogen and progesterone) and activity of receptor tyrosine kinases (RTK) located on the surface of the breast parenchymal cells [5, 6].

Invasive ductal carcinoma (Not Otherwise Specified – NOS) is the most common histopathological type of breast cancer in males [7]. At least 8 out of 10 male breast cancers are invasive ductal carcinomas alone or mixed with other types of invasive or in situ breast cancer [8].

METHODOLOGY
This review paper involved gathering of already published articles and literature documents on male breast cancer. They were then extensively analyzed and presented as it affect African populace most especially Nigeria.

EPIDEMIOLOGY
Male breast cancer is a rare disease. It accounts for only about 1% of all breast cancer [9]. It was estimated that in 2013, about 2,240 new cases of breast cancer in men would be diagnosed and that breast cancer would cause approximately 410 deaths of men in United States [8]. Approximately 350-400 new cases of breast cancer in males are diagnosed annually in the United Kingdom [10]. A man’s lifetime risk of developing breast cancer is 1 in 1000, it can occur at any age, but it is mostly detected in the 60-70 years age group in developed countries [8].
The incidence of breast cancer in men has been increasing globally; one report suggested that incidence has increased 26 percent over the past 25 years [11]. In Africa, various researches conducted in the region have shown a wider increase in male breast cancer occurrence and mortality rate as compared to the developed countries. Incidence of male breast cancer is much higher in sub-Saharan Africa, approximately 5%-15% [12]. When they present, the tumours are often at advanced stages with poorer
prognoses [7]. A meta-analytical study on male breast cancer in African males indicated that the average age of occurrence was 54.6 years, which is 7 years older than the female counterparts [13]
Reasons for the differences in mortality rates between developed countries and African countries include advanced stages at presentation, worse biologic behavior, poor treatment facilities and poor patient acceptance of recommended treatments, which has been attributed to ignorance, superstition, self-denial, fear of mastectomy and unavailability of treatment facilities [14, 15].
In Tanzania for example and areas of central Africa, breast cancer accounts for up to 6 percent of cancers in men [16]. Similar researches in Uganda and Zambia Showed 5% and 15% respectively [17, 18]
Male breast cancer in Nigeria represents 3.7-8.6% of all breast cancers; this is higher than the 1% recorded from other parts of the world [19]. Majority are invasive ductal carcinoma which is characterized by late presentation at advanced stage with attendant poor prognosis [9].

RISK FACTORS
The exact cause of male breast cancer is not known but there are risk factors that make someone susceptible to develop the disease over a period of time. They include:

A. Genetic: Familial disposition just like that of the women breast cancer increases the risk of developing male breast cancer. Men that have first degree relatives with history of breast cancer tend to be susceptible to have the disease in the future. Basham et al [25] stated that 15-20% of male breast cancer cases originate from family history. Men can have mutation on breast cancer gene BRCA1 and BRCA2 but the latter is more common in this sex [26]. A male with BRCA2 mutation carries an increase 6% life time risk of having the disease than 0.1% in the normal population. Other genetic condition that increases susceptibility to the disease is Klinefelter’s syndrome [27].

B. Radiation Exposure: Frequent exposure to ionizing radiation has been associated with an increase risk of developing breast cancer in both men and women [7]. Men with history of undergoing chest x-ray or radiation therapy frequently have a greater chance of having the disease [28]. Therefore, prolong exposure to radiographs may be
harmful to individuals. Even workers that expose themselves to electromagnetic waves and other radiations daily without adequate protection are at risk [29].
C. Hyperestrogenism: Certain conditions can result in abnormally high levels of estrogen in men thereby increasing the risk of developing breast cancer. About 80-90% of male breast cancer has estrogen receptor meaning they are ER positive. Klinefelter’s syndrome and cirrhosis of the liver have been found to cause increase estrogen level in men [30]. Other conditions affecting estrogen in relation to androgen levels are taking exogenous estrogen as medication, mumps ochitis and testicular dysfunction [8]. Obesity also increases risk of breast cancer due to conversion of androgen hormone by the fat cells into estrogen; this means that obese men have higher level of estrogens in their body which subsequently may cause breast cancer [31].

DIAGNOSIS
A number of diagnostic methods are available. They include:
1. Medical history and physical examination: The medical history may give some clues about the cause of any symptom on the patient and tendency of having increase

risk factor(s) of developing breast cancer [8]. A thorough clinical breast examination will assist in locating any lumps or suspicious areas and assess the texture, size, and relationship of the breast to the skin and muscle tissue [32].

2. Biopsy: this involves removal of tissue sample from the body for examination under the microscope. There are different ways of obtaining tissue biopsy namely fine needle aspiration, core needle biopsy and surgical biopsy [33]. Majority of male breast cancers are invasive ductal carcinoma (80-90%) while ductal carcinoma in situ accounted for 10% [34, 35]. Cancer of lobular origin made up of only 1% due to lack of abundant lobules in male breast, others include Paget’s disease (1%), mucinous (1%), medullary and invasive papillary has 2% of occurrence each [36, 37, 38]. Special techniques are employed to further diagnosed breast tissue histopathologically as below:

1. Immunohistochemistry (IHC): This is a method of employing specialized antibodies against specific antigens on the cell membrane or nuclear region of the breast cancer cells. The standard tests include estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor (HER2) and Ki67 tests for invasive breast cancers [37]. About 90% of male breast cancers are ER positive and 16% of the cases over-expressed HER 2 [39].

2. Fluorescent in situ hybridization (FISH): This test uses fluorescent pieces of DNA that specifically stick to copies of the HER2/neu gene in cells, which can then be counted using a fluorescent microscope. Many breast cancer specialists think the FISH test gives more accurate results than IHC, but it is more expensive and takes longer to get the results. When IHC result is 2+, the HER2 status of the tumor is not clear and the tumor is then tested with FISH for confirmation [8, 40].

3. Genetic Testing: Genes are tested on the sample so as to understand the biology of the tumour. Some cancers are fast growing while others are not depending on the type of individual genes they possess. Examples of gene testing methods include Oncotype Dx™ and Mammaprint™ [38].

4. Imaging Tests: Different imaging techniques are use in assisting diagnosis of breast cancer, the methods are diagmostic mammography, ultrasound, magnetic resonance imaging [38]

5. Other Tests: There are several other tests use in assisting diagnosis of breast cancer namely nipple discharge examination, blood tests, X-ray, bone scan, computed tomography and positron emission tomography [8].

PREVENTION
There is need for men to take necessary steps in preventing themselves from having breast cancer by carrying out and observing the following steps:
1. Regular self examination: A person’s best chance of surviving breast cancer is early detection through regular self-examinations. Men should be familiar with the normal feel of their breast tissue so that they can bring any lump or change to the hospital for proper attention.

2. Active participation in exercise activities: It has been realized that increased physical activity is associated with decreased breast cancer risk. This may be because exercise lowers hormone levels, boosts the immune system, and changes metabolism while lack of exercise contributes to obesity.

3. Making healthier food choices: Several researches has shown immense important of particular food substances in preventing development of breast cancer and many other types of cancers. They contained antioxidants and other essential elements that repair and prevent cells from the damaging effects of free radicals and other carcinogens. Examples include tomatoes (lycopene), green vegetables (beta-carotene, folate, Vitamin C, E and K), berries (anthocyanins), onion (quercetin) and sweet potatoes (beta-carotene and Vitamin C).

4. Optimizing Vitamin D source: Vitamin D influences virtually every cell in the body and is one of nature’s most potent cancer fighters. This can be done by appropriate sun exposure or by using Vitamin D supplements.

5. Avoidance of unnecessary exposure to radiation/Environmental pollution: Getting medical imaging studies only when they are necessarily needed and avoidance of environmental pollutions help in cancer prevention.

6. Quitting smoking and sleeping well: Researches has shown cigarette smoking is associated with cancer development because it is injurious to the overall health of an individual. Avoidance of smoking is an essential mean of preventing cancer and having enough sleep maintain someone hormonal balance for a healthy living [41, 42, 43].

CONCLUSION:
Although cancer of the male breast is rare, when it occurs, it presents at an advanced stage especially in resource poor settings. This underscores the need for use of simple and painless preventive measures.

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Prevalence of Bacteriospermia Among Male Partners of Infertile Couples In Bida, Niger State.

Omosigho O P
Medical Microbiology Department, Federal Medical Centre Bida. Niger State .Nigeria

Emumwen E.G, Inyinbor H.E
Medical Microbiology Dept., Federal Medical Centre Bida. Niger State Nigeria.

All correspondents to: Medical Microbiology Department, Federal Medical Centre Bida. Niger State, Nigeria. Email –omosighoop@gmail.com

ABSTRACT

Male urogenital tract infection is an important factor in the management of infertility. This study was carried out to evaluate the prevalence of bacteriospermia, antibiotic susceptibility pattern and its effect on the quality of spermatozoa in male infertility in Bida. Five hundred and six semen samples cultured in the medical microbiology laboratory for three years were analyzed. This study showed a prevalence of bacteriospermia of 39.9% with Staphylococcus aureus having the highest frequency of 30.4%. Bacterial infection has remained one of the important factor in the management of infertility ,this study showed high rate of bacteriospermia in primary infertility 27.7% which is statistically significant (P= 0.000). The prevalence of bacteriospermia in Bida was more pronounced in oligospermia 22.7%. This study found a significant statistical difference between bacteriospermia and age (P=0.000) .In conclusion, the prevalence of bacteriospermia is high in Bida, it is therefore advocated that attention should be given to the treatment of urogenital infection in the management of male factor infertility.

KEY WORDS-Bacteriospermia, Staphylococcus aureus, Antibiotic susceptibility, Male factor, Infertility.

INTRODUCTION

According to World Health Organization (WHO), seminal fluid infection was define as the presence of significant bacteriospermia (= 103 bacterial/ml ejaculate), detection of Nisseria gonorrhoae, Chlamydia trachomatis, Ureaplasma urealyticum, significant leucocytospermia (WHO 1999).
The isolation of microorganisms in seminal fluid especially of infertile men has been widely reported, while the exact role of microbial infection in the etiology of infertility is not very certain owing to the limitation in diagnostic criteria and asymptomatic nature of infection, as some possible effect on the properties of seminal fluid associated with fertility has been suggested (Gregoriov et al .,1989, Merino et al., 1995, Villanueva-Diaz et al., 1999, Purvis and Christiansen1993, Buhharin et a.,l 2000 and Rodin et al., 2003 ).
There is disagreement as to the influence of certain microbial infection on male infertility, several investigation have reported different types of organisms in seminal fluid specimens depending on the method of examination (Macleod and Gold 1951). It was reported that detection of bacteria in semen does not necessary suggest
infection since bacteria isolates in seminal fluid may represent contamination, colonization of urethral orifice or infection. Opportunistic microorganism cause classical infection of urogenital tract and subclinical reproductive tract infection, these infections of the seminal fluid leads to decrease in number of spermatozoa, the suppression of their motility, changes their morphology and fertility capacity (Buhharin et al., 2000).
This study was carried out to evaluate the prevalence of bacteriospermia, antibiotic susceptibility patterns and its effect on the quality of spermatozoa in male infertility in Bida, Niger State.

MATERIALS AND METHOD
Five hundred and six (506) seminal fluids from men investigated for infertility over a period of three years were analyzed. These were seminal fluid of patient referred to the Laboratory from the Gynecology Clinic of Federal Medical Centre, Bida.
The semen was collected after the patient had abstained from sex at least three days. Samples were collected either by self or assisted masturbation into sterile bottle. Patients were educated on proper sample collection to reduce contamination and submitted to the Laboratory within one hour of production. The semen were cultured on Blood, Chocolate and MacConkey agar media and incubated for 24 hours at 37oC while Chocolate agar were incubated under 5% Co2. Emergent colonies were identified according to standard bacteriological method.
The samples were analyzed within one hour of collection or as soon as liquefaction occurred using manual method.
Initial microscopy examination of the appearance, viscosity and volume estimation was done, after which microscopy was carried out to estimate the sperm concentration, motility and morphology according to WHO guideline (WHO 2010).

DISCUSSION

Male genital tract infection is an important etiological factor leading to deterioration of spermatogenesis, impairment of sperm function and/or obstruction of seminal tract (Owolabi et al., 2013). The prevalence of bacteriospermia among male partners of infertile couples in this study is 39.9% with Staphylococcus aureus having the highest frequency of 154(30.4%) followed by Eschericia coli 34(6.7%) and Pseudomonas aeruginosa 7(1.2%) which is in agreement with other studies in Nigeria (Ibekwe and Mbazor 2002 in Abakeliki , Ugboma et al., 2012 in Port Harcout and Emokpae et al ., 2009 in Kano).
Generally ,the risk of infertility increases by age, in this study a high bacteriospermia prevalence rate was obtained among age 36-40 years (12.4%) followed by age 26-30 years (9.2%) and 31-35 years (8.8%) this findings is statistically significant (p=0.000) and agrees with the findings in Port Harcourt Nigeria (Olutimilehin et al .,2012).

Infection has remained one of the important factors in infertility our findings showed a higher rate of bacteriospermia in primary infertile couples 27.7% compared to 12.3% in secondary infertility and it is found to be statistically significant(p=0.000) though, in contrast to the findings of Olujubu et al., (2013)who reported a higher prevalence in secondary infertility.

