International Uroloyy and Nephroloyy 23 (5), pp. 503--509 (1991) Heme Biosynthesis and Porphyrin Studies in Chronic Renal Failure Patients Following Kidney Transplantation M. EL-FAR,* M. SOBH,** M. GHONIEM,** *Faculty of Science, Chemistry Department (Biochemistry Division): **Urology and Nephrology Center, Mansoura University, Mansoura, Egypt (Received November 19, 1990) El-Far and Sobh were the first to describe abnormalities in porphyrin metabolism in Egyptian patients with chronic renal failure (CRF). Our results were confirmed by others. The present investigation aims to study and discuss the nature of those abnormalities and changes in porphyrin metabolism in CRF patients following kidney transplantation. Blood samples and urine were collected from patients (with and without polycythaemia) as well as from normal controls. The activity of heine enzymes such as ALA-S, ALA-D, URO-S, PBGase and URO-D were assayed. Total blood porphyrins as well as enzyme activities such as ALA-S and URO-S were found to be highly significantly increased in all patients, while URO-D activity remained within normal range. The observed elevated erythrocyte porphyrins may be mainly due to increased activity of ALA-S, the rate-limiting enzyme in heine synthesis. The present study is the first of its kind which clearly demonstrates that successful kidney transplantation does not correct or rectify the abnormalities in porphyrin metabolism. Introduction Patients with chronic renal failure (CRF) may develop blisters on lightexposed skin [ 1]. Several reports [2-5] described a group of patients with so-called photosensitive blisters of CRF which are mainly attributed to abnormal porphyrin metabolism. In general, some porphyrins have a natural tendency to concentrate in normal tissues. The interaction of porphyrins with light have been known to cause photosensitivity since the beginning of this century. A series of land-mark contributions was made at our laboratories by using this property of porphyrins to diagnose and treat certain types of cancers [6-11 ]. We were able to introduce not only new porphyrins as tumour localizers, but also novel approaches for combating skin photosensitivity due to retained porphyrins in the skin [12, 13], in order to eliminate photocutaneous sensitivity. El-Far and Sobh [14] were the first to describe abnormalities in porphyrin metabolism in Egyptian patients with CRF. We have reported that free erythrocyte porphyrins (FEP) were significantly elevated in patients with uraemia in comparison to normal controls. On the other hand, we previously showed that VSP, Utrecht Akaddmiai Kiad6, Budapest
504 El-Far et al : Heme biosynthesis activities of some biosynthetic heme enzymes such as 5-aminolevulinic acid dehydratase (ALA-D)were significantly reduced while the activity of leucocyte 5-aminolevulinic acid synthetase (ALA-S) was found to be highly significantly increased in uraemic patients. Our results were recently confirmed by other investigators [1, 15, 16]. The present work was undertaken to increase our knowledge about the disorders of porphyrin and heme metabolism in a group of patients following successful kidney transplantation. To elucidate further the regulation of porphyrins and their role in heme biosynthesis after kidney transplantation, we decided to examine it in a group of transplanted patients with normal haemoglobin and compared them to a similar group of kidney transplant patients with polycythaemia. This is to address some of the unanswered questions concerning porphyrin metabolism in kidney transplant patients with and without polycythaemia to see if any changes, differences or corrections could be observed in porphyrin and heine synthesis after transplantation. Material and methods Among several patients with well functioning kidney grafts following successful kidney transplantation, 14 patients exhibited an abnormal increase in haemoglobin after kidney transplantation. These patients represented one of the three groups under investigation: Group I. Ten normal volunteers used as control. Group II. A second group of 16 patients with well functioning kidney after transplantation was carefully selected and designated as "non-polycythaemic patients" or non-pte group with almost normal haemoglobin (up to 15 g %). Group III. This group of patients was selected with well functioning kidney after transplantation but with high haemoglobin concentration and designated as "polycythaemic patients" or PTE group with increased haemoglobin (more than 15 g %). This group was carefully examined and diagnosed clinically at our center. The following biological measurements were performed on bloed samples collected from patients and controls. Tests were determined on the day of blood collection; samples were kept at 4~ until transferred to the laboratory for analysis I. Measurements of free erythrocyte porphyrins (FEP/100 ml RBC) This was determined spectrofluorometrically by the method of Piomelli [17]. II. Enzyme estimations (1) Leucocyte 5-ALA synthetase (ALA-S) (EC 2.3.1.37) This was determined according to the method of Dowdle et al. [18] and as previously described by us in detail [14].
