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Nephron 1990;55:159-163

Plasma Porphyrins in Chronic Renal Failure Mustafa Gebril'. Cyril Weinkovea. Russ Eadh. Ken McDonaldb, Ross Mortonc 'Department of Medicine (Clinical Biochemistry). University of Manchester, Hope Hospital, '’Dermatology Department, Salford Royal Hospital and T h e Renal Unit, Hope Hospital, Salford, UK

Key Words. Porphyria • Coproporphyrin • Uroporphyrin • Haemodialysis Abstract. Using a quantitative sensitive HPLC fluorometric assay for the measurement of plasma and urine porphyrins, we have calculated the renal clearance of uro- and coproporphyrin in normal volunteers and in patients with congenital erythropoietic porphyria and hereditary coproporphyria (HCP). In patients with porphyria cutanea tarda (PCT), only uroporphyrin clearance was calculated. Plasma porphyrin concentrations were measured in patients with chronic renal failure (CRF). All our CRF patients on haemodialysis (10) had raised plasma uropor­ phyrin, 4 had raised plasma heptacarboxyporphyrins and 2 had raised plasma coproporphyrin, unchanged by haemodialysis. CRF patients with raised uroporphyrins and 7-carboxyporphyrins could be distinguished from PCT in patients with normal renal function, by their higher uro/7-carboxyporphyrin ratio (8.0 vs. 1.7). Renal clearance of coproporphyrin was much greater in HCP than in any of the other groups studied. Since coproporphyrin clearance was less than creatinine clearance in most normal subjects, and because 2 patients with CRF had raised plasma coproporphyrin concentrations, we argue that the kidney is probably not a major source of coproporphyrin, as previously reported. Furthermore, the variability and degree of abnormality in plasma porphyrins in CRF would suggest caution in diagnosing chronic porphyria in patients with renal impairment.

The porphyrias are a group of diseases in which there is a disorder of the metabolic pathway of haem synthesis such that excess porphyrins are produced. Specific en­ zyme defects in this pathway result in different patterns of porphyrin accumulation in plasma and red blood cells, and/or different excretion patterns in faeces and urine. Diagnosis therefore depends on the accurate identifica­ tion of these porphyrins in tissue fluids and excreta [1]. Impaired renal function may interfere with the excre­ tion of porphyrins and lead to raised plasma porphyrins. Several cases of porphyria associated with chronic renal failure (CRF) in patients on haemodialysis have been reported [2-8]. In addition to these cases of clinical and biochemically confirmed porphyria, bullous dermatosis, clinically similar to porphyria cutanea tarda (PCT) but with normal plasma porphyrins, has been observed in patients on haemodialysis [9, 10]. Whereas porphyries with normal renal function may have increased urine porphyrin excretion, plasma por­ phyrin measurement may be the only option in patients

with C RF. It is therefore important to understand the role of the kidney in porphyrin metabolism. We studied plasma and urine porphyrin concentrations in normal volunteers, known porphyries and non-porphyrics with CRF.

Subjects and Methods Subjects Normal volunteers from the laboratory staff provided blood and timed urine specimens for the measurement of porphyrin clearance. Appropriate specimens were also collected from patients known to have one of the following disorders: PCT, congenital erythropoietic porphyria (CEP) and hereditary coproporphyria (HCP). Pre- and postdialysis blood specimens were collected from 10 patients with CRF and compared with normals and the above-mentioned por­ phyries (table I). Materials The HPLC apparatus was as supplied from LDC-Milton Roy (Stone, Staffordshire, UK) with a Schoeffel FS970 fluorometer and Xenon lamp supplied by Kratos Instruments (Ramsey, N.J.). The auto-injector was supplied by Gilson Medical International (Mid-

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Introduction

160

Gebril/W einkove/Ead/M cD onald/M orton

Table 1. Plasma porphyrins (nmol/l) in normals, porphyria and CRF Diagnosis

Age years

Sex

Normal (20) PCT (11) CEP (2) HCP (1) CRF patients on haemodialysis Diabetic nephropathy Analgesic nephropathy Chronic pyelonephritis Radiation nephritis Renal amyloidosis Cause unknown Cause unknown IgA nephropathy Renal amyloidosis Cause unknown

49 61 67 24 68 66 58 58 58 58

m f r m f r m m r f

Uroporphyrin

7-COOH

Coproporphyrin

0-3.8 (1.35) 30-412(157) 727. 1,520 0.7

_

0.4-2.5 (1.4)

