THE INTERNATIONAL JOURNAL OF SCIENCE & TECHNOLEDGE

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1 THE INTERNATIONAL JOURNAL OF SCIENCE & TECHNOLEDGE Investigation of the in Vitro Effects of Some Oral Contraceptives on Human HBSS Erythrocyte Fragility and Gelation Dr. Ngozi Franca Okoye Lecturer, Department of Biochemistry, University of Port Harcourt, Nigeria Augustine Amadikwa Uwakwe Professor, Department of Biochemistry, University of Port Harcourt, Nigeria Edward Obiozor Ayalogu Professor, Department of Biochemistry, University of Port Harcourt, Nigeria Abstract: In this study, the in vitro effect of oral contraceptives namely Microgynon a combined pill (0.15mg levonorgestrel and 0.03mg ethinylestradiol), Primolut -N a mini pill (5mg norethisterone) and postinor a post coital pill on human HbS erythrocyte fragility and gelationon were investigated. The results from this study showed that the drugs inhibited haemoglobin gelation and microgynon showed the highest inhibition (0.010 vs control 0.050) followed by primolut N (0.020 vs control0.080) and postinor (0.060 vs control 0.080). It has been discovered that certain drugs or medications taken for certain ailments are either prosickling or antisickling in nature The results of this study have revealed that the drugs have antisickling properties and might not pose a risk or threat to HbSS subjects who may have cause to use the drug. Keywords: Blood, Ethinylestradiol, Haemoglobin, Levonorgestrel, Norethisterone, Oral contraceptives, Sickle cell. 1. Introduction Sickle-cell anaemia is an inherited disease and it occurs whena person inherits two abnormal copies of the haemoglobin gene, one from each parent. It is a blood disorder originating from the fact that there is an abnormality in the oxygen-carrying protein haemoglobin found in red blood cells. This leads to a rigid, sickle-like shape under certain circumstances. Problems in sickle cell disease typically begin around 5 to 6 months of age. A number of health problems may develop, such as attacks of pain ("sickle-cell crisis"), anaemia, bacterial infections, and stroke. Long term pain may develop as people get older. The average life expectancy in the developed world is 40 to 60 years (Husebye et al., 2014; Biagoli, et al., 2009; Adams, 2005). A sickle cell crisis attack can be set off by temperature changes, stress, dehydration, and high altitude. A person with a single abnormal copy does not usually have symptoms and is said to have sickle-cell trait. Such people are also referred to as carriers. Diagnosis is by a blood test and some countries test all babies at birth for the disease. Diagnosis is also possible during pregnancy. The care of people with sickle-cell disease may include blood transfusion. A small proportion of people can be cured by a transplant of bone marrow cells. (Adams, 2005; Almeida and Roberts, 2005; Anie, 2005).The sickle cell mutation is caused by a single change in the amino acid building blocks of the oxygen-transport protein, hemoglobin. This protein, which is the component that gives red cells their color, has two subunits. The alpha subunit is normal in people with sickle cell disease. The beta subunit has the amino acid valine at position 6 instead of the glutamic acid that is normally present. This alteration is the origin of all the problems that occur in people with sickle cell disease. This change endows Haemoglobin S when deoxygenated with a new property, the capacity to polymerize. This property conspires against an indispensable feature of the red cell. (Chikezie, 2011; Anie, 2005). As of 2013 about 3.2 million people have sickle-cell disease while an additional 43 million have sickle-cell trait. About 80% of sickle-cell disease cases are believed to occur in sub-saharan Africa. It also occurs relatively frequently in parts of India, the Arabian peninsula, and among people of African origin living in other parts of