Efficiency of three different hemodialysis membranes for plasma porphyrin removal

Efficiency of Three Different Hemodialysis Membranes for Plasma Porphyrin Removal Antonio Fontanellas, WC, Jose A. Herrero, MD, Maria J. Moran, PhD, Francisco Coronel, MD, Paloma Sepulveda, BSc, Albert0 Barrientos, MD, and Rafael Enriquez De Salamanca, MD 0 To assess the capability of three diierent membranes to remove porphyrins, plasma and dialysate porphyrin levels were fluorometrically measured in 10 patients with end-stage renal failure who were on hemodiaiysis. Three different hemodiaiysis membranes were used: cuprophan, polyacrylonitffle, and cellulose trlacetate. Total plasma porphyrin concentrations decreased after dialysis, but to a lesser extent when using the cuprophan membrane (19%) than with the poiyacrylonitrile (28%) or cellulose triacetate (20%) membranes (P < 0.01). However, since the free plasma porphyrin fraction remained unchanged, it can be assumed that the equilibrium between proteinbound and non-protein-bound (free) porphyrins is displaced toward the latter fraction. Dialysate porphyrin levels were lower (P < 0.01) when using the cuprophan membrane (10.1 pgkssion) than when using polyactylonitrile (17.8 j&3ession) and cellulose triacetate (21.9 ccg/session). Although most of the plasma porphyrins are protein bound, our results show that hemodialysis can remove significant amounts of non-protein-bound (free) porphyrins. The polyacrylonitrlle and cellulose trlacetate membranes had a greater capacity for porphyrin removal than cuprophan. Thus, two high-permeabilii membranes (polyacrylonittile and cellulose trfacetate) should be used whenever a reduction of plasma porphyrin levels is desired. 0 1995 by the National Kidney Foundation, Inc. INDEX WORDS: High permeability phyria.

membranes;

end-stage

DLASMA porphyrin levels are increased in 1 patients with end-stage renal failure, particularly in those on hemodialysis.14 Since most plasma porphyrins are protein bound (mainly to albumin and hemopexin),5 it has been widely accepted that these porphyrins are unable to dialyze across the membranes used in hemodialysis.6W’0 Using a sensitive fluorometric method we have detected measurable amounts of porphyrins in the dialysate from uremic patients receiving either hemodialysis or peritoneal dialysis.” The aim of the present study was to compare the porphyrin clearance capacity of three different hemodialysis membranes. PATIENTS AND METHODS The study group comprised 10 patients (three women and seven men, aged 40 to 78 years) with end-stage renal failure.

From the Porphyria Unit, Hospital Univcrsitario Dote de Octubre, and Nephrology Service, Hospital Universitario San Carlos, Madrid, Spain. Received June 7, 1994; accepted in revised form August 19, 1994. Presented in part at the 30th European Dialysis and Transplant Association/European Renal Association Congress, Glasgow, Scotland, September 15-18, 1993. Supported by grant no. 93/l 12 from the Spanish Fond0 de Investigaciones Sanitarias de la Seguridad Social. Address reprint requests to Rafael Enriquez de Salamanca. MD, Francisco Gervas 9, 28020 Madrid, Spain. 0 1995 by the National Kidney Foundation, Inc. 0272-6386/95/2501-0006$3.00/O 30

American

Journal

renal disease; free and protein-bound

porphyrins;

por-

All had been on hemodialysis for at least 4 months (median, 24 months) prior to entering the study. Hemodialysis procedure was performed thrice weekly (3.5 hours/session) under similar conditions: bicarbonate buffer, dialysate flow 500 mumin, and blood flow ranging between 350 and 400 mL/min. Over a 3-week period, the patients were dialyzed with a different membrane each week: cuprophan, 2 mZ surface area (CU); polyacrylonitrile, 2 mZ surface area (PAN); or cellulose triacetate, 1.9 m* surface area (CT). The membranes were all of the hollow fiber dialyzer type. Predialysis and postdialysis arterial blood samples were collected at each of the three weekly sessions. In the middle of each session a 2.5-L sample of the dialysate was obtained and lyophilized for porphyrin measurement. Plasma and dialysate porphyrins were fluorometrically analyzed using the Piomelli method.” The ultrafiltration volume was adjusted to the patient’s dry weight at each dialysis session. Since the dialysis procedures were not isovolemic, and taking into account the distribution volume for porphyrins, the postdialysis plasma porphyrin concentrations were corrected for changes in the extracellular volume using the following formula: measured porphyrins divided by the 1 + (body weight change during dialysisl(0.2 x postdialysis body weight)) ratio.” The free (non-protein-bound) fraction of plasma porphyrins was quantified after ultrafiltration through a YMT membrane (Centrifree; Amicon, Danver, MA) with an exclusion limit of 30,000 molecular weight. The individual carboxylated porphyrins in the dialysate were identified by thin-layer chromatography of their methyl esters.14 We measured the blood urea nitrogen (BUN) levels at the midweek dialysis for each of the membranes, at the time points 0 and 210 minutes. The removal efficiency of the hemodialysis for “small molecules” was estimated according to the following formula: Kt/V = In (BUNfiUN21,,).‘5 of Kidney

