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Nephrol Dial Transplant (1996) 11: 492-497 Nephrology Dialysis Transplantation Original Article Is zinc protoporphyrin an indicator of iron-deficient erythropoiesis in maintenance haemodialysis patients? J. Braun1, M. Hammerschmidt1, M. Schreiber1, R. Heidler1 and W. H. Horl2 'Haemodialysis Unit NUrnberg, Ntirnberg, Germany, and department of Nephrology, University of Vienna, Vienna, Austria Abstract Background. Zinc protoporphyrin (ZPP), a metabolic intermediate generated in the red blood cell by incorporation of zinc instead of iron, has been suggested to be a sensitive and specific parameter of absolute iron deficiency in haemodialysis (HD) patients. Methods. We studied 62 HD patients, 29-86 years old, with ZPP levels > 50 umol/mol haeme (normal value of ZPP <40 umol/mol haeme) assessing the value of ZPP as a marker of functional iron deficiency at different cut-off points of ZPP. None of the patients had apparent inflammatory disease, infectious disease, or malignancy. ZPP, haemoglobin, iron and ferritin levels were determined before, and after a 24-week period of once-weekly i.v. administration of 40 mg iron, to determine whether ZPP levels return to normal during adequate iron supplementation (960 mg iron/ patient). Results. There was no significant change in ZPP levels after iron supplementation in patients with a ZPP > 50 umol/mol haeme (96.7+49.8 versus 88.4 + 43.5 umol/mol haeme before and after iron administration respectively, P=n.s.). However, in patients with a ZPP > 90 umol/mol haeme, there was a significant reduction in ZPP levels (141.2 + 54.5 versus 108.0 ± 48.8 umol/mol haeme, /><0.001). Serum ferritin increased significantly in both groups. There was no correlation between ZPP and serum ferritin at any time during the study. There was also no correlation between serum aluminium levels and ZPP and no significant difference in changes in ZPP in patients receiving desferrioxamine therapy compared to those not receiving desferrioxamine therapy. We did find a significant correlation between moderately elevated total blood lead concentrations and ZPP levels at the end of the study. The ZPP levels were not significantly different in the range from 50-110 umol/mol haeme before and after i.v. iron supplementation in the responders (10% increase of haemoglobin or 20% decrease of the recombinant human erythropoietin dose) compared with the non-responders. Conclusions. Our data indicate that ZPP cannot be used to predict the erythropoietic response to iron supplementation. However, ZPP levels may be an indicator of functional iron deficiency due to blockade of the reticuloendothelial iron release in haemodialysis patients. Key words: anaemia; iron deficiency; haemodialysis; zinc protoporphyrin; lead Introduction Iron deficiency is a major cause of anaemia in maintenance haemodialysis (HD) patients, who may also have an impaired response to recombinant human erythropoietin (rHuEpo) therapy. To date, measurement of serum ferritin has been the most powerful test in diagnosing iron deficiency, but it must be interpreted differently for patients with inflammatory diseases. Measuring the percentage of hypochromic red blood cells in the circulation has already been established as a means of assessing the adequacy of iron supplied to the marrow [1]. Measurement of zinc protoporphyrin (ZPP), a metabolic intermediate that is generated in the red blood cells of patients with lead toxicity and iron-deficiency anaemia by incorporation of zinc instead of iron, has been proposed as a sensitive and specific screening test for iron deficiency. ZPP was found to be elevated in HD patients treated with rHuEpo and whose serum ferritin levels were < 50 ug/1 [2]. Hastka et al. concluded that normal ferritin levels in HD patients do not exclude iron deficiency, and therefore ZPP would be useful in deciding who should receive iron supplementation [2]. Two further studies tested the sensitivity and specificity of ZPP as an assay of iron deficiency in HD patients. Fishbane and Lynn [3] considered HD patients with ZPP > 90 umol/mol Correspondence and offprint requests to: Priv.