Study of the Effect of Combined Oral Contraceptive (DUOFEM) on Fibrinogen and Interleukin-6 in Female Wistar Rats.

Toryila J.E, Adelaiye A.B. Achie L.N
Human Physiology Department, Faculty of Medicine, ABU Zaria.
Amadi K, Odeh S.A
Human Physiology Department, Faculty of Medical Sciences Unijos.

ABSTRACT:

BACKGROUND: It is estimated that 38% of all women worldwide between the ages of 15 – 40 years use combined oral contraceptives, which now differ significantly in formula and concentrations from previous ones. Duofem, a third generation combined oral contraceptive contains desogestrol. The formation is anticipated to give lower risks of thromboembolism, myocardial infarction, stroke and autoimmune diseases. This research priority included efforts to discover the effect of combined oral contraceptive (DUOFEM) on fibrinogen and interleukin-6 in female wistar rats.

METHOD: Forty (40) female wistar rats weighing 180-250 g were used for the study. They were divided into four groups of 10 rats each comprising 5 treated and 5 control rats. The treated rats received 0.6mg/kg body weight of COC intragastrically for 36, 48, 60 and 72 days. All groups were given fresh water at ad libitum daily for the period of the experiment. Interleukin-6 was determined using rat ELISA kit ( Karmiya Biomedical company, USA). Fibrinogen was estimated by Clauss Assay (Clauss, 1957)

RESULTS: DUOFEM reduced the serum levels of both fibrinogen and interleukin-6 in all the treated groups compared to controls (P<0.05). Lowest serum levels of fibrinogen and interleukin-6 was observed in group D (72 days). By lowering IL-6 and fibrinogen, estrogen can negatively suppressed the body’s immune response, triggering a predisposition to autoimmune and cardiovascular diseases.

Key words: Combined Oral Contraceptive, Fibrinogen, Interleukin-6

INTRODUCTION

Contraception is an important health issue in preventive medicine because it protects women globally from the effects of unwanted pregnancy and allows them to integrate into society. Combined Oral Contraceptives (COCs) are currently among the most commonly used drugs in developed countries
Long-term use of combined oral contraceptives (COCs) and of hormone replacement therapy (HRT) have been linked to increased blood coagulation, increased risk of cardiovascular diseases and altered immune and inflammatory factors, suggesting an increased risk of chronic immune disorders with an inflammatory component, including cancer (WHO, 2009).
COCs have evolved through various modifications to reduce the dosages of the different hormonal
components to minimize the risk of thrombotic events, such as stroke. The type of oral contraceptives prescribed differs between countries, and both the type of oral contraceptive and the doses of estrogens and progesterone have changed between and within countries over time (Bulur et al, 2012). DUOFEM is an emergency contraceptive product that is designed to prevent pregnancy. It is intended for use by women from eighteen years up to menopause Interleukin-6 (IL-6) is a four-helical cytokine of 184 amino acids. The protein is synthesized by fibroblasts, monocytes, macrophages, T cells and endothelial cells. IL-6 synthesis and secretion is induced during inflammatory conditions such as upon stimulation of Toll-like receptor (TLR)-4 by lipopolysaccharide or upon stimulation of cells by IL-1 or tumor necrosis factor (TNF)-a (Dirk and Stefan, 2016) Interleukin-6 (IL-6) is a multifunctional cytokine which is involved in a broad spectrum of activities such as immune defense, hematopoiesis, and the acute phase response, as well as in the pathogenesis of multiple myeloma. IL-6 has been demonstrated to have a pivotal role in the pathogenesis of rheumatoid arthritis, Castleman’s disease and Crohn’s disease exemplified by the use of an anti-IL-6 biological therapy. IL-6 is also associated with the autoimmune disease systemic sclerosis and has been shown to be directly fibrotic. They are capable of mediating a complex array of pro- and anti-inflammatory effects. IL-6 produces C-Reactive Protein (CRP) which leads to cardiovascular risk (Subhadeep et al., 2008). Estrogen is able to decrease IL-6 expression by blocking the estroblast’s synthesis of IL-6 receptors (Subhadeep et al., 2008). . Straub et al. (2000) and Rachon et al. (2002) report decrease IL-6 level in menopausal women taking hormonal replacement therapy.
Fibrinogen is a key component of the inflammation and clotting pathways and an established risk factor for cardiovascular disease (CVD). Fibrinogen production is upregulated in response to cytokines released during inflammation, infection, neoplasia or tissue damage.(Ernst, 1993). Sex hormone balance is crucial in the regulation of inflammatory and immune responses, as estrogens play a role in enhancing immune function while androgens and progesterone have a suppressive function.

MATERIALS AND METHOD:
Forty (40) female wistar rats weighing 180-250 g were used for the study. They were divided into four groups of 10 rats each comprising 5 treated and 5 control rats. The treated rats received 0.013mg/kg body weight of COC intragastrically for 36, 48, 60 and 72 days. All groups were given fresh water at ad libitum daily for the period of the experiment. Interleukin-6 was determined using rat ELISA kit ( Karmiya Biomedical company, USA). Fibrinogen was estimated by Clauss Assay (Clauss, 1957).

RESULTS:
COC reduced the serum levels of both fibrinogen in all the treated groups compared to controls (P<0.05). Lowest serum levels of interleukin-6 was observed in group D (72 days). 