Diseases,

Vol 25, No 1 (January), 1995: pp 30-33

PLASMA PORPHYRIN REMOVAL DURING HEMODIALYSIS Table 1. Influence

of Three Hemodialysis

31

Membranes

cu Porphyrins

Pre-HD

on Plasma Porphyrin

Levels

CT

PAN

Post-HD

Pre-HD

Post-HD

Pm-HD

Post-HD

1.69 k 0.47

1.35 r 0.39

1.68 t 0.4

1 .17 2 0.28t

1.76 t 0.43

1.29 2 0.32t

1.23 2 0.39 73

0.96 2 0.35 m

1.21 ? 0.36 72

0.83 2 0.27 w

1.30 t 0.37 74

0.92 2 0.36

0.45 2 0.19 27

0.39 ? 0.2 3’3

0.47 k 0.2

0.35 -c 0.14

20

w

0.46 + 0.17 26

0.37 -c 0.17 3ot

Total &dL Protein bound 1.1ddL % Free MdL 46

7’3

Abbreviation: HD, hemodialysis. l P < 0.01, v pre-HD. t P < 0.001, Y pre-HD (NS).

Statistics Differences between prehemodialysis and posthemodialysis plasma porphyrin levels were assessed using Student’s paired r-test. ANOVA was used for other intergroup comparisons. The Pearson correlation coefficient (r) was computed to test associations between pairs of variables. The null hypothesis was rejected when P < 0.05. RESULTS

Posthemodialysis levels of total, proteinbound, and free plasma porphyrins were lower than prehemodialysis ones with all three membranes. However, the fractional percentages of protein-bound and free plasma porphyrins were unchanged (Table 1). After hemodialysis, the plasma porphyrin levels decreased by 19%, 26%, and 30% when using the CU, PAN, and CT membranes, respectively. The CU values differed significantly from those achieved with the PAN and CT membranes (19% v 26%, P < 0.05; 19% v 30%, P < 0.01). The weight change (loss) by ultrafiltration during the hemodialysis procedure was similar in the three groups: CU 1.28 2 0.79 kg, PAN 1.32 + 0.86 kg, and CT 1.35 + 0.81 kg. The Kt/V values were CU 1.18 t 0.2, PAN 1.22 + 0.29, and CT 1.21 2 0.22. These differences were not significant. The amount of porphyrins filtered into the dialysate was also significantly greater when using the PAN or CT membrane than when using the CU membrane (Fig 1). This parameter correlated weakly with the hemodialysis-induced percentage decrease in total plasma porphyrins (r = 0.52, P < 0.001).

The profile of the dialysate porphyrins (Table 2) revealed a predominance of the highly carboxylated forms, octo-(uroporphyrin) and hepta-carboxy1 porphyrin, with low percentages of tetracarboxyl porphyrin (coproporphyrin). DISCUSSION

Plasma porphyrins are largely protein bound (mainly to albumin and hemopexin), with varying affinities.5 Some investigators have suggested that protein binding might prevent the dialysis of porphyrins across standard hemodialysis membranes.6-‘0 However, free plasma porphyrins, which are in equilibrium with the proteinbound fraction,5.‘6 pass across the hemodialysis membranes due to their low molecular weight.

**

24 I

I I

20 1s 10 S 0

cu

PAN

/ CT

Fig 1. Diaiysate porphyrin removal with three different membranes. The asterisks denote a statistically significant difference versus CU. “P < 0.01.

FONTANELIAS

32 Table 2. Dialysate Porphyrin

CU membrane (n = 30) CT membrane (n = 30) PAN membrane (n = 30)

Profile From Patients Dialyzed With Three Dierent

ET AL

Membranes

Uroporphyrin

Hepta

Hexa

Penta

Coproporphyrin

69.30 ” 15.54 63.87 + 11.7 63.90 2 16.02

14.33 -+ 8.81 16.33 k 11.47 14.70 k 8.14

0.13 2 0.7 0.13 !I 0.57 0.80 + 2.94

0.44 2 2.4 0.46 k 1.38 0.80 k 1.92

15.8 2 13.38 19.2 2 11.68 19.8 2 15.65

NOTE. Values (%) are given as means 2 SD.

