[Porphyria and porphyrinuria].

In latent hepatic porphyria normal porphyrins are frequently excreted, but porphyrinuria accompanies many clinical conditions not associated with porphyria. Therefore, the diagnosis of latent porphyria cannot be made by examining for urinary porphyrins alone. We use 16 laboratory methods, some of which which we developed, including sensitive HPLC separation procedures, for determining relevant enzymes, and porphyrins and their precursors in urine, feces and blood. These methods enable use to distinguish between changes in porphyrin metabolism observed in many cases of latent porphyria, as opposed to similar changes which may occur during therapy with certain drugs and in common clinical conditions. During the past 4 years we investigated 406 patients suspected of porphyria. 160 had increased excretion of porphobilinogen, and/or aminolevulinic acid, and/or porphyrins. 87 of them had porphyrinuria only. Without our clinical and laboratory experience, all would have been diagnosed as porphyria. Further examinations revealed that only 14 had porphyria, 8 of whom had porphyrinuria and 6 had normal urinary porphyrins. Overall, there have been 44 families with porphyria diagnosed in our laboratory since 1969, 1 with erythropoietic protoporphyria and the others with hepatic porphyria. In the past decade, the number of patients diagnosed during the latent phase has increased markedly. This points both to increased awareness of porphyria by physicians and to the increased skills of the laboratory staff.

Experimental latent and acute porphyria in the non-fasted rat; preventive effect of propranolol.

This study demonstrates an experimental model of the biochemical pattern of the 'latent phase' of hepatic porphyria subject to 'acute attack', upon application of prophyrinogenic stimuli. The 'latent phase' was achieved by administering 3,5-diethoxycarbonyl-1, 4-dihydrocollidine [DDC], 70 mg kg-1 day, orally to non-fasted rats. A two- and threefold increase in coproporphyrin in urine and protoporphyrin in faeces, respectively, were observed. An 'acute attack' was induced by phenobarbitone (PB), 100 mg kg-1, administered on the third day of treatment with DDC, followed by administration of 2-allyl-2-isopropylacetamide (AIA), 470 mg kg-1, on the fourth day. A fourfold elevation in urinary porphobilinogen (PBG) and delta-aminolevulinic acid (ALA) and further increase of three- and fourfold in urinary coproporphyrin and faecal protoporphyrin, respectively, was observed. The effect of DDC, AIA and PB on the excretion of PBG and porphyrins was found to be synergistic rather than additive. dl-Propranolol, 700 mg kg-1, given to DDC treated rats 'latent phase' reduced the amount of porphyrins excreted in urine and faeces to those observed in control dimethyl sulphoxide (DMSO) treated rats. It also prevented induction of 'acute attack' caused by the combination of PB and AIA. It is shown that dl-propranolol affects a few parameters in the haem biosynthetic pathway. Its beneficial effect in porphyria is probably the result of increasing the concentration of haem in the free haem pool.

Regulation of heme synthesis in the regenerating rat liver.

This investigation shows that the regulation of heme synthesis in the regenerating rat liver does not differ from the regulation in the normal liver. The heme saturation of tryptophan pyrrolase was found to be low, indicating a reduced concentration of heme in the regulatory heme pool of the regenerating rat liver. As expected, ALAS in the mitochondrial fraction was found to be elevated. It was also shown that ALAS in the regenerating rat liver can be induced by the porphyrinogenic drugs AIA and DDC and that heme reduces its activity. The decrease observed in the activity of cytosolic ALAS might be due to impaired synthesis of the enzyme but does not affect the regulation of the heme biosynthetic pathway.

L-Alanine: 4,5-dioxovalerate transaminase does not regulate heme synthesis in rat liver.

L-Alanine: 4,5-dioxovalerate transaminase (ADT) was determined in liver homogenates of rats treated by either inducers of porphyrin synthesis or the repressor, hemin. ADT activity was not induced by the porphyrinogenic agents nor reduced by hemin, indicating that ADT probably has no regulatory role in the heme synthesis pathway. The same conclusion was drawn from similar experiments performed in monolayers of chick embryo liver cells.

Colchicine treatment of abdominal pains in porphyric patients.

Four hepatic porphyria patients were treated with colchicine for abdominal pains. Colchicine administration during prodromal abdominal symptoms prevented the development of these abdominal crises. When colchicine was given after initiation of the crisis the duration and magnitude of the abdominal pains decreased.

The porphyrinogenic effect of simvastatin in experimental systems.

Attacks of acute hepatic porphyria have been previously reported to be frequently associated with transient hypercholesterolemia. This investigation was undertaken in order to establish whether the hypocholesterolemic effect of simvastatin (MK-733) is associated with inhibition of porphyrin metabolism. In two experimental models of acute hepatic porphyria--monolayers of chick embryo liver cells induced by DDC, and diethoxycarbonyl dihydrocollidine (DDC) injected rats--simvastatin was shown to increase porphyrin formation. A similar increasing effect was observed in a system which mimics the latent phase of porphyria, non-induced monolayers of chick embryo liver cells. We conclude that simvastatin is a porphyrogenic drug and should therefore be used with extreme caution in patients with hypercholesterolemia who also have latent porphyria. Its administration should be discontinued, at least temporarily, in patients with hypercholesterolemia during acute attacks of hepatic porphyria.

