Suleiman Ibrahim Eleha

Department of Chemical Pathology, University of Ilorin Teaching Hospital, Ilorin, Nigeria.
Oyedeji Samuel Oyewole
School of Medical Laboratory Science, Obafemi Awolowo University Teaching Hospitals Complex, Ile-Ife, Nigeria.
Muhammed Abdurrasheed Ola
Department of Histopathology, Faculty of Medical Laboratory Sciences, Usmanu Danfodiyo University, Sokoto, Nigeria.
Adesina Adeyemi Adeleke
Department of Chemical Pathology, Obafemi Awolowo University Teaching Hospitals Complex, Ile-Ife, Nigeria
Afolabi O.O
Pathology Department, College of Health Sciences, University of Ilorin, Ilorin, Nigeria.

All correspondences to: Ibegbu A.O, aoibegbu@yahoo.com.

ABSTRACT
Pseudocedrela kotschyi is a medicinally important tree, belongs to the family Maliaceae, and have been widely used by traditional healers for treatment of various illnesses, many of which have not been scientifically scrutinized. Phytochemical screening and acute toxicity study (LD50) arethe two necessary preliminary investigations toward establishing therapeutic usefulness of any plant. The current study was carried out toprovide comparative phytochemical detail of methanol bark extract (BEPK) and aqueous leaves extract (LEPK) of P. kotschyi with their LD50 value, using standard procedure.Results showed that both extracts contained variedphytochemical constituents such as tannins, cardiac glycosides, steroids, and reducing sugars. Maximum phytochemicals was observed in aqueous extract. LD50 value was found to be 1,250 and 1,750 mg extract/kg body weightintraperitoneally respectively in rat, thus BEPK was found to be more toxic than LEPK.

Keyword: Pseudocedrela kotschyi, Cardiac glycosides, LD50, Tanins, Steroids)

INTRODUCTION

Pseudocedrela kotschyi (Schweinf) Harms belongs to the Maliaceae family and comprises a single species, a deciduous, monoecious, medium – sized tree up to 12–20 m tall (Ahua et al., 2007: Oliver-Bever, 1986: Salvina, 1989). P. kotschyi has numerous uses in traditional medicine, particularly its bark, roots and leaves, thus it is an important source of ingredient for local medicine (Kassim et al., 2009). The decoction is used as a wash for ulcers and in Northern Nigeria; the plant also serves as an occasional ingredient for use in arrow poison (Oliver-Bever, 1986). Roots of P. kotschyi are commonly used as chewing sticks in West Africa and it extract have been shown to inhibit the in-vitrogrowth and development of the schizont stage of Plasmodium falciparium, thus the root may provide affordable means of treating malaria (Kassim et al., 2009).Derived natural products such as flavonoids, terpenoids and steroids etc. have received considerable attention in recent years due to their diverse pharmacological properties including antioxidant and hepatoprotective activity (DeFeudis et al., 2003). The quantity and quality of phytochemicals present in plant
parts may differ from one part to another. In fact, there is lack of information on the distribution of the biological activity in different plant parts essentially related to the difference in distribution of active compounds (or active principles) which are more frequent in some plant parts than in others (Solomon et al., 2013). Successful determination of biologically active compounds from plant material is largely dependent on the type of solvent used in the extraction procedure (Solomon et al., 2013).

Acute toxicity refers to those adverse effects occurring following oral or dermal administration of a single dose of a substance, or multiple doses given within 24 hours, or an inhalation exposure of 4 hours (Muhammad et al., 2000). LD50 value (median lethal dose) is one way to measure the short term poisoning potential (acute toxicity) of chemical and in general, the smaller the LD50 value, the more toxic the chemical and the larger the LD50 value, the lower the toxicity (Gadanya, 2011). It is important to know that the actual LD50 value may be different for a given chemical depending on the route of exposure (Oral, dermal, inhalation) (Senin, 2006). It is usually expressed as the amount of chemical administered (e.g. milligrams) per 100grams (for smaller animals) or per kilogram (for bigger subjects) of the body weight of the test animal (Gadanya et al., 2011).

MATERIALS AND METHODS
Plant Materials
Different plant parts; leaves and bark of P. kotschyi were collected from Kisi, Oyo State, South West Nigeria (woody Savannah vegetation). The plant was identified by a plant Taxonomist. The leaves and stem bark were separated from the plants and washed in tap water, rinsed with distilled water. Then the plant parts were shade dried in room temperature till there is no loss of weight. They were ground into coarse powder and stored in room temperature for further study.

Extraction
The methanol and aqueous extracts were prepared by soaking 100 g each of the dry powdered plant materials in 1 L of methanol and water at room temperature for 72 h with occasionalshaking for effective extraction. The extracts were filtered after 72 h, first through a Whatmann filter paper No. 42 (125mm) and then through cotton wool. The extracts were concentrated using a rotary evaporator with the water bath set at 40°C. The percentage yield of extracts ranged from 7–19%w/w.

Animals
Twenty seven (27) healthy Wistar strain albino rats weighing between 150-200 g were obtained from the Laboratory Animal Centre of Osun State University, Osogbo, Osun State Nigeria. The rats were housed in clean metallic cages and kept in a well-ventilated room and allowed to acclimatize to the laboratory environmentalcondition for one week, prior to the commencement of the experiment. They were fed with standard animal pellet and water ad libitum.

Drugs and Chemicals
Hydrochloric acid, Acetic acid, Hydrogen Peroxide, Ferric chloride and the rest of the chemicals utilized were of analytical grade and were purchased from ROVERT Scientific Limited, Edo, Nigeria.

Phytochemical Study
Various phytochemical constituents were tested for,using different chemical tests; Alkaloids (Wagner’s reagent test), Cardiac glycosides (Keller-Killani test), Flavonoids (Alkaline reagent test), Sterols (Liebermann-Burchad’s reaction), Tannins (Ferric chloride test/ Braymer’s test), Phenol (Ferric chloride test), Quinones (HCl test), Saponins (Foam test) and Terpenoids (Salkoki’s test) as described by loan (1994).

Acute Toxicity Study
The method of Fixed Dose Procedure (FDP) (OECD, 1981) was used to establish LD50 value.
This method does not use death as an end point; instead it uses the observation of clear signs of
toxicity developed at one of a series of fixed dose levels to estimate the LD50 (Stallard and Whitehead, 1995).Animals
were fasted over-night prior to dosing (they were allowed access to water).
Following the period of fasting, they were weighed and the test substance administeredintraperitoneally as shown below.