44/35 18/20 12/12 6/11 24/20 29/21 42/29 6/9 56/65 38/38

14-230(97) 17.45 -

-

171,1,300 1.6

1.6/2.2

-

-

-

-

-

-

-

2.0/3.5

-

-

5 /3/3 11/11 3 /-

9.0/4.5 4.7/5.0 1.6/-

Plasma from normals and HCP patients were concentrated up to 40 times. Other plasma samples were not concentrated. Figures in parentheses refer to the number of subjects studied or the mean value of the range. Individual results are recorded when there were less than 3 subjects. 7-COOH = Heptacarboxyporphyrin: - = undetectable concentrations; / = pre- and postdialysis results recorded for the CRF patients.

HPLC Conditions This method was modified from that of Lim and Peters [llj. The separation of the free porphyrin carboxylic acids was achieved by reverse phase chromatography using SAS Hypersil (5 pm) columns with C|Sguard columns. The sample size employed throughout this study was 100 pi. The fluorometric detector was set with an exci­ tation wavelength of 404 nm and used a 580-nm cut-off filter for emission. The mobile phases A and B consisted of 10% acetonitrile in 1.0 mol/l ammonium acetate (pH 5.16) and 10% acetonitrile in metha­ nol, respectively. Both solvents were degassed and filtered (0.45 pm pore size) prior to use. The flow rate was maintained at 1.0 ml/min with the gradient adjusted as follows: linear gradient from 0 to 80% solvent B in 30 min: linear gradient from 80 to 90% solvent B in 5 min and maintained for a further 5 min: 90-0% solvent B in 5 min and maintained for 5 min prior to next injection.

Preparation o f Internal and Marker Standards Stock Protoporphyrin Standard. 5 pg of protoporphyrin was dissolved in 0.1 ml of Protosolv (using an ultrasonic bath to ensure complete solution) and made up to 1.0 ml with 1.5 m ol/l hydro­ chloric acid (HCI). Mixed Porphyrin Standard. One vial of the mixed porphyrin standards containing 10 nmol of each of the six porphyrins (8-, 7-, 6-, 5-,4-carboxylicacid and mesoporphyrin) was reconstituted with 1.0 ml of the stock protoporphyrin standard. Plasma Internal Standard. A stock standard was prepared by dissolving 79.1 mg of meso-tetra (4-carboxyphenyl)-porphine in I litre of 1.5 mol/l HCI to give a I00pmol/l solution. This was stored at 4°C for up to 6 months and diluted to 5 um ol/l with deionised water, prior to use. Urine Internal Standard. A stock standard was prepared by dissolving 50 mg of 2-vinyl 4-hydroxymethyl deuteroporphyrin IX in I litre of 6 m ol/l HCI to give a 100 pm ol/l solution. This was stored at 4°C for up to 6 months and diluted to 5 pmol/l with deionised water, prior to use. Plasma Extraction Methods Dimethyl Sulphoxide Method. 10-50 pi of a freshly diluted plasma internal standard was added to the appropriate volume of plasma (0.5 ml from porphyries and up to 10 ml from normal volunteers) and mixed with 4 ml of a solution of 15% trichloracetic acid (TCA) and dimethyl sulphoxide (1:1 v/v)for2 min. After centrifugation at 1,200 g for 15 min, the supernatant was transferred to a new tube. The precipitate was re-extracted using 2 ml of the TCA/dimethyl sul­ phoxide mixture and both supernatants combined. In the case of porphyric plasma this solution was then filtered through a dispos­ able 0.45- pm filter prior to injection into the HPLC analyser. For

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dleton. Wise.) and fitted with a rheodyne 7010 sample valve and 100-pl sample loop. SAS Hypersil and Bond Elut columns were obtained from Shandon (Runcorn,Cheshire. UK) and Analytichem International (Har­ bor City, Calif.) respectively. Disposable filters were bought from Gelman Science (Ann Arbor, Mich.). Porphyrin standards, the internal standards, meso-tetra (4-carboxyphenyl)-porphine and 2 vinyl-4 hydroxymethyl deuteroporphyrin IX as well as Protosolv were obtained from Porphyrin Products (Logan, Utah). Methanol and acetonitrile were HPLC grade and obtained from May & Baker (Dagenham. UK): Dimethyl sulphoxide (ACS reagent grade) was obtained from Sigma Chemical Co. (Poole, Dorset, UK) and all other chemicals (Analar) from BDH Chemicals, Poole, UK.