The heme biosynthetic pathway in lymphocytes of patients with malignant lymphoproliferative disorders.

The metabolism of heme is impaired in lymphocytes of patients with malignant lymphoproliferative disorders (MLPO). Two of the enzymes of the heme biosynthetic pathway, delta-aminolevulinic acid dehydrase (ALAD) (EC 4.2.1.24) and ferrochelatase (FC) (EC 4.99.1.1) are markedly reduced. The activity of porphobilinogen deaminase (PBGD) (EC 4.3.1.8) is increased. The rate-limiting enzyme of heme biosynthesis in the liver, aminolevulinate synthase (ALAS) (EC 2.3.1.37) remains unchanged although the concentration of total heme in the lymphocytes is markedly reduced. This might reflect a lack of negative feedback inhibition by heme on ALAS activity in this system.

Activity of porphobilinogen deaminase in peripheral blood mononuclear cells of patients with metastatic cancer.

Porphobilinogen deaminase (PBGD), one of the enzymes in the pathway of heme synthesis, was found to be elevated in peripheral mononuclear cells of 60% of patients with epithelial tumors and metastatic spread, but only in 14% of patients with tumor and no evidence of metastases. The combination of both high lactic dehydrogenase and high PBGD afforded a sensitivity of 40%, but a specificity of 96% in diagnosing metastatic spread.

Growth rate determines activity of porphobilinogen deaminase both in nonmalignant and malignant cell lines.

PBGD activity and growth rate were determined in cultures of rat embryo fibroblasts, nontransformed and MLV/MS transformed fibroblastic cell lines; NIH-3T3 cells, and in a mouse lymphosarcoma cell line [L-929]. The two parameters examined correlate positively (P less than 0.001). The results of this investigation would seem to indicate clearly that porphobilinogen deaminase activity is related to growth. However, these experiments do not rule out the possibility that malignant transformation per se also causes changes in porphobilinogen deaminase activity.

Liver cirrhosis and hepatocellular carcinoma after prolonged exposure to TNT: causal relationship or mere coincidence?

Macronodular liver cirrhosis and hepatocellular carcinoma were found in a 61-year-old male who had been exposed for 35 years to Trinitrotoluene (TNT). The question whether TNT had induced these pathological processes is raised.

The heme biosynthetic pathway in the regenerating rat liver. The relation between enzymes of heme synthesis and growth.

Enzymes of heme synthesis, porphyrins and heme content of regenerating rat livers were examined. During the first three days of regeneration the weights of livers of one-third and two-third hepatectomized rats increased 1.5-fold and 2.7-fold and the activity of porphobilinogen deaminase increased 2-fold and 4-fold and was inversely correlated with ferrochelatase activity. delta-Aminolevulinic acid synthase and delta-aminolevulinic acid dehydratase activities were reduced. Concomitantly an increase in the concentration of porphyrins and a decrease in that of heme were observed. The changes in the biosynthetic pathway of heme during rapid growth of the liver are discussed.

Increased porphobilinogen deaminase activity in patients with malignant lymphoproliferative diseases. A helpful diagnostic test.

Patients with malignant lymphoproliferative diseases, ie, chronic lymphocytic leukemia and lymphoma, were found to have higher porphobilinogen deaminase (PBGD) activity in their peripheral lymphocytes than normal control subjects, patients with other malignant neoplasms, and patients with various infectious diseases. The specificity and sensitivity of the test were 99% and 87%, respectively. The sensitivity of the test was 100% in patients with chronic lymphocytic leukemia and 82% in patients with lymphoma. The diagnostic value of PBGD determinations was shown in a prospective study of a group of patients evaluated because of fever of unknown origin, anemia, or other constitutional symptoms with or without lymphadenopathy or a mediastinal mass. The positive and negative predictive values in these patients were 91% and 100%, respectively. Nearly all patients who were in remission had normal enzyme activity. Lymphocyte PBGD determinations also may be of value in determining when to terminate or reinitiate drug treatment.

Lymphocyte urosynthase in non-Hodgkin's lymphoma. An indicator of disease extensiveness.