DISCUSSION AND CONCLUSION
There was decrease fibrinogen levels in all the treated groups compared to the control. This finding differs from that of Eliana and Koni (2014), Peter. (2013) and Carol (2015) who reported increase in fibrinogen level in women taking COC. The finding also differ from that of Akhigbe et al. (2008) who found no significant change in fibrinogen level in female wistar rats treated with COC. Fibrinogen functions as a messenger molecule that coordinates and regulates the body response to inflammation. The association between increase in plasma fibrinogen and thrombosis and the risk of myocardial infarction, atherosclerosis and other cardiovascular diseases are well established. Higher level of fibrinogen raises the risk of stroke (Abdalla et al. 2008).Low fibrinogen levels are associated with low risk of cardiovascular diseases.
There was a significant decrease in serum level of interleukin-6 (IL-6). Estrogen is able to decrease IL-6 expression by blocking the estroblast’s synthesis of IL-6 receptors (Jean et al., 1992). IL-6 produces C – reactive protein (CRP) which leads to cardiovascular risk. Experimental studies have shown strong correlations between the risk of cardiovascular diseases and inflammatory markers such as CRP and tissue neurosis factor-a (TNF a). IL-6 is a pleotropic cytokine which stimulates B-lymphocyte and T-lymphocyte differentiation, and activates macrophages and natural killer cells (NK). Interleukin 6 (IL-6), promptly and transiently produced in response to infections and tissue injuries, contributes to host defense through the stimulation of acute phase responses, hematopoiesis, and immune reactions. IL-6 has been demonstrated to have a pivotal role in the pathogenesis of rheumatoid arthritis, Castleman’s disease and Crohn’s disease exemplified by the use of an anti-IL-6 biological therapy. However, IL-6 is also associated with the autoimmune disease systemic sclerosis and has been shown to be directly fibrotic (Rachon et al.2002).

CONCLUSION:
The inflammatory cytokine interleukin-6 (IL-6) is a main regulator of fibrinogen synthesis. Interleukin 6 (IL-6), promptly and transiently produced in response to infections and tissue injuries, contributes to host defense through the stimulation of acute phase responses, hematopoiesis, and immune reactions. Fibrinogen is a key component of the inflammation and clotting pathways and an established risk factor for cardiovascular disease. By lowering IL-6 and fibrinogen, estrogen can negatively suppress the body’s immune response, triggering a predisposition to autoimmune and cardiovascular diseases.

REFERENCES:

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Vitamins and Micronutrients level among Transfusion-dependent Subjects in Maiduguri, Nigeria.

Waziri Gimba, Bukar Alhaji, Obi Simon Osita, Medugu Jessy Thomus, Bukar Abdullahi Audu,
Department of Haematology, University of Maiduguri Teaching Hospital, Maiduguri
Osaro Erhabor
Department of Medical Laboratory Science, Usmanu Danfodiyo University, Sokoto.
Imoru Momodu
Department of Haematology, Aminu Kano University Teaching Hospital, Kano.
Digban Kestar Awharentomah, Jeremiah Zaccheaus Awortu.
Department of Chemical Pathology, Igbinedion University Teaching Hospital, Okada.
Olaniyan Matthew Folaranmi
Department of Medical Laboratory Science, Achievers University, Owo
All correspondence to: gimbawaziri@gmail.com.

 

ABSTRACT
Background: Blood transfusion has been very beneficial in relieving symptoms of anaemia but is associated with the risk of iron accumulation in tissues. Excess iron, by Fenton chemistry enhances oxidative stress. Antioxidant vitamins and micronutrients supplementation quenches the oxidative stress and obviate tissue damage. Objectives: The aim of the study is to evaluate some antioxidant vitamins and micronutrients level and determine whether supplementation is necessary in the management of transfusion-dependent patients. Methods: Blood samples were collected from 101 patients with ?8 units’ transfusion per year and its equivalent with no history of transfusion as controls. Serum vitamins (A and E), micronutrients (Zn and Cu), serum iron, ferritin and some haematological parameters (Haemoglobin and Haematocrit) were measured from the blood samples. Result: The underlying condition for repeated transfusion was largely SCA (96%) with male preponderance. Antioxidants, vitamins A and E were significantly decreased (p=0.000) compared to the controls, whereas micronutrients, zinc and copper levels were elevated (p>0.05 and 0.05 respectively). There was significant increase in both serum iron and ferritin in the case subjects (p 0.000). Haematocrit and haemoglobin levels was also significantly reduced in the study subjects (P<0.001). Conclusions: Our study demonstrates that the level of antioxidant vitamins was low in the transfusion-dependent subjects. Therefore, its supplementation is necessary to improve the antioxidant defence system and obviate possible tissue damage.