161

Plasma Porphyrins in Chronic Renal Failure

Table 2. Urine porphyrin excretion (nmol/24 h) in normal and porphyric subjects Subjects Normal (86) Mean Range PCT (20) Mean Range CEP (2) HCP (1)

Uroporph

7-COOH

10.8 1.5-38

4.2 0-16

3.838 95-16,100 19,200, 75,000 13

2,140 65-11,400 1,350, 1.700

6-COO H

-

-

187 70-266 755.500 -

COP 1

COPII1

22 7-70

77 6-266

-

-

-

-

6,140. 13,700 491

-

3,200

normal plasma, a concentration stage was necessary and was per­ formed as follows; Bond Elut columns (C|8) were prepared by washing with 6 ml of methanol followed by 6 ml of the ammonium acetate solution. The combined supernatants were applied to the columns and allowed to drain through. The columns were washed with 3 ml of ammonium acetate solution and the porphyrins eluted from the columns with two applications of 0.5 ml methanol with a vacuum pump. The 1.0-ml methanol containing the porphyrins was evaporated to dryness using a Buchler vortex evaporator (40°C) and the porphyrins reconstituted in 250 ul of 1.5 mol/1 HCI.

Table 3. Uroporphyrin clearance in normal and porphyric sub­ jects

Urine Extraction Method (Modifiedfrom the Method o f Doss/ / 2]) 0.2 ml of freshly diluted urine internal standard (5 umol/l) was added to 9 ml of urine and the pH adjusted to 3.5 with 6 mol/1 HCI. 0.5 g talc was added, the tube contents vortex-mixed and then centrifuged at 1,200 g. The supernatant was decanted and the talc cleaned with three 5-ml water washes using sequential vortex mix­ ing, centrifugation and decantation. Talc-bound porphyrins were extracted by vortexing with a mixture of 3 ml acetone and I mol/1 HCI (9:1, v/v). Tubes were centrifuged at 1,200 g for 5 min. I ml of the clear supernatant was transferred to a clean glass tube and the acetone evaporated using a Buchler vortex evaporator. The dried extract was reconstituted in I ml of 1.5 mol/1 HCI and sonicated to ensure complete dissolution. After passing through a 0.45-um dis­ posable filter, 100 pi of the extract was injected onto the HPLC' column.

Mean uroporphyrin clearance with the range, where applicable in parentheses. The number of subjects are recorded in parentheses after each condition.

Identification o f Porphyrin Isomers The porphyrin isomers I and 111 were separated using the H PLC system described above and were checked by co-injection of a known isomer with the sample.

Results Plasma Porphyrin Concentrations. The plasma por­ phyrin concentrations for normal, porphyric patients and patients in CRF pre-and postdialysis are shown in table 1.

Subject

Uroporphyrin clearance ml/min

Normal (6) PCT (5) CEP (1) HCP (1)

9.7 (3.0-31) 31.6(7.5-50) 13 107

Urinary Porphyrin Excretion. Urinary porphyrin excre­ tion in 86 normal subjects, 20 PCT patients, 2 CEP and 1 HCP patient is shown in table 2. Renal Clearance o f Uroporphyrin. The renal clearances of uroporphyrin in normal subjects and patients with PCT, CEP and HCP are shown in table 3. Renal Clearance o f Creatinine and Coproporphyrin. The renal clearances of creatinine and coproporphyrin in normal subjects are shown in figure l.The renal clearance of coproporphyrin in HCP was 1,530 ml/min and in CEP 13 ml/min. Renal clearance of coproporphyrin was not measured in patients with PCT, since the excessive amounts of uroporphyrin and 7-carboxyporphyrin satu­ rated the HPLC columns and did not permit separation of the low concentrations of coproporphyrin in plasma.

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24-hour urine porphyrin excretion in normal subjects and porphyries. The mean (range) are recorded where appropriate, with dashes indicating undetectable concentrations. Figures in parentheses referto the number of subjects studied. Individual results are recorded when there were less than 3 subjects. Uroporph = Uroporphyrin; 7-COOH = heptacarboxyporphyrin; 6-COO H =hexacarboxy porphyrin; COP I = coproporphyrin l;C'O P 111 = coproporphyrin 111.

20 —

*

Creatinine

Coproporphyrin

Fig. I. Differences in the clearance of creatinine and copropor­ phyrin in healthy normal volunteers.

