Folic acid deficiency modifies the haematopoietic response to recombinant human erythropoietin in maintenance dialysis patients

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1 Nephrol Dial Transplant (2006) 21: doi: /ndt/gfi086 Advance Access publication 6 September 2005 Original Article Folic acid deficiency modifies the haematopoietic response to recombinant human erythropoietin in maintenance dialysis patients Helmut Schiffl 1,2 and Susanne M. Lang 2 1 KfH Nierenzentrum Mu nchen-laim, Munich, Germany and 2 Department of Internal Medicine, University of Munich, Munich, Germany Abstract Background. While folic acid deficiency causes macrocytic anaemia in non-renal patients, the relevance of altered folate metabolism in anaemia of end-stage renal disease and its response to rhu-epo is less clear. Methods. Ten haemodialysis patients with macrocytic anaemia due to dietary folic acid deficiency were compared to 10 matched (age, duration of dialysis, degree of anaemia) patients with normocytic normochromic anaemia. Ninteen patients received erythropoietin-alpha intravenously thrice weekly. The study design was a prospective crossover (ABA) comparison of the effects of intravenously administered high doses of folic acid on haemoglobin levels and EPO doses, with 6 months active supplementation (B) and two periods of 6 months duration each without folic acid supplementation (A). Results. The two patient groups did not differ at recruitment. Red blood cell folate levels were normal in patients with normocytic anaemia, but they were subnormal in all patients with macrocytic anaemia. Compared to the first period without folic acid supplementation, patients with macrocytic anaemia had significantly higher haemoglobin levels despite lower EPO doses after 6 months high-dose folic acid, and red cells had become normocytic. The removal of folic acid supplementation resulted in re-occurrence of macrocytosis and in a significantly lower response to rhu-epo. In contrast, high-dose folic acid supplementation had no effect on response to rhu-epo in patients with normocytic anaemia. Conclusions. Folic acid deficiency may occur in elderly haemodialysis patients with poor dietary folate intake without regular oral supplementation and may cause hyporesponsiveness to rhu-epo. Macrocytosis is a simple and cheap indicator for folate deficiency in Correspondence and offprint requests to: H. Schiffl, KfH Nierenzentrum Mu nchen Laim, Elsenheimerstr. 63, D80687 Munich, Germany. hschiffl@hotmail.com end-stage renal disease patients on maintenance dialysis. Keywords: folic acid deficiency; macrocytic anaemia; maintenance haemodialysis; rhu-epo Introduction Normocytic normochromic anaemia is a virtually universal feature among patients with end-stage renal disease (ESRD) on maintenance dialysis. While the primacy of insufficient production of erythropoietin by the diseased kidneys is indisputable, the haematopoietic response to rhu-epo may be affected by various factors related either to uraemia or its treatment. There is ample evidence that iron deficiency, inflammation/infection, hyperparathyroidism or aluminium intoxication may contribute to the severity of renal anaemia and hence increase recombinant human erythropoietin requirements. There is also no doubt that patients with ESRD may develop any of the common causes of anaemia [1]. Folate deficiency has been implicated as a modulating factor in renal anaemia and as a cause of hypo-responsiveness to erythropoietin treatment [2]. However, despite loss of folate into the dialysate [3] and increased folate requirements during rhu-epo treatment [4] relevant folate deficiency is unlikely to occur in well-nourished dialysis patients [5,6]. Furthermore, supplementation of folic acid did not appear to have a beneficial effect on erythropoiesis or on erythropoietin response when serum folate levels were within the normal range [7,8]. Therefore, routine supplementation of folate is not recommended in patients on regular haemodialysis as adjuvant therapy of renal anaemia [9]. The present investigation aimed to identify folic acid deficiency by macrocytic anaemia in an outpatient ß The Author [2005]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please journals.permissions@oxfordjournals.org

