Amiodarone is a pharmacologically safe drug for porphyrias.

Amiodarone (AD) is an effective antidysrythmic drug, however, there can be serious side effects, such as hepatic and neurological alterations, as well as skin photosensitization, as seen in porphyrias. Clinical signs in porphyrias might be triggered by the so-called porphyrinogenic drugs. Without sound basis, Amiodarone has been classified as an unsafe drug for porphyric patients. The aim of this work has been to study the effect of AD, both in vivo and in vitro, on heme metabolism. In the in vivo assays, the activities of 5-aminolevulinate synthetase (ALA-S), ALA dehydratase (ALA-D), porphobilinogenase (PBGase) and PBG-deaminase (PBG-D) in blood, liver, and kidney; hepatic and fecal porphyrins, urinary ALA, PBG and porphyrins in male mice strain CF1 treated with AD (100 mg i.p. daily) for 1 week and 1 month, were measured. No significanat differences were found for any of these parameters in the AD treated animals as compared to controls. In the in vitro experiments human blood, and mice blood, liver, and kidney, were used to measure the activities of ALA-S, ALA-D, PBGase, PBG-D and uroporphyrinogen decarboxylase, in the presence of varying concentrations of AD (0.0172-4.304 mM). AD did not modify any of the enzyme activities. All of the above biochemical parameters were studied in 17 cardiac patients under AD treatment for 3 to 20 years. Neither the activities of the heme enzymes, nor the levels of precursors and porphyrins in urine and plasma were altered. These findings clearly demonstrate that AD is a pharmacologically safe drug and can be used for the treatment of associated pathologies in porphyrias.

Immunological determination of porphobilinogen deaminase as a diagnostic measure in acute intermittent porphyria.

Manifest disease symptoms of acute intermittent porphyria may be provoked by several external factors. Latent gene carriers should be identified at an early stage and informed about preventive measures. Porphobilinogen deaminase activity in red blood cells may be used as one indicator of the carrier state. However, there is an overlap of enzyme activity between healthy controls and carriers of the trait. Thus latent gene carriers cannot always be identified. In the present study a recently reported enzyme-linked immunosorbent assay (ELISA) was used to quantify the concentration of the enzyme porphobilinogen deaminase in erythrocytes in 845 individuals belonging to families with acute intermittent porphyria. Using previous available diagnostic methods 417 of them had been diagnosed as gene carriers, 339 as non-carriers, and 89 were of "uncertain" classification. Of those with "uncertain" diagnosis, 19 had a decreased concentration of porphobilinogen deaminase and could thus be diagnosed as gene carriers. However, 70 cases of the 89 were still "uncertain", which underlines the need for further improvement of the diagnostic methods.

Photodynamic and non-photodynamic action of several porphyrins on the activity of some heme-enzymes.

The action of porphyrins, uroporphyrin I and III (URO I and URO III), pentacarboxylic porphyrin I (PENTA I), coproporphyrin I and III (COPRO I and COPRO III), protoporphyrin IX (PROTO IX) and mesoporphyrin (MESO), on the activity of human erythrocytes delta-aminolevulinic acid dehydratase, porphobilinogenase, deaminase and uroporphyrinogen decarboxylase in the dark and under UV light was investigated. Both photoinactivation and light-independent inactivation was found in all four enzymes using URO I as sensitizer. URO III had a similar action as URO I on porphobilinogenase and deaminase and PROTO IX exerted equal effect as URO I on delta-aminolevulinic acid dehydratase and uroporphyrinogen decarboxylase. Photodynamic efficiency of the porphyrins was dependent on their molecular structure. Selective photodecomposition of enzymes by URO I, greater specificity of tumor uptake by URO I and enhanced porphyrin synthesis by tumors from delta-aminolevulic acid, with predominant formation of URO I, underline the possibility of using URO I in detection of malignant cells and photodynamic therapy.

Preparative free solution isoelectric focusing of human erythrocyte uroporphyrinogen I synthase in an ampholyte pH gradient.

A free solution electrofocusing method for uroporphyrinogen I synthase (EC 4.3.1.8) in an Ampholine pH gradient on a preparative scale is described. Partial purification of the enzyme was achieved in a 4-h focusing run. Enzyme activity was found in the pH range of pH 5.1 to pH 7.0. Complete separation of the most basic and most acidic isozyme from the control and the acute intermittent porphyria (AIP) patient was obtained in this single-step procedure. The level of enzyme activity has been shown to be reduced to about half the normal value in erythrocytes of two patients from a family with AIP. A shift of maximal activity toward the acidic side of the pH gradient was observed with the abnormal enzyme. In contrast to the normal isozyme set with seven isozyme bands, the fluorescence of the three basic bands and the second acidic band was greatly reduced, whereas the intermediate forms showed increased fluorescence intensity.

ELISA for measuring porphobilinogen deaminase in human erythrocytes.