INTRODUCTION
Blood transfusion is an important therapeutic approach especially in patients with refractory anaemia (Khaled et al., 2013, Antoine et al., 2015). One unit of red cell transfused provides more than 200 times the normal iron intake through intestinal absorption (Crichton and Ward, 2003) and there is no major mechanism of its excretion from the body (Hoffbrand et al., 2006, Kohgoet al., 2008). Hence, in transfusion dependence, iron released from transfused red cells inevitably saturate storage sites (Salibaet al., 2015) and the unbound (free) iron molecules are available to catalysethe conversion of molecular oxygen to highly reactive oxygen species (ROS) by Fenton chemistry (Adhamet al., 2014).In addition, ROS arealso generated from the normal metabolic processes, but neutralized by the body’s antioxidant defence (Bilbiset al., 2010, Mahdi, 2014). The rate of generation of ROS and its clearance by the antioxidant defence is delicately balanced (Pamplona, 2008) and If the production exceeds the capacity of the antioxidant systems to scavenge these species, then oxidative stress ensues (McCord, 2000).Withoxidative stress resulting from antioxidant depletion, peroxidative damage to cells is evident especially in tissues that stores iron such as liver, pituitary gland, pancreas and heart (Brissotet al., 2011).Antioxidantdefence systems are intimately involved in the prevention of cellular damage (Asare et al., 2009, Lobo et al., 2010). There are several enzymes and non-enzymatic defence such as vitamins and micronutrients (Simseket al., 2005) that scavenge free radicals and greatly enhance resistance to oxidative insult (Okochi and Okpuzor, 2005). This defence is significantly diminished in oxidative stress (Dhawan et al., 2005, Rashidiet al., 2011) due to increased consumption of the antioxidants (Mainasaraet al., 2016). However, antioxidants supplementation has been reported to improve antioxidant status and precludeoxidative damage by eliminating ROS (Prasad, 2009; Arruda et al., 2013;Tukuret al., 2015).Since transfusion-dependence is prone to oxidative stress and data concerning antioxidant vitamins and micronutrients in transfusion-dependent subjects in this environment are quite limited,it is imperative to measure the exogenous non-enzymatic antioxidant status and determine whether supplementation may be necessary.

METHODS
This cross-sectionalstudy was conducted at the University of Maiduguri Teaching Hospital (UMTH) and State Specialist Hospital Maiduguri, Borno State. The ethical committees of the hospitals approved the study protocol and written informed consent was sought for and obtained from all participants. A total of 101 subjects age 20-36 years who had =8 units of red blood cells transfusions per year; and an equivalent number of age and sex unmatched normal subjects who had no history of blood transfusion were recruited for the study. Hospital records were used to determine patient’s eligibility for participation. The case subjects had history of continuous treatment with blood transfusionfor the pass at least two years.
Demographic characteristics of the patients were obtained usinga structured questionnaire. About 5 mL of blood was aseptically collected from each subject and, 3 mL and 2 mL were separately dispensed into plain and EDTA bottles respectively. The serum was harvested by centrifugation of the samples at 4000 RPM for 5 minutes and stored in cyro-vial at -20 0C until analysis.
Serum ferritin was measured using immune-enzymatic assay kit from Monobind Inc. Lake Forest, C 92630, USA while serum Iron was analysed by NITRO-PAS colorimetric method as described by Makino and colleagues (1988) using Iron Nitro-Pas mono-reagent, Centronic GmbH Am Kleinfeld 11, 85456 Wartenberg, Germany.The anticoagulated blood samples collected were tested some haematological parameters using Auto Haematology Analyser LW D3600, Shenzhen Landwind Industry Co., Ltd. Spectrophotometric methods described by Hashim and Schuttringer, (1966) and Bassey et al.,(1946) were used to analysed serum vitamin E and vitamin A respectively, while serum zinc and copper were colorimetrically estimated as described by Knoellet al. (2009) and Bartnikas, (2012) respectively using Quanti-ChromTM Assay Kits from BioAssay Systems, Corporate Place, Hayward, USA.
Statistical Package for Social Sciences (SPSS) for windows, Version 22.0 (SPSS Inc., Chicago, IL, USA) was used to express data generated as Mean ± SD. Statistical comparison of observed variables were made using student’s t-test. The level of significance (P-value) was set at less than 0.05.

RESULT
Demographic and clinical characteristics of the study subjects are presented in table 1. Demographic information showed that the age of the case subjects was not significantly different (P>0.05) compared to that of the controls. The subjects were gender unmatched but predominantly male in both case and control participants (79.2% and 56.4% respectively). The underlying clinical condition for transfusion dependence was largely sickle cell anaemia (96%). Despite the regular transfusion therapy, significant decrease in haematocrit and haemoglobin levels (20.17± 4.17 and 6.56± 1.24 respectively) was noticed in the case subjects compared to the control participants (P0.000). Although iron overload was not reported in the case subjects, serum iron indices (serum iron and ferritin) were significantly (P 0.000) elevated compared to the controls. Serum antioxidant vitamins A, E and copper were significantly lower (P0.000 and P0.05 respectively) in the case subjects compared to the controls. However, serum zinc was moderatelyincreased but there was no statistically significant difference compared with the controls (P >0.05).

DISCUSSION
Disorders with refractory anaemia are managed with repeated blood transfusion and dependence on itis associated with iron accumulation that may leads to iron overload (Cid et al., 2014). Excess iron accumulation is accompanied by increased generation of reactive oxygen species (ROS) which may outweigh body’s antioxidant defence. Disruption of pro- and anti-oxidant balance is inevitably followed by oxidative stress (Simseket al., 2005;Vaculinet al., (2010)). Non-enzymatic antioxidant supplementation has been found to be beneficial in the prevention of oxidative stress (Rashidiet al., 2011).
The present study revealed that the serum iron indices (serum iron and ferritin)were significantly elevated(P <0.05)compared to the controls, which is consistent with several other studies (Rahu et al., 2008;Simseket al., 2005;Attiaet al, 2011). Higher ferritin content was directly linked to the accumulation of iron in the tissues of the patients. However, iron overload was not observed in the case subjects largely due to the low transfusion rate. This finding is inconsistent with Cid et al., (2014);Jokhioet al., (2009) and Gao et al.,(2014)who showed many patients with iron overloaded due to much higher transfusion rate.