Bacteriospermia was more pronounced in couples with oligospermia 22.7% followed by normospermia 10.5% and azoospermia 6.7% similar to the findings in Kano by Emokpae et al .,2009, while Owolabi et al ., in Ile ife reported 50.2% seminal infection in normospermia ,20.3% in oligospermia and 4.4% in azoospermia.
Antibiotic susceptibility pattern of bacteriospermia in Bida from our study shows that all isolates are 70-90% susceptible to Levofloxacin, Ciprofloxacin, Pefloxacin and Azithromycin while many are resistant to Gentamycin , Cefuroxime, Ceftazidime and Cotrimoxazole.

In conclusion, the prevalence of bacteria in semen may affect fertility in several ways including damage of spermatozoa, hampering their motility, altering the chemical composition of fluid (Mogra et al., 1981). Bacterial infection in this study is high and we advocate that proper attention should be given to the treatment of bacteriospermia in the management of male factor cause of infertility.

 




Pattern of Abnormal Liver Enzymes Activities in Diabetic Patients in Zaria.

Bakari AG
Department Of Medicine Ahmadu Bello University Teaching Hospital, Zaria
Lawal N; Akuyam SA ; Anaja PO
Department of Chemical Pathology Ahmadu Bello University Teaching Hospital, Zaria

All correspondence to: Lawal N nasiruacademy@gmail.com

ABSTRACT

Type 2 diabetic patients are at an increased risk of developing liver diseases owing to the nature of the disease and its inherent complications. Elevated serum activities of the liver enzymes are the most frequent indicators of liver disease. The purpose of this study was to determine the pattern of abnormal serum liver enzymes activities in type 2 diabetic individuals. The study comprised of 170 type 2 diabetic patients attending Medical Outpatients Department of Ahmadu Bello University Teaching Hospital, Zaria. Diabetes mellitus (DM) was confirmed according to the new diagnostic criteria based on 2 fasting or 2 random plasma glucose levels of more than 7.0 mmol/L and 11.1 mmol/L respectively. A concise history of the patients, physical examination and laboratory findings were recorded on a proforma. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were measured using the kinetic method of IFCC. Serum gamma glutamyl transferase (GGT) activities were measured using the kinetic method of SZASZ. Serum alkaline phosphatase (ALP) activities were measured using the colorimetric method of King and Amstrong. The concentrations of serum FBG and RBG were measured using glucose oxidase method of Trinder.
One hundred and eighteen (69.4 %), 46 (27.1 %), 26 (15.3 %) and 51 (30.0 %) of patients had mild increases in serum levels of AST, ALT, GGT, and ALP respectively. In addition 42 (24.5 %) patients had both mild increases of AST-ALT and 10 (5.9%) had mild increases of all the liver enzymes activities. It can be concluded from the findings of the present study that there is chronic mild increases in serum liver enzymes activities in diabetic patients therefore, liver function tests (LFTs) be included into routine laboratory investigations of DM in Nigerian hospitals.

KEY WORDS: Diabetic, Serum, Liver Enzymes

INTRODUCTION

Diabetes mellitus (DM) is a systemic disease caused by absolute or relative deficiency of insulin and is manifested by disorders of carbohydrates, lipid and protein metabolism1. The prevalence of diabetes is high in patients who have liver disease such as non alcoholic fatty liver disease (NAFLD), chronic viral hepatitis, haemochromatosis alcoholic liver disease and cirrhosis. Similar studies have shown that DM plays a significant role in the initiation and progression of liver injury (Hickman IJ and MacDonald GA, 2007).
Onyemelukwe and Bakari (1998) observed that chronic liver disease was responsible for secondary diabetes mellitus in 15 cases (2% of total and 36 % of secondary diabetes mellitus). Of this number, 10 were secondary to liver cirrhosis, 3 with schistosomal liver fibrosis and 1 case each secondary to chronic active
hepatitis and chronic persistent hepatitis. All cases except Schistosomal liver fibrosis tested positive to serum hepatitis B surface antigen.
The hallmark of the disease is fasting hyperglycemia (WHO; Geneva, 1999) and studies have shown that liver plays a critical role in carbohydrate homeostasis and insulin degradation therefore, it is not surprising that it’s function may be affected by DM (Hanley et al. 2004). Association exists between DM and liver injury (Meltzer A and Everhart JE 1997). There are evidences have revealed that patients with type 2 DM have two times the risk of developing liver diseases than their healthy counterparts (Karen Barrow, 2005). Hsiao et al;2007 reported that alanine aminotransferase (ALT), aspartate aminotransferase (AST) and gamma glutamyltransferase (GGT) were associated with insulin resistance (IR) as glycaemic status progresses in the impaired fasting glucose group.

Liver disorders among diabetics is similar to that of alcoholic liver disease including fatty liver (steatosis), steatohepatitis, fibrosis and cirrhosis. Elevated serum activities of the liver enzymes such as aspartate aminotransferase (AST), alanine aminotransferase(ALT), alkaline phosphatase (ALP) and gamma glutamyltrasferase(GGT) are the most frequent indicators of liver disease and occur in diabetics more frequently than in healthy individuals Hsiao et al; 2007.The aim of the present study was to determine the pattern of abnormal liver enzyme activities in type II diabetic patients in Zaria , Northern Nigeria.

MATERIALS AND METHODS
The study was conducted in Ahmadu Bello University Teaching Hospital (ABUTH), Zaria, Nigeria. This study was approved by the ethical committee of the ABUTH, Zaria in accordance with the declaration of Helsinki. A total of 170 diabetic patients attending Medical-outpatients Department (MOPD) and 80 apparently healthy individuals were studied. The criteria for diagnosis of type 2 DM was the American Diabetes Association Criteria (2004), fasting blood glucose of 7.0 mmol/L on two occasions or random blood glucose of 11.1 mmol/L with diabetic symptoms. The diabetic patients were provided with conventional diabetes care/control measures. At the MOPD, arrangement was made with the Physicians whereby subjects who satisfy the study inclusion criteria were selected. Informed consent for inclusion into the study was obtained from the subjects. The nature of the study was explained to the subjects by using an appropriate language. A full medical history was obtained from the subjects by the Physician followed by clinical examination. The findings were documented in the Proforma. Blood specimens were taken into plain tubes,
using sterile technique. The blood was centrifuged and the serum was carefully drawn into sample bottles and then stored frozen at -200C until the time for analysis. The samples were analyzed for plasma glycatedheamoglobin (GHbA1c) using the method of Triveli et al; 971 and Fasting Blood Glucose (FBG) as well as Random Blood Glucose (RBG) using the method of Trinder 1964..
Statistical analysis was performed using statistical package for social sciences (SPSS) for Windows, version 15.0. Data were presented as Mean±SEM. plasma glycated heamoglobin (GHbA1c) levels and serum Fasting Blood Glucose (FBG) as well as Random Blood Glucose (RBG) levels were compared with those of the apparently individuals using two tailed student t-test. A p-value of equal to or less than 0.05 (p=0.05) was considered as statistically significant.

RESULTS
The results of clinical parameters are presented in table I. The differences in weight and body mass index (BMI) between diabetic subjects and controls were statistically significant. Plasma GHbA1c and serum FBG as well as RBG in diabetic patients and controls are presented in table II. The mean values of plasma GHbA1c and serum FBG as well as RBG were significantly higher in diabetic patients than the control subjects (p<0.01).

The result of pattern of abnormal liver enzymes activities are presented in Table II. One hundred and eighteen (69.4 %) , 46 (27.1 %), 26 (15.3 %) and 51 (30.0 %) of patients had mild increases in serum levels of AST, ALT, GGT, and ALP respectively. Similarly, in the control subjects, 33 (41.3 %), 1 (1.3 %) 27 (36 %) 11 (13.8 %) individuals also had mild increase in serum levels of AST, ALT, GGT and ALP respectively.

DISCUSSION

The results obtained in the present study showed that the percentage prevalence of abnormal serum liver enzymes activities were significantly higher in diabetic patients than in control subjects. The finding of the present study was similar with those of Salmela et al; 1984 and Erbey et al; 2000 as seen in (table II). Even though there were variations in the values of percentage prevalence reported by the above mention authors as well as the present study however, the values were higher in diabetic patients than in control subjects.

The high percentage prevalence of elevated liver enzyme activities in type II diabetic patients seen in the present study suggest that the liver is diseased. It was reported that elevation of serum activities of the liver enzymes such as aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP) and gamma glutamyltrasferase (GGT) are the most frequent indicators of liver disease (Monica et al 2005 and Meltzer A, Everhart JE 1997) therefore, the high percentage prevalence of elevated liver enzyme activities in type II diabetic patients as well as the higher mean level liver enzyme activities in type II DM than control subjects seen in the present study further confirm the abnormalities of liver function in type II DM. The hallmark of type 2 DM is hyperglycaemia and it leads to fat accumulation in the liver14. Fat accumulation is known to be directly toxic to hepatocytes . An elevation of liver enzymes such as ALT and GGT even within the normal range, reflects deposition of excess fat in the liver (Meybodi et al 2008 and Maryam et al 2008). This may be responsible for the increased serum activities of transaminases seen in the present study. Zachary, 2007stated that fat accumulation in the liver also stimulates the release of inflammatory cytokines such as tumor necrosis factor-α (TNF) and interleukine-6 (IL-6) which may contribute to hepatocellular injury. This might also result to increased serum activities of transaminases in the present study.

Hyperglycemia leads to myoinositol depletion in several tissues susceptible to diabetic complications (for example liver), although the exact mechanism is unclear. The fall in myoinositol levels in the cell membrane alters the functioning of Na+/K+ ATPase pump and the integrity of the cell membrane is impaired (Malnick et al; 2003). Clinical observations and experimental studies have shown that subtle changes of cell membrane are sufficient to allow passage of intracellular enzymes such as AST and ALT to the extracellular space with subsequent elevation of their levels in plasma (Malnick et al; 2003). This could be another reason for increased serum activities of transaminases in the present study.

Monica et al; 2005 reported that ALT, ALP and GGT were associated with prevalent type 2 DM and AST with prevalent IGT). NAFLD is a complication in 32%-78% of patients with type 2 DM and 50% of these patients may have NASH. Some studies demonstrated that hyperglycemia and hyperinsulinaemia can promote fatty infiltration of the liver (De Marco et al 1999).
CONCLUSION
It can be concluded from the findings of the present study that there were high prevalence abnormal liver enzyme activities in diabetic patients therefore, liver function tests (LFTs) should be included into routine laboratory investigations of DM in Nigerian hospitals.

ACKNOWLEDGMENT
We acknowledged the assistance of University Board of Research of Ahmadu Bello University, Zaria. We thank Dr IS Aliyu, Head of Chemical Pathology Department of Ahmadu Bello University Teaching Hospital (ABUTH), Zaria for his constant advice and encouragement during this study. We are also grateful to Dr Muazu Babura of the Department Medicine of ABUTH, Zaria for his assistance during the course of this work. We also thank Mal. MA Auwal and of the Department of Chemical Pathology ABUTH, Zaria for his assistance in analysis and other logistics.




An Unusual Occurrence: A case of venous thromboembolism in pregnancy associated with heterotaxy syndrome

Narendranath Epperla, Patrick Foy

Division of Hematology and Oncology, Medical College of Wisconsin, 9200 W Wisconsin Avenue, Milwaukee, WI, USA

Erika Peterson
Division of Obstetrics and Gynecology, Medical College of Wisconsin, Milwaukee, WI, USA

Correspondences to: Narendranath Epperla · ·nepperla@mcw.edu

ABSTRACT
Background: Heterotaxy is a relatively uncommon congenital anomaly that is usually diagnosed incidentally on imaging studies in adults. We present an unusual case of venous thromboembolism in a 26 year old pregnant female with Heterotaxy syndrome.

Case presentation: A 26 year-old pregnant female at 13 weeks gestation suffered cardiac arrest with successful cardiac resuscitation and return of spontaneous circulation. The cardiac arrest was secondary to massive pulmonary embolism requiring thrombolytic therapy and stabilization of hemodynamics. She had extensive evaluation to determine the etiology for the pulmonary embolism and was noted to have an anatomic variation consistent with heterotaxy syndrome on imaging studies. After thrombolysis the patient was treated with UFH and then switched to enoxaparin without complication until 25 weeks of gestation when she experienced worsening abdominal pain with associated headaches, lightheadedness and elevated blood pressures needing elective induction of labor. The infant died shortly after delivery. The anticoagulation was continued for additional 3 months and she was subsequently placed on low dose aspirin to prevent recurrent venous thromboembolic episodes. She is currently stable on low dose aspirin and is into her third year after the venous thromboembolism without any recurrence.

Conclusion: To our knowledge, this is the first reported case of venous thromboembolism in pregnancy associated with heterotaxy syndrome. A discussion on pathophysiology of venous thromboembolism in pregnancy and heterotaxy syndrome has been undertaken along with treatment approach in such situations.

Keywords: Venous thromboembolism; Inferior vena cava; Low molecular weight heparin

BACKGROUND

Heterotaxy is an uncommon polymalformative syndrome characterized by congenital anomalies that arise from disorderly arrangement of asymmetric thoracic and abdominal viscera and blood vessels [1]. The estimated prevalence is approximately 1 in 10,000 live births. Morbidity and mortality rates are high approaching nearly 70 % due to major cardiac malformations (especially in heterotaxy with asplenia patients). Five to ten percent of patients with minor or no cardiac anomalies will survive to the adulthood. Some will remain asymptomatic and can be diagnosed incidentally on imaging studies. Heterotaxy has been reported to be associated with VTE and is felt to be related to abnormal lower extremity venous system. Infact in the last few years some authors considered it as a VTE risk factor. However VTE in pregnancy with heterotaxy syndrome has not been
reported.
Herein we present a case of 26 year-old pregnant female who suffered cardiac arrest related to massive pulmonary embolism requiring cardiac resuscitation. Imaging studies incidentally discovered anatomic variation consistent with heterotaxy syndrome. She was anticoagulated for 6 months with low molecular weight heparin (LMWH) and subsequently placed on low dose aspirin to prevent recurrent VTE. A detailed discussion of the pathophysiology of VTE in pregnancy and heterotaxy syndrome has been undertaken with a focus on treatment approach in such situations.