The decrease in plasma porphyrin levels induced by hemodialysis must therefore be due to the removal of the non-protein-bound fraction. Nevertheless, since the relative proportions of bound and free plasma porphyrins remained unchanged, it should be assumed that the equilibrium between the free and bound porphyrins is moved toward the free porphyrin fraction. Contrary to other reports,“3-9’17 but confirming our previous findings,” we have observed that hemodialysis lowers plasma porphyrin concentrations. These conflicting results might be due to differences in the removal efficiency of the dialysis membrane used. The hemodialysis procedure as performed with our three membranes can be considered to be highly efficient for the depuration of small molecules, as the Kt/V values show. Therefore, the observed greater efficiency of the PAN and CT membranes in removing plasma porphyrins could be due to a greater permeability of those membranes for middle molecules. When the hemodialysis is not isovolemic, hemoconcentration usually has not been considered by other researchers,‘V3-9*17 and this phenomenon may be a confounding factor in the evaluation of the results. The plasma porphyrin concentration may be falsely increased after hemodialysis because of the contraction of the porphyrin distribution volume during ultrafiltration. Therefore, the posthemodialysis measured plasma porphyrin values should be corrected, as suggested by Bergstrom and Wehle.13 The greater efficiency of PAN and CT membranes for reducing plasma porphyrin levels is therefore likely to be due to their greater permeability. Unlike previous reports,1S6S’o*17 we have confirmed that hemodialysis effectively removes porphyrins by detection of substantial concentrations of porphyrins in the dialysate. This difference might be explained by the fact that the dialysate porphyrin concentrations are lower than the

detection limits of most analytical methods. It therefore would be appropriate to concentrate the dialysate before measuring porphyrins with sensitive fluorometric techniques. The greater permeability of the PAN and CT membranes achieved a higher dialysate porphyrin “excretion” than that observed with the CU membrane. However, even when the former membranes are used, the porphyrin excretion achieved by a single hemodialysis session (10 to 25 pg) is much less than that of a normal kidney (approximately 80 pg/d). Normal urine contains various carboxylated porphyrins, mainly coproporphyrin.” In chronic renal failure the urinary excretion of coproporphyrin, but not that of uroporphyrin, progressively decreases. This suggests that urinary coproporphyrin is not only filtered by the kidney, but also in part is synthesized by the organ.“*” End-stage renal failure is associated with an increase in plasma porphyrin levels, mainly uroporphyrin. Although the molecular weight of uroporphyrin (830.7) is higher than that of coproporphyrin (654.7), it has much less affinity for plasma proteins: the dissociation constant for uroporphyrin is 100 pmol/L, compared with 5 pmol/L for coproporphyrin. This might explain the finding of high relative proportions of uroporphyrin and low ones of coproporphyrin in the dialysate, a profile that is the reverse of that in normal urine. Some uremic patients on hemodialysis suffer from bullous dermatosis, a cutaneous lesion resembling porphyria cutanea tarda. Although the plasma porphyrin levels may be markedly elevated in these patients, the etiopathogenesis of this dermatosis remains obscure. However, true porphyria cutanea tar-da has been reported in some patients on hemodialysis.6‘10 In these cases the plasma porphyrin levels are extremely high, producing severe photocutaneous involvement. Carson et al** have recently demonstrated that