El-Far et al. : 1-1eme biosynthesis 505 The results were expressed in nmol 5-ALA produced per mg protein of leucocyte per hour. (2) Erythrocyte ALA dehydratase (ALA-D) (EC 4.2.1.24) The technique of Weissberg et al. [19] was used in which 5-ALA was incubated with blood haemolysate for one hour at ph 7.0. The porphobilinogen (PBG) formed was assayed with Ehrlich's reagent. The unit of ALA-D activity is defined as the amount of enzyme necessary to convert 1 nmol/ml/min of ALA to PBG/ml of red blood cells. (3) Erythrocyte uroporphyrinogen-i-synthetase (URO-S) (EC 4.3.1.8) The method of Piepkorn et al. [20] was used in which 5-ALA was incubated with blood haemolysate. This substrate is converted to PBG by the enzyme ALA-D to provide the working substrate for URO-I-S. The units are expressed as nmol porphyrin formed per ml of erythrocytes per hour. (4) Erythrocyte porphobilinogenase (PBGase) PBGase is a convenient description of the combined activities of the enzymes URO-I-S and URO-III isomerase in converting PBG to uroporphyrinogen-iii. The method used was described by Baffle et al., in which a buffered solution of PBG at ph 8.2 was incubated at 45~ for one hour, the porphyrin formed being measured spectrofluorometrically. The results were expressed as nmol porphyrin formed per ml of erythrocytes per hour. (5) Erythrocyte uroporphyrinogen decarboxylase ( URO-D) (EC 4.1.1.37) The technique of Moore et al. was used, in which PBG was incubated with erythrocyte haemolysate to allow the enzymes URO-I-S and URO-III isomerase to produce the working substrate, uroporphyrinogen-iii, ph then adjusted to 7.2 and incubation allowed to proceed for a further hour. The end product, coproporphyrin-iii, was fractionated from uroporphyrin. The results were expressed in terms of nmol coproporphyrin formed per ml of erythrocytes per hour. Total urinary ALA and PBG This was determined by spectrofluorometric methods. Creatinine content in each sample was estimated routinely and results were expressed as mg ALA/mg creatinine or mg PBG/mg creatinine.
506 El-Far et al : tieme biosynthesis Statistics and calculations All tests were carried out in duplicate. Student (t) distributions were estimated in all experiments in which data from at least six cases were available. Differences were said to be almost significant (A.S.), significant (S) or highly significant (H.S.) when the corresponding level of probability (P) was 0.05-0.01, 0.01-0.001 and less than 0.001, respectively, while (N. S.) means not significant. Means S. E. are also shown. It is worth mentioning that not all patients had the complete range of tests; their number is indicated in Table 1. Results and discussion Table 1 summarizes all biological results in the different groups. In another study on erythrocyte porphyrins (EP) in haemodialysis patients, several investigators [1, 14, 21, 22] had reported elevated mean values. In our group of patients in the present study, both PTE and non-pte, we observed a highly significant increase in EP. This could be attributed to the highly significant increase in the leucocyte 5-ALA-S activities in both groups when compared to normals. We have previously reported an increase in 5-ALA-S activity, rate-limiting enzyme, in haemodialysis patients with CRF [14]. Other investigators [16] showed that in haemodialysis patients with CRF, serum ALA values were 4 times higher than in normal patients. This would confirm our findings of increased ALA-S activities present in haemodialysis patients before and after transplantation. Thus, we conelude that kidney transplant patients with and without polycythaemia do have the same elevated values of ALA-S and consequently increased production of erythrocyte porphyrins. Increased serum ALA under physiological conditions could be enzymatically converted to porphobilinogen (PBG)and uroporphyrin- (-ogen) after entry into red cells, thus causing increased erythrocyte porphyrins (Table 1). It is well known that synthesis of porphyrins from glycine and acetic acid is possible only in immature red blood cells, whereas in human