A Study on Glucose-6-Phosphate Dehydrogenase Enzyme Activity

Öz

Aim:Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common erythrocyte enzyme deficiency in the world. It is the most common cause of hemolytic anemia in our country especially in the Mediterranean region. Most patients with enzyme deficiency have no clinical symptoms. However, hemolytic state develops in the red blood cells due to drugs, infections, favism, newborn period which cause oxidative damage. In our study, we aimed to investigate G6PD enzyme activity and kinetic properties.

Material and Method:G6PD enzyme activity was measured qualitatively with a fluorescence spot test and quantitatively in a G6PD deficient blood sample and in a control blood sample, and partial enzyme purification was performed to compare kinetic characteristics. 

Results: No fluorescence was observed qualitatively in the blood sample with enzyme deficiency. Enzyme activity measured quantitatively in the sample with G6PD deficiency was statistically significantly lower than the control (p <0.05). The kinetics of the partially purified deficient enzyme were found to have lower Km values (G6P and NADP) than in the normal enzyme. The other kinetic characteristics of enzyme of deficient G6PD activity in comparison with normal enzyme were; a higher optimum pH, more activity with the substrate analogs and a lower thermostability.

Conclusion:G6PD enzyme deficiency is important in the diagnosis and monitoring of the patient in terms of public health and preventive medicine.

Anahtar Kelimeler

• Glucose-6-phosphate dehydrogenase,enzyme deficiency,kinetic properties

Glukoz-6-Fosfat Dehidrogenaz Enzim Aktivitesi Üzerine Bir Çalışma

Öz

Amaç: Glukoz-6-fosfat dehidrogenaz (G6PD) eksikliği, dünyada en çok rastlanan eritrosit enzim eksikliğidir. Ülkemizde özellikle Akdeniz bölgesinde görülen hemolitik anemilerin en sık nedenidir. Enzim eksikliği olan hastaların çoğunda klinik belirti yoktur. Ancak alyuvarlarda oksidatif hasara yol açan ilaçlar, enfeksiyonlar, bakla yenmesi, yenidoğan dönemi gibi nedenlerle hemolitik tablo gelişir. Çalışmamızda G6PD enzim aktivitesi ve kinetik özelliklerini araştırmak amaçlandı.

Gereç ve Yöntem: G6PD enzim aktivitesi floresan spot testi ile kalitatif olarak ve kantitatif olarak ölçülen G6PD eksikliği olan bir kan örneği ve bir kontrol kan örneğinde kısmi enzim saflaştırması yapılarak kinetik özellikleri karşılaştırıldı. 

Bulgular: Enzim eksikliği bulunan kan örneğinde kalitatif olarak floresans gözlenmedi. G6PD eksikliği olan örnekte kantitatif olarak ölçülen enzim aktivitesi kontrole göre istatistiksel olarak anlamlı düzeyde düşük bulundu (p<0.05). Kısmen saflaştırılan hasta G6PD enziminin G6P ve NADP için Km ve Vm değerleri normal enzime göre daha düşük bulundu. Hasta enziminin diğer kinetik özellikleri; normal enzime oranla alkalen pH’da daha yüksek aktivite, substrat benzerlerini daha yüksek oranda kullanabilme ve daha düşük termostabilite idi.

Sonuç: Halk sağlığı ve koruyucu hekimlik açısından G6PD enzimi eksikliği hastanın tanısı ve izlenmesinde önem taşır.