Discussion We had hoped that the measurement of plasma por­ phyrins would be sufficient to diagnose all the chronic porphyrias. However 1 patient with HCP, diagnosed on the basis of high urinary coproporphyrin 111 excretion, was found to have a normal plasma concentration of this porphyrin (table I). This could be explained if copropor­ phyrin was produced in the kidney as claimed by Day and Eales [5] and excreted into the urine before accumu­ lating in the plasma. However they reported high plasma coproporphyrin concentrations in their 2 patients with HCP [13]. This stimulated us to examine the renal han­ dling of porphyrins in patients with CRF. In agreement with other workers [3,5,13] we found no significant difference between pre- and posthaemodialysis samples. Seubert et al. [14] found that uroporphyrin I was the main isomer in patients with CRF, unlike Day and Eales [5] who found mainly isomer III. We could only detect uroporphyrin I and not uroporphyrin III in the plasma of our patients. Anderson et al. [8] had found raised plasma uropor­ phyrin in 58% of their renal failure patients. However, by concentrating the plasma of our renal failure patients, we were able to demonstrate that they all had raised plasma

uroporphyrins (2-20 times the upper limit of normal), similar to the results reported earlier by Day and Eales [5]. Using our sensitive fiuorometric assay for the mea­ surement of plasma and urine porphyrins we calculated the renal clearance of porphyrins in normal (fig. 1) and porphyric subjects. In contrast to Day and Eales [5] we found that the majority of our normal subjects had a greater creatinine than coproporphyrin clearance. We cannot exclude the possibility that our 15% TCA/ dimethyl sulphoxide extraction procedure may be more effective in extracting protein-bound porphyrins than the 5% H2S 04/methanol solution used by Day and Eales [5]. This would give a higher total plasma concentration (free plus protein-bound porphyrin) and a consequent under­ estimation of calculated porphyrin clearances, since one expects only the non-bound porphyrins to be excreted in the urine. However this should not effect the difference in plasma coproporphyrin concentrations between normal and CRF subjects. The HCP patient was found to have a coproporphyrin clearance greater than the creatinine clearance. It could be argued that the kidneys of patients with HCP were maintaining normal levels of circulating coproporphyrin by active tubular secretion and that, if we infused copro­ porphyrin into normal subjects, they too would increase their renal coproporphyrin clearance. This may not be an ethical experiment in view of the light sensitivity induced by this compound. Interestingly coproporphyrin clearance was low (13 ml/min), in the I patient with CEP in whom it was measured. These patients produce an excess of copropor­ phyrin I, which is found in excess in both plasma and urine. In HCP the plasma coproporphyrin was normal but the urine excretion was high, with a calculated clear­ ance of 1,530 ml/min. This would suggest that the copro­ porphyrin III found in HCP is handled differently to coproporphyrin I. If coproporphyrin III was produced in the kidney we would expect to find low plasma concentrations in pa­ tients with CRF, as previously reported [5]. 5 of our 10 patients had detectable plasma coproporphyrin, while 2 patients had higher than normal concentrations (up to 4 times the upper limit of the reference range). We do not know what happens to the coproporphyrin in CRF patients with undetectable plasma copropor­ phyrins. It could be argued that the excess coproporphyr­ ins in these patients are excreted in the faeces. We did not measure faecal porphyrin excretion in our subjects. There are major methodological problems in ensuring adequate faecal collections and avoiding dietary or bac­

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G ebril/W einkove/Ead/M cD onald/M orton

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Plasma Porphyrins in Chronic Renal Failure

terial contributions to faecal porphyrins. Furthermore, since the major component of faecal porphyrin is of exogenous origin [15], we doubt the validity of faecal coproporphyrin measurement as an index of faecal excretion. 4 of the renal failure patients had raised plasma heptacarboxyporphyrins (normally undetectable), but found classically in PCT. To further complicate the problem, patients with CRF may develop skin lesions which are clinically and histologically indistinguishable from PCT [16] (pseudoporphyria). It is important to distinguish pa­ tients with PCT from those with CRF and skin lesions. We found that patients with CRF have a higher ratio of uroporphyrin to heptacarboxyporphyrin (8.0 vs. 1.7) than patients with true PCT. Day and Eales [5] also com­ mented on the higher uro/7-carboxyporphyrin ratio in CRF than in patients with uncomplicated PCT (i.e. with­ out renal failure). None of our CRF patients had light sensitivity. Day and Eales [5] reported that 2 of their patients (D.T. and L. de S.) suffered from both PCT and CRF and had a high uro/7-carboxyporphyrin ratio, indistinguishable from CRF without light sensivitity. The diagnosis of PCT was made on the finding of isocoproporphyrin in the faeces, an established diagnostic marker [17]. This would argue against the value of a high uro/7-carboxyporphyrin ratio as a distinguishing feature between CRF alone and CRF with PCT. In summary, we have demonstrated that all our CRF patients have increased plasma uroporphyrin concentra­ tions, which was unchanged after haemodialysis. 2 of these patients had increased plasma coproporphyrin concentrations, which does not support the thesis that the kidney is essential for coproporphyrin synthesis. Despite plasma porphyrins approaching concentrations found in PCT, none of our renal failure patients were photosensi­ tive. Our data and that of other workers imply that plasma porphyrin concentrations must be interpreted with caution in patients with CRF.