Case presentation
A 26 year old otherwise healthy, G1P0 female at 13 weeks gestation was admitted to medical intensive care unit after she experienced cardiac arrest at home. She had reported shortness of breath earlier that day and then collapsed.

During initial paramedic evaluation, she was pulseless and found to be in pulse less electrical activity (PEA). She was successfully resuscitated with return of spontaneous circulation (ROSC) in approximately 7 min and was brought to the hospital. Emergency department (ED) evaluation revealed the patient to be nonresponsive, and she was immediately intubated. Her exam was remarkable for cold and cyanotic extremities, without palpable distal pulses but strong femoral pulse. Shortly after her arrival to ED, the patient became pulse less again needing resuscitation and ROSC on 2 occasions.

Initial blood work was remarkable for severe metabolic acidosis (HCO3 of 10) and elevated troponin I (2.77 ng/ml, normal range 0–0.34 ng/ml). EKG showed right bundle branch block while bedside 2 D echocardiogram demonstrated right heart strain pattern. Bedside ultrasound (USG) showed no significant free fluid with live intrauterine gestation. Bilateral lower extremity compression venous dopplers of the entire venous system were negative for any evidence of deep venous thrombosis. She underwent computerized tomography (CT) of the chest which revealed a nearly occlusive thrombus in the bilateral lower lobar arteries extending into the segmental arteries (Fig. 1). The main pulmonary arterial trunk was noted to be enlarged with evidence of right heart strain (Fig. 2). Head CT scan was negative for any acute intracranial abnormalities. She was administered intravenous tissue plasminogen activator (TPA) (50 mg after ROSC the second time) in the ED with improvement in her hemodynamics. Hypothermia protocol was concurrently initiated. She was subsequently transferred to medical intensive care unit on unfractionated heparin (UFH), bicarbonate and norepinephrine drips. The patient was extubated two days following and was gradually weaned off blood pressure support. UFH drip was titrated based on aPTT and 6 days later she was transitioned to LMWH (dalteparin 10,000 units subcutaneously once daily in view of her insurance issues). She had aggressive supportive care and after a 2 week hospital stay, she was discharged to rehabilitation facility on Dalteparin. A month later dalteparin was changed to enoxaparin 55 mg (1 mg/kg) s/q twice daily (monitored with anti Xa levels).
At approximately 20 week’s gestation, the patient had a fetal ultrasound that revealed singleton gestation with massive hydrops, fetal contractures and ventriculomegaly/hydranencephaly. It was presumed that these findings were secondary to early hypoxic injury and she was counseled about poor prognosis. During her 25th week gestation, the patient noted worsening abdominal pain with associated headaches, lightheadedness and weakness. She had elevated blood pressures and was suspected to have mirror syndrome. Enoxaparin was discontinued 24 h prior to the planned procedure with initiation of UFH drip. The patient underwent elective induction of labor with palliative care services. The infant died shortly after delivery. The patient experienced increased vaginal bleeding secondary to retained products of conception and underwent dilatation and curettage with achievement of hemostasis. Enoxaparin at previous dosage (55 mg s/q twice daily) was restarted 24 h after the delivery without any major or untoward complication. Platelet count and anti-Xa levels were checked while the patient was on enoxaparin. Anti-Xa levels were in therapeutic range (0.8–1, goal 0.6–1.0 IU/ml) and platelet counts remained within normal limits.
In order to identify the cause of her VTE the patient had further work up including thrombophilia testing and CT chest, abdomen/pelvis. Thrombophilia testing revealed the patient to be wild type for Factor V and Prothrombin gene. Protein C and S antigen levels were normal. Antithrombin activity was initially low (71, normal range 75–125 % ACT) at the time of initial VTE but subsequently normalized (107). Antiphospholipid antibody testing including lupus anticoagulant, anti-cardiolipin antibodies and beta 2 glycoprotein I antibodies was negative. CT chest, abdomen and pelvis showed constellation of imaging findings compatible with heterotaxy syndrome. These included left-sided superior vena cava (SVC) draining into the left coronary sinus, hemiazygous vein drains into left SVC, absent azygous vein, absent (interrupted) inferior vena cava (IVC) below the hepatic IVC with persistent left IVC which drains into left coronary sinus, polysplenia, bilateral left lung and benign hepatic hemangioma (Fig. 3A-C).

The patient completed 6 month duration of anticoagulation with LMWH and was subsequently placed on ASA 81 mg. Prior to discontinuation of anticoagulation therapy the patient had CT pulmonary angiogram which showed normal diameter of the main pulmonary artery without any evidence of acute or chronic pulmonary emboli. She is currently stable on low dose ASA and is into her third year after the VTE without any recurrence. She has had no further pregnancies.

DISCUSSION
Heterotaxy or situs ambiguous refers to malposition and dysmorphism of viscera and blood vessels, often with indeterminate atrial arrangement. This abnormal arrangement of body organs is different from the orderly arrangement seen in situs solitus or situs inversus. Heterotaxy syndrome can be associated with either asplenia or polysplenia. Our patient had heterotaxy with polysplenia and hence will limit our discussion to this abnormality and hereon will be referred to as heterotaxy.
Heterotaxy implies that the patients have bilateral bilobed lungs, bilateral pulmonary atria, a centrally located liver, a stomach in indeterminate position, interrupted IVC with azygous continuation and multiple spleens. After polysplenia, the most frequent finding in heterotaxy is the hypoplasia of the IVC with absence of the intrahepatic segment and direct continuation with the azygous venous system. Heterotaxy commonly occurs as a sporadic condition but it can be inherited.
Varied timing of embryological causative factors during the development of fetus can explain the wide range of anomalies in the heterotaxy syndrome. Cardiac anomalies are mostly the result of abnormal embryological development around day 28 of embryogenesis, as it is during this period that connection between primitive heart and venous channels occurs. The failure of fusion of fetal lobules leads to individualization of multiple splenic nodules, which are located along the greater curvature of the stomach, as the spleen develops in the mesogastric region. IVC is a complex vascular structure that is developed during weeks 6–8 of embryogenesis. During this period three pairs of primitive venous channels (posterior cardinal, subcardinal and supracardinal veins) form the mature venous system (IVC) through a complex sequential process. Various malformations including partial or even complete absence of IVC can result from the failure in the developmental steps related to embryonic dysontogenesis that can affect separate segments or even the entire IVC. Thus mutations in genes that control left-right patterning and teratogenic exposures in early embryonic period underlie majority of heterotaxy cases.
The occurrence of DVT seems to be higher in patients with heterotaxy compared to the general population. It is postulated that stasis related to the abnormal venous drainage of the lower extremity venous system due to interrupted IVC, and increased platelet aggregation related to the anomalous drainage of the splenic venous system into the azygous system seems to be the possible culprits for heightened risk of pulmonary thromboembolism in these patients.
VTE is 5 times more frequent in pregnant women than in non-pregnant women of similar age. This is because pregnancy induces a state of venous stasis and
Fig. 3. A. CT scan of the chest with contrast.
Coronal section showing bilateral bilobed lungs
(yellow arrows depict the major fissure). B. CT
scan of the abdomen. The arrow points to multiple
splenic lobules suggestive of polysplenia.
C. CT scan of the abdomen. Coronal section
showing azygous continuation of inferior vena cava

hormonal and hematological changes leading to an increased risk for VTE. Venous stasis occurs from progesterone mediated increased venous distension in the first trimester and the compressive effect by the enlarging uterus on the common iliac vein in the late second and third trimesters. In addition there is an imbalance between the procoagulant (increased circulating levels of fibrinogen, von Willebrand factor, VII, VIII, IX, X and XII and increased generation of fibrin) and anticoagulant factors (decreased Protein S levels and fibrinolysis) through the pregnancy. Though pregnancy is considered to be a relative contraindication to systemic thrombolysis (recombinant TPA or streptokinase) in hemodynamically unstable patients with pulmonary embolism, the risk of complications for pregnant females treated with thrombolytic agents may be similar to that in the non-pregnant population.

In our case, the exact etiology for VTE is unclear. It may be related to pregnancy alone or mechanical factors from distorted venous system anatomy or a combination of both. Because the patient’s VTE occurred early in pregnancy, uterine enlargement is unlikely to have contributed to venous thrombosis. Effects of pregnancy in heterotaxy have been poorly described.

Challenges to the clinical management of our patient include the duration of anticoagulation and future pregnancy. Based on the 2012 American College of Chest Physicians (ACCP) guidelines, anticoagulation with LMWH is recommended for at least 3 months from the initial pulmonary embolism and 6 weeks postpartum. In our case, the patient was into her 3rd month of anticoagulation when she had induction of labor and delivery; hence the anticoagulation was continued for additional 3 months for a total of 6 months. Subsequently she was placed on low dose aspirin (81 mg) to prevent recurrent VTE (especially given her heterotaxy syndrome) based on the Warfarin and Aspirin (WARFASA) and Aspirin to Prevent Recurrent Venous Thromboembolism (ASPIRE) trials as well as the individual patient data analysis of WARFASA and ASPIRE trials performed by the INSPIRE Collaboration. Currently there is no evidence about the risk of VTE recurrence and anticoagulation duration in similar patients. According to the 2012 ACCP guidelines the decision regarding the duration of anticoagulation in a patient should be made after careful evaluation of both the risk of VTE recurrence and bleeding risk. Given the congenital prothrombotic risk factor (interrupted IVC related to heterotaxy syndrome), severity of the presentation and apparently low bleeding risk extended anticoagulation treatment with targeted oral anticoagulation maybe a suitable alternative to aspirin with periodic assessment of the bleeding risk.

Our patient had a massive pulmonary embolism with cardiac arrest that created a great sense of apprehension both in the patient and her family regarding future pregnancy. Unfortunately, there is paucity of data to either support or refute future pregnancy associated with her condition. Previous VTE alone is not a contraindication to future pregnancy provided anticoagulation is available. However patients with heterotaxy syndrome and VTE are undoubtedly at increased risk of VTE compared to other pregnant women. Hence future pregnancies in this patient group should be considered high-risk, and requires multi-disciplinary management. We recommend full intensity anticoagulation with LMWH (likely enoxaparin 1 mg/kg two times daily) with regular monitoring of anti-Xa levels prior to her pregnancy. It is important to measure anti-Xa levels 4 h after the last dose and be aware of the different targets based on which LMWH regimen is used (once-daily [goal 1.0–2.0 IU/mL] or twice-daily [goal 0.6–1 IU/mL]). In addition we recommend that there is a detailed discussion between the physician and the patient regarding the role of thrombolytic therapy in the event of recurrent VTE and possible termination of pregnancy in the worst case scenario.

CONCLUSION
To our knowledge this is the first reported case in the English literature that shows pregnancy associated VTE in a person with heterotaxy. After completion of anticoagulation therapy for the initial thrombotic event, the patient needs secondary prevention for VTE recurrence and low dose aspirin was the chosen treatment in this case. Although there are no published guidelines we believe that there are no contraindications for future pregnancy in this special group provided the patient is on anticoagulation prior to the pregnancy.




Epidemiology of Breast Cancer among Male in University of Abuja Teaching Hospital Gwagwalada

Dangana .A,

Hematology Laboratory, University of Abuja Teaching Hospital, Abuja
Ishaku .H, Victoria .I,

Histology Laboratory, University of Abuja Teaching Hospital, Abuja
Christy .C.F,

College of Health Sciences, Pathology Department, University of Abuja
Egenti B.N

College of Health Sciences, Community Medicine Department, University of Abuja

All correspondence to: isalemit@yahoo.co.uk

ABSTRACT
Breast Cancer is the most common non-communicable disease and non-cutaneous malignancy. it’s a rare disease among men which account for less than 1% of all cancers in men. We examined the trends in the prevalence rate of breast cancer in men among tissues submitted to histopathology laboratory university of Abuja Teaching Hospital. A total of 544 data collected consisting of men between the age 17-86years with the mean aged group of 56years and was analysed using Epi-Info version 6.1. it was found that the prevalence of breast cancer among men was 4(2.6%), fibroadenoma197(36.8%),fibrocytic disease 120(22.4%), granulomatouse mastitis 14(2.6%)lactating adenoma 11(2.1%),sclerosing adenosis 8(1.5%), the highest prevalence rate was found between the aged group of 39-48years(50%) followed by 39-48years(25%) and 79-88years (25%) respectively.

KEYWORD: Breast Cancer, Male.