In the 1st phase:There were 2 groups of 3 rats per group. Group 1 was treated with the BEPK extract at doses of300mg/kgand Group 2 was treated with1000 mg/kgof LEPK .

In the 2nd phase: There were 2 groups of 3 rats per group. Group 1 was treated with the BEPK extract at doses of750 mg/kg and Group 2 was treated with1,500mg/kg of LEPK
In the 3rd phase: There were 2 groups of 3 rats per group. Group 1 was treated with the BEPK extract at doses of1000 mg/kg and Group 2 was treated with 1,750mg/kg of LEPK
In the 4th phase: a group of 3 ratswere further treated with BEPK at dose of 1,250 mg/kg.

After the substance has been administered, food was withheld for a further 4 hour. They were closelyobserved in the first 4 hours and then hourly for the next 12 hours followed by hourly intervals for the next 56 hours (overall 72hrs) after the administration to observe any death or changes in general behaviours and other physiological activities (OECD, 2001).

DISCUSSION
The medicinal value of plants lies in some chemical substances that produce a definite physiological action on the human body (Montilla et al., 2005). The most important of these bioactive constituents of plants are alkaloids, tannins, flavonoids, and phenolic compounds (Mir et al., 2013).
Phytochemical are naturally synthesized in all parts of the plant body; bark, leaves, stem, root, flower, fruits, seeds, etc. i.e. any part of the plant body may contain active components.Results of comparative study obtained for qualitative phytochemicals screening of BEPK and LEPKare presented in table 1. Ten phytochemicals werescreened for; alkaloid, cardiac glycosides, carbohydrate, flavonoids, phenols, saponins, sterols, tannins, terpenoids and quinines. All were found present in aqueous leaf extracts, while quinine and terpenoids were absent in methanol stem bark extract. This finding corroborates the reports of Asase et al.,(2008); Musa et al.,(2008) and Ojewale et al.,(2013).
Remarkably, the concentrations of flavonoids, alkaloid and tannins were higher in the leaf extract than the methanol stem bark extract. This suggests that the leaf offers a wider array of phytochemicals than the bark.
The presence of those selected phytochemical constituents in the plant made the plant to be a potential source of crude drug that can positively serve as source of modern drugs. This view was supported by Ahmadiani et al., (2000), who reported that tannins and/or flavonoids of medicinal plant origin were found to possessed significant
pharmacological activities: antidiarrheal, analgesic and anti-inflammatory among others in the animal body systems.Diets containing tannins at low dosages (0.15 – 0.20%), have been shown to improve wellbeing of the human body (Shiavone et al., 2008). Saponins enhance nutrient absorption and aid in animal digestion. Cardiac glycosides could improve circulation and heart function in congestive heart failure (El-olemy et al., 1994).

Acute Toxicity (LD50 Estimation)
The LD50 for BEPKwas estimated as1,250 mg/kg i.p. in rat (Table 2). While LEPK was found to have median lethal doses of 1,750 mg/kg i.p. in rat (Table 3). These values indicated that BEPK was slightly toxic to the test rats.Though the stating dose for LEPK was 1000 mg/kg, this isaccording to (OECD, 2000).
However, both the LD50 values were significantly lower than earlier value gotten by Ojewale et al., (2014) the reason for this might be due to difference in the solvents used for the extractions.
The World Health Organization bases its ranking of hazards chemicals on the lowest published rat oral LD50 in mg/kg body weight and in it ranking, any substance with oral LD50 values in the range of 200 – 2000mg/kg body weight oral falls into category II and classify as moderately toxic substances.
Thus, the indication is that both BEPK and LEPK are slightly toxics but could be used safely on human at lower doses (Adoun et al., 2012). The result also indicated that LEPK is less toxic in relation to BEPK, though both are relatively safe to the experimental model used, at the doses indicated.

Conclusion
Both methanol bark and aqueous leave extracts of Pseudocedrela kotschyishowed some of their chemical componentssuch as flavonoids, alkaloids, saponins, tannins, glycosides, phenol and sterols. There is no doubt thatthese plants are reservoir of potentially useful chemical compounds which serve as drugs, provide newer leads andclues for modern drug design. The LD50 of both the Leaves and Bark has been established in this study. So, its slighttoxicity at the higher doses should be taken into consideration during usage. Also, further clinical and pharmacological study should be conducted using lower dosage to investigate the unexploited potentials of this plant.

The post A Comparative Phytochemical and Acute Toxicity Studies of the Bark and Leave Extracts of Pseudocedrela Kotschyi appeared first on Nigerian Biomedical Science Journal.

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.

The post Effect Of Ethanolic Extract Of Ocimum gratissimum (scent leave) Leaves On Sodium Nitrite Induced Changes In The Liver Of Adult Wistar Rats appeared first on Nigerian Biomedical Science Journal.

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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The post Breast Cancer In Men: A Review of Epidemiology, Risk Factors, Diagnosis And Prevention In Africa. appeared first on Nigerian Biomedical Science Journal.

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.

The post Prevalence of Bacteriospermia Among Male Partners of Infertile Couples In Bida, Niger State. appeared first on Nigerian Biomedical Science Journal.

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.

The post Pattern of Abnormal Liver Enzymes Activities in Diabetic Patients in Zaria. appeared first on Nigerian Biomedical Science Journal.

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.

The post An Unusual Occurrence: A case of venous thromboembolism in pregnancy associated with heterotaxy syndrome appeared first on Nigerian Biomedical Science Journal.

Enabulele, O.I.
Faculty of Life Sciences,Department of Microbiology, University of Benin Edo State, Nigeria.

Tchounga, K.S., Ukaji, D.C., Ajugwo’ A.O.
Department of Medical Microbiology/ Medical Mycology, Faculty of Medical Laboratory Science,
Madonna University Elele Campus, Rivers State,Nigeria
All correspondence to: ukajidamian@yahoo.com

ABSTRACT
Serotype and biotype of Vibrio cholerae isolated from stool samples collected from patients with acute diarrhea and water samples were determined during September to November 2004 cholera outbreak in Uzebba in Edo–State, Nigeria. A total of 137 stool samples and 50 water samples were investigated. The samples were subjected to standard recommended microbiological techniques and confirmation of isolates by seroagglutination using Vibrio cholerae polyvalent O1 and O139 antisera and monovalent Ogawa and Inaba antisera, while biotyping was carried out by agglutination test and sensitivity to polymyxin B. Out of 137 stool samples, 63((45.98%) were found to be positive for Vibrio cholerae serogroup O1, and of 63Vibrio cholerae O1, 60(95.24%) were Vibrio cholerae serogroups O1 serotype Inaba, biotype classical and 3(4.76%) were found to be Vibrio cholerae serogroup O1, serotype Ogawa, biotype classical. Only stream water samples yielded growth of Vibrio cholerae serogroup O1, with 3(23.7%) out of 13 stream water samples. Well and bore hole water samples yielded no growth of Vibrio cholerae. Out of three Vibrio cholerae O1 isolated from stream water samples, 2(66.67%) were found to be positive, for Vibrio cholerae serogroup O1, serotype Inaba and classical biotype, while 1(33.33%) was Vibrio cholerae serogroup O1, serotype Ogawa and El -Tor biotype. This study demonstrated that some Vibrio cholerae O1 serogroup O1 isolated from stream water and stool samples had the same serotypes and biotypes. Therefore, there is need to perform a strong epidemiological surveillance for emergence and distribution of Vibrio cholerae O1.

Key words: Vibrio cholerae O1, serotype, biotype, water supplies, stool samples.

INTRODUCTION
Cholera (frequently called Asiatic cholera or epidemic cholera) is an acute diarrheal illness caused by a Gram negative slightly curved rod Vibrio cholerae (Ryan and Ray, 2004). Cholera is a worldwide problem, especially in developing countries. It has been very rare in developed nations for hundred years; however the disease is common today in other parts of the world, including the India sub-Asian continent and sub-Sahara Africa. Cholera is a major health threat in poor nations that frequently results in mortality. Although more than 100 serogroups of Vibrio cholerae exist, only two cause human disease. Vibrio cholerae O1 of which there are two biotypes (Classical and EL Tor); classified into serotypes Ogawa and Inaba and rarely Hikojima and Vibrio cholerae O139, which emerged in 1992. Cholera world wide is usually characterized by painless diarrhea and vomitting.
In 1991, there were more than a million cases in Central and South Canada. Several new cases have since been reported in Louisiana and other Gulf Coast areas (Werdlow
et al., 2002). African countries have in recent years experienced more epidemics and (WHO, 1999). A total of 29321 cholera cases and 10586 deaths were reported to WHO in 1998, with Africa accounting for the largest part with 72% of the global total (WHO, 1999). An explosive outbreak of cholera occurred among Rwanda refugees in Goma. Democratic Republic of Congo involved about 70,000 cases and caused about 12,000 deaths in 1994 (Siddique et al., 1995). In 1995, West Africa reported 64% cholera cases and 61% cases of cholera deaths (WHO, 1999). Between July and November 2001, several outbreaks were reported in the region, including 897 cases with 47 deaths in Cote d’voire and 575 cases with 21 deaths in Kano (WHO, 2001). In 2004, another outbreak involving 1316 cases and 76 deaths were again reported in Kano, while Edo-State (Uzebba) witnessed an outbreak of cholera involving 300 reported cases with 50 deaths (WHO, 2004). The Onslaught of cholera in Africa continent continues unbeaten as another one was reported in Nigeria involving 400 cases in Jigawa State, resulting in 15 deaths in October 2009 (WHO, 2009).

Strategies for prevention and control of this infectious disease depend on understanding the origin, transmission and other characteristics associated with it epidemiology. Therefore, this study was carried out to determine the prevalent serogroup, serotypes and biotypes of Vibrio cholerae responsible for the cholera outbreak in Edo-State, (Uzebba) between September to November 2004.

Materials and Methods
Stool samples were collected from patients with acute diarrihea. During the outbreak, exclusion criteria included patients using antimicrobial agents within the previous two weeks to avoid cases of antibiotic associated diarrhea. Water samples were collected in clean containers containing alkaline peptone water (APW) from various sources of water supply (streams, boreholes and wells).

Sample Processing
Stool samples collected in plain containers were cultured directly onto Thiosulphate Citrate Bile Salt Sucrose (TCBS) agar plates and incubated at 37oc for 24 hours. Water samples inoculated in APW were incubated at 37oc for 4 to 6 hours, after the incubation period, from the inoculated APW, plating was done onto TCBS agar and the plates incubated at 37oc for 24 hours. The isolates were purified by sub-culturing single colonies on sterile Nutrient agar plates which were then incubated at 37oc for 24 hours. Following standard morphological and biochemical tests according to Buchanan and Gibbons (1974), characteristic colonies grown on the selective TCBS agar were then confirmed for identification. The series of biochemical tests commonly used to identify V. cholerae (Baumann and Schubert, 1984; West and Cowell, 1984) were originally designed for clinical samples in order to specifically detect pathogenic vibrios. The series of biochemical tests include: Gram staining, oxidase , glucose and L-arabinose , methyl red , voges-proskauer and ornithine tests were performed.

Serological Test
Serological identification of isolates was done using slide agglutination test (Sakazaki and Donovan, 1984; Shimada et al., 1994), using polyvalent Vibrio cholerae O1 and Vibrio cholerae O139 antisera and monovalent Ogawa and Inaba antisera.

Biotyping
Biotyping of Vibrio cholerae isolates was done by using agglutination with chicken red blood cells and polymixin in B (50 units) sensitivity test (WHO, 1987).

RESULTS
A total of 137 stool samples collected from hospitalized patients during cholera outbreak from September to November 2004 in Uzebba (Edo – State), and water samples 50(13 stream water, 20 borehole water and 17 well water samples) were studied.

Table 1 shows the frequency of isolation, serotyping and distribution of Vibrio cholerae. Isolated from stool and water samples in Uzebba. Out of 137 stool samples, 63(45.98%) were found positive for Vibrio cholerae O1 and 60(95, 24%) out of 63, belonged to Inaba serotype, while 3(4.76%) were Ogawa serotype. None of the samples yielded growth of Vibrio cholerae O139. Water samples were also analyzed bacteriologically, 3((23.07%) out of 13 water samples collected from streams yielded growth of Vibrio cholerae O1 and borehole and well water samples yielded no growth. Table 2 shows the distribution of Vibrio cholerae O1 biotype isolated from stool and water samples. All vibrio cholerae O1 from stool samples both Inaba and Ogawa serotypes were Classical biotype, two Vibrio cholerae O1 Inaba serotype isolated from stream water samples were Classical biotype, while only Vibrio cholerae O1 Ogawa isolated from stream water sample was EL Tor biotype.
DISCUSSION
Cholera continues to be an important pubic Health problem among many poorer and vulnerable communities despite the fact that bacteriology, epidemiology and public health aspects of the disease were described in detail over a centuary (Shears, 2001). In the study, we isolated and identified 66 Vibrio cholerae out of which 63 isolates were from stool samples and 3 isolates from water samples.It was observed after serological test that the isolates were Vibrio cholerae O1 and that there was no serogroup O139 isolates. These findings are in agreement with the report of Tamang et al.,( 2005) reported that Vibrio cholerae O1 were predominant in Nepal, also similar reports were presented by Urassa et al.,( 2000);Inaet al.,(2007) reported that during his study in Manhica District hospital Southern Mozambique, all isolates were Vibrio cholerae O1. During this study, it was noticed that two serotypes Inaba and Ogawa co-existed with Vibrio cholerae O1 serotype Inaba being the most predominant with 62(93.94%) while 4(6.06%) were Vibrio cholerae O1 Ogawa serotype; and these isolates were both from stool and water samples. Our findings are in accordance with reports of Mercy et al.,( 2004) who isolated Vibrio cholerae O1 in Ghana with Inaba serotype having the highest occurrence over Ogawa serotype. Similar report was presented by Shukla et al., ( 2006 ) who recorded an emerged predominance of Vibrio cholerae O1 serotype Inaba over Ogawa between 2004 – 2006 in East Delhi.

The co-existence of the two serotypes recorded during our study was not in conformity with Inacio et al., (2007) who reported that only Vibrio cholerae O1 serotype Ogawa was isolated in Manhica District hospital Southern Mozambique. Our findings also contradicted claims by Hossein et al., ( 2005) that after a six year study on Vibrio cholerae in South Eastern Tran, all Vibrio cholerae O1 isolated were Ogawa serotype. EL Tor was the predominant biotype causing outbreak up till 1998. But during our study in Uzebba, the Classical biotype emerged predominant that although that one of the isolates from water sample was EL Tor biotype. The high emergence of Classical biotype in Uzebba could be justified by the
reports of Rafi et al., ( 2004) also isolated Vibrio cholerae O1 Classical biotype between 2000 – 2001 in Rawalpindi. This high occurrence of Vibrio cholerae O1 Inaba and Classical biotype in our study is not in accordance with the reports of Bradley et al.,(1997), Jacques et al.,( 2002) and Iwanaga et al.,( 2004), who reported that most of the strains of Vibrio cholerae O1 isolated from Madagascar, Bangladesh, Tanzania, Zaire, Latin America, Southern and Eastern regions of India were Ogawa serotype and EL Tor biotype. Our study revealed that high percentage of Vibrio cholerae isolated from both stool and water samples were Vibrio cholerae O1 Inaba and Classical biotype, which is also contrary to the findings of Hossein, et al.,(2005) that all strains of Vibrio cholerae O1 isolated between July and September 1998 in Goa belonged to EL Tor biotype, 53(66%) of them being Ogawa serotype, while 21(26%) were Inaba serotype.

Some isolates obtained from stream water samples when serotype and biotype were reported as Vibrio cholerae O1 Inaba serotype Classical biotype same as some of the isolates obtained from patient’s stool samples during the outbreak. One could be tempted to say that by referring to the phenotypic and genotypic characteristics of the isolates, this stream water that has been one of the sources of water supply in Uzebba community must have played an important role in the spread of cholera outbreak in that region. We can also say that outbreak in Uzebba is defined by social and environmental factors.The importance of aquatic reservoir depends on sanitary conditions of the community(CEDECO,2010. Cholera is spread mainly through drinking fecal – contaminated water. . While contaminated water remains the major route for cholera transmission (Shapiro, et al., 1999) food and utensils are also important (Rabbani and Greenough, 1999), emphasizing the importance of hygiene within the house hold (Fotedal, 2001).

Effective food hygiene measures include cooking food thoroughly and eating it while still hot; preventing cooked foods from being contaminated through contact with raw food or drinking water is important (Seas and Gotuzzo, ensuring proper management of excreta to avoid contamination of other water sources were important measures to reduce cholera transmission.   Education of the population at risk regarding appropriate hygienic practice is always recommended. Identification of local customs that place people at risk is also important in order to eliminate such practices (Sears and Gotuzzo, 2000). A greater understanding of the pathogen, its biology, ecology, epidemiology and strategies for treatment and prevention are essential to guide policies and programmes for the control of cholera. . Adequate measures to improve hygiene and sanitation and supply of safe potable water are needed to prevent any future outbreak of cholera in Uzebba.

The post Serotypes and Biotypes of Vibrio Cholerae O1 Isolated from Stool and Water Samples in Uzebba (Edo – State). appeared first on Nigerian Biomedical Science Journal.

Atiegha C. Igoni M , Victor I.,
Department Of Medical Laboratory Sciences, College Of Health Technology Otuogidi-ogbia , Bayelsa State.
Ayaowei I.T.
Department Of Health Information Management, College Of Health Technology Otuogidi-ogbia , Bayelsa State.
Eseimokumo Me.
Department Of Pharmarcy Technician Studies, College Of Health Technology Otuogidi-ogbia , Bayelsa State.

All correspondence to: IGONI M ; Bayelsa State College of Health Technology: e mail; achristo40@gmail.com.

ABSTRACT
The treatment of tuberculosis has evolved from streptomycin to the DOTS strategy .Even as at that it is a serious challenge. This retrospective study was carried out to evaluate the effectiveness of the method. A total of sixty seven(67) Tuberculosis cases were evaluated at the Tuberculosis Treatment centre Nembe comprehensive Health Centre, Bayelsa state , it covered a period of four (4) years, from 2009 to 2012. Out of sixty seven cases that continued their treatment regiment to the end sixty four persons were cured and in only seven instances persons shows the ineffectiveness of the drugs by testing positive to the bacilli during chemotherapy. It is therefore recommended for community and family based treatment.

Key words: DOTS, Cured, treatment completed and treatment failure.

INTRODUCTION
Tuberculosis, a major health challenge and leading infectious killer is caused mainly in man by Mycobacterium tuberculosis. The bacilli enters the body by inhalation of droplets or dust particles containing it. It occurs as pulmonary tuberculosis or non- pulmonary tuberculosis. In pulmonary tuberculosis the pulmonary aveoli and surrounding lymph glands are lodged by the bacilli resulting in lesion characterized by acute inflammatory reactions with accumulation of fluid and white blood cells around the aveoli, while the non pulmonary tuberculosis includes the renal and urogenital tuberculosis, military tuberculosis and tuberculosis meningitis with varying symptoms1.
In sub-saharan Africa, cases of tuberculosis have increased dramatically, overwhelming control programs 2. This could be as a result of the fact that tuberculosis infection control interventions are not routinely implemented in contrast to what happens in high income countries with low prevalence of tuberculosis where infection control policy is routinely observed3. However the launch of directly observed treatment shortcourse (DOTS) in 1995 by the world health organization (WHO) has shown to be an effective intervention that will lead to reduced tuberculosis transmission and decreasing number of tuberculosis cases4,5. Also it is among the most cost-effective global health interventions available today6,7.
The DOTS strategy is based around a short-course treatment regimen for a minimum of six months of four drugs in combination (2 months of Isoniazide, Rifampicin ,Pyrazinamide and Ethambutol), a good management practice, sputum smear microscopy for diagnosis and the direct observation of doses to ensure adherence. Treatment success under DOTS concerns two outcomes- cured and treatment completed: “cured” if patient finish the treatment regimen with negative bacteriology at the end of the treatment whereas “treatment completed” refers to patients who have finished the regiment in full without showing evidence of treatment failure and without negative bacteriology. This treatment regiment however use to suffer from a number of drawbacks, with the combination of available drugs, the duration of treatment cannot be reduced below six months without a significance in relapses although re-infection with a different strain of mycobacterium tuberculosis can cause tuberculosis recurrence, which is considered to be an important measure of efficacy of tuberculosis treatment8,9. Also when treatment is given under sub-optimal conditions, regiments are associated with high rate of patients non-adherence specially in low income countries which harbor high burden of diseases10. The consequence of this is increased mortality and creation of clinic infections, drug resistant cases. The World Health Organization estimates in 2004, 424,203 cases of multidrug resistant tuberculosis globally among which 181,408 cases occurred in patients who had already been treated with standard (first line) therapy11.
This study was therefore carried out to determine the effectiveness of DOTS (Directly Observed Treatment Short course) on tuberculosis in the Nembe Local Government Area of Bayelsa state south-south Nigeria.

MATERIALS AND METHODS
Setting Comprehensive Health Centre Nembe, is situated in Nembe, the headquarters of Nembe local government area, about eighty kilometers south-east of Yenagoa the capital of Bayelsa State, Nigeria.

Subjects
The subjects of this report are those patients that reported to the TB treatment centre and those patients who were got through surveillance in the surrounding communities. A total of sixty seven positive cases were involved in the study. These cases were got over a four year period (from 2009 – 2012)

Laboratory analysis
Patients were given sterile wide mouthed plastic containers which they voided sputum into. Three specimens were collected from them over a two days period (spot, early morning and spot). Sputum analysis was done using the Ziehl-Neelson technique as described by cheesbrough12.

Method of drug administration
The strategy adopted here is the short course regiment that lasts for six months. Upon visit to the treatment center, Zeihl-Neelson(AFB) test is carried out. Treatment commences on patients who are newly diagnosed i.e. their sputum show smear positive. After the initial test, a second test (Zeihl- Neelson) is conducted upon revisit at the end of the second month of chemotherapy. The same thing applies
upon a second and third revisit which is at the end of the fifth and sixth months respectively.
For category 2 patients i.e. those who defaulted treatment or had a relapse, treatment is for seven months. Treatment on the intensive phase is for three months after which the second AFB test is done. The third and fourth AFB tests are done at the end of the fifth and seventh month of chemotherapy. Patients that absconded during treatment were not included in this work.

In 2009 eighteen new cases of sputum positives were recorded (six scanty and twelve +s). Upon first revisit i.e. after intensive phase, all eighteen persons were tested smear (AFB) negative. On second revisit the same feet was recorded. In the last revisit seventeen tested negative while one patient was AFB positive: table 1.

In 2010, seventeen persons were tested positive with varying degree of intensity of infection ( one scanty, eighty +s, four ++s and four +++s). sixteen persons tested smear negative at the end of the first revisit while person was tested positive. At the end of the fifth month of chemotherapy all fifteen persons tested smear negative (second revisit). After six months of chemotherapy one patient was tested smear positive while the other sixteen persons that continued their treatment to the end were tested negative. Conclusively, eleven persons were cured while one person was termed a “Treatment Failure”: table 2
Table 3 represents the summary of the 2011 treatment outcome. There were ten sputum smear positive cases in
2011. Eight of them were graded +, while one patient had 2+ and another 3+. Seven persons tested AFB negative upon the first revisit and one patients sputum was positive. In the second and third revisits, all eight patients had there AFB test negative. i.e. in 2011 eight persons were cured of tuberculosis in the treatment center.
Two scanty cases, twelve +s and eight cases of 2+s were recorded in the year 2012, twenty two cases in all. Three scanty cases were recorded in the first revisit stage, while nineteen tests were negative. In the second and third revisit all twenty two tests were smear negative. in 2012 twenty two persons were cured of tuberculosis in the treatment center. Table 4

Discussion
In this four years under study, the success of this method of treatment has been so tremendous. Out of sixty seven cases that continued their treatment regiment to the end sixty four persons were cured and in only seven instances persons shows the ineffectiveness of the drugs by testing positive to the bacilli during chemotherapy. When the result was subjected to the chi-square test, it shows that there is a significant difference between those that are cured of TB by the dots strategy in the five years under study and those that were not cured by the strategy p<0.05, (234.845, df 3, assymp. Sig (2-sided) 0.000

Conclusion:
Since with the DOTS strategy it is very difficult for patients to skip treatment, (which in most times is the cause of resistance), the researcher recommends that this strategy should be adopted, be it community based or family based)

Acknowledgment
We wish to express our heartfelt thanks to God almighty for his abundance grace to carry out this research and the strength to stand it all, without him nothing would be done. We also greatly appreciate the provost of the Bayelsa State College of Health technology, Dr Adias T. Charles for his inspiration and motivation. Also, the research and manpower development department of the College was involved with the funding. Finally we thank the staffs of the comprehensive health centre Nembe, Bayelsa State.

The post Diagnosis And Treatment Of Tuberculosis By The Directely Observsed Treatment Shortcourse (DOTS): A Case Study Of Nembe Comprehensive Health Centre TB Treatment Unit, South-South Nigeria. appeared first on Nigerian Biomedical Science Journal.

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

1. Department of Human Physiology, Faculty of Medicine, Ahmadu Bello University, Zaria, Nigeria.
Musa, K.Y.,

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

Ayaowei IT
Department Of Health Information Management, College of Health Technology Otuogidi-ogbia , Bayelsa State.
Helmy, A.
1. Biotechnology Centre, Misr University for Science and Technology, Cairo, Egypt.

All correspondents to: M.I.A. Saleh, Department of Human Physiology, Faculty of Medicine, Ahmadu Bello University,
Zaria, Nigeria. E-mail: alhajisaleh@yahoo.com

ABSTRACT
Nigella sativahas been used for medicinal purposes for centuries, both as a herb and when the seeds are powdered or 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 .This study was aimed at investigating the effect of the n-butanol seed fraction of this plant as it affects gastric mucosal integrity and basal gastric secretions using the indomethacin –induced model. Phytochemical screening revealed the presence of flavonoids, alkaloids,, saponins, glucocinolates amongst others, whereas acute toxicity studies revealed a median lethal dose above 5000mg/kg. The rats were grouped into 6 (n = 5), with the extract fraction administered at 50, 100 and 200mg/kg subcutaneously, followed by pyloric ligation with indomethacin and cimetidine used as the standard drug. For the mucosal integrity study, ulcer and preventive indices were analysed, while volume of gastric juice, titratable acidity,acid output and pepsin concentration were assessed for basal gastric secretions. The three experimental doses of the extract at 50,100 and 200mg/kg showed a dose –dependent decrease in both ulcer and preventive indices. It also showed a significant (p<0.05) decrease in volume of gastric juice, titratable acidity, acid output and pepsin concentration in dose-dependent manner with the three experimental doses administered with the highest reduction at the 200mg/kg dose. The results obtained suggest that this fraction down regulated all those parameters which might be attributed to the presence of the phytoconstituents present in this fraction. Therefore, the extract fraction of this plant possesses gastroprotective and antisecretory effects further explaining the folkloric use of this plant in the therapy of peptic ulcer disease.
KEYWORDS: Nigella sativa, gastroprotection, basal secretions, NSAIDs

Introduction:

Peptic ulcers are breakages or discontinuities that can occur in the mucosal epithelial lining of either the stomach, small intestine, large intestine or the Merkel’s diverticulum along the gastrointestinal tract (Falase and Akinkugbe, 2010). They are known to occur world wide (Cemek et al., 2010) and are major causes of morbidity and mortality (Chaturvedi et al., 2007). The pathophysiology of peptic ulcer has been centralized on an imbalance between aggressive and protective factors. Factors such as stress, cigarette smoking, nutritional deficiencies, inadequate dietary habits, hereditary predisposition and frequent ingestion of non-steroidal anti-inflammatory drugs (NSAIDs) all are known to increase
the gastric ulcer incidences (Klein et al.,2010).In developing countries, usually 50-90% of the populations are infected with Helicobacter pylori, which is the main organism responsible for majority of peptic ulcer cases, and children acquire the infection soon after being weaned. Many natural products and modern synthetic drugs have been used to treat the peptic ulcer disease, but so far a complete cure has not been achieved or discovered, and exploration of new anti-ulcer drugs has remained a field of active research (Bandyopadhyay et al.,2001).Although there are many products in the market for the treatment of gastric ulcers, including antacids, proton-pump inhibitors, anticholinergics and H2-receptor antagonists, most of these drugs produce several adverse reactions,such as hypersensitivity reactions, arrythmias, impotence, gynaecomastia, nephrotoxicity, and haemopoetic changes(Chang and Leung.,2002; Scholl et al.,2005).Development of tolerance and incidence of relapses and side-effects on clinical evaluation make their efficacy arguable, further promoting non-drug compliance to therapy (Santin et al., 2011). In addition, most of these medications are expensive, which further restricts their use (Santin et al., 2010).
This has been the basis for the development of new antiulcer drugs, which include herbal drugs (Altinkaynak et al., 2003). Herbs are used in many domains including medicine, nutrition, flavouring, beverages, dyeing, repellants, fragrances and cosmetics (Djeridane et al., 2006). 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 diaphoretic(Gupta et al., 2009).The aim of the present study was to evaluate the effect of the n-butanol fraction of this plant seeds on gastric mucosal damage and secretions.
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 authentication was 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 of the plant material:
Nigella sativadried 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 methanol and partitioned with n-butanol to obtain the n-butanol (n-BuOH) fraction. The fraction was further concentrated in-vacuo and the residue obtained. The extract yielded about 80% of the residue.

2.3 Phytochemical screening of the fractions:
The preliminary analysis for the extract was conducted for the presence of flavonoids, alkaloids, saponins, steroids, glycosides, anthraquinones, resins, reducing sugars and other phytochemicals 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. The rats were treated with the n-butanol fraction of the extract at doses of 10, 100 and 1000mg/kg subcutaneosly. 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 fraction at appropriate doses subcutaneosly. 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 (Lek Pharma, Slovenia), Indomethacin (Liomethacin(R))(Cheisi, Egypt),
Thiopental Sodium (Abbott Laboratories, UK), Phenol Red (BDH Poole, England), Sodium Hydroxide (NaOH) (BDH Poole, England) for the preparation of 0.01N NaOH solution, Phosphate Buffered Saline (PBS),Casein Substrate Solution 1% (w/v) (Sigma-Aldrich, USA), Hydrochloric Acid (HCl) 0.1N(Sigma-Aldrich, USA), Trichloroacetic Acid Solution 6% w/v (Sigma-Aldrich, USA). All other chemicals and reagents were analytical grade.
2.6 Experimental animals:
A total of ninety adult 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 internationally accepted principles of laboratory animal use and care. 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 Experimental design:
Group I: Study of gastric mucosal damage
Group IA; Normal saline (Negative Control)Five rats received normal saline (1ml/kg/rat S.C).
Group IB: Indomethacin-treated (Positive Control) Five rats received indomethacin (20mg/kg S.C) for the study of gastric mucosal damage.
Group IC: Cimetidine-treated Ten rats for the study of the effect of two different doses of cimetidine 50 and 100mg/kg S.C on gastric mucosal damage (5 rats for each dose)
.Ten rats for the study of effect of two different doses of cimetidine (50mg and 100mg/kg) S.C, given 30 minutes prior to indomethacin administration on gastric mucosal damage (5 rats for each dose).
Group ID: Nigella sativa extract treated Fifteen rats for the study of the effect of n-Butanol (BuOH) fraction, each at three different doses (50, 100 and 200mg/kg S.C),when given 30 minutes prior to indomethacin on gastric mucosal damage (5 rats for each dose).
Group II: Study of basal gastric secretion
Group IIA: Normal saline (Negative Control) Five rats received normal saline (2 ml/rat S.C).
Group IIB: Indomethacin-treated (Positive Control)Five rats received indomethacin (20 mg/kg S.C), followed by pyloric ligation for the study of basal gastric secretion.
Group IIC: Cimetidine-treated Ten rats for the study of the effect of two different doses of cimetidine, 50 and

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

2.8 Induction of gastric ulceration
After 48 hours of starvation, the animals were weighed and maintained in their individual cages. Then, indomethacin 20mg/kg was injected subcutaneously and the animals were then deprived of both food and water for 7 h (Urushidani et al., 1979). The animals were later sacrificed by decapitation (Satoh et al., 1983). Their stomachs were opened along the greater curvature, rinsed slowly with running water, then stretched out as much as possible by the use of pins on No.1 Whatman’s filter paper on a ceiling board.

2.9 Quantitative assessment of mucosal damage
The ulcerated areas in each stomach were measured with a transparent (mm) ruler scale, the result of each group were expressed as ulcer index (U.I) in mm of mean ulcer ± standard error of mean (Scepovic and Radmanovic, 1984).
The degree of ulceration was expressed as ulcerindex (U.I). It was calculated by multiplying ulcer score by 100 (Robert et al., 1968). Ulcer score for each group was calculated by dividing the total number of ulcers in each group by the total number of rats in that group (Robert et al., 1968).The percentage preventive index was calculated according to the method of Hano et al.(1976), which is expressed as:
Preventive Index (%) = (U.I. Indomethacin – U.I. Extract/drug plus) indomethacin x 100
U.I. Indomethacin

2.10 Collection of gastric secretion
The gastric juice was collected according to the technique of Shay et al. (1954) as modified by Levine (1965), where oesophageal ligation was avoided. The animals were fasted for 48 hrs to ensure complete emptying of the stomach, but allowed water ad libitum. Each animal was weighed at the end of the fasting period. Light anaesthesia was sodium thiopental 10 mg/kg intraperitoneal (Juliane et al., 2009), abdomen of each rat was opened via a midline incision and the stomach exteriorated. A pyloric ligature was made using a thread with care to avoid damage to the blood vessels or traction to the stomach. The abdomen was then closed by suture, cleaned thoroughly with distilled water or saline, and the animal was allowed to recover.
After 3 h, the rats were sacrificed by decapitation, abdomen of each of the animals were opened. The oesophagus was ligated, and the stomachs removed and washed with distilled water or saline. An opening along the greater curvature was made (Nwafor et al., 2000) and the gastric content drained into a graduated centrifuge tube, the volume noted, then centrifuged at 1,006 x g for 15 minutes.

2.11 Analysis of the gastric juice
Determination of each sample volume after centrifugation
The volume of 3 hours gastric secretion was measured after being subjected to centrifugation at 1,006 x g for 15 mins.
Determination of titratable acidity: A given volume of the gastric juice (1ml) was titrated against 0.01N NaOH. An end point of pH 7.0 as determined colorimetrically at 280nm by phenol red was used (Grossman, 1973; Davenport, 1977).The values were calculated as micro-equivalents per litre (Meq/L), which is equal to the number of millitres (ml) of 0.01N NaOH required to neutralize 1ml of gastric juice.
Titritable Acidity =
Volume of 0.01 N NaOH (mol) which neutralized 1ml of gastric juice
10
Determination of acid output: This was calculated by multiplying the volume (ml) of the gastric juice of each animal by the titritable acidity in that animal.
Determination of pepsin concentration: Pepsin concentration which is the major factor involved in the proteolytic activity of gastric secretion was determined in terms of the amount of protease enzymes produced after incubation of the substrate for 30 minutes with pepsin. It was determined by the spectrophotometric method devised by Jongensen (1954) and Hawk et al. (1960).
2.13 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).
3.0 Results:
The phytochemical screening revealed the presence of the following phytoconstituents as depicted in table I below:

The Phytochemical Analysis of N-Butanol Fraction of Nigella sativa L. seed extracts. Acute Toxicity Studies
The toxicity studies of the n-butanol seed extract of Nigella sativa in the first phase after being observed for 24hr, the rats did not show any signs and symptoms of toxicity or death. In the second phase, none of the rats produced any toxic symptoms or mortality up to the dose level of 5000mg/kg body weight, hence, they were considered safe for further pharmacological screening.

DISCUSSION AND CONCLUSION
To evaluate the gastroprotective effect of N-butanol fraction of Nigella sativa, the model of acute ulcer induced by a non-steroidal anti-inflammatory compound indomethacin was performed.
In this model, it was found that treatment with normal saline (2ml/rat) which was isotonic with the plasma as a control, showed no significant ulcer or lesion index with a preventive index of 97%. The group that received indomethacin 20mg/kg alone produced the highest lesion index when compared with all the treatment groups. There were gastric mucosal congestion, oedema, haemorrhage, lamina epithelial necrosis, leucocytic infilteration, blood vessels congestion with foci of necrotic tissues in the lesions. These results are consistent with previous studies that reported similar histopathological derangements and mucosal oxidative stress effects that involves weakening of gastric mucous, leading to formation of lesions in the gastric epithelium (Cyer, 2000; Chang and Leung, 2002; Ryo et al., 2006; Valcheva-Kuzmanova et al., 2007; El-Moselhy et al., 2009).
It was observed that the group that received 50 and 100mg/kg of the standard drug cimetidine alone, showed a significant decrease in gastric lesions (p<0.05) compared to the indomethacin 20mg/kg, cimetidine plus indomethacin group in the ulcer index with preventive indices of 73 and 79%, respectively. This corroborated with the studies of Marivane et al. (2011) who reported a decrease in lesion index on the gastric mucosa of rats treated with the H2-receptor antagonist cimetidine compared to indomethacin.
With regard to the effects of three different graded doses of the N-butanol fraction of Nigella sativa, it was found that treatment with the 50, 100 and 200mg/kg significantly reduced the lesion index compared with the control group (p<0.05) in a dose-dependent manner in this indomethacin-induced model. The highest dose of 200mg/kg N-butanol fraction produced the highest preventive index of 96% above that of the standard drug 50 and 100mg/kg cimetidine. A similar finding was reported by Marivane et al. (2011) that reported a significant reduction in lesion index, total injured area and the percentage injured area when extract of Brassica oleracea was used on indomethacin ulcer model.
Both Brassica oleracea and Nigella sativa are found to contain some flavonoids, especially quecertin and kacempferol mainly in glycosodic form and these are secondary metabolites that are widely distributed in nature with several biological activities including gastroprotective potentials (Martin et al., 1998). Croton urucurana with high flavonoid content also exhibited same mucosal cytoprotective potentials (Esmeraldino et al., 2005; Alves et al., 2008). The flavonoid content of this fraction had prevented and exerted a protective effect possibly by its inherent ability to scavenge free radicals, inhibit lipid peroxidation, increase mucous and prostaglandin contents of the gastric mucosa (Alanko et al., 1999).
Through phytochemical analysis of the fraction of N-butanol, apart from flavonoids, the presence of terpenoids, tannins, cardiac glycosides, steroids, saponins were detected amongst others. To further support and corroborate the possible fractions for the mucosal cytoprotection exhibited by this fraction of N. sativa containing flavonoids are the studies by Alcaraz and Hoult, 1985 that flavonoids increase mucosal prostaglandin content, inhibit histidine decarboxylase thereby decreasing histamine secretion (Bromer and Landry, 1985).
The presence of saponins in the fractions of N. sativa to improve on mucosal integrity has been reported in several other studies where plants containing saponins have been shown to possess antiulcer activity in several experimental ulcer models. Among these, saponins isolated from the rhizome of Panax japonicus and the fruit of Kochia scoparia (which contain approximately 20% of saponins) have been demonstrated to possess gastroprotective properties (Matsuda et al., 2003) in conformity with this study. The protective activities of all these saponins are not due to inhibition of gastric acid secretion, but probably due to activation of mucous membrane protective factors (Borreli and Izzo, 2000). Moreover, several plants containing high amount of saponins have been shown to possess antiulcer activity in several experimental bioassays, probably acting as an activator of mucus membrane stabilizing factors (Morikawa et al., 2006). Similarly, presence of tannins, terpenoids in N. sativa fractions further validate the cytoprotective property in the gastric mucosa observed in our study as reported by Al-Rehaily et al. (2002), where several saponins, tannins, terpenoids were found to possess gastroprotective properties. Additionally, Terpenoids are a widespread class of secondary compounds with several pharmacological activities, including anti-inflammatory and antiulcer activities (Arrietta et al., 2003). Plant extracts of Eleagnus angutifolia, Hibiscus esculentus, Papaver rhoeas, Phlomis grandiflora, Rosa canina all with a high flavonoid and saponin contents as reported by Ilhan et al. (2003) showed potent in vivo gastroprotective activity similar to that of Nigella sativa.
The interference of the N-butanol fraction of the extract using same model was also evaluated on parameters of basal gastric secretion. This method is an important procedure that reveals the possible changes of gastric secretory physiological parameters relating to volume of gastric secretion, titratable acidity, acid output relating to pH and the most important proteolytic enzyme in the stomach pepsin. The findings suggest that this fraction interfered with these major basal secretory indices of gastric juice.
Considering that the volume of gastric juice which is mainly acidic encompasses mucus, hydrochloric acid, pepsinogen, bicarbonates, intrinsic factor and protein plays a vital role in the aetiopathogenesis of gastric mucosal integrity, it is plausible to consider this fraction as a putative cytoprotective agent. This assumption was made based on the observation that the different quantities or volume of gastric juice obtained in this study showed a general inhibitory pattern with regard to its production in the stomach in this fraction evaluated. There was a significant reduction in the volume of gastric juice at the 100 and 200mg/kg extract treated groups when compared with the control. These results are in agreement with the studies of Muriel et al., (2008), who reported a significant decrease in volume of gastric juice on a similar ulcer model after using Green propolis.
With regard to the titratable acidity, there was no significant difference in all the three doses of N-butanol evaluated when compared with the control. Acid output significantly decreased in a dose related manner when compared with control. This decrease in stomach acidity facilitated the healing of gastric ulcers, because exposing the mucosa to high concentrations of acid favours mucosal epithelial damage (Laine et al., 2008). Contact between stomach acid and the mast cells of the submucosa and lamina propria causes mast cell degranulation and the release of histamine, which stimulates hydrochloric acid secretion by parietal cells and produces inflammation and acute oedema at the site of contact (Rodrigues et al., 2008). Overall, concentration of hydrogen ions in the gastric juice decreases reflective of high pH, further aggravating the aggressive factors (Lullmann et al., 2000). Consistently, hyperacidity is known to result due to uncontrolled hypersecretion of hydrochloric acid from parietal cells of gastric mucosa through the proton pump H+-K+ ATPase (Kishor et al., 2007). The results of 50mg/kg N-butanol was insignificant. Proteolytic activity as pepsin concentration significantly decreases (p<0.05) in a dose-dependent manner compared to control. A similar finding was reported by Halter et al. (1988) and Hatazawa et al. (2006).
In all the cimetidine treated groups, the volume of gastric juice, titratable acidity and pepsin concentration all significantly reduced compared to control. Gastric acid decimation by cimetidine has been attributed to its ability to antagonize the binding of histamine to the H2 receptor on the parietal cell membrane (Banji et al., 2010).The reported results have validated the folkloric use of N. sativa in the therapy of peptic ulcer disease. N. sativa offers protection against NSAIDs-induced gastric ulceration and down-regulate basal acid secretions to promote mucosal cytoprotection. The presence of phytoconstituents in this medicinal plant might be responsible for those pharmacological actions. In this context, extracts and active principles from plants could serve as leads for the development of new drugs. Therefore, this plant specie(s) have a great potential to be used as a gastroprotective agent in combination with others or alone.

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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.

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.

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.

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