The result of the current study indicate that the subjects were significantly deficient of serum antioxidant vitamins A and E.Similar results have been described in other studies by Tesoriereet al., (2001); Das et al.,(2004); Dhawan et al.,(2005) in patients with thalassemia and other transfusion-dependent entities. Ironically, non-transfused (non-iron overloaded) patients with SCD have also demonstrated low levels vitamins A and E due to chronic inflammation (Buisonet al., 2004).Similarly, Arruda et al.,(2013) reported deficiency of vitamin E in70% of non-transfusion dependent SCA patients. There was no iron overload in the present study that could promote iron-induced oxidative stress that consumes the antioxidant vitamins. However, given that 96% of the case subjects were SCA patients, the vitamin deficiency observed could be pursuant to increased pro-oxidant production in the sickle red blood cells (Titus et al., 2004).
In addition to the vitamins, abundant evidence demonstrates the role of micronutrients in protecting against free radical-induced oxidative damage (Rostanet al., 2002). Although Zincdeficiency is not relatively rare in patients with SCD (Prasad, 2002), our findings revealed that serum zinc is elevated but not significant (P>0.05) compared to the controls. Similarly, Mansi et al., (2009); Rashidiet al.,(2011);Mehdizadehet al., (2008) documented increased zinc level in transfusion-dependent thalassemia patients. Regular blood transfusion which predisposes to iron overload has been suggested to increase zinc level and prevent the zinc deficiency(Alexander et al., 2000). However, the subjects in the current study were regularly transfused but there was no iron overload noticed. Nile et al.,(2008) documented that zinc deficiency induces iron accumulation or conversely increase in zinc level lessen iron accumulation. Therefore, elevated zinc level observed in the current study might have been induced by decreased iron accumulation (absence of iron overload) orabnormal glomerular filtration of zinc as seen in chronic haemolysis (Mansi et al., 2009), obvious symptom of most underlying clinical entities of the present study. This phenomenon can be exploited in transfusion-dependent subjects to prevent iron overload and its attendant consequences by maintaining normal zinc level. Copper level was significantly high (P0.05)compared the control group and this agrees with some studies (Irshaidet al., 2009; Malakaret al., 2014). Copper has an antioxidant property (Shaziaet al., 2012) and its deficiency is quite rare (Turnlundet al., 1998).
This study revealed that haemoglobin and haematocrit were significantly (p<0.05) lower compared to the controls and is in accordance with the outcome of studies by Mansi et al., (2009)and Karim et al., (2016).Anaemia is the hallmark of transfusion dependency and is reported even in non-chronically transfused subjects with SCD (Fung et al., 2007).

Conclusion and Recommendations
Our findings indicated that there was no iron overload that may induce oxidative stress which could consume the antioxidants vitamins. However, the antioxidant vitamins were deficient. Therefore,administration of antioxidants vitamins may help strengthen the antioxidant defencesystem and obviate possible tissue damagearising from pro-oxidants.Further study is suggested on the status of free radicals and endogenous antioxidants to determine whether the deficiency is coupled with enhanced free radicals’ generation and reduced endogenous antioxidants.

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Haematological Profile of Alcoholics Residing in Port Harcourt Metropolis, Rivers State, Nigeria

Eze, Evelyn M., Jacob, Ransom B. and Christian, Serekara G.
Department of Medical Laboratory Science, Rivers State University, Port Harcourt
All correspondence to: Email: evelyn.eze@ust.edu.ng.

ABSTRACT
A study of haematological profile of alcoholics residing in Port Harcourt Metropolis was conducted on 150 subjects, comprising of 100 heavy alcohol consumers and 50 non-alcoholics who served as control with age ranging between 20-50 years (mean 35years). Male constituted 94(63%) and females 56(37%) of the studied population. Standard venepuncture technique was used to collect blood sample from each subject into ethylene diamine tetra acetic acid ((K3EDTA) anticoagulant containers and analysed for haemoglobin concentration, white blood cells, packed cell volume (PCV), total and differential WBC count, platelet count, erythrocyte count, mean cell volume, mean cell haemoglobin, mean cell haemoglobin concentration, mean platelet volume(MPV), platelet distribution width(PDW) and plateletcrit(PCT) using automated methodology.The results obtained were analysed using statistical package for social science (SPSS 21.0). Mean and standard deviation (S.D) were done for all the haematological variables and subjected to test of significance. Student t-test was used to compare the result between the alcoholics and non- alcoholics. For validation of observations, values below p<0.05 were considered statistically significant. The results obtained shows that the alcoholics had significantly lower values (P < 0.05) for erythrocytes, platelets, mean platelet volume, PCV, leucocytes, and haemoglobin concentration and significantly lower values (P < 0.05) in MCV, MCH and MCHC in alcoholics compared to non-alcoholics. Heavy alcohol intake has serious effects on haematological parameters which could predispose the consumers to infection and bone marrow malfunction or even stroke. Hence, it is important to establish baseline indices for these parameters on the basis of level of alcohol intake and determine its deleterious effect on these indices. It is equally recommended to include red cell indices as one of the routine laboratory tests required in the management of alcoholic patients.