INTRODUCTION

Cancer is a term used for diseases in which abnormal cells proliferate without control.Breast cancer in men is a rare disease that accounts for less than 1% of all cancers in men and less than 1% of all diagnosed breast cancers,Magno et al,2009It is a diagnosis for which optimal management is not clearly established and treatment guidelines are scarce. The medical literature regarding breast cancer in men consists mainly of case-control and retrospective studies, and there are no randomised prospective data for this disease. Recent emphasis therefore has been placed on extrapolating data derived from studies of breast cancer in women and using those data as a benchmark for treating men—what’s good for the goose is good for the gander.Most types of cancer cells eventually form a lump; growth or mass called a tumor, and are named after the part of the body where the tumor originates. Breast cancer begins in breast tissue, which is made up of glands for milk production, called lobules, and the ducts that connect the lobules to the nipple. The remainder of the breast is made up of fatty, connective, and lymphatic tissue.Edge, et al,2010 Breast Cancer constitutes a major publichealth issue globallywith over 1 millionnewcases diagnosed annually, resulting in over 400,000annualdeath. Veronesi et al,2005, Omaret al,2013.There is an international/geographical variation in the incidence of Breast Cancer. Incidence rates are higher in the undeveloped countries than in the developing countries. Parkin ,et al,2005, Pages et al,2001. In Africa, Breast Cancer has overtaken cervical cancer as the commonest malignancy affecting women and the incidence rates appear to be rising. Vorobiofet al,2001, Omar et al,2013.In Nigeria breast cancer has increased from 116 per 100,000 in 2001. Adebamowo and, Ajayi. 2000. The activities of breast cancer society in some parts of the globe regarding cancer control and prevention may also be responsible for the better quality of live for victims of cancer in those countries Braunstein, 1993,Stratton et al,1997. Breast Cancers in developing countries are diagnosed when they are at advanced stages, which may be responsible for the higher mortality. The cost of treating Breast cancer in the tropics is prohibitive for most breast cancer victims; this is largely due to poverty and low per capita income as most people live below $1 per day, Thomaset al,1992, Althuiset al,1997.Cancers of cervix and breast have become preventable and have been well controlled In developed nations. Health education to increase awareness on breast self examination, may also contribute to early detection and prevention of invasive breast cancer,Adebamowo and, Ajayi ,2000. Agbo,et al,2013. The aim of this work is to determine the epidemiology and prevalence of breast cancer in menamong breast tissues submitted to histolopathology of university of Abuja Teaching Hospital, and also to determine the prevalence and proportion of breast cancer amongst the age group.

New Picture

This is a retrospective study comprises of 544 samples of breast tissue submitted to the histopathology laboratory of university of Abuja Teaching Hospital Gwagwalada Abuja Nigeria between 2010-2014.
Inclusion: Samples that have patient’s detail such as age, sex and diagnosiswere used
Exclusion: samples without age, sex ,and all females were excluded from the study.
Statistical analysis: Result of data were captured in excel and then analyzed using epi-info version 6.1

Methodology
Histological examinations
Breast tissues received registered, grossed and was processed in automatic tissue processor containing 12
beakers, The tissue was processed by allowing the tissue to passed through the following stages Fixation, Dehydration,Clearing,Impregnation,Embedding,Sectioning,Staining,Mountingthe tissues were passed from beaker 1,2,3 contains formalin where the tissues was allowed to stay for 30min each, beaker 4,5,6 contains alcohol and was allowed to stay for 1hour each, beaker 7,8,9 10 contains xylene and was allowed to stay for 1hr each while the last 2 beakers which is 11,and 12 contains wax and was allowed to stay in beaker 11 for 2hours (infiltration) and 12 for 2hours (impregnation).

Staining procedures for Haematoxylin and Eosin
Princples.
The haematoxylin is a basic dye and has affinity for the nucleus DNA & RNA which is acidic,the orange G6 has affinity for the acidophilic cells of the cytoplasm of superficial cells while the eosin azure has affinity for the cytoplasm of intermediate, parabasal and basal cells.

Methods
The smears was remove from fixatives and rinsed in descending grades of alcohol (80,70,50%),for 8secs each Stain in Harris alum-Haematoxylin for 4mins.
Wash in tap water for 1-2 mins, and was differentiated in 1% acid alcohol briefly.
It was then wash and blue in tap water for 3-5mins, and then transferred to two changes of 95% alcohol for a few seconds each.Stain in OG6 for 2minutes, Rinsed in two changes of 95% alcohol, and Stain in Eosin azure for 2-4mins.
It was rinsed in two changes of 95% alcohol .complete dehydration in absolute alcohol and cleared in xyleneand was mounted in DPX and examined to study the architecture of the tissues.
Any tissue examined and aberrations in the structural integrity of the tissues seen and a case of malignancy established, immunohistochemistry employed to profile the markers.

Principles of Immunohistochemistry
Immunohistochemistry (IHC) is a wide-used biological technique that combines anatomy, physiology, immunology and biochemistry. Developed from the antigen-antibody binding reaction, immunohistochemistry can be considered as a method that visualizes distribution and localization of specific antigen or cellular components in separated tissues, or tissue sections. Compared to other bio-techniques that are based on the antigen-antibody reaction such as immunoprecipitation, or western-blot, immunohistochemistry provides in situ information which promises a more convincing experimental result.
Major components in a complete immunohistochemistry experiment:
1) Primary antibody binds to specific antigen;
2) The antibody-antigen complex is formed by incubation with a secondary, enzyme-conjugated, antibody;
3) With presence of substrate and chromogen, the enzyme catalyzes to generate colored deposits at the sites of antibody-antigen binding.

New Picture (1)

Protocol

Prepare formalin-fixed, paraffin-embedded tissue sections (Step 1-8):
1. The freshly dissected tissue was fixed (<3mm thick) with 2% paraformaldehyde from 1h to overnight at room temperature.
2. The tissue was rinsed in running tap water for 5 min.
3. The tissue was then dehydrated through 70%, 80%, 95% alcohol, 5 min each, followed with 3 times of 100% alcohol, 5 min each.
4. The tissue was cleared in xylene for 2 times, 5 min each.
5. The tissue was immersed in paraffin for 3 times, 5 min each.

6. And Embeded in a paraffin block. The paraffin tissue block was then stored at room temperature for years.
7. The paraffin-embedded tissue block was section at 5-8 µm thickness on a microtome and floated in a 40°C water bath containing distilled water.
8. The sections were transferred onto glass slides suitable for immunohistochemistry. The slides were allowed to dry overnight and it was stored at room temperature until ready for use.
Immunostain formalin-fixed, paraffin-embedded tissue sections (Step 9-29):
9. The slides were Deparaffinizein xylene for 2 times, 5 min each.
10. The slides were transferred to 100% alcohol, for 2 times, 3 min each, and then transfer once through 95%, 70% and 50% alcohols respectively for 3 min each.
11. The endogenous peroxidase activity were blocked by incubating sections in 3% H2O2 solution in methanol at room temperature for 10 min to block endogenous peroxidase activity.
12. It was rinsed with PBS for 2 times, 5 min each.
13. The blocking buffer was drained from the slides.
14. 100 µl of diluted primary antibody was apply appropriately (in antibody dilution buffer, e.g. 0.5% bovine serum albumin in PBS) to the sections on the slides and incubate in a humidified chamber at room temperature for 1 h.
15. The slides were then washed with PBS for 2 times, 5 min each.
16. 100 µl of diluted biotinylated secondary antibody was apply appropriately (using the antibody dilution buffer) to the sections on the slides and incubate in a humidified chamber at room temperature for 30 min.
17. The slides were washed with PBS for 2 times, 5 min each.
18. 100 µl diluted Sav-HRP conjugates was apply appropriately (using the antibody dilution buffer) to the sections on the slides and incubate in a humidified chamber at room temperature for 30 min (keep protected from light).
19. The slides were washed with PBS for 2 times, 5 min each.
20. 100 µl DAB substrate solution was applied (freshly made just before use: 0.05% DAB – 0.015% H2O2 in PBS) to the sections on the slides to reveal the color of antibody staining.The color was allowed to development for < 5 min until the desired color intensity is reached. (Caution: DAB is a suspect carcinogen. Handle with care. Wear gloves, lab coat and eye protection.)
21. The slides were washed with PBS for 3 times, 2 min each.
22. The slides were Counterstain by immersing in Hematoxylin for 1-2 min.
23. The slides were rinsed in running tap water for 10 min.
24. Theslides were dehydrated through 4 times of alcohol (95%, 95%, 100% and 100%), 5 min each.
25. The slides were cleared in 3 times of xylene and coverslip using mounting solution. The mounted slides were stored at room temperature permanently.
26. The color of the antibody staining in the tissue sections were then observe under microscopy.

RESULT
Out of 544 data collected for breast tissues, 19 (3.6%) were males, Breast cancer showed a prevalence rate of 15

DISCUSSION
Of the 544 breast tissues submitted to histopathology laboratory of University of Abuja Teaching Hospital Gwagwalada, male study subjects shows a breast cancer prevalence rate of 4(2.6%) and the age range of the study subject 7-86,with the highest prevalence rate at 49–58yrs,(50%) followed by 39-48yrs and 79-88(25% and 25%) respectively which is agreement with a study carried out by (Kidman et al., 2000) in Jos with the age ranging from 12 -85 years which shows that Male breast cancer rate was 2% and also with 2.5% recorded in Benin by (Okobiaet al., 2001) and 1.47% recorded in Nnewi by (Anyanwu, 2000) respectively.
This result is not in agreement with the results obtained from Zaria (Hassan et al., 1995) and Jos which recorded 8.6% and 9% respectively. Also different from results obtained in Tanzania and Zambia which shows a prevalence rate of 6% and 15% respectively according to(Singgal et al, 2006) and (Ihekwaba, 1994). The high incidence of male breast cancer in Nigeria and Africa compare to Western countries have been attributed to hyperestrogenism due to endemic liver infections in Africa and environmental changes and lifestyle (Pere et al., 2007).
The definite etiology of male breast cancer is idiopathic just like other cancers. Factors such as alteration of hormonal milieus, family history and genetic alterations are known to affects its occurrence.Various studies have also shown that conditions that alter the estrogen-testosterone ratio in males predispose to breast cancer (Balleriniet al, 1990 and Casagrandeet al, 1988). Among these conditions, the strongest association is with Klinefelter Syndrome. Males with this condition have a fifty times increased risk and account for 3% of all breast cancer (Hultbornet al, 1997). Any condition associated with increased estrogen levels like cirrhosis and exogenous administration of estrogen (either in transsexuals or as therapy for prostate cancer) have been implicated as causative factors (Symmers, 1968, and Pritchard et al., 1988).
Androgen deficiency due to testicular disease like mumps, undescended testis, or testicular atrophy has been linked to the occurrence of breast cancer in men (Thomas et al., 1992) and (Mabuchi etal., 1985). Occupational exposure to heat and electromagnetic radiation, causing testicular damage and further leading to the development of male breast cancer have been postulated (Stenlund and Floderus, 1997) and (Pages et al., 2001). Hereditary breast cancers are known to occur in males.
Studies have found that gremlin mutations in BRCA1 and BRCA2 account most for this. (Stratonet al, 1997).Gynaecomastia has also been implicated as a risk factor (Braunstein, 1993).
The peak level incidence among Male was between 40 -86 years with mean age of 57 years which is similar to other results obtained in Nnewi, Nigeria(Anyanwu,2000) with a mean age of 60yrs and 66yrs obtained in Spain respectively (Ihekwaba,1994). KaiyumarContractor,et al,2008 also found average age mean of diagnosis to be 60yrs which is similar to the mean age of this study.
The burden of breast cancer among Nigerian women is becoming overwhelming, this may be due to the cultural practice in this country where most women seek alternative treatment and healing (ranging from herbal to religious help believing in the say that “It never my portion” or people attacking them in their villages and poverty rather than coming to die in the hospital. This partially explains why most women present with advance disease as our figure has shown (97.6%) thereby leaving them only the palliative option of treatment. Aggressive awareness campaign is the only way to change these attitudes.

CONCLUSION
The rise in breast cancer cases in this study population is an indication of inadequate or ineffective control measures to curtail the disease or due to diversion of global attention to HIV/AIDS and Tuberculosis in the country. Therefore, there is urgent need to step up activities through non-governmental agency to promote advocacy, national policy on training of personnel for clinical and self-breast examination, and nationwide screening program (Mammography) in order to enhance early detection, control the upward trends and reduce the mortality rate of breast cancer .Breast cancer aggressive awareness campaign should be increased to allow earlier detection so as to reduce the mortality and morbidity rate.




Evaluation of The Antioxidant Activity of Ethyl Acetate Fraction of Nigella sativa(L.) Seed Extract On Gastric Mucosal Integrityin Rats.

Saleh, M.I.A, Isa., A.I, Mabrouk, M. A.,  Mohammed, A., Alhassan, A.W

Department of Human Physiology, Faculty of Medicine, Ahmadu Bello University, Zaria, Nigeria.
Heba, D.
Animal Science Unit, El-Kasr el-Ain, Cairo University, Cairo, Egypt.
Musa, K.Y.