2 134 H. Schiffl and S. M. Lang dialysis population and to characterize the response of combined renal and folic acid deficiency anaemia to rhu-epo therapy in a small group of patients. Subjects and methods Subjects Ten patients (4 men, 6 women, aged years) with macrocytic anaemia were identified from an outpatient population of 136 patients maintained on regular dialysis therapy. None of these patients had symptoms of malabsorption or known gastrointestinal disorders, a myeloproliferative syndrome, viral infection or liver disease. No patient was taking drugs known to cause megaloblastic anaemia or was a heavy alcoholic. None of the female patients was pregnant. They all had serum cobalamin, LDH, liver enzymes and bilirubin levels within the normal range. None of the patients had glossitis, neurological or psychiatric symptoms. Nine patients received erythropoietin alpha (Erypo, Ortho Biotec; Cilag Janssen, Neuss, Germany) intravenously thrice weekly at the end of a haemodialysis session. One more patient had macrocytic anaemia with haemoglobin levels consistently over 11 g/dl and was not treated with rhu-epo. The study patients were matched with 10 dialysis patients with normocytic normochromic anaemia. Matching criteria were age, duration of dialysis and degree of anaemia. The 10 control patients received erythropoietin alpha (Erypo, Ortho Biotec, Cilag Janssen, Neuss, Germany) intravenously thrice weekly after haemodialysis for partial correction of their renal anaemia. None of the 20 dialysis patients had iron deficiency judged by determinations of serum iron, ferritin and transferrin, infection (by CRP levels), severe hyperparathyroidism (by intact PTH concentrations), excess aluminium levels or occult blood loss (haemoccult testing). Oral medications included antihypertensive drugs (beta blockers, angiotensin-converting enzyme inhibitors, calcium antagonists in six patients of each group), calcium acetate, aluminium hydroxide and sevelamer. To replete iron stores ferrum(iii) sodium gluconate (Ferrlecit, Aventis, Frankfurt a. Main, Germany) was given intravenously according to the individual patient s needs to maintain serum ferritin levels between 300 to 500 ng/ml and transferrin saturation values at 20 to 40%. To avoid iron overload (i.e. serum ferritin over 800 ng/ml and/or transferrin saturation over 50%) maintenance intravenous iron therapy was withheld and restarted with a reduced dose (50%) when serum ferritin levels fell below 800 ng/ml. Haemodialysis Haemodialysis was performed thrice weekly for 4 5 h, using machines with volumetrically controlled ultrafiltration (MTS 4008, FMC, Bad Homburg v.d.h., Germany), new synthetic high-flux membranes (APS 650, polysulfone, Asahi Medical Co, Tokyo, Japan) and ultrapure bicarbonate-based dialysate (by additional one step online filtration). for the study gave written consent. In Germany, folic acid preparations are classified as dietary supplements and not as drugs and are available without prescription. The laboratory monitoring tests were either routinely performed in the haemodialysis unit or recommended for evaluation of anaemia or responsiveness of rhu-epo. There were no additional samples drawn for study purposes. The investigations were performed as an unblinded prospective cross-over comparison (ABA-sequence) of the effects of intravenously administered high-dose folic acid (folic acid, Hevert Arzneimittel, Nußbaum, Germany). The study protocol included three phases of 6 months duration each: an observation phase without intravenous folic acid supplementation (A), a second phase (B) with high-dose folic acid supplementation (20 mg thrice weekly at the end of a dialysis session), and a final phase without supplementation (A). Folic acid was administered intravenously to guarantee compliance and to avoid differences in bioavailability. Laboratory investigations Blood specimens from each patient were collected at recruitment and at the end of each study period into lightprotected tubes. Serum and whole blood aliquots were frozen immediately and stored at 20 C until analysis. Serum folate, erythrocyte folate and serum vitamin B12 were quantified by an automated chemiluminescence microparticle immunoassay on the Architect system (Abbott Diagnostics, Abbott Park, Illinois, USA). Conditions specified by the manufacturer were used to prepare whole blood haemolysate [10]. Complete blood cell count, iron indices, intact parathyroid hormone and serum aluminium were measured by routine methods at recruitment. Blood cell counts were repeated every 6 weeks, iron indices were evaluated every 12 weeks, Kt/V urea was calculated every 6 months. Reference values for serum folic acid were ng/ml, for whole blood folate ng/ml and for serum vitamin B12 levels pg/ml. The Architect folate assay and Architect B12 assay precision (total coefficient of variation in percent) was below 7% for low and normal serum folate levels, for low or normal erythrocyte folate concentrations and for normal vitamin B12 concentrations. The normal range for the mean corpuscular volume of erythrocytes was 83 to 95 fl, macrocytosis was defined by MCV values greater than 96 fl. Dietary record A 7 day dietary record assessed nutrient intakes of the study patients at recruitment and after each of the three study periods. Folate values were calculated with the FOODOPT software, which is based on the German Food Code and Nutrient Database. The calculated folate intake was compared with the corresponding recommended dietary allowance for the particular age and sex. The current German recommendation for both men and women is 400 mg folate per day. This amount is based on the folate content for raw materials [11]. Study design The study was conducted according to the Declaration of Helsinki and the ICH GCP guidelines. All patients enrolled rhu-epo dosage Target haemoglobin concentrations in patients receiving rhu-epo were 11 to 12 g/dl. rhu-epo dosages were

