Asian Journal of Medical Sciences 3(6): 234-236, 2011 ISSN: 2040-8773 © Maxwell Scientific Organization,2011 Submitted: September 21, 2011 Accepted: November 16, 2011 Published: December 25, 2011

Comparative Stabilizing Effects of Some Anticoagulants on Fasting Blood Glucose of Diabetics and Non-diabetics, Determined by Spectrophotometry (Glucose Oxidase)

Nwangwu C.O. Spencer, Josiah J. Sunday, Omage K. Erifeta. O. Georgina, Asuk A. Agbor, Uhunmwangho S. Esosa and O. Jenevieve Department of Biochemistry, College of Basic Medical Sciences, Igbinedion University, Okada, Edo State, Nigeria

Abstract: The comparative stabilizing effects of the anticoagulants; Fluoride oxalate, EDTA and Heparin on fasting blood glucose level were determined, using the spectrophotometry (glucose oxidase) method. Fasting blood samples were taken from ten (10) diabetic patients and ten (10) non-diabetic people, and the blood glucose levels determined at 30 min intervals for a maximum time of 2 h. Our results showed that the rate at which plasma glucose changes with time varies with specific anticoagulants. With Fluoride oxalate and Heparin, it increased by 1.77 and 6.67%, respectively, while with EDTA it decreased by 4.0%, within the first 30 minutes for non-diabetics. For diabetics, within the same period, with Fluoride oxalate and Heparin it increased by 1.9 and 3.7%, respectively, while with EDTA it decreased by 3.6%. Blood glucose levels were however shown to increase significantly (p<0.05) at 60 min (1 h) with the three anticoagulants in diabetics and non-diabetics. But at 120 min (2 h) plasma glucose in Fluoride oxalate, EDTA and Heparin decreased by 16.4, 17.5 and 8.5%, respectively for non-diabetics and by 10.6, 9.8 and 8.6%, respectively for diabetics. These showed that Fluoride oxalate had a better stabilizing effect on plasma glucose within the first 30 minutes while Heparin is a better stabilizer at after 120 min.

Key words: Anticoagulants, EDTA, fluoride oxalate, glucose oxidase, heparin, plasma glucose

INTRODUCTION (EDTA), fluoride oxalate etc. Heparin acts by forming a complex with anti-thrombin; this complex inhibits factor Blood glucose refers to the amount of glucose present X (stuart prower) and thrombin which is required in the in a mammal’s blood. Normally, the blood glucose level formation of fibrin. EDTA acts as a chelator and binds to is maintained at a reference range between 4 to 6mM. calcium which is required for coagulation thereby making Glucose can be measured in whole blood, serum or calcium unavailable and inhibiting clotting. Fluoride plasma. An anticoagulant is a substance that prevents the oxalate acts as an inhibitor of glycolysis. clotting of blood. Anticoagulants can be used Heparin is a naturally occurring anticoagulant endogenously or/and exogenously. The endogenous produced by basophils and mast cells (Guyton and Hall, anticoagulants help prevent formation of hard clots in the 2006). Heparin, a highly sulfated glycosaminoglycans, is blood by decreasing the ability of the blood to clump widely used as an inject-able anticoagulant and has the together. They are also called “blood thinners”. They highest negative charge density of any known biological include medications that slow-down blood clotting time. molecule (Cox and Nelson, 2004). Native heparin is a The exogenous anticoagulants are used during analysis of polymer with a molecular weight ranging from 3 kDa to blood samples. As medication, anticoagulants are 50 kDa, although the average molecular weight of most administered to patients to stop thrombosis (blood clotting commercial heparin preparations is in the range of 12 kDa inappropriately in the blood vessels). This is useful in the to 15 kDa (Bentolila et al., 2008). Heparin is a linear primary and secondary prevention of deep vein poly-disperse polysaccharide consisting of repeating units thrombosis, pulmonary embolism, myocardial infarction of alpha 1÷4 linked pyranosyluronic acid and 2-amino-2- and strokes in those who are predisposed (Schrezenmeier, deoxyglucopyranose (glucosamine) residues (Comper, 1995). 1981). The exogenous anticoagulants are compounds that EDTA is a colorless, water-soluble solid produced on have been developed using several mechanisms of action. a large scale for many applications. Its prominence as a They include heparin, Ethylene Diamine Tetra-acetate chelating agent arises from its ability to “sequester” di-

Corresponding Author: Omage Kingsley, Department of Biochemistry, College of Basic Medical Sciences, Igbinedion University, Okada, Edo State, Nigeria 234 Asian J. Med. Sci., 3(6): 234-236, 2011 and tricationic metal ions such as Ca2+ and Fe3+. After were then centrifuged and the plasma separated from the been bound by EDTA, metal ions remain in solution but blood cells. exhibit diminished reactivity. The compound was first described in 1935 by Ferdinand Munz, who prepared it Glucose determination: The concentrations of glucose in from ethylenediamine and chloroacetic acid (Munz and the plasma were determined immediately after collection Robert, 1987). Today, EDTA is mainly synthesized from spectrophotometrically, using Glucose-oxidase test kit ethylenediamine (1,2-diaminoethane), formaldehyde (from Randox Laboratory Limited, U. K.). The procedure (methanal), and sodium cyanide. EDTA is used to was repeated at every 30 min interval for 2 h. detoxify metal ions in chelation therapy for e.g. mercury and lead poisoning (Debusk et al., 2002). Similarly, it is RESULTS AND DISCUSSION used to remove excess iron from the body. EDTA exhibits low toxicity with LD50 of 2.0-2.2 g/kg. It has been found The assay of glucose in blood samples stored in to be cytotoxic and weakly genotoxic in laboratory bottles containing anticoagulants is a common practice in animals. Oral exposures have been noted to cause this part of the world. When blood samples are collected, reproductive and developmental effects (Lanigan and they are stored in their native state by preserving them in Yamarik, 2002). anticoagulant bottles (Oduola et al., 2006). Our results In testing for blood glucose level, sodium fluoride is showed that though their native state was preserved, the used as a glycolytic inhibitor to preserve the blood blood glucose level when assayed in different glucose level combined with anticoagulant, potassium anticoagulant bottles at different time varies. Trinder oxalate. It has long been recognized that blood sample is (1969) reported that plasma glucose levels remain quite subjected to in vitro glycolysis, which is enhanced by the stable for 6 h at room temperatures (25-35ºC). This presence of leucocytes and diminished by sample however is not in agreement with our results as it seems refrigeration (Totsoi, 2004). Most researchers have that plasma glucose levels are not exactly stable within concluded that addition of sodium fluoride to blood the stated time. It was observed that the rate at which sample is capable, in at least some parts of decreasing ex- plasma glucose changes with time varies with specific vivo glycolysis which results to decline in glucose anticoagulant. With fluoride oxalate and heparin, it concentration of the blood sample (Chan et al., 1989). increased by 1.77 and 6.67%, respectively, while with Fluoride oxalate is an anticoagulant which binds to EDTA it decreased by 4.0% within the first thirty (30) ionized calcium, thereby preventing blood from clotting. min for non-diabetics (Table 1). Within the same period, Fluoride oxalate’s ability to prevent clotting by binding to for the diabetics (Table 2), with fluoride oxalate and Ca2+ is not as strong as that of EDTA. heparin it increased by 1.9 and 3.7%, respectively while In many laboratories in this part of the world, blood with EDTA it decreased by 3.6%. This suggests that samples are collected and kept with the intention to test fluoride oxalate has stronger stabilizing ability for blood for glucose. These blood samples may not be properly glucose within the first 30 min. This may be due to the stored, and when tested for glucose, a wrong result may ability of fluoride ion to inhibit the activities of the be gotten. On another note, anticoagulants may be used glycolytic enzyme enolase, thereby stopping further indiscriminately giving room for errors in results. To this breakdown of glucose. effect, it became necessary to investigate the changes in Our results also showed that at 120 min, plasma blood glucose concentrations with time, in blood samples glucose in fluoride oxalate, EDTA and heparin decreased stored or collected in different anticoagulants, using the by 16.4, 17.5 and 8.5%, respectively for non-diabetics spectrophotometry method (Glucose-oxidase kit). (Table 1) and by 10.6, 9.8 and 8.6%, respectively for diabetics (Table 2). This shows that although fluoride MATERIALS AND METHODS oxalate was most stable in the first 30 min, heparin had better stabilizing ability after 120 min. This suggests that Specimen type: Fasting blood samples were gotten from the anticoagulant action of heparin in binding to ten (10) diabetic and ten (10) non-diabetic patients at antithrombin thereby forming a complex that inhibits the Igbinedion University Teaching Hospital (IUTH). procoagulant proteinases, stuart prower factor and thrombin (Linhardt and Gunay, 1999); is also effective in Anticoagulants: The three (3) anticoagulants used are maintaining the glucose level of blood samples to an Fluoride oxalate, EDTA and Heparin. extent. Blood glucose levels increased significantly at 60 Specimen collection: About 6 mL of the subjects’ blood min with all three anticoagulants in diabetics and non- were collected via venipuncture and put into three sample diabetics likewise. The reductions were less in diabetic bottles (2 mL in each) containing the three anticoagulants; blood samples which showed 10.6, 9.8 and 8.6%, Fluoride oxalate, EDTA and Heparin. The blood samples decreases for fluoride oxalate, EDTA and heparin

235 Asian J. Med. Sci., 3(6): 234-236, 2011

Table 1: Time-dependent changes in the mean blood glucose (mmol/L) of non-diabetics in the presence of anticoagulants Time (min) ------Anticoagulants 0 30 60 90 120 Floride Oxalate 4.51±0.25 4.50±0.23 4.70±0.20 3.92±0.18 3.77±0.22 Edta 4.44±0.24 4.27±015 4.77±0.19 3.88±0.17 3.67±0.19 Heparin 4.37±0.22 4.73±0.14 4.65±0.15 3.75±0.15 3.98±0.23 Values are expressed as mean ± SEM; n: 10

Table 2: Time-dependent changes in the mean blood glucose (mmol/L) of diabetics in the presence of anticoagulants Time (min) ------Anticoagulants 0 30 60 90 120 Floride Oxalate 11.08±0.69 11.29±0.81 11.38±1.13 9.87±0.84 9.88±0.76 Edta 9.87±0.91 9.87±0.75 10.55±0.85 9.15±0.86 8.89±1.08 Heparin 10.85±0.88 11.25±0.86 11.55±0.85 10.03±0.81 9.92±0.81 Values are expressed as mean ± SEM; n: 10 respectively after 120 min when compared to the non- Comper, W.D., 1981. Heparin: Polysaccharides; diabetics. This could be due to the presence of compounds Structure-Activity Relationship. Biochemistry. or macromolecules in the blood of these patients, which Gordon and Breach Science Publishers. 7th Edn., may include their medications. New York, U.S.A., pp: 266. Our findings suggest that blood glucose analysis Cox, M. and D. Nelson, 2004. Lehninger Principles of results would be most reliable when blood samples are Biochemistry. Freeman worth Publishers, New York, stored in fluoride oxalate bottles for the first 30 min. pp: 1100. However, if the blood samples must be stored for up to Debusk, R., et al., 2002. Ethylenediaminetetraacetic Acid 120 min, then heparin is a better choice. (EDTA). University of Maryland Medical Center Website. CONCLUSION Guyton, A.C. and J.E. Hall, 2006. Textbook of Medical Physiology. Elsevier Saunders, pp: 464. Our results showed that although Fluoride oxalate Lanigan, R.S. and T.A. Yamarik, 2002. Final report on had stronger stabilizing effect on plasma glucose than the safety assessment of EDTA, Calcium Disodium EDTA and Heparin in the first 30 min, Heparin had better EDTA, Dipotassium EDTA, TEA-EDTA, stabilizing effect after 120 min. This suggests that Tetrasodium EDTA, Tripotassium EDTA, Trisodium Lithium Heparin is the best choice of anticoagulant for EDTA, HEDTA, and Trisodium HEDTA. Int. J. analysis of sample which will be stored for 2 h. Toxicol., 21(2): 95-142. ACKNOWLEDGMENT Linhardt, R.J. and N.S. Gunay, 1999. Production and chemical processing of low molecular weight The Authors are grateful to the authorities of heparins. sem. Thromb. Hem., 25(3): 5-16. Department of Biochemistry, Igbinedion University, Munz, C. and P.V. Robert, 1987. Air-water phase Okada (Edo State, Nigeria) and the authorities of the Equillibria of volatile organic solutes. J. Am. Water Department of Chemical Pathology Laboratory, Works Assoc., 79: 62-69. Igbinedion University Teaching Hospital, Okada, for Oduola, T., G. Adeosun, E. Ogunyemi, F. Adenaike and providing the necessary laboratory facilities. A. Bello, 2006. Studies on G6PD stability in blood stored with different anticoagulants. Inter. J. REFERENCES Hematol., 2(2). Schrezenmeier, H., 1995. Anticoagulant-induced Pseudo- Bentolila, A., V. Israel, H. Christine and J.D. Abraham, thrombocytopenia and Pseudo-leucocytosis. Thromb. 2008. Synthesis and heparin-like biological activity Haemost., 73: 566-613. of amino acid based polymers. Polym. Adv. Totsoi, E., 2004. Glycolysis in blood of normal subjects Technol., 11(8- 12): 377-387 and of diabetic patients. J. Biol. Chem., 1924: 60-69. Chan, A.Y., R. Swaminathan and C.S. Cockram, 1989. Trinder, P., 1969. Determination of glucose in blood Effectiveness of Sodium Fluoride as a preservative of using glucose Oxidase with an alternative oxygen glucose in blood. Clin. Chem., 35: 315-317. receptor. Ann. Clin. Biochem., 6: 24.

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