Department of Pharmacognosy and Drug Development, Faculty of Pharmaceutical Sciences, Ahmadu Bello University, Zaria, Nigeria

All correspondence to: alhajisaleh@yahoo.com

ABSTRACT
Nigella sativaL. is one of the most extensively studied plants both phytochemically and pharmacologically.It is an annual flowering plant, native to Southwest Asia and indigenous to the Mediterranean region, Middle-East, but now widely found in India.In the UnaniTibb system of medicine, N. sativa is regarded as a valuable remedy for a number of diseases including gastric diseases. The present study was carried out to investigate the gastroprotective and antioxidant effect of ETAC fraction of Nigella sativa L. (Family; Ranunculaceae) seed extract on indomethacin-induced gastric ulcer model in adult male albino Wistar rats with the fraction of the extract used at doses of 50, 100 and 200 mg/kg using cimetidine as standard drug. Phytochemical screening of the fraction revealed significance and variations in the presence of flavonoids, alkaloids, tannins, saponins, steroids/triterpenes, glycosides/glucosinolates and free anthraquinones in the fraction, while acute toxicity studies revealed a lethal dose (LD50) above 5000 mg/kg. The extract was administered subcutaneously thirty minutes prior to indomethacin (20 mg/kg). The various parameters studied were malondialdehyde, superoxide dismutase and catalase activities. Pretreatment with the fraction at doses of 50, 100 and 200 mg/kg with the 50 and 100mg/kg doses led to a decrease, though not significant in catalase(CAT) and superoxide dismutase (SOD) activities,while malondialdehyde (MDA) concentration significantly increased (P<0.05)with regard to the extract treated groups. This inhibition of lipid peroxidation and the generation of MDA and related substances from lipids that react with thiobarbituric acid was found to be inhibited by this extract. The inhibition was found to increase when the concentration of the extract was increased to 200mg/kg. Thus, it appears that the anti-oxidant property of this extract could counteract oxidative damage caused by NSAIDs. In conclusion, the gastroprotective properties of this fraction evaluated may be attributed to its reducing effect on oxidative damage and neutrophil infilteration in tissues due to the presence of phytochemicals like flavonoids, alkaloids and tannins, amongst others, present in the seed extracts with various antioxidant and other biological activities.

Keywords: Nigella sativa, Free Radicals, Antioxidant, NSAID’s,

1.0 INTRODUCTION

Peptic ulcer disease is one of the most common gastrointestinal disorders which causes a high rate of morbidity and at times even mortality particularly in the developing world (Falk, 2001).The pathophysiology of these disorders has focused on an imbalance between aggressive and protective factors in the stomach such as acid-pepsin secretion, mucosal barrier , mucus secretion , blood flow, cellular regeneration, prostaglandins, and epidermal growth factors.Stress, smoking, nutritional deficiencies, and ingestion of non-steroidal anti-inflammatory drugs are all factors which increases the gastric hyperacidity and gastroduodenal ulcer incidences (Lima et al., 2006;Jainu and Devi, 2006).An imbalance between antioxidants and reactive oxygen species results in oxidative stress, leading to cellular damage including those of the gastric mucosa that could lead to peptic ulceration (Burkeret al., 2003).Cooperative defence systems that protect the body from free radical damage include the anti-oxidant nutrients and enzymes. The antioxidant enzymes include catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx),and indirectly, glutathione reductase.Their roles as protective enzymes are well known and have been investigated extensively in both in vivo and in vitro model systems.The first three enzymes directly catalyse the transformation of peroxides and superoxides to non- toxic species.Glutathione reductase reduces oxidized glutathione to glutathione; a substrate for GPx.The consequences of oxidative stress are serious,and in many cases are manifested by increased activities of enzymes involved in oxygen detoxification (Kyounget al., 2003).Identification of new anti-oxidants remains a highly active research area, because recently reactive oxygen species generated in the cells of aerobically respiring organisms due to many factors have been implicated in the pathogenesis of many human sufferings like Parkinson’s disease, cancer and gastric diseases (Gutteridge, 1998; Mau et al.,2001; Gulcinet al.,2002b) and anti-oxidants may reduce the risk of various acute and chronic diseases caused by free radicals (Kyounget al.,2003). For the discovery of new leading agents which would be used for the benefit of mankind, we focused our study on the free radical scavenging potentials of Nigella sativa. The plant Nigella sativa has been used for medicinal purposes for centuries, both as a herb when pressed into oil in Asia, Middle East and Africa. It has been traditionally used for a variety of conditions and treatments related to respiratory health, stomach, intestinal, kidney, liver, circulatory and immune system support, and for general well-being. The seeds are used as carminative, aromatic, stimulant, diuretic, antihelminthic,galactagogue and to increase sweat. They are used as a condiment in curries. A tincture prepared from the seeds is useful in indigestion, loss of appetite, diarrhoea, dropsy, amenorrhoea, dysmenorrhoea and in the treatment of worms and skin eruptions. Externally, the oil is used as an antiseptic. To arrest vomiting, the seeds are roasted and given internally (Gupta et al., 2009).

2.0 MATERIALS AND METHODS:

2.1 Plant material:
Nigella sativadried seeds were obtained during the month of July, 2011 from Sabon-Gari market in Zaria. Botanical identification and authentification were done by Mr. U.A Gallah at the Herbarium section of the Department of Biological Sciences, Ahmadu Bello University, Zaria. A voucher herbarium specimen (No: 101201) was deposited at the herbarium for future references.

2.2 Extraction and fractionation of the plant material:
Nigella sativa seeds weighing about 2kg were crushed and pounded with pestle and mortar. The powder was extracted with aqueous ethanol (70%) in a Soxhlet Extractor, concentrated using rotaryevaporator at reduced pressure, suspended in ethanol and partitioned with ethyl acetate(ETAC) to obtain the ethyl acetate fraction. The fraction obtained was further concentrated in-vacuo and the residue obtained. The extract yielded about 80% of the residue fraction.

2.3 Phytochemical screening of the fraction:
The ethyl acetatefraction was analysed for the presence of
flavonoids, alkaloids, saponins, steroids, glycosides, anthraquinones, resins and reducing sugars using standard procedures for analysis (Evans, 2002 and Harborne, 2007).
2.4 Acute toxicity studies:
Lethal Dose (LD50) determination was conducted using the method of Lorke (1983). In the initial phase, male rats were divided into three groups of 3 rats each, making a total of 9 rats, for the ethyl acetate group.The rats were treated with the ethyl acetate fraction of the extract at doses of 10, 100 and 1000mg/kg subcutaneously. Animals were observed for 24 hours and the number of death(s) or those that showed neurological signs were recorded. In the second phase, the animals were grouped into 4 groups of one rat each and treated with the ethyl acetate fraction at appropriate doses subcutaneously. The rats were observed for 4 h for deaths or neurological signs, and the final LD50 was calculated as the square root of the highest non-lethal dose in which the animal survived multiplied by the lowest lethal dose in which the animal died.

2.5 Drugs and chemicals/reagents:
Cimetidine (LekPharma, Slovenia), Indomethacin (Liomethacin(R))(Cheisi, Egypt),
Thiopental Sodium (Abbott Laboratories,UK), Trichloroacetic Acid Solution 6% w/v (Sigma-Aldrich,USA),Phosphate Buffered Saline (PBS) Solution at pH 7.4 (Life Technologies,USA), Heparin (Pfizer Pharmaceuticals, USA), Potassium Phosphate at pH 7.5 (Sigma-Aldrich,USA), Thiobarbituric Acid 25mmol/L (Sigma-Aldrich,USA), Hydrogen Peroxide (H2O2) 500mM/L (OCI Chemical Corporation, USA), Ethylene DiamineTetraacetic Acid (EDTA) (DOW ChemicalCompany,USA), Trichloroacetic Acid Solution 6% w/v (Sigma-Aldrich,USA).

2.6 Experimental animals:
A total of fifty eightadult male albino wistar rats were used in this study. The animals were obtained from the Animal House, Faculty of Medicine, El-Kasr el-Ain, Cairo University, Egypt. Their weights ranged from 180 – 240g. They were maintainedunder a similar conditions of humidity, temperature and light/dark cycle respectively and each of the animal was kept in a single individual cage, with wide-meshed galvanized wire bottoms to decrease coprophagy as much as possible.The rats were given access to food and water ad libitum for two weeks to acclimatize, prior to the commencement of the experiment. The rats were treated in accordance to the Principles of Laboratory Animal Care, and experimental protocol was approved by the Animal Ethical Committee in accordance with the guide for the care and use of laboratory animals.At the time of the experiment, all treatments were conducted between 9:00 and 10:00 (GMT+1) h to minimize variations in animal response due to circadian rhythm. The animals were divided into the following groups and subgroups for gastric mucosal damage and gastric secretion studies respectively.

2.7 Experimentaldesign:
Group 1: Normal saline. Five rats received normal saline (1ml/kg/rat subcutaneously (S.C)).
Group 2: Indomethacin-treated.Five rats received indomethacin (20 mg/kg S.C)
Group 3: Cimetidine-treated. Ten rats for the study of the

for the study of the effect of cimetidine, 50 and 100 mg/kg S.C, given 30 mins prior to indomethacin administration (5 rats for each dose).
Group 5: Nigella sativa-treated. Fifteen rats for the study of the effect of ethyl acetate fraction, each at three different doses (50, 100 and 200 mg/kg S.C), when given 30 minutes prior to indomethacin administration (5 rats for each dose).

2.8 Collection of blood samples
Blood samples were collected using heparinized capillary tubes from the retro-orbital plexus of each rat, and kept in EDTA bottles. Immediately, the blood was centrifuged at 1,006 x g at 6oC for 10 minutes, the plasma was obtained and preserved at -20oC until use.

2.9 Collection of tissue samples
From each of the already dissected rats, the liver tissues were removed and placed in tissue sample bottles containing normal saline, ready for sample preparation as a homogenate for oxidative activity assay.

2.10 Determination of anti-oxidant enzyme activities.

2.11 Catalase assay: Determination of CAT activity was carried out according to the method described by Sinha (1972), Passatiet al., (1980) and Achi (1984), with Catalase Assay Kit (Bio-diagnosticR), Egypt. CAT. No. MD 25 17.Distilled water 0.9 ml was added to 0.1ml of tissue homogenate and mixed thoroughly. 2.5ml of phosphate buffer was put into a small conical flask; 0.5 ml of tissue homogenate was added; and 2.0ml of H2O2 also added, and a stop watch was started. The reaction mixture thoroughly mixed and the reaction stopped after every 60 seconds for 3 min with dichromate/acetic acid solution. The mixture in the flask was heated in a water bath for 10 minutes at 80oC. Absorbance was read at 570nm. Catalase activity was expressed as unit/mg of protein.

2.12 Superoxide dismutase assay
Determination of SOD activity was carried out according to the method described by Fridovich (1987), with Superoxide DismutaseAssay Kit (Bio-diagnosticR), Egypt. CAT. No. SD 25 21.To 0.1ml of tissue homogenate was added0.9 ml of distilled water to make 1:10 dilution of tissue homogenate. An aliquant mixture of 0.2 ml of the diluted microsome was added to 2.5ml of 0.05M carbonate buffer. The reaction started with the addition of 0.3ml of 0.3 mM adrenaline. The reference mixture contained 2.5ml of 0.05 M carbonate buffer, 0.3ml of 0.3mM adrenaline and 0.2 ml of distilled water. Absorbance was measured at intervals of 30 s up to 150 s at 480nm. SOD activity was expressed as unit/mg protein.

2.13 Determination of Lipid peroxidation (Malondialdehyde):
Lipid peroxidation was evaluated according to the procedure described by Ohkama and Ohishi (1979), as modified by Varshney and Kale (1996), with Lipid Peroxide Assay Kit (Bio-diagnosticR), Egypt. CAT. No. MD 25 29.To 1.0 g of stomach tissue, 10ml of 1/150 phosphate buffer (pH 7.0) was added and homogenized. 0.5ml tissue homogenate, 0.5ml saline and 1.0ml of 10% TCA was added.The mixture was centrifuged at 3000rpm for 20 min.0.25 ml of 0.1M TBA was added to the content and mixed well.The mixture was incubated for 1 h at 95oC and allowed to cool down and the supernatant 2.0 ml was measured using spectrophotometer at 532nm. The level of lipid peroxides was expressed as MDA nmol/mg of protein.

2.14 Statistical analysis
All data were expressed as Mean ± S.E.M (standard error of the mean) using SPSS Version 20. Statistical evaluation was done by analysis of variance (ANOVA) followed by post-hoc analysis by Duncan and Scheffe. Values of p<0.05 were considered significant (Microcal Software Inc., Northampton, USA).
RESULTS

Table 1: The Phytochemical Analysis of Ethyl acetate Fraction of Nigella sativa L. seed extract.

Phytochemical Tests Ethyl acetate
Test for flavonoids +++
Test for alkaloids ++
Test for tannins +
Test for saponins –
Test for steroids Trace
Test for glycosides –
Testforanthraquinones +
Test for reducing sugars +
Test for resins –

+ :Presence of the constituents – – Absence of the constituents

Effect of Ethyl acetateFraction on Markers of Oxidative Stress
As shown in table 2, administration of indomethacin 20mg/kg, the values obtained for catalase, superoxide dismutase and malondialdehyde were 4.76 ± 0.16u/mg, 6.21 ± 0.62u/mg and 4.53 ± 2.19nmol/mg of protein respectively. The catalase significantly increased compared to the normal saline control.The cimetidine 50mg/kg and 100mg/kg alone recorded 1.29±0.05u/mg and 5.98±0.30u/mg of catalase activity with the cimetidine 50mg/kg significantly (p<0.05) decreasing compared to indomethacin control, where as the cimetidine 100mg/kg significantly increased compared to the normal saline control. The superoxide dismutase and malondialdehyde in those groups are insignificant. Cimetidine 50mg/kg when administered with indomethacin 20mg/kg, catalase, superoxide dismutase and malondialdehyde values were 1.59 ± 0.13u/mg, 3.94 ± 0.11u/mg and 4.47 ± 2.52nmol/mg respectively, with the catalase significantly (p<0.05) decreasing, compared to the indomethacin control. In cimetidine 100mg/kg plus indomethacin catalase decreases significantly lower than the indomethacin control with a value of 4.15 ± 0.14u/mg, whereas superoxide dismutase and malondialdehyde were 2.75 ± 0.28u/mg and 3.816 ± 1.45 respectively, and both are insignificant.
In the ethyl acetate fraction 50, 100 and 200mg/kg plus the indomethacin 20mg/kg, catalase, superoxide dismutase and malondialdehyde values were 1.30 ± 0.15u/mg, 4.16 ± 0.51u/mg and 12.58 ± 1.82nmol/mg respectively for ethyl acetate 50mg/ug, while 1.83 ± 0.08u/mg, 3.56 ± 0.16u/mg and 14.30 ± 1.40nmol/mg were obtained for the ethyl acetate 100mg/kg respectively. For ethyl acetate 200mg/kg, 5.13 ± 0.47u/mg for catalase, 2.72 ± 0.28u/mg for superoxide dismutase and 4.50 ± 1.16nmol/mg for
malondialdehyde. The catalase level activity for ethyl acetate 200mg/kg was significantly higher than the control, ethyl acetate 50mg/kg and 100mg/kg groups.