and animal tissues, porphyrins are synthesized from ALA. Thus we assume that ALA produced in excess would be transported to organs where it serves as the substrate for porphyrin synthesis. On the other hand, the reduced excretion in urine of ALA and PBG found in PTE and non-pte patients (Table 1) would support this notion. The increased levels of ALA due to highly significantly increased 5-ALA synthetase become a source of porphyrin synthesis and thus ALA or PBG will not be detected in the urine as they have been consumed in porphyrin synthesis. Our results are in agreement with previous findings of Anderson et al. [1] who demonstrated a highly significant increase in EP concomitant with a low urinary porphyrin in a group of patients with CRF on haemodialysis. Others [16] reported that urinary ALA was reduced in another group of patients with CRF on haemodialysis. So, the increased activity of ALA-S presented in transplant patients (Table 1) would in turn result in overproduction of ALA and thus in
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508 El-Far et al : tteme biosynthesis increased EP in those patients. On the other hand, it is well known that ALA-D, the second enzyme of the heme biosynthesis pathway, is found to be present in some tissues in excess of the rate-controlling enzyme, ALA-S, but has not been shown so far to be rate-limiting for heme biosynthesis in tissues. The diverging data we are presenting on ALA-D activity for PTE and non-pte patients cannot be fully explained, although we know that a high degree of ALA-D inhibition must be achieved in various tissues before significant inhibition of heme biosynthesis is achieved. Thus porphyrin synthesis cannot be completely blocked due to decreased ALA-D activity. The elevated URO-I-S activity and normal URO-D activity together with elevated EP in our group of patients would support this assumption (Table 1). Recently Gebril et al. [23] showed that patients with CRF on haemodialysis had raised plasma porphyrins, especially uroporphyrins, which supports our findings. In both PTE and non-pte patients we have found URO-I-S activity statistically highly significantly increased above normal values. This may be due to the fact that in lymphocytes (URO-I-S) there seems to be a rate-limiting enzyme. Thus, increased ALA-S observed in the two groups under investigation would be concomitant with an increase in URO-I-S. Both enzymes may be responsible for the enhancement of biosynthetic heine and porphyrin production. Increased porphyrins may also be due to the deficiency of ferrochelatase enzymatic activity which catalyzes the insertion of ferrous iron into the protoporphyrin ring to produce heme. Thus increased porphyrins in CRF and in transplant patients would cause a certain degree of photosensitivity. Finally, we would recommend that the oral charcoal treatment we have described recently [12, 13] could be used as a safe and novel approach in combating photosensitivity which might be seen in some cases with CRF or in transplant patients. Summing up, the present study demonstrates that in patients with CRF after kidney transplantation, several parameters of heme biosynthetic pathway (ALA-S, URO-I-S, EP) are more significantly elevated than those found in control subjects. Furthermore, they are in agreement with and similar to the same disturbances in porphyrin metabolism demonstrated in CRF by us and others, as previously discussed. Our present study is the first of its kind, which clearly demonstrates that successful kidney transplantation does not correct or rectify the abnormalities in porphyrin and biosynthetic heine metabolisms. Acknowledgements Dr. Mohamed EI-Far wishes to express his gratitude to Prof. R. Benson, Chief of Urology at Mayo Clinic, USA, for providing him with some chemicals to finalize this study. Accomplished secretarial work of Miss Heind Sharabi is greatly appreciated. References 1. Anderson, C. D., Rossi, E., Garica-Webb, P. : Porphyrin studies in chronic renal failure patients on maintenance hemodialysis. Photodermatology, 4, 14 (1987).