3 Folic deficiency and haematopoietic response 135 adjusted by a 25% reduction or 25% augmentation to the closest 1000 units if haemoglobin levels rose above 13 g/dl or fell below 10 g/dl. Dose adjustments were made on a caseby-case basis at each assessment time point by the attending nephrologists, who were not necessarily identical with the study physicians. Table 1. Characteristics of patients with macrocytic anaemia (MCV fl) and normocytic anaemia (MCV fl) at recruitment. Macrocytic anaemia Normocytic anaemia Statistics Data are presented as mean±sd. The Mann Whitney U-test was applied to study differences between the two groups and the Wilcoxon rank test was used for the paired differences within the groups. A P-value of less than 0.05 was considered to reflect statistical differences. Results Characteristics at recruitment The two patient groups were comparable regarding mean age, distribution of gender, causes of end-stage renal disease, body mass index and time on haemodialysis. All patients were adequately dialyzed. They had similar mean PTH levels, none of the patients had excess hyperaluminaemia. The number of patients taking angiotensin-converting enzyme inhibitors was identical. All patients were iron-repleted, none of the patients had biochemical evidence of infection/ inflammation. Serum folate and vitamin B12 levels were above the lower values of the normal range in each patient and the mean concentration of the two vitamins did not differ among groups. All patients with macrocytic anaemia had red cell folate levels below the cut-off. Normocytic anaemia was associated with normal red cell folate levels in all cases. Pre-dialysis haemoglobin levels did not differ between groups, but there was a need for higher rhu-epo doses in macrocytic patients (Table 1). Dose of rhu-epo and folic acid supplementation All 20 patients completed the three study periods. High-dose folic acid was well-tolerated by all participants, folic acid deficiency was not associated with clinical signs other than macrocytic anaemia. During the first period, patients with macrocytic or with normocytic anaemia exhibited no significant changes in haemoglobin concentrations or erythrocyte cell volume. There was a need for higher doses of rhu-epo (units per week related to patients body weight) in patients with macrocytic anaemia during the first 6 months of the study. In the second period, intravenous supplementation with high-dose folic acid was well-tolerated by all patients and resulted in supra-physiologic serum folate concentrations. Supplementation with high doses of folic acid caused a normalization of the erythrocyte cell volume and a significant reduction in rhu-epo dose (to reach similar target haemoglobin concentrations) in the nine patients with macrocytic anaemia Number of patients Age (years) 56 (9) 54 (12) Gender (m/f) 4/6 5/5 Time on HD (months) 62 (14) 56 (12) Body mass index (kg/m 2 ) 23 (5) 23 (2) Kt/V urea 1.3 (0.1) 1.2 (0.1) ipth (pg/ml) 101 (33) 88 (22) Ferritin (ng/ml) 544 (82) 602 (104) Transferrin saturation (%) 27 (3) 29 (4) Serum folate(ng/ml) 12 (4) 10 (5) Red blood cell folate (ng/ml) 122 (17) 480 (144)* Serum vitamin B12 (pg/ml) 622 (144) 580 (192) CRP (mg/dl) 0.3 (0.2) 0.2 (0.1) ACE-Inhibitors (patients/n) 3/10 3/10 MCV (fl) 106 (3) 88 (3) Haemoglobin (g/dl) 10.9 (0.2) 11.0 (0.2) rhu-epo dose (IU/kg/week) 79 (12) 48 (9) Data are given as mean (±SD). *P ¼ 0.05 vs corresponding value. A B A A B A Macrocytic anaemia Normocytic anaemia Fig. 1. Haemoglobin levels in patients with macrocytic or normocytic anaemia at the end of the three study periods. Data are given as mean±sd. *P<0.05 vs pre-treatment value. receiving rhu-epo. By contrast, in normocytic patients neither the response of anaemia to rhu-epo nor erythrocyte cell volume was changed by high-dose folic acid (Figures 1 and 2 and Table 2). During the last 6 months of the study without intravenous administration of folic acid, there was a re-occurrence of macrocytosis and an increase in rhu-epo requirements in the nine previously macrocytic anaemic patients, but no significant changes were observed in the patient group with normocytic anaemia (Figures 1 and 2 and Table 2). In the patient with macrocytic anaemia who was not on rhu-epo, high doses of folic acid induced a sustained increase in the haemoglobin level from 11.2 to 13.4 g/dl and a reduction in MCV from 103 fl to 93 fl.