DISCUSSION AND CONCLUSION
Recent clinical researches shed a light on the NSAID-induced mucosal injuries and the evident roles played by reactive oxygen species (ROS) via the free radical scavenging pro and antioxidant property of several plant species used in folkloric medicine.
Involvement of ROS in pathogenesis of gastric ulceration was first evident from the studies on ischaemiare-oxygenation-induced gastric mucosal injury (Yoshikawa et al., 1989; Yuda, 1993; Perry et al., 1996). The results of these experiments are in line with those previous reports. In this respect, the ETAC-treated groups, both 50 and 100mg/kg showed decreased catalase activity compared to control, decreased SOD activity that was dose-dependent and increased MDA concentration. Indomethacin-induced gastric ulceration was accompanied with a severe oxidative stress in gastric tissue causing damage to key biomolecules such as lipids, which was apparent from the stimulated lipid peroxidation that led to the accumulation of MDA as well as reduction in the gastric activity of CAT. As shown in the present results, the ETAC fraction of N.sativa significantly reversed the indomethacin-induced changes in SOD, CAT and MDA. This significant reduction in lipid peroxide levels along with significant increase in CAT level suggest increased antioxidant activity and prevention of lipid peroxidation by the ETAC fraction of Nigella sativa. Preventive anti-oxidant enzymes such as SOD and CAT are the first line of defence against reactive oxygen species (ROS) (Yogenderet al., 2007). Flavonoids and other phenolic compounds of plant origin have been reported as scavengers and inhibitors of lipid peroxidation (Formica and Regelson,1995) and are known to have the capacity to sequester endogenously and exogenously produced ROS and free radicals, thereby inhibiting mucosal damage (Repetto and Llesuy, 2002). This inhibition of lipid peroxidation and the generation of MDA and related substances from lipids that react with thiobarbituric acid was found to be inhibited by this extract. The inhibition was found to increase when the concentration of the extract was increased. Thus, it appears that the anti-oxidant property of this extract could counteract oxidative damage caused by NSAIDs. The cimetidine treated groups were insignificant.
It has been shown that compounds isolated from Nigella sativa have appreciable free scavenging properties (Buris and Bucar, 2000). These might be attributed to various mechanisms among which are prevention of chain initiation, binding of transition metal ion catalysts, decomposition of peroxide, prevention of continued hydrogen abstraction, reductive capacity and radical scavenging ability (Diplock, 1997; O’Ktayet al., 2003). The ethyl acetate fraction exhibited free-radical scavenger activity as well as primary antioxidant that react with free radicals, which might possibly limit free radical damage, occurring in the body. ETAC fraction of Artemesiaapiacea was found to possess strong antioxidant activity and the major metabolite isolated was flavonoid cacticin (Lee et al., 2002). Similarly, the extracts from Culcitumreflexumhave been reported to possess photo-protective and anti-oxidant activities and their major compounds were flavonoids (Aquino et al., 2002). In line with the present findings, many researchers have indeed demonstrated the in vivo and in vitro anti-oxidant and free radical scavenging activities of flavonoids (Cao et al., 1997; Rice-Evans et al., 1997; Brown et al., 1998; Plumb et al., 1999; Pietta, 2000). Matsuda et al. (2003) reported that flavonoids are the major secondary metabolites class with several descriptions of antiulcer, antioxidant and other gastroprotective properties. Essential oils which are mixtures of wide variety of volatile terpene hydrocarbons (aliphatic and cyclic) were reported in N.sativa(Magiatiset al.,2002). Several terpenoid-containing extracts from plants commonly used in non-orthodox medicine to treat different gastric illnesses have been reported (Arrietaet al., 2003; Schmerda-Hirschmannet al.,2005; Morikawaet al.,2006). According to Esteveset al. (2005), the essential oil from Cascariasylvestris, which contains sesquiterpinebicy-clogermacrene as the major component, displays mucosal cytoprotectiveproperty. Baccharisdra-cunculifolia essential oil also showed a similar activity (Klopellet al., 2007; Lemosetal., 2007).

CONCLUSION
These studies suggested that the ETAC fraction of Nigella sativa protects against free radicals and possessed appreciable free radical scavenging property by promoting oxidant/antioxidant balance,hence it might play a role in combating gastric ailments like peptic ulcers.




Assessment of Glucose and Lipid Profile of Women on Contraceptives in Maiduguri North Eastern Nigeria

R.M. Gali, F. Dalhatu, Y.P. Mamza, F. Zakari
Department of Medical Laboratory Science, College of Medical Sciences, University of Maiduguri, Borno State Nigeria.
S. Kwari
Department of Obstetrics and Gynaecology, College of Medical Sciences, University of Maiduguri, Borno State.
R.Y. Genesis
Department of Chemical Pathology, University of Maiduguri Teaching Hospital Borno State Nigeria.

All correspondents to: Dr (Mrs) R M Gali, Department of Medical Laboratory Science, College of Medical Sciences,
University of Maiduguri. E-Mail rmgali@yahoo.com

ABSTRACT
Objectives: This study assessed the effect of contraceptives on glucose and lipid profile of women on contraceptives (oral and injectable) in family planning unit of State Specialist Hospital, Maiduguri, Borno State Nigeria.
Methods: Ninety women made up of Fifty(50) women) on oral and injectable contraceptives and forty (40) mothers who are not on any contraceptive had their Glucose determined by glucose oxidase method, total cholesterol, triglyceride and high density lipoprotein determined by enzymatic reaction while Freidwald’s formular was used for low density lipoprotein estimation.
Results: Fasting glucose level was significantly higher in contraceptive women than in non-contraceptive women (5.54±3.35mmol/L and 4.15±0.70mmol/L respectively; p=0.018). Total cholesterol was significantly higher in contraceptive women than in non-contraceptive women (5.33±1.31mmol/l and 4.60±1.12mmol/l; p=0.007). In addition, low-density lipoprotein were significantly higher in contraceptive women than in non-contraceptive women (3.43±1.22mmol/l and 2.65±1.14mmol/l; p=0.007). Whereas triglyceride and high-density lipoprotein in both group were statistically not significant (p>0.05). The comparison of oral and injectable contraceptives showed no significant differences in all the parameters studied except high density lipoprotein which is higher in oral contraceptive women than in women using injectable contraceptives (1.70±0.73mmol/l and 1.37±0.37mmol/l; p=0.043). There was no significant differences in Glucose concentration of women on oral contraceptives and those on injectable contraceptives (P>0.05),
Conclusion: In conclusion, glucose, total cholesterol and low-density lipoprotein are elevated in this study. Therefore, regular tests for lipid profile and glucose is necessary for women on contraceptives to avoid risk of developing diabetes mellitus and cardiac vascular disorders.

KEYWORDS: Glucose, lipid profile, contraceptives.

INTRODUCTION

Birth control in developing countries has decreased the number of maternal deaths by 40% and could be prevented by 70% if fully demands for birth controls were met1,2. Birth control methods include barrier methods, hormonal birth controls, sterilization, and behavioral methods. Hormonal contraceptives are referred to as birth control methods that act on the endocrine gland. Hormonal contraceptives work by inhibiting ovulation and fertilization3. Hormonal contraceptives are available in a number of different forms. The oral and injectable methods are by far the most popular methods. Altogether, 18% of the world`s contraceptive users rely on hormonal method4. The use of injectable hormonal contraceptives has risen rapidly
in many countries5. The estimated number of injectable users doubled from 1995 to 2005; by 2015, about 40 million women are expected to use this method5. Some studies indicated that intake of oral contraceptives could cause metabolic changes in carbohydrate, protein, and lipid macromolecules6,7. Progestin exerts an adverse influence on lipid metabolism, they have ability to counteract the estrogen-induced changes in low density lipoprotein and high density lipoprotein levels8. Women are prone to develop dyslipidemia due to long-term use of hormonal contraceptives9. Hormonal contraceptives induce the body to produce more of the stress hormone cortisol (glucocorticoid), which has effects on lipid and glucose metabolism. Therefore, this study aims at assessing the effect of contraceptives on glucose and lipid in women in this environment.

MATERIALS AND METHODS
This study was carried out in State specialist hospital (family planning unit) Maiduguri Borno State Nigeria. A total of 90 women were recruited for this study. Fifty women who were randomly selected at the family planning unit and forty women who were not on any contraceptives were regarded as control group. The subjects were apparently healthy and were within the reproductive age. Eleven (11) of them were on combined oral contraceptives, whereas 39 were on injectable contraceptive (Medroxyprogesterone). Ethical clearance was obtained from the research and ethical committee of State Specialist Hospital Maiduguri. Oral consent of the mothers was sought after explaining the purpose of the research. Questionnaire was administered to every woman that was eligible for the research.
A fasting blood sample of 5ml volume was withdrawn from each patient; 2ml of blood was collected into fluoride oxalate vacutainer bottle and 3ml into plain container. The plasma was used for the estimation of fasting glucose using oxidase-peroxidase enzymatic colorimetric method as described by Trinder10 and the serum was used for the estimation of triglyceride (TG) by enzymatic method as described by Fossati11, total cholesterol by enzymatic method as described by Meiattini12 and HDL by enzymatic method as described by Warnick13. All the test kits are manufactured by Randox laboratories limited, Ardmore, Diamond road, Crumin.co. Antrim UK. Low-density
lipoprotein (LDL) was calculated using Freidwald’s formular as described by Freidwald14. Statistical analysis of data was done with SPSS version 18 to determine mean, standard deviation, Anova and student t- test. The level of confidence was set at p<0.05.

RESULTS
There were significant differences between means of fasting plasma glucose, total cholesterol, and low-density lipoprotein of contraceptive women with that of the control group. Fasting plasma glucose level was significantly higher in contraceptive women than non-contraceptive women (5.54±3.35mmol.l and 4.15±0.70mmol/l; p=0.018).There is significant high level of total cholesterol in women on contraceptives than non contraceptive (5.33±1.31mmol/l and 4.60±1.12mmol/l; p=0.007). Also low-density lipoprotein is significantly higher in women on contraceptive than the women that are not on contraceptive (3.43±1.22mmol/l and 2.65±1.14mmol/l; p=0.007). On the other hand, there was no significant differences in the levels of triglyceride and high-density lipoprotein in both women on contraceptive and the control group. The comparison between women on oral contraceptive and those on injectable contraceptives showed no significant difference in all the parameters study except high-density lipoprotein, which is significantly higher in women on oral contraceptives than those on injectable contraceptives. No significant change was seen in all the parameters on duration of the use of contraceptives.

were slightly higher in oral contraceptive users than in injectable contraceptive women but are statistically non significant. However, high-density lipoprotein cholesterol was significantly increased in oral contraceptive women as compared to those injectable contraceptive women (Table 2), and this may be due to the effect of estrogen in combined oral contraceptive pills. This study is in accord with the findings of Sitruk-ware19 and Hogan et al20 whose studies revealed high levels of high-density lipoprotein cholesterol in oral contraceptive women.
This study grouped the subjects for the duration of the use of contraceptives into two; less than five years using contraceptive (45 subjects) and five to ten years of using contraceptives (5 subjects). Our findings revealed no significant changes in all the parameters used on duration of contraceptives use (Table 3), this study contradicts the findings of Berenson et al18 who stated that over 3 years of using injectable contraceptives, there was increased in levels of glucose and insulin. In addition, our results contradict with other studies who stated that the levels of triglyceride, LDL-cholesterol and VLDL-cholesterol increased with duration of oral contraceptives intake 16-18. Although this study revealed increased in low-density cholesterol due to duration, the increase is not statistically significant. On the other hand, there is decreased in high density cholesterol in 5-10 years of contraceptives use (Table 3) but not statistically significant. This study was not able to prove the claim by Santos et al9 who state that Women are prone to develop dyslipidemia due to long-term use of hormonal contraceptives.
In conclusion, contraceptives are good for birth control that help families to plan their lives, this should be used with caution, and regular check up is necessary to avoid risk of diseases such as diabetes mellitus and cardiovascular diseases that may be detrimental to human health.