Keywords: Haematological profile, alcoholics, haematological variables, Port Harcourt

INTRODUCTION

Alcohol abuse constitutes one of the major socio-economic and health problems in the world today, the abuse cut across gender, race and ethnicity and the effect vary with age. It has become a public health concern among Nigerian youths and adults strata of the society. Alcohol as an organic compound is composed of a hydroxyl functional group bound to carbon atom (Nic et al., 2006, Oyedeji et al., 2013). Alcohol addiction and dependence have become increasingly serious health and social problems with many clinical studies indicating that adolescence is a key period for the development of this addiction (Crew et al., 2007) and it has been generally acknowledged that both genetic and environmental factors contribute to the propensity to drink alcohol (Pohorecky, 1991).
Alcohol is the foremost contributing factor to the global bondage of avoidable diseases, disabilities and mortality among high, middle and low income earners. Heavy intake of alcohol is one of the key factors to premature deaths and has tremendous impact on public health. Alcohol
consumption has been linked to more than sixtymedical conditions and is also linked to categories of disease whose relative impact on the global burden is predicted to increase (Das et al., 2006; Daset al., 2011).

Indeed, there are variations in the biological response to alcohol consumption, which could be attributed to genetic differences. In low concentrations, alcohol reduces inhibitions and as blood alcoholic concentration increases in high concentration greater than 0.35grams/100 milliliters of blood (equivalent to 0.35grams/210 liters of breath) it affects the metabolism of many tissues and organs of the body. The organs most affected are the liver, bone marrow and the brain and the biochemical makers of alcohol abuse are mostly enzymes of the liver such as gamma glutamyltransferase (GGT), aspartate aminotransferase (AST) and alkaline phosphate (ALP) among which the GGT is the most sensitive (Adias et al., 2013; Das et al., 2011; Spoerke et al., 2010; Ejilemele and Orluwene, 2010).

In Nigeria and other developing countries in the world so many alcohol – related accidents and mortality may have occurred without reliable documentation. Chronic alcoholism is the third leading cause of mortality even though many alcohols related death go unrecorded particularly in developing countries (Latvala, 2005) Chronic alcoholism has significant serious consequences on the haematopoietic systems involving the various blood cells, their progenitors in the bone marrow and clotting components (Erhabor et al., 2014; Arjun and Chaitali, 2015; Das et al., 2005; Costa et al., 2007). It is estimated that 3.5% of the global burden of disease is attributed to alcohol (Rehm et al., 2003). Nutritional deficiencies are not only necessitated by poor dietary habits of alcohol abusers but by the effect of alcohol on the absorption, storage and utilization of several vitamins.

On the other hand, some findings from Ajani et al., (2012) revealed that wine may provide additional benefits over and above. Other researchers have been so intrigued by the possible benefits of low-dose alcohol that they have gone so far as to consider mechanisms by which alcohol can have salutary cardiovascular effects. Those mechanisms include: increased levels of high-density lipoprotein cholesterol and a decreased tendency to thrombosis. Coalescing these reported biological benefits with epidemiological findings, some medical organizations have stated that low levels of alcohol consumption may be considered safe or may be a legitimate item of discussion between physician and patient (Oduola et al., 2005).

Alcohol abuse have tremendous impact on transport medium in cardiovascular system and the system of effect has been established but there is considerable evidence which have adverse effect on serum proteins, blood cells and their progenitors in the bone marrow (Laurent and Edwards, 2014; Nikajet al., 2014).

Alcohol has numerous adverse effects on the various types of blood cells and their functions (Nikaj et al., 2014 Erhabor et al., 2014; Laurent and Edwards, 2014). Heavy alcohol consumption can cause generalized suppression of blood cell production and the production of structurally abnormal blood cell precursors that cannot mature into functional cells. Alcoholics frequently have defective red blood cells that are destroyed prematurely, possibly resulting in anaemia (Nikaj et al., 2014 Erhabor et al., 2014; Laurent and Edwards, 2014). Alcohol also interferes with the production and function of white blood cells, especially those that defend the body against invading bacteria. Consequently, alcoholics frequently suffer from bacterial infections. Alcohol adversely affects the platelets and other components of the blood-clotting system. Heavy alcohol consumption thus may increase the drinker’s risk of suffering a stroke

Haematological abnormalities are regularly seen among heavy drinkers and haemolysis is considered to be one of the most common causes (Arjun and Chaitali, 2015). A full haematological examination of the alcoholics include, red blood cell count, haemoglobin concentration, packed cell volume, mean cell volume, mean cell hemoglobin concentration, white blood cell count, platelet count and
cell morphology.
The aim of this study was to thoroughly assess some haematological parameters among alcoholics residing in Port Harcourt, Rivers State, Nigeria as a way of generating scientific proofs for strategic interventions that can help curb or forestall deleterious effects of alcohol in Nigeria.

Materials and Methods
Study Population
This cross sectional study was carried out on a population of 150 adults comprising of 100 heavy alcohol consumers (subjects) and 50 non-alcohol consumers (non-alcoholics) that were apparently healthy and served as controls. Consumers of at least two bottles (120 centilitres) of alcoholic beverages (beer, red wine and local gins) daily for at least a period of four years were designated as alcoholics and strictly none alcoholics with neither current nor past history of alcohol drinking were designated as controls. The subjects were between the ages of 20-60 years for both test and control. The samples were sourced from different parts of Port Harcourt, Rivers State, Nigeria.

Consent and Eligibility of Subjects
Informed consent was obtained from all the subjects and none of them was clinically ill at the time of study while unwilling subjects were excluded. All the subjects were administered questionnaire to determine the amount of alcohol they consumed daily and the duration before their blood samples were collected.