4 136 H. Schiffl and S. M. Lang rhu-epo dose (IU/kg/week) Withdrawal of intravenous folic acid supplementation resulted in the re-occurrence of macrocytic anaemia in the non-rhu-treated patient (Hb 10 g/dl, MCV 103 fl). Discussion * A B A A B A Macrocytic anaemia Normocytic anaemia Fig. 2. rhu-epo doses in patients with macrocytic or normocytic anaemia at the end of the three study periods. Data are given as mean±sd. *P<0.05 vs pre-treatment value. Table 2. Iron status indices, erythrocyte corpuscular volume and red blood cell folate concentration at the end of the three study periods (mean±sd) Ferritin (ng/ml) TSAT (%) MCV (ml) RBC folate (ng/ml) Macrocytic anaemia (n ¼ 9) No folic acid (A) 578 (62) 29 (2) 104 (2) 104 (12) Folic acid suppl. (B) 520 (54) 31 (3) 94 (4)* Above normal range No folic acid (A) 596 (72) 27 (2) 106 (2) 125 (14) Normocytic anaemia (n ¼ 10) No folic acid (A) 610 (48) 31 (3) 89 (3) 642 (122) Folic acid suppl. (B) 570 (66) 30 (4) 90 (2) Above normal range No folic acid (A) 535 (53) 28 (2) 88 (4) 680 (140) TSAT, transferrin saturation. *P ¼ 0.05 vs corresponding value. The major results of the present investigation demonstrate that folate deficiency with subsequent folateinduced megaloblastosis impairs the haematopoietic response to erythropoietin in a small group of patients with renal anaemia undergoing maintenance haemodialysis. The numeric estimate of the incidence of folate deficiency in patients with end-stage renal disease treated with non-transplant renal replacement techniques has not been determined. Current routine folate supplementation for haemodialysis patients with lowdose folic acid (1 2 mg/day) or treatment of hyperhomocysteinaemia in this patient population with high-dose folic acid (5 15 mg/day) may have resulted in a low incidence of this form of vitamin deficiency [9]. From our 136 outpatient haemodialysis population, 10 patients presented with megaloblastic anaemia and showed intracellular folate deficiency, as evidenced by low RBC levels. The utility of red cell indices, particularly of the classical parameter mean cell volume or red blood cell distribution width as indicators for folate deficiency was studied by Fialon et al. [12] in regular dialysis patients treated with rhu-epo. These authors conclude that red blood cell parameters may be an inexpensive sensitive test to detect vitamin B12 or folate deficiency in iron-repleted haemodialysis patients. Assessment of folate status should include serum folate and RBC folate levels [13]. Serum folate may be the earliest indicator of folate deficiency, but does not reflect folate body stores. While serum folate levels respond quickly to changes in diet and low folate levels may reflect low dietary intake during the previous 2 3 days, RBC folate levels do not fluctuate with changes in diet during the lifespan of the erythrocytes. RBC folate levels are usually low in patients with folate deficiency. The incidence of folate deficiency in CAPD patients or haemodialysis patients is dramatically overestimated by using common cut-off values of normal serum folate concentrations [14]. Bamonti-Catena and co-workers found low serum folate levels in 63% of their patients, but more than 80% of these patients had RBC folate concentrations within the normal range [15]. In our elderly (8 of 10 were aged 60 years and more) patients, folate deficiency was caused by low dietary intake as in the majority of cases in the general population. Major dietary sources of folic acid are enriched bread and grains, fortified cereals, raw cruciferous or green leafy vegetables or citrus fruits (oranges). The dietary record demonstrated that all our patients with macrocytic anaemia failed to achieve the recommended daily folate intake of 400 mg/day. None of the patients took oral vitamin supplements. In Germany, cereals, with few exceptions, are not enriched with folate. Moreover, our patients cooked foods in water with excessive heat and destroyed the folic acid to some extent. Poor dietary intake may be aggravated by loss of appetite in an elderly patient with end-stage renal disease. Folate may also be lost during the dialysis process, particularly with high-flux haemodialysis [3]. Resistance to erythropoietin therapy remains an important problem in the treatment of anaemia in patients with end-stage renal disease. High intravenous folate supplementation corrected the haematological features of folate deficiency. There was a fall in MCV back to the normal range, and a rise in haemoglobin allowing reduction of rhu-epo to reach target concentrations. These observations are in line with the anecdotal observation that correction of vitamin B12 and folate deficiency improved the responsiveness to erythropoietin in a patient who developed macrocytic anaemia during rhu-epo therapy [16]. Furthermore, Pronai et al. reported that folic acid supplementation