ACKNOWLEGDMENTS
We wish to thank all staff of Family planning unit of State Specialist Hospital Borno State and Department of Chemical Pathology University of Maiduguri Teaching Hospital Borno State Nigeria for their support and
DISCUSSION
The major health risks of oral and injectable contraceptives are glucose impairment and cardiovascular diseases particularly coronary artery disease, stroke and venous thrombolism. Hypercholesterolemia is associated with endothelial cell dysfunction, elevated oxidant stress, and creation of a strongly pro-inflammatory conditions15.
In this study, the total cholesterol and low-density lipoprotein cholesterol levels in women on contraceptive are higher than non-contraceptive women and are statistically significant (Table1). This study agrees with the findings of Yesmin and Asare16,17 who reported increase total cholesterol, and low density lipoprotein. The increase in serum total cholesterol in women taking contraceptives may be due to impaired lipoprotein metabolism and higher levels of low-density lipoprotein may be due to increase lipoprotein synthesis rather than impaired lipolytic catabolism in association with accumulation of cholesterol16. In this study, there is no significant difference in triglyceride level in women on contraceptive and non contraceptive women (Table1), which is in line with findings of Asare17 and contradicts the findings of Yesmin16, who stated that there is elevated triglyceride in women on contraceptive than non contraceptive women. He attributed that increased serum triglyceride might be due to increase production and transportation of very low-density lipoproteins that endogenously synthesize triglyceride in the blood; although our finding did not indicate that.
There was no difference in high-density lipoprotein, which is in accordance with findings of Asare17. In this study, fasting plasma glucose was significantly high in contraceptive women than non contraceptive women, this finding is similar to those of Berenson et al18 who reported high glucose level in women on contraceptives. They attributed this high glucose level to compensation for increased insulin resistance and glucocorticoid-like activity of progestogen.
Glucose level in oral contraceptive women was slightly higher than in injectable contraceptive women but is statistically non-significant. Also the total cholesterol, triglyceride, and Low Density Lipoprotein cholesterol




Human Brucellosis: Seroprevalence amongst Patients attending the General Out-patient Department (GOPD), Federal Teaching Hospital Gombe, Nigeria

Kudi A.A., Ahmed R.A., Baba-Ali F.

Bio-global Diagnostic & Research Laboratories, Opp. State Specialist Hospital Main Gate, Jekadafari Road, Gombe
Uba A., Tahir F., Yusuf I.Z.

Department of Medical Microbiology & Immunology Federal Teaching Hospital Gombe, Gombe State
Bubalu J.L

Faculty of Science, Biological Science Department Abubakar Tafawa Balewa University, Bauchi

All correspondence to: Kudi A.A, kclifeglobal@yahoo.co.uk

ABSTRACT
Background
Brucellosis is a disease of domestic, livestock and wild animals with serious zoonotic implications in man. Its spread is worldwide and transmission to humans is by contact with fluids from infected animals or derived food products such as unpasteurized milk and cheese. The clinical picture of the disease in man is so strange and protean; easily bewildered with other infectious and noninfectious diseases, leading to diagnostic delays and late onset of therapy. The intention of this study was to determine and document the prevalence of Brucellosis in patients presented with acute febrile illnesses resembling malaria and/or typhoid infections using the serological technique.
Methods: A retrospective study of a two-year laboratory records of cases of brucellosis among patients with febrile illnesses resembling malaria and/or typhoid fever seen at the General out-patient department (GOPD), Federal Teaching Hospital, Gombe between 2012 and 2014. Blood samples routinely collected from 246 patients were allowed to clot and the sera extracted were tested for Brucella antibodies using Rose Bengal plate test (RBPT). Standard test protocol described by Alton et al., (1975) and Morgan et al., (1978) was followed. Results obtained were statistically analysed using tables and chi-square test at 95% confidence level.
Results: Out of the 246 samples analysed, 56.6% were from the males, while 43.4% from females between the ages 0-60yrs. Our record shows 32.5% positivity for brucellosis, while the remaining 67.5% were negative. Among the males tested, 14.5% were found positive for the disease, while among the females 18.0% was recorded, (p>0.05). Majority (13.3%) that tested positive for brucellosis were between the ages 31- 40 years. The least (3.6%) affected were seen in ages 21-30 and 51-60 years.
Conclusion: Brucellosis among the population tested was considered to be high. Symptoms clinically thought to be malaria and/or typhoid fever were seen to be cases of brucellosis. There was no significant association between sex and age in the rate of infection (p>0.05). Routine laboratory test request for Brucella antibody among patients with febrile illness in our Community is recommended. Co-ordinated public enlightment on zoonosis, with special emphasis to brucellosis in Gombe State was encouraged.

Key words: Brucellosis, Zoonosis, Febrile illnesses, Pyrexia of unknown origin, Gombe

INTRODUCTION

Brucellosis is an infectious disease caused by bacteria of the genus Brucella (Colmenero et al., 1990; Memish et al., 2000; Al Dahouk et al., 2003; Kaltungo et al., 2014). The disease is caused by several species of Gram-negative facultative intracellular bacteria of the genus (Sofian et al., 2008). It is a neglected debilitating zoonosis and is recognized as an occupational hazard with a high prevalence in developing countries
(Mabel et al., 2013). Primarily, it is a disease of domestic, livestock and wild animals with serious zoonotic implications in man; causing huge economic losses to the livestock industry. Cattle, goats, pigs, sheep, horses and dogs play an important role in the transmission of this disease to man. It is defined as a contagious systemic bacterial disease of ruminants, characterized by inflammation of the genital organs and fetal membranes, abortion, sterility and formation of localized lesions in the lymphatic system and joints (WHO, 1971, CDC, 2005).

Brucellosis remains a main source of disease in humans and animal husbandry worldwide (Corbel, 1997).

The clinical picture of brucellosis in man is so strange and protean that it can be easily bewildered with other infectious and noninfectious diseases, leading to diagnostic delays and late onset of therapy (Al Dahouk and Nockler, 2011). Malta, Rock, Gilbraltar, Crimean, Cyprus or Mediterranean fever, Bang’s disease, intermittent typhoid or Typho-malarial fever, undulant fever, etc, are just various synonyms for brucellosis (Al- Dahouk et al, 2003). Those suffering from the disease show unspecific symptoms, e.g. fever, chills, malaise, arthralgia, headache, tiredness and weakness. Therapeutic failure and relapses, chronic courses and severe complications like bone and joint involvement, neurobrucellosis and endocarditis are characteristic for the disease (Al- Dahouk et al, 2003).
About half a million human brucellosis cases are reported annually. However, according to the WHO (1997) estimates, the true frequency of the disease is 10-25-times higher than the reported number. The highest annual incidence rates are reported from the Middle Eastern countries, such as Syria, Iraq, Iran, and Saudi Arabia (Pappas et al., 2006). In Iran, where Brucellosis is endemic, the incidence of the disease is up to 34 per 100,000 per year in certain areas (Najafi et al., 2011). In Nigeria, it was reported that human brucellosis is hardly diagnosed in hospitals despite suggestions that the magnitude of the infection may be greater than appreciated (Njoku 1995; Rajis et. al, 2003).
Detailed studies confirming the problem of brucellosis in Nigeria’s livestock have been documented by several authors (Esuruoso, 1974; Falade, 1974; Falade, et al., 1974; Falade et al., 1975; Okon, 1980, Chukwu, 1987; Brisibe et al., 1993; Ajogi, 1997; Ajogi et al., 1998; Ogundipe et al., 1994; Ishola et al., 2001); with evidence of the spread of the disease in all parts of the country which is usually accompanied by severe economic losses. Serological prevalence rate of between 0.20% and 79.70% have been reported in various parts of the country to date. The infection has been reported in various animal species in Nigeria (Esuruoso and Hill, 1971; Esuruoso, 1974; Falade, 1974; Falade and Shonekan 1981; Falade et al., 1975; Okoh et al., 1978; Adamu and Ajogi, 1995). These demonstrate how brucellosis has been identified as an endemic and problematic disease in Nigeria. However, the infection is not static; it is evident from previous studies that prevalence varies at different times and locations. This is especially apparent where there is no control policy, like Nigeria. There is a pattern of low and high prevalence in specific areas of the country and prevalence variability also arises between herds in the same area (Nuru and Dennis, 1975). Although prevalence in brucellosis has been shown to be low in most dairy and private farms, it is actually on the increase among nomadic and semi-nomadic herds which contribute about 95% of all annual food population in Nigeria (Rikin, 1988).

Making a diagnosis of brucellosis may be difficult because of the unspecific symptoms and signs shared with other febrile illnesses, slow growth rate of the causative agent in blood culture, and the complexity of sero-diagnosis (Colmenero et al., 1990; Memish et al., 2000; Al Dahouk et al., 2003). Presumptive diagnosis of brucellosis can be
made by the use of several serological tests to brucella antibodies, but the “gold standard” remains isolation and identification of the bacterium. Evidence of the infection either through serological or cultural examinations has been demonstrated in domestic livestock and humans in Nigeria (Ocholi et al., 1993). Most of the disease reports originated from Government herds where screening tests were easily carried out, while some originated from settled Fulani herds and private farms (Ocholi et al., 1993). The general situation is that the disease is more prevalent in Government-owned farms than in the nomadic herds (Esuruoso, 1974). Epizootiological investigations also revealed that results obtained varied depending on the region, area or animal group sampled (Ocholi et al., 1993). The distribution of the disease among humans is not well known but serological evidence has shown that the disease exists worldwide. Evidence of the presence of the disease in humans in Nigeria has also been published (Collard, 1962, Alausa, 1977, Alausa and Osoba, 1977, Falade, 1974 and Falade, 2002). It is a zoonosis and the disease in man is highly debilitating, though not considered to be fatal (Falade, 1974). Collard (1962) documented the first case of human brucellosis in Nigeria where Brucella antibodies were demonstrated in the sera of healthy persons in various parts of the country.
The intention of this study was to determine and document the prevalence of Brucellosis in patients presented with signs and symptoms of being ill (acute febrile illness) resembling malaria and/or typhoid infections in our hospital using the serological technique retrospectively. We suggest that similarities in signs and symptoms of diseases have often led to under diagnosis or misdiagnosis of brucellosis in humans. Most often, as the case may be, patient illness is dismissed as malaria, typhoid or pyrexia of an unknown origin (PUO); in that case, the disease go undiagnosed and untreated, leading to considerable suffering for those affected (John et al, 2002). We view brucellosis as one of the neglected zoonosis and most under reported or misdiagnosed disease in most hospitals in our locality. We found no known documented report of brucellosis among humans in Gombe and its environs; the few found reports were among domestic animals but not humans. Access to current documented cases of brucellosis in humans in our environment may be of paramount assistance in patients’ management thereby, contributing to quality healthcare delivery; the gap we made attempt to close.

MATERIALS AND METHODS
Study Area
Gombe, the capital city of Gombe State is located in the Guinea Savannah of North-Eastern part of Nigeria in West Africa. The State is bordered by Borno State to the East, Yobe State to the North-East, Taraba State to the South, Adamawa State to the South-East and Bauchi State to the West. It occupies a total land area of 20,265 square kilometres and has a population of about 2,353,879. The State is made up of 11 Local Government Areas (Jibrin, 2003, GomSACA, 2008). The principal ethnic groups of State are the Tera, Tangale, Fulani, Waja and Bolewa. The people are predominantly farmers and as is the culture, they keep and rear domestic animals and poultry for economic purposes.

Ethical Consideration and Confidentiality
Approval for this work was obtained from the Federal Teaching Hospital (FTH), Gombe Research and Ethics Committee. Patients’ consent was obtained at the time of sample collection to run the test. Throughout the test analysis, confidentiality of health information of the patient was maintained.

Sample Collection
Whole blood samples were collected from 246 patients with febrile illnesses resembling malaria and/or typhoid seen at the General out-patient department (GOPD), Federal Teaching Hospital, Gombe from 2012 to 2014. The samples were routinely collected in the Medical Microbiology & Immunology Laboratory of the same hospital by the attending Phlebotomist. From each patient, 4mls of whole blood was collected in a sterile screw capped non-anticoagulant 5ml-capacity disposable (plastic) bottles using sterile disposable syringe with a 21G needle. The blood was allowed to clot and then centrifuged at 1500 rpm to separate the serum from the red blood cells. Using a sterile disposable Pasteur pipette, the serum extracted was transferred into a sterile screw capped vial, labeled with a laboratory number that correspond with the subject identification. The labeled serum sample was ready for analysis (Alton et al., 1975). Where there was delay in the analysis, the sample was stored at -20oC until required for testing. Demographic characteristics of each patient were noted at the time of sample collection.

Serological Testing
Each sample was screened for Brucella antibodies using Rose Bengal Plate Test (RBPT). The test was carried out using the standard protocol described by Alton et al., (1975) and Morgan et al., (1978). Procedure: Serum samples and antigen were brought to room temperature (22 ± 4°C). Approximately 25µl of each serum was placed on a white tile and an equal volume of antigen was placed near each serum spot. Both were mixed thoroughly using a clean wooden rod and read for agglutination immediately after a 4-minute period. The agglutination reactions were recorded as positive (+) or negative (-) depending on whether there was agglutinations or not. The reagents in the kit were reconstituted and the test procedure was carried out according to manufacturers’ instructions. Individuals were considered as positive based on a positive RBPT result. The necessary quality control (QC) was carried out on the reagent used using known positive and negative controls. All safety precautions were followed according to the manufacturer’s instructions on the kit insert.

Statistical Analysis
Data collected were analysed using tables and chi-square tests. Results were considered as significant if the chi-square p-value was < 0.05 otherwise, non-significant if the p-value was > 0.05.

DISCUSSION
Similarities in signs and symptoms of diseases have often led to under diagnosis or misdiagnosis of brucellosis in humans. It was postulated that making a diagnosis of brucellosis is difficult because of the unspecific symptoms and signs shared with other febrile illnesses (Colmenero et al., 1990; Memish et al., 2000; Al Dahouk et al., 2003). The clinical picture of brucellosis is so strange and protean that it can be easily bewildered with other infectious and noninfectious diseases (Al Dahouk and Nockler, 2011). As the case may be, patient illness is dismissed as malaria, typhoid or pyrexia of unknown origin (PUO). This leads to diagnostic delays and late onset of therapy; therefore, laboratory investigation is always needed to confirm etiologic agent (Al Dahouk and Nockler, 2011).