the world. In 2013, it resulted in 176,000 deaths, up from 113,000 deaths in 1990 (Chikezie et al., 2013, Husebye et al., 2014; Biagoli, et al., 2009; Adams, 2005). The polymerization of deoxy Hbs is the primary and indispensable event in the molecular pathogenesis of sickle cell disease. The polymer has the form of an elongated rope- like fibre which usually aligns with other fibres forming a fascile and distorting the red cell into the classic crescent or sickle shape, among other many abnormal shapes, and resulting in a marked degree in cell deformability (Chikezie et al., 2013; Kabanova, et al., 2009). Haemoglobin-S can polymerize when oxygenated and depolymerize when reoxygenated infinitely, however, the sickle erythrocyte membrane can withstand only a finite number of these cycles before it is irreversibly deformed and recognized via the many abnormal sickle shapes that circulate in patients (Anie, 2005). 27 Vol 5 Issue 7 July, 2017

2 Polymerization alone does not account for the physiology of sickle cell disease. Changes in red cell membrane structure and function, disordered red cell volume control, also contribute to the physiology of the disease. (Anie, 2005; Ulkeret al., 2009). Oral contraceptives (OCs) are drugs taken orally for the prevention of pregnancy. Nowadays, OCs are widely available and used for family planning. It is estimated that the drugs are now used by more than 100 million women throughout the world (Okoye and Uwakwe, 2016; Kuhl and Goethe, 1990; CHPE, 1984; Bremner, et al 1977; Kay et al., 1974). The Population Reference Bureau (2005) reported that 12% of Nigerian married women are on contraceptives out of which 8% use modern methods of contraception. The National Cancer Institute (2003) stipulated that there are currently two types of OCs available. The most commonly prescribed OC contain two man made versions of the female hormones (estrogen and progesterone) that are similar to the hormones the ovaries normally produce. The second type of OC available is called the mini pill. It contains only a progestogen. The oral contraceptives: Microgynon a combined pill (0.15mg levonorgestrel and 0.03mg ethinylestradiol), Primolut- N a mini pill (5mg norethisterone) and postinor a post coital pill (75mg levonorgestrel) are among the most common drugs used for contraception and also for other non - contraceptive benefits (Okoye et al., 2012; Briggs, 1980). These oral contraceptives have been known to have some side effects ranging from nausea to cancer (Okoye and Uwakwe 2016; The National Cancer Institute 2003). Initial oral contraceptive formulations contained very high levels of synthetic estrogen and progesterone, based on the assumption that these levels were necessary to prevent pregnancy (Skouby and Jesperson, 1990). It is worthy to note that because of problems associated to oral contraceptives, the manufacturers of these oral drugs have continually decreased hormone levels in order to provide formulation with maximum efficiency and minimum side effects (Okoye et al., 2011; Kaunitz, 2004; Grimes et al., 1993). It is also known that most sickle cell patients usually experience severe painful crisis and over the years research has been ongoing to find out the drugs that sickle cell patients can tolerate. Furthermore, it has been discovered that certain drugs or medications taken for certain ailments are either prosickling or antisickling in nature. Several works have been carried out in the past to determine the effect of various drugs taken for other ailments on sickle cell haemoglobin gelation. However, not much information exit in literature to reveal the effect of oral contraceptives on HbS erythrocyte fragility and gelation. Therefore, the objective of this work is to investigate the effects of three different types of oral contraceptives with varying hormonal levels on the sickle cell erythrocyte fragility and gelation. 