El-Far et al : tteme biosynthesis 509 2. Gilchrest, B., Rowe, J. W., Mihm, M. C.: Bullous dermatosis of hemodialysis. Ann. Intern. Med., 83, 480 (1975). 3. Keczkes, K., Farr, M. : Bullous dermatosis of chronic renal failure. Br. J. DermatoL, 95, 541 (1976). 4. Coles, G. A., Verrier, Jones K. : Uraemic bullae. Br. Med. J., 525 (1976). 5. Anderson, G., Larsson, L., Skogh, M.: UVA photosensitivity in photosensitive bullous disease of chronic renal failure. PhotodermatoL, 2, 111 (1985). 6. EI-Far, M. A., Pimstone, N. R.: Tumor localization of uroporphyrin isomer I and III and their correlation to albumin and serum protein binding. CellBiochem. Func., 1, 156 (1983). 7. El-Far, M. A., Pimstone, N. R. : A comparative study of 28 porphyrins and their abilities to localize in mammary mouse carcinoma: Uroporphyrin I superior to hematoporphyrin derivative. In : Porphyrin Localization and Treatment of Tumors. Alan R. Liss, Inc., New York 1984, p. 557. 8. El-Far, M. A., Pimstone, N. R.: The interaction of tumor localizing porphyrins with collagen, elastin, gelatin, fibrin and fibrinogen. CellBiochem. Func., 3, 115 (1985). 9. El-Far, M., Pimstone, N. : Selective in vivo tumor localization of uroporphyrin isomer I in mouse mammary carcinoma. Superiority over other porphyrins in a comparative study. Cancer Res., 46, 4390 (1986). 10. El-Far, M., E1-Zahab, M., Ghoneim, M., Ibrahim, E. : Tumor localization of newly developed hematoporphyrin (DHP) using a b/adder tumor model: a novel hematoporphyrin derivative. Biochimie, 70, 251 (1988). 11. El-Far, M., Abd E1-Hamid, N., Ghoneim, M. : Selective in vivo tumor localization of heptacarboxylic porphyrin isomer I in a bladder tumor model: a novel technique to modulate porphyrin localization. Biochimie, 70, 1379 (1988). 12. El-Far, M., Ghoneim, M. : Photoradiation therapy of tumors with hematoporphyrin derivative: Role of low density lipoprotein in porphyrin localization and elimination. J. Tumor Marker Oncology, 4, 121 (1989). 13. El-Far, M., Sobh, M., Ghoneim, M. : Synthesis and in vivo biological evaluation of some newly developed porphyrins as bladder tumor markers. J. Turnout Marker Oncology, 4, 99 (1989). 14. El-Far, M., Sobh, M. : Haem biosynthesis in chronic renal failure J. MRI, 7, 89 (1986). 15. Buchet, J. P., Lauwerys, R., Hassoun, A., Dratwa, M.: Effect of albumin on porphyrin metabolism in hemodialyzed patients. Nephron, 46, 360 (1987). 16. Gorcheim, A., Webber, R. : 5-Aminolaevulinic acid in plasma, cerebrospinal fluid, saliva and erythrocytes: Studies in normal, uraemic and porphyric subjects. Clin. ScL, 72, 103 (1987). 17. Piomelli, S. : A micromethod for free erythrocyte porphyrins: The FEP test. 3". Lab. Clin. Med., 81, 932 (1973). 18. Dowdle, E. B., Mustard, P., Eales, L.: Delta-aminolevulinic acid synthetase activity in normal and porphyric human liver. S. Aft. Med. J., 41, 1093 (1967). 19. Weissberg, J. B., Lipschutz, F., Oski, F. A. : Delta aminolevulinic acid dehydratase activity in circulating blood ceils : A sensitive laboratory test for the detection of childhood lead poisoning. N. EngL J. Med., 289, 565 (1971). 20. Piepkorn, M. W., Hamernyik, P., Labbe, R. F. : Modified erythrocyte uroporphyrinogen synthetase assay and its clinical interpretation. Clin. Chem., 24, 1751 (1978). 21. Day, R. S., Eales, L. : Porphyrin in chronic renal failure. Nephron, 26, 90 (1980). 22. Tschudy, D. P., Ebert, P. S., Hess, R. A., Frykholm, B. C., Atsmon, A. : Porphyrin levels in plasma and erythrocytes of chronic hemodialysis patients. Oncology, 40, 148 (1983). 23. Gebril, M., Weinkove, G., Ead, R., McDonald, K., Morton, R.: Plasma porphyrins in chronic renal failure. Nephron, 55, 159 (1990). 7*