-Doz. Dr J. Braun, haeme, or ferritin < 50 ng/ml, to be iron-deficient KfH Dialysezentrum NOrnberg, Virnsbergerstr. 43, D-90431 (normal value of ZPP <40 umol/mol haeme) and NUrnberg, Germany. found ZPP > 90 umol/mol haeme to be the most 1 1996 European Dialysis and Transplant Association-European Renal Association Zinc protoporphyrin an indicator of iron-deficient erythropoiesis sensitive (90%) and specific (87%) indicator for iron deficiency in HD patients. In contrast, Ahmed et al. [4] found that ZPP was the most specific and the least sensitive test in 22 HD patients (8 patients were HFV+), divided into responders (10% increase of haematocrit over 2 months) and non-responders to rHuEpo. We therefore investigated a group of 62 HD patients with ZPP levels > 50 umol/mol haeme, without apparent inflammatory disease, infectious disease, or malignancy, to assess the value of different cut-off points of ZPP as a marker of functional iron deficiency. In addition we examined whether elevated ZPP levels returned to normal following treatment of haemodialysis patients with i.v. iron. Subjects and methods We measured ZPP levels in 148 HD patients without apparent inflammatory disease, infectious disease, or malignancy. Exclusion of malignancy or inflammatory disease was based on lack of signs and symptoms. All patients were negative for antinuclear antibodies, c-ANCA, p-ANCA and rheumatic factor and had a normal white blood cell count. There were 62 HD patients (31 male, 31 female) with ZPP levels > 50 umol/mol haeme who completed the study. These patients were 29-86 years of age (60+15 years, m + SD) and had been on maintenance HD from 14 to 184 months (59 + 37 months, m±SD) without surgical treatment, gastrointestinal blood loss, or unusual blood loss during the study. Some of the patients had been on oral or i.v. iron therapy prior to the start of the study. During the study period, each patient received an intravenous dose of 40 mg iron III sodium gluconate complex (3.2 ml ampoule Ferrlecit) for 24 weeks, once a week after the end of the HD session, resulting in a total i.v. iron load of 960 mg within 6 months. ZPP, serum iron (Fe), serum ferritin (Fer) and haemoglobin (Hb) were measured at the beginning (ZPP1, Fel, Ferl, Hbl), 12 weeks (ZPP2, Fe2, Fer2, Hb2), and 24 weeks after the start of the study (ZPP3, Fe3, Fer3, Hb3). Vitamin B12, folic acid, and aluminium were measured before the start of the study. All patients included in the study had normal folic acid (normal range 4-40nmol/l) and normal vitamin B12 levels (normal range 120-700 pmol/1) and were negative for faecal occult blood loss, as determined by three consecutive tests. Blood lead concentrations were measured at the end of the study. Of the 62 patients, 12 were treated with intravenous desferrioxamine due to elevated aluminium levels and enhanced mobilization of aluminium after a 0.5 g desferrioxamine (Desferal) test dose. To avoid elimination of iron with desferrioxamine, 0.5-2.0 g desferrioxamine was administered once a week at the end of HD therapy and i.v. iron was administered following the next HD therapy. ZPP measurement was performed in a front-face haematofluorometer (AVIV Biomedical Company, Lakewood, NJ) with washed erythrocytes to remove interference from EDTA blood [5]. The haematofluorometer measures the ZPP-haeme ratio as a function of the amount of light emitted by ZPP at 594 nm versus the amount of excitatory light absorbed by haemoglobin at 420 nm. The instrument was calibrated to measure ZPP in umol/mol haeme. ZPP in non-anaemic healthy volunteers from our laboratory and the dialysis unit was 22.7±9.8 umol/mol haeme (m + SD, « = 25). Serum ferritin was measured by enzyme-linked immuno- 493 adsorbent assays (normal range in non-renal patients 30-300 ug/1 for men and 25-150 ug/1 for women). Total