Our findings in this study show that more males (56.6%) presented with febrile illness that resembles malaria and/or typhoid fever at our Hospital’s out-patient department (GOPD) than females (43.4%). However, there was no significant difference between the sexes in the clinical presentation (p>0.05). As high as 32.5% prevalence rate of brucellosis in this study was recorded among the patients tested. This finding seems to agree with other workers who reported – though in domestic animals, serological prevalence rate of brucellosis of between 0.20% and 79.70% in various parts of Nigeria (Esuruoso and Hill, 1971; Esuruoso, 1974; Falade, 1974; Falade and Shonekan 1981; Falade et al., 1975; Okoh et al., 1978; Adamu and Ajogi, 1995). Other researchers recorded in humans close to the range of 28 to 57% among high risk group such as abattoir/butchers workers (Falde 1974; Ocholi 1993; Edu, 2005). In contrast however to our findings, Maryceline et al., (2001) recorded as low as 5.2% sero-positivity among
patients with PUO in Maiduguri, northeastern Nigeria. Perhaps geographical location and other demographic factors play some roles to this variation. The distribution of the disease among humans is not well known but serological evidence has shown that the disease exists worldwide and it is a zoonosis (Falade, 1974). Our findings therefore, has further agrees with other published reports that the presence of the disease in humans in Nigeria exist (Collard, 1962; Alausa, 1977; Alausa and Osoba, 1977; Falade, 1974; Falade, 2002). The first case in Nigeria was documented by Collard (1962) among apparently healthy persons in various parts of the country.
Other workers further reported cases among abattoir workers, nomads and those handling meat and other animal products. Mabel et al. (2013) recorded 32.7% seroprevalence among butchers in Abuja Nigeria; this is almost comparable to our findings in this study. Asanda and Agbede (2001) suggested that infection seems more associated with humans engaged in livestock and livestock product activities than those engaged in other productive ventures. Occupational status of the subject in this study was not determined; we therefore acknowledged this as one of the limitations, however, we could assume that contact with infected animals or animal products, such as meat, milk and other animal products among the population was inevitable, hence the high prevalence recorded. It was reported that the infection is not static; the prevalence varies at different times and locations (Rikin, 1988).
We noticed in this study that the females have the highest prevalence of brucellosis with 18.0% as against the males (14.5%). However, there was no significant difference in the rate of infection among the sexes (p>0.05). Our findings here tally with the report of Maryceline et al., (2001) where sex has no significance role in the distribution of brucellosis. This is in contrast with the findings recorded by Marbel (2013) among the abattoir workers and butchers in Abuja Nigeria, where males were significantly more infected with Brucella. This should be expected since males (in terms of occupation) are more involved in the handling of animals and animal products such as raw meat, unpasteurized milk and cheese. Such activities have been documented as significantly associated with Brucella infectivity (Hannah et. al., 2011); butchering in particular was noted to be a male-dominated activity thereby, the males stand at high risk of being infected with Brucella.
Our findings indicated that the highest seropositivity (13.3%) was in the age group 31-40 years, while the least was found in the ages between 21-30 and 51-60 years. However, in contrast to our findings, other workers reported individuals who tested positive to the brucellosis test were within the ages 18-25 years, while the least affected were those above 41 years of age (Marbel, 2013). The differences in the rate of infection among the ages in this study was found to be statistically significant (p<0.05). Reasons for the difference in the rate of infection was not immediately known to us, but it could be speculated that the ages 31-40 years is the most productive and active years thereby, frequency in contact with infected animals/products was high, as such, chances of getting infected therefore, most likely was significantly increased. Maryceline et al., (2001) however reported that age does not significantly affect the distribution of brucellosis.he limitation to the current study as observed and stated earlier was lack of determining the level of association of the subject with animals/animal product and brucellosis to confirmed zoonosis. However, infection with Brucella has always been reported to be the primary risk factor associated with keeping sheep, goats and other small animals in the household (Hannah et. al., 2011). It was generally observed that keeping and rearing goats, sheep, pigs and Cattles in our community is the practice for economic reasons; we may therefore, speculate that the high seropositivity (32.5%) recorded in our study was somewhat expected. The study was also limited in determining the level of knowledge of brucellosis in the community. Howbeit, with the high prevalence rate of infection recorded in this study, it is most likely that the level of awareness among the subjects, especially the zoonotic role in transmission, control and preventive measures was very low. In such situation therefore, and with the fact that infection is often subclinical, with high morbidity for both humans and animals (Colmenero et al. 1996), we again speculate that the disease is wide spread and diagnosis is either missed, under diagnosed or unreported in most of our healthcare settings.
Conclusion
The study reveals that among the population tested for brucellosis, there was no significant association between sex and age in the rate of infection. To the contrary however, other workers have reported significant difference in the rate of infection among the male abattoir workers/butchers (Falde 1974; Ocholi 1993; Edu, 2005). Among all that presented with febrile illnesses in our Centre, 32.5% would have been thought, dismissed or treated as having malaria, typhoid fever or PUO, but for the serological test which indicated brucellosis. This shows the significance of Brucella antibody testing on all patients presented with acute febrile illnesses in our healthcare facilities. We therefore advocate regular or routine laboratory test requests for diagnosis of brucellosis as is the case with other endemic infectious diseases, such as malaria and typhoid fever in our Community. The practice may enhance adequate and prompt diagnosis of acute febrile illnesses. Co-ordinated public enlightment and education programme generally on zoonosis, with special emphasis to brucellosis’ prevention and control should be instituted by the authorities concern in Gombe State and North eastern Nigeria as a whole.

 




Evaluation of Serum Proteins in Pregnancy

Jemikalajah J.D.
Department Of Medical Microbiology And Parasitology, Faculty of Clinical Science, Delta State University, Abraka.
Adu M.E.
Department Of Medical Laboratory Services, Antiretroviral Therapy Centre, Central Hospital, Agbor.

All Corresponding to: adumatthew10@yahoo.com

ABSTRACT

Pregnancy induced changes in the maternal system in order to accommodate the foetus. One of such changes is the anabolism and catabolism of plasma proteins. The effects of pregnancy on serum proteins were investigated in eighty (80) pregnant women attending antenatal clinic in central hospital, Auchi and forty (40) apparently healthy women as controls. Total protein, albumin, and globulin as well as albumin/globulin ratio were examined. There was decreased total protein, albumin and albumin/globulin ratio and increased globulin in pregnant women when compared with non- pregnant women. Also there was significant difference when different trimesters were compared. These changes are attributed to increased plasma volume as well as synthesis of pregnancy induced plasma proteins. We advocate the routine examination of serum protein during pregnancy.

Keywords: Serum proteins, Pregnancy, Globulin, Auchi, Trimester

INTRODUCTION

Pregnancy involves the sequence of events that take place after fertilization of an ovum, thus enabling the fertilized ovum to eventually develop into a full- term foetus (Guyton and Hall, 2000). During pregnancy estrogen and progesterone are produced in large quantities and exert some influence on a pregnant woman. These hormones induce an increase in the metabolism of the woman as she prepares to carry the baby (Bassi et al., 2011). Physiological response in the metabolism of the expectant mother is due to foetal requirement of oxygen and food substance, growth of the uterus and preparation for lactation. The pregnant women experience physiological changes to support fetal growth and development (Blackburn and Loper, 1992, Taylor 1995). The level of estrogens (estradiol) and progesterone increase progressively during pregnancy (Vanthiel and Gavaler, 1987). These sex hormones have effects on hepatic metabolic, synthesis, and excretory functions (Vigil and Gratia, 2004, Bacq, 1999, Marpeau et al., 1999). The phenomenon of hemodilution secondary to the increase in plasma volume decreases the serum protein concentrations. Consequently, certain changes in values of liver function tests occur during normal pregnancy (Everson 1998, Alonso 2006). There are scanty literatures on the serum proteins of pregnant women especially in the locality studied; we therefore sought to determine the serum proteins of pregnant women in Auchi, Edo State, Nigeria.
MATERIALS AND METHODS
Study Area
This study was carried out in Central Hospital, Auchi, Edo State Nigeria. It is a secondary health institution that serves as a referral centre for other primary health institution in this locality.
Sample Collection
A total of 120 samples were collected from 80 pregnant women attending ante-natal clinic and 40 non pregnant women as controls after informed consent. Five milliliters of venous blood was collected aseptically into a plain container and allowed to clot. This was spun at 3000rpm for 10minutes to obtain a clear serum which is kept frozen until required for analysis. Ethical approval was obtained from the ethics committee of Central Hospital, Auchi.
Biochemical Analysis
Total serum protein was determined spectropho-tometrically using Biuret method (Doumas et al., 1981) while serum albumin was determined by bromocresol green method (Doumas et al., 1981). Serum globulin was calculated by subtracting albumin from total protein. The Albumin Globulin ratio was also determined by dividing Albumin with Globulin. All reagents were products of Randox Laboratories UK. In all test, manufacturer instructions were strictly adhered to.
Statistical Analysis
The groups mean ± SD was calculated for each analyte and significant difference between means evaluated

using the student t-test. Statistical Package for Social Science SPSS version 16.0 software (SPSS Inc., Chicago, IL USA) for windows was used, with P<0.05 considered as statistically significant.

RESULTS
The mean serum total protein of pregnant women was observed to be decrease (6.23±0.69) than the control subjects (6.63±0.43). Mean serum albumin level of pregnant women was found to be 3.43±0.61as against controls which is 3.93±0.36.Table 1 also show the globulin level of both pregnant women and controls to be 2.75±0.29 and 2.70±0.24 respectively. There was decreased Albumin: Globulin ratio observed in pregnant women (1.27±0.29) when compared with control subjects (1.46±1.50).
A/G = Albumin: Globulin Ratio
Fig 1 shows the mean serum proteins of different trimesters with first trimester having an increased total protein when compared with both second and third trimester. There is an increased serum albumin: globulin ratio observed in the third trimester when compared
with first and second trimester.

DISCUSSION
Pregnancy is a period between conception and delivery and has been associated with increased dietary protein requirements in humans. During this period of rapid growth, the foetus and placenta accrue proteins very rapidly (Lewis et al., 2010). Pregnancy induces major physiological, hormonal and biochemical changes to achieve an optimal outcome for the baby and its mother. Our study show significant decreased in total protein of pregnant women when compared with controls. This decrease can be attributed to changes in plasma volume as opined by Varley et al., (1980). The fall in protein concentration seen during pregnancy may be due to the dilution of the plasma, since total protein concentration is inversely related to plasma water concentration (Paaby 1960).

The albumin level of pregnant women was found to be significantly decreased when compared with non-pregnant women. This is in accordance with the findings of Coryell et al., (1950) and Horme et at., (1970) who attributed the decrease in albumin to increased plasma volume found in pregnancy. However, Laurell et al., (1968) stated that albumin synthesis which is normally stimulated due to hypoalbuminemia is inhibited during pregnancy by progesterone and estrogen which is abundant in pregnant women.

There was no significant difference observed in the globulin levels of pregnant and non pregnant women. This is in consonance with the findings of Adedeji et al., (2012) who observed that concentration of serum globulin is not affected by pregnancy. The Albumin/Globulin ratio of pregnant women was significantly decreased when compared with non pregnant women. This similar observation was reported by Adedeji et al., (2012). This difference is attributed to the decreased albumin as occasioned by increased plasma volume.
Our results revealed that the mean concentrations of serum proteins exhibited some variations with advancing gestational age. There was significant increased observed in the total protein of first trimester when compared with second and third trimester. Also there was significant increase in the total protein of second trimester when compared with third trimester. This is in tandem with Adedeji et al., (2012) who did similar work on protein profile of pregnancy. Shimokawa and his colleagueset al et al.,(1980) demonstrated that certain serum protein fraction of molecular weight of 185,000 Dalton are elevated in relative concentration during the first trimester and decrease to non- pregnant levels after 25th week of gestation. There was no significant difference observed between the first trimester and second trimester in albumin level when compared but there exist a significant difference when both first and second trimester where compared with third trimester. This is in agreement with previous findings of Adedeji et al., (2012).This difference is attributed to increased plasma volume being experienced in the third trimester as well as nutritional needs of the foetus. The globulin fraction of first trimester and second trimester was observed not to be significantly different but an increased significant difference was observed when third trimester was compared with both first and second trimester. This difference may be due to the synthesis of various pregnancy induced proteins such as ?- fetoprotein, placental isoenzyme of alkaline phosphatase, oxytocinase, human chorionic gonadotropin and the pregnancy associated plasma proteins. The concentrations of these proteins rise during gestation and disappear within few days or weeks of postpartum as opined by Joseph et al., (1978). The albumin: globulin ratio of the third trimester was significantly increased when compared with both first and second trimester whereas there was no significant difference observed between first and second trimester in the albumin: globulin ratio. This may be probably due to imbalance between albumin and globulin concentration which has been earlier attributed to increased plasma volume and synthesis of pregnancy induced plasma proteins.

In conclusion, alteration in function of any sites will affect the appropriate serum protein fractions. Changes in serum protein during normal pregnancy can also be attributed to varying factors like increase in plasma volume and wide individual physiological variations at any given stage of pregnancy. However, a considerable amount of albumin passes through the glomerular filtrate daily during pregnancy and most of this is reabsorbed in the renal tubules, being broken down in the process and are lost to the body. It is therefore imperative to have a protein profile analysis during pregnancy.
Conflict of Interest: None