Anahtar Kelimeler

• Glukoz-6-fosfat dehidrogenaz,enzim eksikliği,kinetik özellikler

Kaynakça

1. Beutler E. Glucose-6-phosphate dehydrogenase deficiency. N Engl J Med 1991;324:169-174.
2. Beutler E. Red cell metabolism: a manual of biochemical methods. 3rd ed. New York: Grune and Stratton, 1984.
3. Şevkinaz Konak, Mümin Polat. Glukoz 6 Fosfat Dehidrogenaz Enzim Eksikliği; Tanı ve Tedavi MAKÜ Sag. Bil. Enst. Derg. 2015, 3(2): 77-83.
4. İlgen Şaşmaz. Glukoz-6-fosfat dehidrogenaz enzim eksikliği Türk Ped Arş 2009; 44 Özel Sayı: 35-8.
5. Tetik E, Aral YZ, Türkmen MK, Bozkurt G. Glukoz-6-fosfat dehidrogenaz enzim eksikliği olan çocuklarda G6PD S218F Akdeniz mutasyonu sıklığı. ADÜ Tıp Fakültesi Dergisi 2014;15:1–8.
6. Acıpayam C, Orhaner BB, Karal Y. Screening of Glucose-6-phosphate dehydrogenase defciency incord blood Journal of Clinical and Analytical Medicine 2014, DOI: 10.4328/JCAM.2390.
7. Beutler E, Westwood B, Prchal JT, Vaca G, Bartsocas CS, Baronciani L. New glucose-6-phosphate dehydrogenase mutations from various ethnic groups. Blood. 1992;1;80: 255-6.
8. Say S, Özand P, Berkel İ. Erytrocyte glucose-6-phosphate dehydrogenase deficiency in Turkey. Acta Paediat Scand 1965; 54: 319-24.

9. Kus S. G6PD Çorum. A new variant of glucose 6 phosphate dehydrogenase. J Islamic Acad Sci 1989;2:31-3.
10. Luzatto L. Glucose-6-phosphate dehydrogenase: Genetic haematological aspects. Cell Bio Fun 1987; 5: 101-7.
11. Beutler E. The genetics of glucose-6- phosphate dehydrogenase deficiency. Semin Hematol 1990; 27: 137-64.
12. Beutler E. Hematology. Ed: Williams JW, Beutler E, Erslev JA, Lichtman AM. Third Ed., Mc Graw-Hill Inc., New York, 1983.
13. WHO Scientific Group. Standardization of procedure for the study of G6PD. WHO Techn Rep Ser 1967;366:9-51.
14. Burtis CA, Ashwood ER. Tietz Textbook of Clinical Chemistry, Second Edition, W.B. Saunders Company, Philadelphia; 1994. p. 2020-2021.
15. Davis BJ. Disc electrophoresis. II. Method and application to human serum proteins. Ann NY Acad Sci 1964;121:404-27.
16. Vuopio P, Harkonen M, Johnsson R, Nuutinen M. Red cell glucose-6-phosphate dehydrogenase deficiency in Finland. Annals Clin Res 1973;5:168-73.
17. Yıldız ŞM, Arıyürek SY, Aksoy K. Glukoz-6-Fosfat Dehidrogenaz Genindeki Akdeniz Mutasyonunun Mikroarray Tekniğiyle Saptanması Turk J Biochem.,2010; 35 (1) ; 63–66.
18. Luzzatto L, Poggi V. Glucose-6-phosphate deficiency. In: Orkin SH, Nathan D, Ginsburg D, Look AT, Fisher DE, Lux SE (eds). Hematology of Infancy and Childhood. 7th edition. Philadelphia: WB Saunders Company, 2009: 883-907.
19. Hafez M, Amar ES, Zedan M, et al. Improved erythrocyte survival with combined vitamin E and selenium therapy in children with glucose-6-phosphate dehydrogenase deficiency and mild chronic hemolysis. J Pediatr 1986;108:558-561.
20. Schulpis KH, Reclos GJ, Parthimos T, Parthimos N, Gavriilidis A, Tsakiris S. Lcysteine supplementation protects the erythrocyte glucose-6-phosphate dehydrogenase activity from reduction induced by forced training. Clin Biochem 2006;39:1002-1006.
21. Georgakouli K, Deli CK, Zalavras A, et al. α-lipoic acid supplementation up-regulates antioxidant capacity in adults with G6PD deficiency. Food Chem Toxicol 2013;61:69-73.
22. Minucci A, Giardina B, Zuppi C, Capoluongo E. Glucose-6- phosphate Dehydrogenase laboratory assay: how, when, and why? IUBMB Life 2009; 61: 27-34.
23. Reclos GJ, Hatzidakis CJ, Kruithof RA. G-6-PD Diagnosis: Modification of the standard method eliminates the need for an additional hemoglobin determination. Pharmakeftiki 1999;12: 25-31.
24. Reclos GJ, Hatzidakis CJ, Schulpis KH. Glucose-6-phosphate dehyrogenase deficiency neonatal screening: preliminary evidence that a high percentage of partially deficient female neonates are missed during routine screening. Journal of Medical Screening. 2000;7: 46-51.