blood lead concentrations (normal range < 350 ug/1) and serum aluminium concentrations (normal range < 10 ug/1) were measured by use of atomic absorption spectrometry in the laboratories of Dr Schmidt-Gayk in Heidelberg. Statistics We used SPSS software (SPSS Inc., Chicago, Illinois) for data base management and statistical analysis. The statistical techniques used were linear regression analysis, Levene test of variance, and the Mann-Whitney U test as a nonparametric test. P<0.05 was considered statistically significant. Results The mean ZPP level in 148 HD patients without inflammatory disease or malignancy was 76.40 + 49.74 umol/mol haeme (m + SD) (Figure 1). There is clearly a right-sided tail, reflecting the generally higher ZPP levels in these patients. In the 62 patients with ZPP levels > 50 umol/mol haeme who completed the study, there was no reduction of ZPP (96.7+49.8 versus 88.4±43.5 umol/mol haeme), whereas serum ferritin increased significantly (167.0+138.7 versus 280.2 + 202.4 ug/1, P<0.001) after 24 weeks of iron supplementation. There was no significant correlation between ZPP levels and serum ferritin levels at any time during the study. In 35 of the 62 patients we found ZPP3<ZPP1 and in 27 patients, ZPP3>ZPP1, whereas serum ferritin increased significantly in both these groups. Only in those patients with ZPP levels > 90 umol/mol haeme did i.v. iron supplementation lead to a significant reduction of ZPP (141.2 ±54.5 versus 108.0 + 48.8 umol/mol haeme, P<0.001). Table 1 summarizes the parameters of the patients receiving rHuEpo (« = 36) compared with those not receiving rHuEpo (n = 26). We reduced the rHuEpo in those patients whose haemoglobin increased during iron supplementation to 20.0 60.0 40.0 »0.0 100.0 140.0 100.0 220.0 260.0 300.0 340.O 120.0 160.0 200.0 240.0 290.0 320.0 ZPP(pmd/mol Haeme) Fig. 1. Distribution of zinc protoporphyrin (ZPP) levels in 148 haemodialysis patients without apparent inflammatory or infectious disease or malignancy. J. Braun et al. 494 Table 1. Values ±SD of 36 HD patients on rHuEpo therapy compared with 26 HD patients without rHuEpc> therapy Non-rHuEpo patients (n = 26) rHuEpo patients (n = 36) Hb (g/dl) Fe (ug/1) Ferritin (ug/1) ZPP (u.mol/mol haeme) Baseline 24 weeks Baseline 24 weeks 9.8 + 0.7 51.4±18.5 208.0 ±162.4 92.9±41.3 10.0+1.0 (P = n.s.) 64.2 + 21.3 (P<0.05) 339.3±221.1 (.P<0.001) 88.3 ±39.7 (/>=n.s.) 11.7 + 1.2 46.9 + 17.5 109.6 + 67.1 99.7 + 59.0 12.2 + 1.3 (P = n.s.) 53.0+15.5 (P = n.s.) 200.3 + 140.1 (P<0.001) 83.5+42.9 (/> = n.s.) Significances are calculated for the differences between the parameters at baseline and after 24 weeks of i.v. iron supplementation. keep haemoglobin nearly constant, at a target value of 10 g/dl. The rHuEpo doses after 24 weeks of iron supplementation decreased significantly (P< 0.001) from 4638±2319IU/week (mean 73 IU/kg body weight/week) to 3166+1889 IU/week (mean 50 IU/kg body weight/week). There was no reduction of ZPP, whereas serum ferritin increased significantly. In the responders (10% increase in haemoglobin or 20% decrease in rHuEpo dose), the initial haemoglobin level was significantly lower (9.8 + 0.9 versus 11.1+ 1.4 g/dl, P < 0.001) and the duration of HD treatment was significantly shorter than in the nonresponders (69.3 + 38.8 versus 46.8 + 32.6 months, P<0.05). Stepwise analysis of different ZPP cut-offs, from 50 to 110 umol/mol haeme, revealed that there was a significant difference only in the initial haemoglobin levels between responders and non-responders, but there were no significant differences in the ZPP levels at the start (Table 2) and at the end of i.v. iron supplementation (Table 3), which would have been useful in predicting the erythropoietic response. However, there was a significant inverse correlation between the relative decrease in ZPP levels and the relative increase in serum ferritin levels (r = 0.30, P = 0.017). Of the 62 patients with serum aluminium concentrations of 34.0 + 33.2 ug/1 (m + SD), 12 were on desferrioxamine therapy at the beginning of the study. We found no significant reduction of ZPP either in the desferrioxamine-treated patients (94.1+26.5 versus 83.1 ±32.6 umol/mol haeme, P=n.s., n = 12) or in the non-treated patients (95.9 + 52.9 versus 87.3+42.8 umol/mol haeme, P = n.s., n = 50), whereas the serum iron and serum ferritin levels increased significantly in both groups. Haemoglobin was kept constant in the desferrioxamine treated patients and increased from 10.56 + 1.31 to 11.07 +1.56 g/dl (P<0.05) in the non-treated patients. 