PLASMA PORPHYRIN REMOVAL DURING HEMODIALYSIS

conventional hemodialysis is ineffective in removing porphyrins in this condition, while highflux polysulfone dialyzers achieve a significant porphyrin clearance. Therefore, in patients with porphyria cutanea tarda on hemodialysis, highly permeable membranes should be used since they will enable a greater and quicker efficacy of the well-known treatment with recombinant human erythropoietin.23*24 In conclusion, the higher the permeability of membranes used in hemodialysis, the greater will be the removal of plasma porphyrins. Whenever a reduction of plasma porphyrin levels seems desirable, high-permeability membranes should be used. REFERENCES 1. Day RS, Eales L: Porphyrins in chronic renal failure. Nephron 2690-95, 1980 2. Topi GC, D’Alessandro Gandolfo L, De Costanza F, Cancarini GC: Porphyria and pseudo-porphyria in hemodialyzed patients. Int J Biochem 12:963-967, 1980 3. Poh-Fitzpatrick MB, Sosin AE, Benis J: Porphyrin levels in plasma and erythrocytes of chronic hemodialysis patients. J Am Acad Dermatol 7:100-104, 1982 4. Gebril M, Weinkove C, Ead R, McDonald K, Morton R: Plasma porphyrins in chronic renal failure. Nephron 55:159-163, 1990 5. Morgan WT, Smith A, Kostelo P: The interaction of human serum albumin and hemopexin with porphyrins. Biochim Biophys Acta 624:271-285, 1980 6. Lichtenstein JR, Babb EJ, Felsher BF: Porphyria cutanea tarda (PCT) in a patient with chronic renal failure on haemodialysis. Br J Dermatol 104:575-578, 1981 7. Topi GC, D’Alessandro GL, Cancarini GC, De Costanza F, Griso D, Ravelli M: Porphyria cutanea tarda in a haemodialyzed patient. Br J Dermatol 104:579-580, 1981 8. Kiistler VE, Heinicke HJ: Porphyria cutanea tarda, pseudoporphyrie und hanmdialyse chronisch niereninsuffizienter. 2 Gesamte Inn Med 41:48-52, 1986 9. Yaqood M, Smyth J, Ahmad R, McClelland P, Fahal I, Kumar KAS, Yu R, Verbov J: Haemodialysis-related porphyria cutanea tarda and treatment by recombinant human erythropoietin. Nephron m428-43 1, 1992 10. Hebert AA, Farmer KL, Poh-Fitzpatrick MB: Perito-

33 neal dialysis does not reduce serum porphyrin levels in porphyria cutanea tarda. Nephron 60~240, 1992 11. Fontanellas A, Coronel F, Santos JL, Herrero JA, Moran MJ, Guerra P, Tomero F, Emiquez De Salamanca R: Heme biosynthesis in uremic patients on CAPD or I-ID. Kidney Int 45:220-223, 1994 12. Piomelli S: Free erythrocyte porphyrins in the detection of undue absorption of Pb and of Fe deficiency. Clin Chem 23:264-289, 1977 13. Bergstrom J, Wehle B: No change in corrected p2. microglobulin concentration after cuprophane haemodialysis. Lancet 1:628-629, 1987 14. Day RS, De Salamanca RE, Eales L: Quantitation of red cell porphyrins by fluorescence scanning after thin-layer chromatography. Clin Chim Acta 89:25-33, 1978 15. Barth RH: Direct calculation of Kt/V. A simplified approach to monitoring of hemodialysis. Nephron 50:191195, 1988 16. Santos JL, Moran MJ, MuAoz JJ, Fontanellas A, De Salamanca RE: Influence of PH on the porphyrin binding to plasma proteins. Honn Metab Res 14:140, 1992 17. Poh-Fitzpatrick MB, Masullo AS, Grossman ME: Porphyria cutanea tarda associated with chronic renal disease and hemodialysis. Arch Dermatol 1163191-195, 1980 18. Garcia-Patrilla J, Ortega R, Pefia ML, Rodicio JL, De Salamanca RE, Olmos A, Elder GH: Porphyria cutanea tarda during maintenance haemodialysis. BMJ 280~1358, 1980 19. Enriquez De Salamanca R, Peiia ML, Chinarro S, Olmos A, Mingo D, Molina C, Muiloz JJ: Quantitative and qualitative porphyrin excretion in normal subjects. Int J Biothem 14:251-254, 1982 20. Day RS, Eales L, Disler PB: Porphyrins and the kidney. Nephron 28:261-267, 1981 21. Mart&ek P, Jirsa M, Kordac V: Role of the kidneys in porphyrias. Nephron 32:277-278, 1982 22. Carson RW, Dunnigan EJ, DuBose TD, Goeger DE, Anderson KE: Removal of plasma porphyrins with high-flux hemodialysis in porphyria cutanea tarda associated with endstage renal disease. J Am Sot Nephrol 2:1445-1450, 1992 23. Anderson KE, Goeger DE, Carson RW, Lee SMK, Stead RB: Erythropoietin for the treatment of porphyria cutanea tarda in a patient on long-term hemodialysis. N Engl J Med 322:315-317, 1990 24. Peces R, Emiquez De Salamanca R, Fontanellas A, Sanchez A, De La Torre M, Caparros G, Ferteras I, Nieto J: Successful treatment of haemodialysis-related porphyria cutanea tarda with erythropoietin. Nephrol Dial Transplant 9:433-435, 1994