2. Materials and Methods Microgynon was purchased from Schering AG Germany. Primolut- N purchased from Medipharm (Pvt) Ltd., Lahore Licencee of Schering AG. Federal Republic of Germany. Postinor was purchased from Chemical works of Gedeon Ritcher Ltd. Budapest Hungary Determination of Erythrocyte Fragility and Gelation Sample collection for in vitro tests. Blood samples were collected from a lady aged 28years into a heparin bottle. The blood samples were confirmed as HbSS using standard haemoglobin electrophoresis and was supplied by RAHA laboratories. All experiments were carried out with fresh heparinized blood. Reagents: Normal saline, phenyl alanine, distilled water, sodium chloride, sodium metabisulphite, deionized water. Principle: Haemoglobin-S undergoes gelation when deprived of oxygen. Sodium metabisulphite was used as a reductant. Method: Haemoglobin-S gelation (polymerization) experiment. The haemoglobin polymerization (gelation) experiment was based on the method described by Noguchi and Schechter (1985). Control experiment: Sodium metabisulphite (4.8ml), 0.1ml of HbSS haemosylate and 0.1ml of normal saline were quickly mixed in a cuvette and the absorbance read at 540nm, and at 1 minute intervals for 5 minutes. Test experiment: Sodium metabisulphite (4.8ml), 0.1ml of HbSS haemosylate and 0.1ml of test compound were quickly mixed in a cuvette and the absorbance read as was the control. 3. Statistical Analysis All data were subjected to statistical analysis. Values are reported as mean ± standard deviation (SD) while one way ANOVA was used to test for differences between treatment groups. The results were considered significant at p-values of less than 0.05, that is, at 95% confidence level (p<0.05). 4. Results The results of the effect of microgynon on Hb gelation are presented below. The results showed that the drug decreased Hb gelation. The differences in dosage and time were statistically significant on the effect of the drug on the Hb gelation levels at 95.0% confidence level (P < 0.05). 28 Vol 5 Issue 7 July, 2017

3 Microgynon Erythrocyte fragility and Hb gelation (optical density) standard = Control ± 0.05 ± 0.05 ± 0.05 ± 0.05 ± 0.05 ± 0.36 ± 0.04 ± 0.04 ± 0.04 ± 0.03 ± 0.03 ± 0.01* 0.72 ± 0.03 ± 0.03 ± 0.03 ± 0.02 ± 0.02 ± 0.01* 1.40 ± 0.02 ± 0.02 ± 0.02 ± 0.02 ± 0.01 ± 1.80 ± 0.02 ± 0.02 ± 0.01 ± 0.01 ± 0.01 ± 3.60 ± 0.01 ± 0.01 ± 0.01 ± 0.01 ± 0.01 ± Table 1: In vitro effect of microgynon on human erythrocyte fragility and Hb gelation *Statistically significant at 95% confidence level, (P < 0.05). The results of the effect of primolut - N on Hb gelation are presented below. The results showed that the drug decreased Hb gelation. The differences in dosage and time were statistically significant on the effect of the drug on the Hb gelation levels at 95.0% confidence level (P < 0.05). Primolut - N Erythrocyte fragility and Hb gelation (optical density) standard = Control ± 0.05 ± 0.05 ± 0.05 ± 0.07 ± 0.08 ± 1 ± 0.02 ± 0.02 ± 0.02 ± 0.03 ± 0.04 ± ± 0.02 ± 0.02 ± 0.02 ± 0.03 ± 0.03 ± 0.01* 4 ± 0.01± 0.01 ± 0.01 ± 0.01 ± 0.02 ± 0.02* 5 ± 0.01 ± 0.01 ± 0.01 ± 0.02 ± 0.02 ± 0.01* 10 ± 0.01 ± 0.01 ± 0.01 ± 0.02 ± 0.02 ± Table 2: In vitro effect of Primolut - N on human erythrocyte fragility and Hb gelation *Statiscally significant at 95% confidence level, (P < 0.05). The results of the effect of postinor on Hb gelation are presented below. The results showed that the drug decreased Hb gelation. The differences in dosage and time were statistically significant on the effect of the drug on the Hb gelation levels at 95.0% confidence level (P < 0.05). Postinor Erythrocyte fragility and Hb gelation (optical density) standard = Control ± 0.05 ± 0.05 ± 0.05 ± 0.07 ± 0.01* 0.08 ± 0.01* 1.50 ± 0.02 ± 0.03 ± 0.04 ± 0.04 ± 0.04 ± 0.01* 3.00 ± 0.02 ± 0.03 ± 0.03 ± 0.04 ± 0.04 ± 0.01* 6.00 ± 0.02 ± 0.02 ± 0.04 ± 0.05 ± 0.01* 0.06 ± 0.02* 7.50 ± 0.02 ± 0.02 ± 0.04 ± 0.05 ± 0.05 ± 0.01* ± 0.01 ± 0.02 ± 0.04 ± 0.04 ± 0.01* 0.02 ± Table 3: In vitro effect of postinor on human erythrocyte fragility and Hb gelation *Statiscally significant at 95% confidence level, (P < 0.05). 5. Discussion This study has looked at the in vitro effect of microgynon, a combined oral contraceptive pill (0.15mg levonorgestrel and 0.03mg ethinylestradiol), primolut N a mimi pill (5mg norethisterone) and postinor a post coital pill (0.75 levonorgestrel) on erythrocyte fragility and gelation. The results of the in vitro studies of the erythrocyte fragility and gelation are presented on Tables 1 to 3.The drugs inhibited haemoglobin gelation and microgynon showed the highest inhibition (0.010 vs control 0.050) followed by primolut N (0.020 vs control0.080) and postinor (0.060 vs control 0.080). It has been discovered that certain drugs or medications taken for certain ailments are either prosickling or antisickling in nature (Uwakwe and Akhidue, 2000; Uwakwe etal 2002). 29 Vol 5 Issue 7 July, 2017

4 Sickle cell disease is genetic disorder which has over the years proved difficult to manage which results from the abnormal haemoglobin S. So it is of immense benefit that the side effects of most medications or drugs on the sickle cell patients are realised before they are prescribed these drugs. The case of the oral contraceptives is made even more crucial as it is taken by an otherwise healthy individual. This study is in accordance with the several works that have been carried out in the past to determine the effect of various drugs taken for other ailments on sickle cell haemoglobin gelation (Chikezie, et al., 2013; Ibegbulem, et al., 2011; Miera, et al., 2011; Uwakwe and Akhidue, 2000; Uwakwe and Onyeike, 2005). The biochemistry of haemoglobin S and the hypothesized pathophysiologic mechanisms of complications provide principles for treatment of complications in sickle cell syndromes. These principles must be applied in preventing and treating almost every complication observed during the clinical course of patients. Also good medical as well as nutritional management has helped immensely in reducing the incidence of death among sicklers. Most of these treatments are aimed at alleviating one or more of the complications that accompany crisis (Chikezie, 2011; Bianchi, et al., 2009; Ekekeet al., 2000, Uwakwe et al., 2002). At present time, the important aspect of treatment is supportive care with fluids and analgesics and the judicious use of transfusion. Most currently some chemicals have been found to be effective in preventing or managing sickle cell disease in various way. These includes nutritional supplement sicklevit an antisickling formula that have been formulated. It exerts its effects in reducing the frequency of crisis occurring and act as a maintenance formula(lee, 2015; Chikezie and Uwakwe, 2011; Lambrou, et al., 2009; Ballas, 2005). The results of this study have revealed that the drugs have antisickling properties and might not pose a risk or threat to HbSS subjects who may have cause to use the drugs as contraceptives or for other health benefits. 6. Acknowledgement Thanks to the staff of RAHA laboratories, Rumumasi Port Harcourt. 7. References i. Adams, RJ (2005). TCD in sickle cell disease: An important and useful test paediatric radiology (35): ii. Almeida, A, Roberts I (2005). Bone Involvement in sickle cell disease. British Journal of Haematology 129: iii. Anie, KA (2005). Physiological complications in sickle cell diseases. British Journal of Haematology 129: iv. Ballas, SK, Lusardi, M (2005) Pain Management of sickle cell disease. Haemotology clinics of North America 19: v. Bianchi, N, Zucatto, C, Lampronti, I, Borgatti, M, Gambari, R (2009). Fetal haemoglobin inducers from natural world. A novel approach for identification of drugs for the treatment of β thalassemia and sickle cell amaemia. 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