100 200 300 400 Total Blood Lead ((jg/1) Fig. 2. Correlation between the total blood lead concentration and ZPP level at the end of the study (r = 0.382, P = 0.002, n = 62, 95% confidence intervals). and the reduction of rHuEpo dose were not significantly different between the two groups. Discussion It has previously been shown that ZPP is a valid parameter in assessing iron deficiency in both healthy blood donors [6] and HD patients [2]. Therefore we selected 62 HD patients with ZPP levels > 50 umol/mol haeme to study the effect and time course of weekly i.v. iron administration on ZPP levels. All patients were without apparent inflammatory disease, infectious disease or malignancy as it is well known that ZPP levels are elevated in these disease states [7]. It was unfortunate that 12 of the 62 HD patients with a ZPP level > 50 umol/mol haeme had some evidence of aluminium overload and required desferrioxamine, as there is clearly an interaction between aluminium and There was a correlation between the whole blood iron. However, there were no other differences between lead concentrations and the ZPP3 levels at the end of the desferrioxamine-treated patients and the nonthe study. Mean total blood lead concentration in the treated HD patients. 62 patients was 190.91 ±57.56 ug/1 (m±SD) The elimination half-life of ZPP from the blood (Figure 2). compartment is closely related to the half-life of erythWe found significantly lower ZPP levels in those rocytes and the production rate of new ZPP in the patients with total blood lead concentrations bone marrow compartment. The half-life of erythro< 200 ug/1 (« = 40) than in those patients with cytes in HD patients has been found to be shorter higher lead concentrations (86.2 ±38.1 versus than in non-renal patients [8]. The study period of 6 115.5 ±65.8 umol/1, P<0.05). Haemoglobin, ferritin months, therefore, was sufficient to achieve significant Zinc protoporphyrin, serum ferritin, serum iron, and haemoglobin levels (a) in responders (b) and non-responders to i.v. iron supplementation at the start of the study ZPP 1 mol Non-responders Fer 1 Responders Fe 1 Hb 1 Non-responders Responders Non-responders Responders Non-responders R 98.4 + 58.5 94.7 ±39.4 199.3 ±169.2 132.6 + 86.4 50.9 ±18.4 46.5±17.9 11.1 + 1.4 9 122.2 + 63.0 113.4±38.4 213.5±193.6 127.0±75.6 50.9 + 18.8 42.1 ±12.0 11.3 + 1.4 9 144.1+69.1 137.9±32.8 219.6 + 233.3 134.3 + 79.3 47.7 + 20.5 44.1 + 11.3 11.2±1.5 9 181.7 + 77.1 146.7 ±29.4 257.4±313.5 141.6 + 76.4 46.1 ±15.5 42.6±11.7 11.6±1.8 9 s. n = 30 vs. n=19 s. n = ll n=9 n values + SD; *P<0.001; **i><0.01; ***/><0.05 compared to non-responders. increase in haemoglobin or 20% decrease in recombinant human erythropoietin dose. Zinc protoporphyrin, serum ferritin, serum iron, and haemoglobin levels (a) in responders (b) and non-responders to i.v. iron supplementation at the end of the study Fer 3 ZPP 3 ol s. H = Non-responders Responders Fe3 Hb3 Non-responders Responders Non-responders Responders Non-responders R 85.0 + 46.2 92.0 ±40.9 320.5 + 228.9 235.7 + 160.7 59.5+16.4 56.3 ±25.8 10.9±1.5 1 102.2 + 50.7 106.3 ±42.2 346.0 ±238.3 253.9± 173.2 60.0 ±17.2 55.6±23.3 11.1 ± 1.8 1 109.4 + 60.2 106.4 + 33.3 359.3 + 274.7 281.0+198.8 59.3+20.3 61.0±27.7 10.9±2.0 1 127.4 + 74.3 112.7 + 31.0 316.4 + 317.6 245.3+98.7 52.2 + 22.2 60.2 + 30.2 11.7 + 2.1 1 30 VS. M=19 . n=ll n=9 n values ±SD. None of the differences were statistically significant. increase in