5 Folic deficiency and haematopoietic response 137 improved erythropoietin response and normalized mean corpuscular erythrocyte volume in 11 patients with chronic renal failure receiving either conservative treatment or maintenance dialysis [17]. Despite normal serum folate and vitamin B12 levels, these patients had developed macrocytosis and partial resistance to rhu-epo. Unfortunately, the authors did not measure RBC folate concentrations. In contrast we and others [7,8,18,19] found no improved erythropoietin response even after high-dose folic acid administration to dialysis patients with normocytic or elevated MCV but normal red cell folate concentrations. Folate deficiency may occur in elderly nonsupplemented end-stage renal disease patients maintained on dialytic therapy with poor dietary intake of food not enriched with folate or water cooked food. Vitamin B12 and red blood cell folate levels should be assayed in all cases showing megaloblastic anaemia, as correction of dietary folate deficiency by oral supplementation is simple, effective and will improve the responsiveness to erythropoietin in such patients. There is a need for further investigations to characterize the incidence of folic acid deficiency in maintenance dialysis patients and the optimal route and dose of folic acid supplementation in patients with poor dietary folic acid intake. Acknowledgements. The study was supported in part by a grant from OrthoBiotech. Conflict of interest statement. None declared. References 1. Hampers CL, Streiff R, Nathan DG, Snyder D, Merrill JP. Megaloblastic hematopoiesis in uremia and in patients on longterm hemodialysis. N Engl J Med 1967; 276: Drueke T. Hyporesponsiveness to recombinant human erythropoietin. Nephrol Dial Transplant 2001; 16 [Suppl 7]: Leblanc M, Pichette V, Geadah D, Ouimet D. Folic acid and pyridoxal-5 0 -phosphate losses during high-efficiency hemodialysis in patients without hydrosoluble vitamin supplementation. J Ren Nutr 2000; 10: Duclos J, Sieberth HG, Mann H. [Is it necessary to supplement with folic acid patients in chronic dialysis treated with erythropoietin?]. Rev Med Chil 1993; 121: Fodinger M, Veitl M, Skoupy S et al. Effect of TCN2 776C>G on vitamin B12 cellular availability in end-stage renal disease patients. Kidney Int 2003; 64: Tamura T, Johnston KE, Bergman SM. Homocysteine and folate concentrations in blood from patients treated with hemodialysis. J Am Soc Nephrol 1996; 7: Ono K, Hisasue Y. Is folate supplementation necessary in hemodialysis patients on erythropoietin therapy. Clin Nephrol 1992; 38: Westhuyzen J, Matherson K, Tracey R, Fleming SJ. Effect of withdrawal of folic acid supplementation in maintenance hemodialysis patients. Clin Nephrol 1993; 40: Teschner M, Kosch M, Schaefer RM. Folate metabolism in renal failure. Nephrol Dial Transplant 2002; 17 [Suppl. 5]: Owen WE, Roberts WL. Comparison of five automated serum and whole blood folate assays. Am J Clin Pathol 2003; 120: Wolters M, Hermann S, Hahn A. B vitamin status and concentrations of homocysteine and methylmalonic acid in elderly German women. Am J Clin Nutr 2003; 78: Fialon P, Leaute AG, Sassier P, Vallot C, Wone C. Use of red blood cell indices (MCV, MCH, RDW) in monitoring chronic hemodialysis patients treated with recombinant erythropoietin. Pathol Biol (Paris) 1993; 41: Galloway M, Rushworth L. Red cell or serum folate? Results from the National Pathology Alliance benchmarking review. J Clin Pathol 2003; 56: De Vecchi AF, Bamonti-Catena F, Finazzi S et al. Homocysteine, vitamin B12, and serum and erythrocyte folate in peritoneal dialysis and hemodialysis patients. Perit Dial Int 2000; 20: Bamonti-Catena F, Buccianti G, Porcella A et al. Folate measurements in patients on regular hemodialysis treatment. Am J Kidney Dis 1999; 33: Breen CP, Macdougall IC. Correction of epoetin-resistant megaloblastic anaemia following vitamin B(12) and folate administration. Nephron 1999; 83: Pronai W, Riegler-Keil M, Silberbauer K, Stockenhuber F. Folic acid supplementation improves erythropoietin response. Nephron 1995; 71: Blumberg A, Zehnder C, Huber A. Folic acid supplementation rarely improves erythropoietin response. Nephron 1998; 78: Wang MC, Huang J-J, Liao LH et al. Effect of high-dose folic acid on hemodialysis patients with poor erythropoietin response. Dial Transplant 2000; 29: Received for publication: Accepted in revised form: