| Abstract|| |
Anemia is a common feature among patients with chronic renal failure (CRF). Low activity of the erythrocyte enzyme glucose-6-phosphate dehydrogenase (G6PD), which plays a major role in protecting red blood cells against oxidative agents, has been described as one of the contributing factors to anemia in patients with CRF treated with hemodialysis (HD). In this study, blood samples were randomly collected from 65 patients on HD and investigated for G6PD deficiency using the methemoglobin reduction test. The hemoglobin (Hb) concentration, packed cell volume (PCV), red blood cells (RBCs) count and reticulocyte count were determined in all the samples. Our results showed that 39 of 65 patients (60%) on HD had low G6PD activity and 26 (40%) patients had normal activity; 59% of the patients with low G6PD activity were males. The mean Hb, PCV and RBCs counts were lower in patients with low G6PD activity than in those with normal G6PD activity, but the difference was not statistically significant. Likewise, no statistically significant difference was found in the reticulocyte count in patients with low G6PD activity and in those with normal G6PD activity. The low G6PD activity that was found in a large proportion of patients on HD seems to be the result of enzyme inhibition rather than deficiency. No statistically significant difference was found in anemia parameters between patients with and without G6PD deficiency.
|How to cite this article:|
Ali EW, Ahmed EM. The role of erythrocyte enzyme glucose-6-phosphate dehydrogenase (G6PD) deficiency in the pathogenesis of anemia in patients on hemodialysis. Saudi J Kidney Dis Transpl 2013;24:1153-6
|How to cite this URL:|
Ali EW, Ahmed EM. The role of erythrocyte enzyme glucose-6-phosphate dehydrogenase (G6PD) deficiency in the pathogenesis of anemia in patients on hemodialysis. Saudi J Kidney Dis Transpl [serial online] 2013 [cited 2020 Oct 23];24:1153-6. Available from: https://www.sjkdt.org/text.asp?2013/24/6/1153/121271
| Introduction|| |
Anemia is an almost invariable manifestation of chronic renal failure (CRF) in both pre-dialysis stage and in patients maintained on dialysis. Often, it contributes substantially to the morbidity of the condition. Anemia results directly from failure of the endocrine and excretory functions of the kidney. In addition, renal failure is associated with retention of a variety of potentially toxic substances, including some that may inhibit erythropoiesis and others that may shorten erythrocyte survival. ,
The pentose phosphate pathway (PPP) is the major generator of reducing power within the red blood cells (RBCs), which protects erythrocytes against oxidative substances through two reactions catalyzed by the enzyme glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD). 
There is considerable evidence that patients on hemodialysis (HD) are in a continuous state of oxidative stress, perhaps provoked by bioincompatibility of the dialyzer membrane, which may induce the formation of reactive oxygen species.  An impaired defense mechanism against oxidative stress has been reported in patients with CRF who are being treated with HD. Glutathione (GSH) levels and the activity of G6PD have been reported to be low in these patients. , Erythropoietin therapy has been suggested to have a positive anti-oxidant effect in patients on HD. 
The aim of this study was to determine the frequency of G6PD deficiency among Sudanese patients on HD and assess whether the presence of G6PD deficiency has a role in aggravating the anemia of patients on HD or not.
| Subjects and Methods|| |
This descriptive, cross-sectional study was conducted in three different dialysis centers at Khartoum state and included 65 patients diagnosed to have end-stage renal disease (ESRD) who were on treatment with regular HD, twice per week.
Venous blood samples were collected from all patients in ethylene diamine tetra-acetic acid (EDTA) bottles. Packed cell volume (PCV) was determined for all samples and adjusted to 40% before screening for G6PD deficiency. All samples were investigated for the presence of G6-PD deficiency within 1 h after collection using the methemoglobin reduction test: 2 mL of the patient's blood was added to a test tube containing 0.2 mL of a combined reagent (sodium nitrite, dextrose and methylene blue) and incubated at 37°C for 90 min. The control tubes were prepared by adding 2 mL of blood to a similar tube without regents (normal reference tube) and to another tube containing 0.1 mL of sodium nitrite - dextrose mixture without methylene blue (deficient reference tube). Both control tubes were also incubated for 90 min at 37°C. After incubation, 0.1 mL of the sample was transferred from the test sample, the normal reference tube and the deficient reference tube into 10 ml of distilled water in three separate, clear glass test tubes with identical diameter, mixed and the color was compared in all the tubes. The test sample was considered positive if its color was similar to that of the deficient reference tube (brown) and considered negative if the color was similar to that of the normal reference tube (clear-red). Estimation of hemoglobin (Hb) concentration, PCV and RBCs count was performed using an automated hematology cell counter (Sysmex KX-21). For reticulocyte count, two drops of the dye solution "brilliant cresyl blue" were taken in a 75 × 10 glass test tube to which two drops of the patient's blood were added and mixed. The mixture was incubated at 37°C in a water bath for 20 min. After incubation, the mixture was resuspended and a thin film was made and air dried and the reticulocytes and RBCs were counted using an oil immersion lens in ten microscopic fields. The reticulocytes were then calculated as a percentage of the total RBC count. 
Informed consent was taken from all patients before sample collection. Data of this study were analyzed using the statistical package for social sciences (SPSS) computerized program.
| Results|| |
This is a descriptive, cross-sectional study conducted at the Khartoum state to assess the role of the erythrocyte enzyme G6PD deficiency in the pathogenesis of anemia among patients on HD. The study included 65 patients, 43 male (66 %) and 22 female (34%), with an age range of 21 - 63 years.