Sample Collection and Preparation
3ml of fresh venous blood was collected from each subject using a standard venepuncture technique into ethylene diamine tetra acetic acid ((K3EDTA) anticoagulant containers and analysed for haemoglobin concentration, white blood cells, packed cell volume (PCV), total and differential WBC count, platelet count, red blood cells count, mean cell volume, mean cell haemoglobin, mean cell haemoglobin concentration, mean platelet volume (MPV), platelet distribution width (PDW) and plateletcrit(PCT). Each sample was mixed thoroughly with K3EDTA to prevent lyses and ensure anticoagulation. Samples were analyzed within 2 hours of collection.

Method
The Mindray automated blood cell analyzer performs haematology analyses according to the radio frequency and direct current (RF/DC) detection method was used.

Principle of the Test
This method detects the size of the blood cells by changes in direct-current resistance, and the density of the blood cell interior by changes in radio-frequency resistance. A blood sample was aspirated and measured, diluted to the specified ratio, and sent to the applicable detector chamber. Inside the chamber is a tint hole called an “aperture,” on both sides of which are electrodes. Between the electrodes flow the direct current and radio-frequency current. Blood cells suspended in the diluted sample pass through the aperture, changing the direct-current resistance and radio-frequency resistance between the electrodes. The size of the blood cell is detected via changes in the direct-current resistance, and the density of the blood cell interior (size of the nucleus) is detected via changes in the radiofrequency resistance, with such detections coming in the form of electrical pulses. Based on the size of these pulses, a two dimensional distribution (scatter gram) of the blood-cell size and internal density can be drawn.

Statistical Analysis
Data were analysed using the SPSS 21.0 Windows statistical package and a student t-test was used to test for difference in haematological parameters between alcoholics and non-alcoholics. P<0.05 was considered as statistically significant.

RESULTS
The results obtained from the study are presented in Table 1.
Table 1: Haematological Parameters of the Study Population 

DISCUSSION
Alcohol exerts a direct toxic effect on the bone marrow resulting in vascuolization of the bone marrow and thrombocytopenia (Erhabor et al., 2010). It also affects the function of leukocytes and platelets (Nikaj et al., 2014; Laurent and Edwards, 2014). Haematological functions are affected directly, and can also cause chronic liver disease and other metabolic derangement (Arjun and Chaitali, 2015, Das et al., 2005).

Result from this study showed that alcohol has a wide spread effect directly or indirectly on haematological system which affect the leukocytes, erythrocytes and thrombocytes production which is also in consonant with the findings of Erhabor et al., (2010); Nikaj et al., (2014); Laurent and Edwards, (2014). Alcohol exerts a direct toxic effect on the bone marrow resulting in vascuolization of the bone marrow and thrombocytopenia. Haematological functions are affected directly, and can also cause chronic liver disease and other metabolic derangement (Arjun and Chaitali, 2015; Das et al., 2005).

Values obtained for haematological parameters in alcohol drinkers showed a significantly difference in haemoglobin concentration and packed cell volume while platelet is reduced which could be attributed to ethanol which is the basic constituent of alcohol that has been reported to cause neutrophil and lymphocyte impairment with increased frequency and severity of infections in alcoholics. However, there are differences in the effect of alcohol from one individual to another.

According to other research by Oduola et al., (2004) when the beverage is sipped, the peak of blood -alcohol concentration gets lower than when it is drunk, the activity of the enzyme in metabolism also aggravates as the percentage of alcohol to the blood is not constant after consumption but drops steadily when alcohol is ingested in combination with food. Nutritional differences are not only necessitated by poor dietary habits of alcohol abusers but by the effect of alcohol on the absorption, storage and utilization of several vitamins.
This study has clearly shown that alcohol intake has serious effects on some haemotological parameters which could predispose the consumers to infection and bone marrow malfunction. Therefore, educating the society of the haematological health risk of the social behaviour is advocated.

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Evaluation of Ameliorative Effects of A Novel Insulinotropic Agent, Archachatina Marginata Haemolymph on Physiological Lipoproteins in Streptozocin induced Diabetic Rats

Adeboye S.K
Department of Chemical Pathology, Obafemi Awolowo University Teaching Hospital Complex, Wesley Guild Hospital Unit, Ilesa, Osun State, Nigeria.
Department of Chemical Sciences, Joseph Ayo Babalola University, IkejiArakeji.
Ogundajo A.T
Department of Chemical Pathology, Obafemi Awolowo University Teaching Hospital Complex, Wesley Guild Hospital Unit, Ilesa, Osun State, Nigeria.
Department of Biochemistry, Ekiti State University, Ado Ekiti, Ekiti State.
Ajayi O.O
Department of Chemical Pathology, Obafemi Awolowo University Teaching Hospital Complex, Wesley Guild Hospital Unit, Ilesa, Osun State, Nigeria.
All correspondence to: Dr Ogundajo A.T, Department of Biochemistry, Ekiti State University,
Ado Ekiti, Ekiti State. E-mail : adetap1977@gmail.com

INTRODUCTION

Diabetes mellitus is a group of chronic metabolic disorder characterized by a high blood glucose concentration caused by insulin deficiency, often combined with insulin resistance (Teixeira et al., 2000). Diabetes mellitus is a major cause of disability and hospitalization and it results in significant financial burden (Vats et al., 2002).Diabetes mellitus is associated with an increased risk of thrombotic, atherosclerotic and cardiovascular disease. About 70- 80% of deaths in diabetic patients are due to vascular disease (Chattopadhyay and Bandyopadhyay, 2005). Hyperglycemia, the primary clinical manifestation of diabetes, is thought to contribute to diabetic complications by altering vascular cellular metabolism, vascular matrix molecules and circulating lipoproteins (Chattopadhyay and Bandyopadhyay, 2005). Globally, as of 2013, an estimated 382 million people have diabetes worldwide, with type 2 diabetes making up about 90% of the cases. This is equal to 8.3% of the adults’ population with equal rates in both women and men (Vos et al., 2012). Worldwide in 2012 and 2013, diabetes resulted in 1.5 to 5.1 million deaths per year, making it the 8th leading cause of death (Cooke and Plotnick, 2008). The increasing number of ageing population, consumption of calories rich diet, obesity and sedentary life style have led to a tremendous increase in the number of diabetics world-wide. Pharmacological means (insulin and oral hypoglycemics) as well as non-pharmacological means (diet and exercise) may be used in the management of diabetes mellitus. The doubts about the efficacy and safety of the oral hypoglycemic agents have prompted a search for safer and more effective drugs in the treatment of diabetes (Reaven, 1983). Recent findings from our laboratory suggested that Archachatina marginata haemolymph potentiates hypoglycemic effect by mimicking insulin in streptozotocin -induced diabetic rats where Archachatina marginata haemolymph of 2ml regimen significantly improved the insulin concentration over the 1ml regimen (Adeboye et al., 2017). This is also reflected in the gradual and consistent reduction in the blood glucose concentration as opposed to the consistent increase in the diabetic control group. Since Diabetes mellitus is associated with a large number of lipid abnormalities with evidence confirming the pivotal role of hyperlipemia, mainly elevated blood cholesterol, particularly LDL cholesterol and VLDL cholesterol in the development of atherosclerosis-related disease( Pyorala et al.,1987), this study evaluate the ameliorative effect of Archachatina marginata haemolymph on physiological lipoproteins in streptozocin induced diabetic rats.

Key Words: Diabetes mellitus, Lipoproteins, Snail Haemolymph, Streptozocin, Albino rats

MATERIALS AND METHOD

Streptozotocin (STZ) was obtained from Sigma-Aldrich (Germany). All other chemicals used were of analytical grade and obtained from FLUKA, BDH (Germany) and other standard commercial suppliers.
Animals Healthy female albino rats were acquired from the animal holding unit of the Institute for Advanced Medical Research and Training (IMARAT), University College Hospital, University of Ibadan. Animals were maintained with food and water ad libitum and under a 12-h light/12-h dark cycle. African Giant Snails were bought from Ipetu-Ijesa Market and taken to the department of animal science, Joseph Ayo Babalola University, IkejiArakeji for authentication. The “principle of laboratory animal care” (National Institute of Health-NIH publication No. 85- 23) guidelines and procedures were followed in this study (NIH publication revised, 1985). The ethical committee of the department of chemical sciences, Joseph Ayo Babalola University, Ikeji Arakeji approved the research work.
Preparation of Archachatina marginata Snail Extract

The whole snails were washed with copious amount of tap water and then rinsed with distilled water. The apex of the snail was opened by method adopted from Akinloye and Olorode (2000) and the haemolymph collected into a clean beaker. It was stored at 40 C

Animal Studies.
The rats were set into four groups (n=5).
Group 1- Non Diabetic Control (NDC). Rats treated with rat diet and water ad libitum.
Group2- Diabetic Control (DC). Induced rats
Group3- Diabetic rats with 1ml administration of snail haemolymph (DSS1)
Group4- Diabetic rats with 2ml snail haemolymph administration (DSS2)

After the initial two weeks period of acclimatization, the animals were fasted overnight but allowed access to water ad libitum. The blood glucose concentration of each rat was taken and thereafter, they were weighed and then administered with 55mg/kg STZ in citrate buffer (pH 4.5) peritoneally (Garza-Rodea et al., 2010) except non-diabetic control group (NDC) that received citrate buffer only.

The rats were returned to their respective cages and fed glucose laden water to guide against the immediate hypoglycaemic effect of STZ administration. After three days, the blood glucose levels of the rats were again checked to ascertain successful diabetes inducement with the presence of hyperglycaemia. Rats with blood glucose level of 200mg/dl and above were considered diabetic and used as diabetic animals in subsequent studies.
Treatment with Archachatina marginata Snail Haemolymph
The snail haemolymph was administrated orally using the gastro-enteral cannula. 1ml of haemolymph was administered to rats in DSS1while 2mls was administered to those in DSS2. The diabetic control, DC, received 2mls distilled water. Administration was done once a day in the morning for fourteen (14) consecutive days
Blood Sample Collection
At the end of the fourteenth day administration, the animals were fasted overnight. The rats were sacrificed by cervical dislocation before slicing their carotid artery with blade and the flowing blood samples were collected into appropriately labeled Lithium heparin bottle for lipid profile analysis. The blood samples were spun at 3,000 rpm for 10 minutes using the centrifuge. The plasma was carefully transferred to appropriately label plain tube with the aid of a Pasteur pipette. Samples were immediately stored at – 4°C till analysis.

Determination of Concentration of Plasma Total cholesterol
Plasma total cholesterol concentration was estimated spectrophotometrically according to the method of Burtis and Edward (1999).