haemoglobin or 20% decrease in recombinant human erythropoietin dose. 496 changes in ZPP levels if the production rate of ZPP was decreased due to iron supplementation. Currently we do not know all the factors influencing ZPP synthesis in HD patients. Patients undergoing maintenance HD therapy have signs of T-cell activation and increased production of interleukin 1, tumour necrosis factor, and interleukin 6. Those cytokines have been incriminated in acute and chronic inflammatory reactions associated with HD [9]. The elevated ZPP levels might therefore be a consequence of these inflammatory processes and the presence of uraemic factors that could potentially inhibit ferrochelatase. Indeed, several abnormalities of the haeme biosynthesis pathway have been observed in patients with chronic renal failure [10]. The observation that whole blood lead levels correlate with ZPP indicates that this parameter must be considered as a confounding factor when interpreting the results. The linear correlation between the moderately elevated total blood lead concentrations found in HD patients in this study and the ZPP3 levels does not imply a cause-and-effect relationship, but elevated lead concentrations may contribute to the disturbed erythropoiesis. Steassen et al. [11] stated that renal insufficiency by itself might be responsible for the increased blood lead concentrations resulting in increased ZPP levels. Colleoni et al. [12] found significantly higher mean blood lead concentrations in HD patients than in healthy subjects. In contrast, two studies [13,14] found that the lead burdens in renal failure patients were not different from those patients with normal renal function and no unusual exposure to lead. The sensitivity of ZPP as a screening test for lead poisoning is at the moment a matter of controversy [15]. Iron deficiency seems to be an additional contributor to increased ZPP levels in those patients with ZPP levels > 90 umol/mol haeme, whereas in patients with ZPP levels < 90 umol/mol haeme ZPP levels remain unchanged during iron supplementation. Our results would support those of Fishbane and Lynn [3,16], who considered HD patients with ZPP levels > 90 umol/mol haeme and serum ferritin levels <100ng/nl, to be iron deficient. These investigators found that among patients with ferritin higher than 100 ng/ml but ZPP >90 umol/mol haeme, seven of 10 patients (70%) responded positively to therapy with i.v. iron (sustained 5% increase of haematocrit or a decrease of rHuEpo dose of more than 2000 IU per treatment). In our patients with ZPP > 90 umol/mol haeme, a positive response was noted in 11 of 24 (45%), but we also found positive reactions in 30 of 62 (48%) patients with ZPP levels > 50 umol/mol haeme. We regarded patients whose haemoglobin increased by 10%, or whose rHuEpo dose was reduced by 20%, as positive responders to i.v. iron supplementation. ZPP levels were not significantly different in responders and non-responders at the beginning of the study; a finding that was independent of the ZPP cut-off in the J. Braun et al. range from 50 to 110 umol/mol haeme. Therefore ZPP concentration cannot be used to predict responders and non-responders to iron supplementation and does not provide additional information that would allow optimization of iron therapy on an individual level. Red cell protoporphyrin was studied in 1983 by Moreb et al. [17] prior to the introduction of rHuEpo. These authors found that red cell protoporphyrin was not predictive of iron deficiency. In our study, there was also no difference between initial ZPP levels and those measured after i.v. iron supplementation in rHuEpo-treated and non-treated patients. Fishbane and Lynn [16] overestimated the sensitivity of ZPP as an indicator of absolute iron deficiency. They selected iron-depleted patients (serum ferritin levels < 100 ng/ml) with ZPP >90 umol/mol haeme, and therefore the positive predictive value of 83% for determining ZPP level changes in response to intravenous iron