Low G6PD activity was found in 39 of the 65 (60%) patients on HD, while 26 patients (40%) were found to have normal G6PD activity. Twenty-three (59%) of the patients with low G6PD activity were males and 16 (41%) patients were females. The mean age was higher in patients with low G6PD activity compared with those with normal G6PD activity, but the difference was not statistically significant (mean: 37 and 23 years, respectively; P-value = 0.383).
The mean Hb concentration, PCV and RBCs count were lower in patients with low G6PD activity than in those with normal G6PD acivity, but the difference was not statistically significant [Table 1]. Similarly, no statistically significant difference was found in the mean reticulocyte count between patients with low G6PD activity and those with normal G6PD activity; it was higher than the normal range in both groups [Table 1].Long-term HD is known to have a positive effect on G6PD activity; the number of patients with low G6PD activity was lower after 40 months on HD, and this number continued to decrease as the duration of HD increased [Figure 1].
|Table 1: Mean hemoglobin concentration, packed cell volume, red blood cells count and reticulocyte count in patients with low glucose-6-phosphate dehydrogenase activity compared with those with normal glucose-6-phosphate dehydrogenase activity.|
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|Figure 1: Effect of duration on hemodialysis on glucose-6-phosphate dehydrogenase activity.|
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| Discussion|| |
This study was conducted to evaluate the role of low G6PD enzymatic activity in the pathogenesis of anemia among patients on HD at three different dialysis centers in Khartoum state.
Our results revealed that 60% of the patients on HD had low G6PD activity while 40% of the patients had normal G6PD activity. This finding agrees with the findings of Gauhan et al who reported that patients on HD had significantly low G6PD activity.  It also agrees with the findings of Weinstein et al who investigated the impaired defense mechanism against oxidative stress in HD patients and found that GSH levels were lower in patients with ESRD as compared with healthy controls, and there was an inverse correlation between GSH level and the degree of hemolysis.  G6PD is an important enzyme for the production of GSH; we believe that decreased GSH in these patients might have resulted from decreased G6PD activity.
We found that 59% of the patients with low G6PD activity were males and 41% were females. The hereditary type of G6PD deficiency anemia is inherited as an X-linked disease thus usually affecting males, while females are just carriers of the disease.  The presence of a high proportion of females with low G6PD activity in our study suggests that the presence of a low activity of G6PD in patients on HD is mainly caused by inhibition rather than by deficiency of the enzyme. This is consistent with the findings of Shainkin-Kestenbaum et al, who reported that the presence of creatinine, guanidine- propionic acid (GPA) and guanidine- butyric acid (GBA) was demonstrated in the RBCs of uremic patients; they were found only in trace amounts in the RBCs of normal controls. Both creatinine and GPA inhibit G6PD in vitro in physiological concentrations. 
The Hb level, PCV and RBCs count were lower in patients with low G6PD activity than in those with normal G6PD activity, but the difference was not statistically significant. This indicates that the presence of G6PD deficiency in patients on HD, to some extent, may aggravate the anemia in these patients. The lack of a significant difference in these parameters may be because most of our patients were treated with erythropoietin, which has been reported to have an anti-oxidant effect. 
We found a significant inverse correlation between duration on HD and frequency of patients with low G6PD activity; the number of patients with low G6PD activity was reduced with increasing dialysis duration. This result agrees with the findings of Ghauhan et al, who reported that after adequate dialysis, G6PD can undergo an adaptive alteration that, however, appears reversible. 
In conclusion, low G6PD activity was found in a large proportion of patients on HD. It was found in both sexes, with the mean age being higher in patients with low G6PD activity compared with those with normal G6PD activity. No statistically significant difference was found in the anemia parameters and reticulocyte count in the two groups. Long-term HD seems to have a positive effect on G6PD activity in CRF patients.
| References|| |
|1.||Lee G, Forester J, Lukens J, Paraskevas F, Greer J, Rodgers G Wintrobe's clinical haematology. 10 ed. Maryland: Williams & Wilkins; 1998. p. 1497-503. |
|2.||Tarif N. Anemia management in patients with chronic renal disease: Current recommendations. Saudi J Kidney Dis transpl 2002; 13:331-5. |
|3.||Hoffbrand A, Catovsky D, Tuddenham E. Post-graduate haematology. 5 ed. Ljubljana: Blackwell Publishing Ltd.; 2005. p. 133-50. |
|4.||Loughery CM, Young IS, Kightbody JH, Mc-Master D, McNamee PT, Trimble ER. Oxidative stress in hemodialysis. Q J Med 1994; 87:679-83. |
|5.||Weinstein T, Chagnac A, Korzets A, et al. Haemolysis in hemodialysis patients: Evidence for impaired defense mechanisms against oxidative stress. Nephrol Dial Transplant 2000; 15:883-7. |
|6.||Chauhan DP, Gupta PH, Nampoothiri MR, Singhal PC, Chugh KS, Nair CR. Determination of erythrocyte superoxide dismutase, catalase, glucose-6-phosphate dehydrogenase, reduced glutathione and malonyldialdehyde in uremia. Clin Chim Acta 1982;123:153-9. |
|7.||Inal M, Kanbak G, Sen S, Akyüz F, Sunal E. Antioxidant status and lipid peroxidation in hemodialysis patients undergoing erythropoietin and erythropoietin-vitamin E combined therapy. Free Radic Res 1999;31:211-6. |
|8.||Lewis S, Bain B, Bates I. Dacie and Lewis: Practical Haematology. 9 ed. London: Churchill Livingstone; 2001. p. 19-181. |
|9.||Shainkin-Kestenbaum R, Giat Y, Berlyne G The Toxicity of guanidino compounds in the red blood Cell in uremia and the effects of hemodialysis . Nephron 1982;31:20-3. |
Elshazali Widaa Ali
Faculty of Medical Laboratory Sciences, Al-Neelain University, Khartoum