The activity of lymphocyte uroporphyrinogen synthase (URO-S) was examined in 51 non-Hodgkin's lymphoma (NHL) patients at various follow-up periods. Mean +/- SD activity (pmol porphyrin/mg protein/hr) at diagnosis (n = 24), on relapse (n = 14) and during active disease (n = 14) were 31.7 +/- 19.8, 31.7 +/- 27.2 and 29.4 +/- 18.5, respectively. These values were significantly higher than the enzyme activity during remission (14.1 +/- 4.0), which was in the normal range (14.5 +/- 3.8). Abnormally high activity was found in 65.4% of determinations at diagnosis, on relapse and during active disease, compared to 5.5% during remission (P less than 0.001). Significant association of abnormal URO-S activity was found with advanced clinical stage (P less than 0.01), spleen enlargement (P = 0.048), involvement of bone marrow (P = 0.02), as well as lymphoma cell spread to peripheral blood (P = 0.03). Highly significant correlation (r = 0.65, P less than 0.001) was found between URO-S activity and serum lactic dehydrogenase (LDH) levels. Excessively high levels of URO-S activity were found only in patients with lymphoma cells in peripheral blood. No association was found with histopathologic classification and liver size. The authors conclude that URO-S activity is a biochemical indicator for patients in all stages of NHL and seems to be a specific marker for the extensiveness of the disease.

The influence of carbamazepine on the heme biosynthetic pathway.

Carbamazepine, a drug which is widely used in neurological diseases, has a porphyrogenic effect in chick embryo liver cells in culture. It increased the concentration of cellular porphyrins by 80-fold and delta-aminolevulinate synthase activity by 4-fold. The increase in the accumulation of porphyrins preceded that of ALAS activity. Measurements of the activities of aminolevulinate dehydrase, porphobilinogen deaminase, and uroporphyrinogen decarboxylase showed that C inhibits UROD up to nearly 50% and PBGD activity up to 20%, but does not affect the activity of ALAD. The pattern of accumulation of porphyrins, mainly uro- and heptacarboxylporphyrin, is compatible with an inhibition of UROD. We may, therefore, conclude that the porphyrogenic effect of C in monolayers of chick embryo liver cells is the result of its inhibitory effect on the activity of UROD.

The porphyrogenic effects of calcium channel blocking drugs.

Treatment of monolayers of chick embryo hepatocytes with the calcium channel blocking drugs nifedipine and verapamil resulted in a decrease in the activity of uroporphyrinogen decarboxylase, an increase in the activity of delta-aminolaevulinate synthase and accumulation of porphyrins with uroporphyrin and heptacarboxylic porphyrin predominating. Diltiazem, another calcium channel blocking drug, did not affect uroporphyrinogen decarboxylase activity and had a slight effect only on the accumulation of porphyrins. Experiments with nifedipine and verapamil in the presence of various concentrations of calcium indicate that the porphyrogenic effect is apparently not related to blocking of calcium channels.

Impairment of induction of delta-aminolevulinic acid synthase by gluconeogenic amino acids and carbohydrates in vitro.

This study was undertaken in a system of chick embryo liver cells incubated in Earle's Basal Salt Solution with hormones. Impairment of induction of delta-aminolevulinic acid synthase (ALAS) by allyl-isopropylacetamide (AIA) was observed in the presence of glucose. Fructose and various gluconeogenic substances including gluconeogenic amino acids had a similar effect. Leucine, which is purely ketogenic, did not influence induction of ALAS. SH-containing amino acids increased induction of ALAS by AIA. The glucose analogues 3-O-methylglucose and 2-deoxyglucose did not impair induction of ALAS by AIA. The inhibitory effect of glycerol, fructose, and glycine was not affected by 3-O-methylglucose but was reversed by 2-deoxyglucose. The results indicate that the salutary effects of proteins on acute attacks of hepatic porphyria are probably caused by their gluconeogenic properties and that glucose-6-phosphate, or metabolite of glucose-6-phosphate that is not in the glycolytic pathway, is the active agent that leads to the glucose-like effect.

Evidence of abnormality of lymphocyte uroporphyrinogen synthase in family members of patients with lymphoproliferative diseases.

Patients with active lymphoproliferative diseases (LPD) were shown to have high activity of lymphocyte uroporphyrinogen synthase (L-UROS), the enzyme which converts porphobilinogen to uroporphyrinogen. The mean L-UROS activity of 64 first-degree relatives of patients with LPD was significantly higher than that of a control group and 45% of these relatives had pathological values of L-UROS. L-UROS activity was also determined in the spouses of 2 patients and was pathologically elevated in both. The pattern of pathological values among family members may indicate the presence of a communicable agent.

Inhibition of experimental porphyria with colchicine.

Colchicine at the concentrations of 5 X 10(-7) - 5 X 10(-6) M decreased significantly both delta-aminolevulinic acid synthase activity and accumulation of porphyrins in monolayers of chick embryo liver cells induced by allyl-isopropylacetamide, by 3,5-diethoxycarbonyl-1,4-dihydrocollidine or by phenobarbitone. No effect was noted in non-induced cells. In rats, colchicine 0.3 mg/kg, reduced significantly the allyl-isopropylacetamide induced increase in the activity of delta-aminolevulinic acid synthase in the liver and the concentration of urinary porphyrins while it did not affect these parameters in non-induced rats.