163

2 Poh-Fitzpatrick MB, Bellet N. Deleo VA. et al: Porphyria cuta­ nea tarda in two patients treated with hemodialysis for chronic renal failure. N Engl J Med 1978:299:292-294. 3 Poh-Fitzpatrick MB. Masullo AS, Grossman ME: Porphyria cutanea tarda associated with chronic renal failure and hemodi­ alysis. Arch Dermatol 1980:116:191-195. 4 Topi GC, D’Alessandro Gandolfo L, De Costanza F, Cancarini G C : Porphyria and pseudo-porphyria in hemodialyzed patients. Int J Biochem 1980:12:963-967. 5 Day RS. Eales L: Porphyrins in chronic renal failure. Nephron 1980;26:90-95. 6 Harlan SL. Winkelmann RK: Porphyria cutanea tarda and chronic renal failure. Mayo Clin Proc 1983;58:467-471. 7 NcColl KEL, Simpson K, Laiwah AY. et al: Haemodialysis-re­ lated porphyria cutanea tarda - Treatment failure with charcoal hemoperfusion. Photodermatology 1986:3:169-173. 8 Anderson CD, Rossi E, Garcia-Webb P: Porphyrin study in chronic renal failure patients on maintenance hemodialysis. Photodermatology 1987;4:14-22. 9 Brivet F, Drucke T, Guillemette J. et al: Porphyria cutanea tarda-like syndrome in hemodialyzed patients. Nephron 1978: 20:258-266. 10 Anderson CD, Larsson L, Skogh M: UVA photosensitivity in photosensitive bullous disease of chronic renal failure. Photo­ dermatology 1985:2:111-114. 11 Lim CK, Peters TJ: Urine and faecal porphyrin profiles by reversed-phase high-performance liquid chromatography in the porphyrias. Clin Chim Acta 1984:139:55-63. 12 Doss MO: Porphyrins and porphyrin precursors; in Curtius HC, Roth M (eds): Clinical Biochemistry. Principles and Methods. Berlin, De Gruyer, 1974, pp 1323-1371. 13 Day RS, Pimstone NR, Eales L: The diagnostic value of blood plasma porphyrin methyl ester profiles produced by quantita­ tive TLC. Int J Biochem 1978:9:897-904. 14 Seubert S, Seubert A, Rumpf KW, Kiffe H: A porphyria cutanea tarda-like distribution pattern of porphyrins in plasma, hemodialysate and urine of patients on chronic hemodialysis. J Invest Dermatol 1985:85:107 109. 15 Beukeveld GJ, Wolthers BG, Van Saene JJ, et al: Patterns of porphyrin excretion in faeces as determined by liquid chroma­ tography. Reference values and the effect of flora suppression. Clin C'hem 1987;33:2164-2170. 16 Harber LC, Bickers DR: Porphyria and pseudoporphyria. J Invest Dermatol 1984:82:207-209. 17 Elder G H : Differentiation of porphyria cutanea tarda symptom­ atica from other types of porphyria by measurement of isocopro­ porphyrin in faeces. J Clin Pathol 1975:28:601-607.

Acknowledgements We are grateful to Dr. S. Waldek, consultant physician in the Renal Unit. Hope Hospital for advice and access to his patients.

References I Moore MR, McColl K.EL, Rimmington C, Goldberg A: Disor­ ders of porphyrin metabolism. New York, Plenum Medical Book Company, 1987.

Dr. C. Weinkove Department Medicine (Clinical Biochemistry) Clinical Sciences Building Hope Hospital Salford M6 8HD(UK)

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Accepted: October II, 1989

Plasma porphyrins in chronic renal failure.

Using a quantitative sensitive HPLC fluorometric assay for the measurement of plasma and urine porphyrins, we have calculated the renal clearance of u...
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