can only be valid for this particular group of patients. Elevated ZPP levels in HD patients with adequate iron stores may indicate a functional iron deficiency due to a blockade of reticuloendothelial iron release [18], or a disturbed activity of ferrochelatase, an enzyme which catalyses the addition of iron to protoporphyrin. From our results we conclude that nearly 50% of the HD patients with ZPP levels > 50 umol/mol haeme in the presence of adequate iron stores (serum ferritin > 100 ng/ml) will respond to i.v. iron supplementation. Thus i.v. iron supplementation may overcome functional iron deficiency in these patients. However, ZPP does not seem to be a suitable parameter in deciding when to stop i.v. iron supplementation in HD patients with functional iron deficiency, as the elimination halflife of ZPP from the blood compartment is too long to allow one to make this decision, and the production rate of ZPP may be influenced by unknown factors which are independent of iron stores. References 1. Macdougall I, Cavill I. Detection of functional iron deficiency during erythropoietin treatment: a new approach. Br Med J 1992; 304: 225-228 2. Hastka J, Lassere JJ, Schwarzbeck A, Hehlmann R, Strauch M. Zinkprotoporphyrin als Alternative zu Ferritin bei Steuerung der Eisensubstitution erythropoietinbedilrftiger Dialysepatienten. Nieren-Hochdruckkrankheiten 1991; 12: 697-700 3. Fishbane S, Lynn RI. The utility of zinc protoporphyrin (ZPP) as an assay for iron deficiency in hemodialysis patients J Am Soc Nephrol 1993; 4: 425 4. Ahmed U, Fadia A, Baskin S, Lasker N. What is the best laboratory indicator of iron availability in hemodialysis patients. J Am Soc Nephrol 1993; 4: 423 5. Hastka J, Lassere JJ, Schwarzbeck A, Strauch M, Hehlmann R. Washing erythrocytes to remove interferents in measurement of zinc protoporphyrin by front hematofluorometry. Gin Chem 1992; 38: 2184-2189 6. Schifman RB, Rivers SL, Finley PR, Thiers C. RBC zinc protoporphyrin to screen blood donors for iron deficiency anemia. JAMA 1982; 248: 2012-2015 7. Hastka J, Lassere JJ, Schwarzbeck A, Strauch M, Hehlmann R. Zinc protoporphyrin in anemia of chronic disorders. Blood 1993; 81: 1200-1204 Zinc protoporphyrin an indicator of iron-deficient erythropoiesis 8. Eschbach JW. The anemia of chronic renal failure: pathophysiology and the effects of recombinant erythropoietin. Kidney Int 1989; 35: 134-148 9. Descamps-Latscha B, Herbelin A. Long term dialysis and cellular immunity: A critical survey. Kidney Int 1993; 43 [suppl 41]: S135-S142 10. Fontanellas A, Coronel F, Santos JL et al. Heme biosynthesis in uremic patients on CAPD or hemodialysis. Kidney Int 1994; 45: 220-223 11. Staessen JA, Lauwerys RR, Buchet JP et al. Impairment of renal function with increased blood lead concentrations in the general population. N EnglJ Med 1992; 327: 1515-1516 12. Colleoni N, Arrigo G, Gandini E, Corigliano C, D'Amico G. Blood lead in hemodialysis patients. Am J Nephrol 1993; 13: 198-202 13. Batuman V, Wedeen RP. Impairment of renal function with 497 14. 15. 16. 17. 18. increasing blood lead concentrations. N Engl J Med 1992; 327: 1394 Emmerson BT. Lead stores in patients with renal insufficiency. Nephron 1991; 58: 233-234 Turk S, Schonfeld DJ, Cullen M, Rainey P. Sensitivity of erythrocyte protoporphyrin as screening test for lead poisoning. N Engl J Med 1992; 326: 137-138 Fishbane S, Lynn RI. The utility of zinc protoporphyrin for predicting the need for intravenous iron therapy in hemodialysis patients. Am J Kidney Dis 1995; 25: 426-432 Moreb J, Popovetzer MM, Friedlander MM, Konijin AM, Hershko C. Evaluation of iron status in patients on chronic hemodialysis: relative usefulness of bone marrow hemosiderin, serum ferritin, transferrin saturation, mean corpuscular volume, and red cell protoporphyrin. Nephron 1983; 35: 196-200 Krantz SB. Pathogenesis and treatment of the anemia of chronic disease. Am J Med Sci 1994; 307: 353-359 Received for publication: 22.12.94 Accepted in revised form: 23.10.95