Determination of Concentration of Plasma high density lipoprotein cholesterol
Plasma high density lipoprotein cholesterol concentration was estimated spectrophot-ometrically according to the method of Burtis and Edward (1999).

Determination of Concentration of Plasma Low density lipoprotein-cholesterol
Plasma Low-density lipoprotein-cholesterol is analysed mathematically using Friedwald Equation.

Determination of Concentration of Plasma Triacylglyceride
Plasma triacylglyceride concentration was estimated spectrophotometrically according to the method of Burtis and Edward (1999).

STATISTICAL ANALYSIS
All data were subjected to one way analysis of variance. The mean and standard error of mean were used for statistical analysis. The statistical significance between the control and each treated groups were determine using student t-test. The level of significance was set at P< 0.05.

RESULTS
Effects of Snail Haemolymph on Blood triglyceride in STZ-Induced Diabetic Rats
Triglyceride concentration is significantly elevated between NDC and other groups whereas the decrease between DC and DSS1/DSS2 are not significant statistically (p<0.05).NDC=13.8 ± 0.11; DC=45.90 ±7.10; DSS1=36.21 ± 1.0; DSS2=33.55 ± 1.0.

Results are represented as Means ± SEM of five independent determinations of Triacylglycerol in mg/dl. Bars carrying alphabets have significant differences ( p<0.05) while those with same superscripted alphabets are not significant statistically (p>0.05).

Effects of Snail Haemolymph on Serum Total Cholesterol of STZ-induced Diabetic Rat Total cholesterol concentration is significantly increased (p<0.05) when NDC is compared with other Groups. There is a significant decrease also between DC and DSS1/DSS2, DSS1 and DSS2(p?0.05). NDC= 14.25 ± 2.5; DC= 60.55 ± 1.5; DSS1= 48.41 ± 0.9; DSS2= 42.83 ± 0.2.

Results are represented as Means ± SEM of five independent determination of total cholesterol in mg/dl. Bars carrying different superscript alphabets have significant differences statistically p< (0.05).

Effects of Snail Haemolymph on LDL of STZ-induced Diabetic Rats

LDL concentration is significantly increased (p<0.05) when DC/DSS1/DSS2 was compared with NDC and when DC was compared with DSS2. However DC and DSS1, DSS1 and DSS2 show no significant differences (p>0.05).NDC = 9.07 ± 2.50 mg/dl; DC =39.95± 5.00 mg/dl; DSS1 = 31.45 ±5.50; DSS2 = 27.56 ± 1.50 mg/dl.

Results are represented as Means ± SEM of five independent determination of LDL- cholesterol in mg/dl. Bars carrying different superscript alphabets have significant differences statistically (p<0.05).

Effects of Snail Haemolymph on Serum HDL in STZ induced Diabetic Rats
HDL concentration significantly increased (p<0.05) when DC/DSS1/DSS2 were compared with NDC .However there was no significant differences between DC and DSS1 and DSS1 and DSS2 a (p>0.05). NDC=1.91±0.80; DC=7.28±0.25; DSS1= 5.41± 0.09; DSS2=4.78±0.7.

Results are represented as Means ± SEM of five independent determination of HDL- cholesterol in mg/dl. Bars carrying different superscript alphabets have significant differences statistically (p<0.05).

DISCUSSION
Hyperlipidemia is a recognized complication of DM characterized by elevated levels of cholesterol, Triglyceride and phospholipids and changes in lipoprotein compositions (Segal et al., 1984). Risk of hypertension and other coronary heart diseases are associated with DM as precipitated by hyperlipidemia. Snail haemolymph has been found to reduce both systolic and diastolic blood pressure at a slow rate (Dede et al., 2003).
Snail haemolymph administration brings an apparent decrease in the lipid panel of the induced rat. Statistically, triglycerides concentration increased significantly in induced animals and are not decreased significantly by the administration of snail haemolymph in both regimen of 1ml and 2 ml though there is apparent decrease observed. Significantly, total cholesterol concentration was reduced by treatment with the snail haemolymph. The reduction by increasing the regimen to 2mls over 1 ml is also significant (p<0.05). A parallel had been drawn between hyperlipidemia and hypertension. This decrease may not be in variance with the findings of Dede et al., (2003) that snail haemolymph reduce high blood pressure by exerting hypolipidemic effect.
Low density lipoprotein concentration was increased also by induction of DM with STZ. The effect of snail’s haemolymph however is apparent but not significant when 1 ml was administered but significantly reduced when 2mls was administered. Lowering of LDL being the bad cholesterol is expected to be part of the target of any useful medication for treatment of DM and its complications.
HDL pattern is in variance with the early works (Berbera et al., 1997 Murali et al., 2002) on streptozotocin induced DM with the significant increase in concentration which is lowered by administration of haemolymph. HDL is the good cholesterol which is desirable as they transport cholesterol to the liver for metabolism and possible excretion of excess cholesterol as conjugates. This lowering effect needed to be investigated further.

CONCLUSION
The reduction observed in lipid profile of the treated rats, thereby reducing the risk of coronary heart disease and hypertension is an encouragement. The results summarily express hope at higher concentration of administered haemolymph as significant improvement was observed in all parameters tested in the 2mls administered haemolymph over 1ml and apparent improvement of 1ml over the non-treated diabetic control (DC). The indiscriminate lowering of HDL as observed also call for caution in the administration of snail haemolymph on diabetes.

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