An ELISA method has been developed to quantitate human porphobilinogen deaminase in erythrocyte lysate. The antiserum used in the assay was raised against the erythropoietic form of human porphobilinogen deaminase. The IgG fraction was characterized by use of immunoblotting technique, rocket immunoelectrophoresis and immunotitration and shown to be monospecific. The measuring range of the method was from 4 ng to 50 pg. Intra- and inter-assay coefficients of variation were 6% and 7%, respectively. Erythrocyte lysates from 97 apparently healthy individuals were assayed giving a mean erythrocyte porphobilinogen deaminase protein concentration of 150 +/- 28 SD (micrograms/g Hb) and a specific enzyme activity of 750 +/- 140 SD (nkat/g). Eight patients with acute intermittent porphyria were also investigated. A decreased concentration of enzyme protein, i.e. 84 +/- 13 SD (micrograms/g Hb) with a normal specific activity, was found.

Protoporphyrinaemia and decreased activities of 5-aminolevulinic acid dehydrase and uroporphyrinogen I synthetase in erythrocytes of a Vitamin B6-deficient epileptic boy given valproic acid and carbamazepine.

Carbamazepine (CBMZP) has been implicated as an inhibitor of the activities of 5-aminolaevulinic acid dehydratase (ALA-D) and uroporphyrinogen I synthetase (URO-S). In an epileptic boy undergoing long-term treatment with valproic acid (VPA), 1.3 g/d, CBMZP, 0.9 g/d and folic acid, 7.5 mg/d, decreased activities of ALA-D and URO-S coincided with increased levels of erythrocyte protoporphyrin (EP) in the absence of Pb poisoning, iron depletion and erythropoietic protoporphyria. A progressive fall in plasma pyridoxal 5'-phosphate (B6-P) to 7.7 nmol/L (lower reference limit, 14.6 nmol/L) prompted implementation of pyridoxine HCl (B6-HCl), 87.5 mg/d followed by administration of both B6-HCl and preformed B6-P (50 mg/d each). This permitted the eventual withdrawal of VPA and a net reduction of CBMZP to 450 mg/d. During these manipulations, ALA-D and URO-S activities, EP and urinary porphyrins and their precursors were measured serially. An assay system utilizing red cell ALA-D for generation of porphobilinogen (PBG) from added ALA at pH 7.4 was used for determination of ALA-D and URO-S activities in separate aliquots of the same assay mixture both in the absence and presence of Zn and dithiothreitol (DTT). One unit (U) for ALA-D = 1 nmol PBG/L RBC/s; for URO-S = 1 nmol porphyrin/L/s; minimum normal level for ALA-D = 135 U; for URO-S = 6 U. B6-HCl alone entailed increases in ALA-D and URO-S prior to any reduction of CBMZP. After administration of both B6-HCl and B6-P and withdrawal of VPA, the overall increase in ALA-D was from 54.59 to 197.2 U (-Zn; -DTT) and from 50.76 to 217.3 U (+Zn; +DTT). The overall increase in URO-S was from 2.67 to 8.90 U (-Zn; -DTT) and from 3.02 to 8.66 U (+Zn; +DTT). During stepwise reduction of VPA, EP remained elevated to values as high as 2.48 mumol/L (upper reference limit, 1.33 mumol/L). Only after permanent withdrawal of VPA did concentrations of EP fall to normal levels. Values for porphyrins and their precursors in urine were normal throughout. Since both VPA and B6-P are strongly protein-bound, it is suggested that VPA displaced B6-P from protective protein binding sites and that the resulting deficit in B6-P (rather than CBMZP) reduced ALA-D and URO-S activities via primary reduction of ALA-synthetase activity. Increases in EP emerge as a hitherto unappreciated effect of VPA warranting further investigation.

Acute intermittent porphyria in two patients on anticonvulsant therapy and with normal erythrocyte porphobilinogen deaminase activity.

1. Acute intermittent porphyria (AIP) is sometimes termed a 'pharmacogenetic' disease. patients with genetic deficiency of the enzyme porphobilinogen deaminase are liable to develop acute attacks of porphyria if exposed to a variety of drugs. 2. Two patients are reported who had no evidence of deficiency of erythrocyte porphobilinogen deaminase yet developed typical attacks of AIP while on anticonvulsant therapy. 3. Normal activity of erythrocyte porphobilinogen deaminase does not completely exclude porphyria. 4. Acute porphyria should be suspected if clinical deterioration occurs during therapy with anticonvulsants, or other porphyrinogenic drugs, even in the absence of an underlying genetic defect in haem synthesis in peripheral blood cells.

Erythrocyte uroporphyrinogen I synthase activity as an indicator of acute porphyria.

The pre-clinical diagnosis of acute intermittent porphyria (AIP) is important because acute attacks can be brought about by drugs, liver toxins, hormonal changes and diet. There also may be no obvious precipitating agent. The discovery that the activity of uroporphyrinogen I synthase (URO-S) activity in the red blood cells of patients with AIP is half that found in normal persons is of great value in diagnosing this disorder and also appears useful in detecting patients with the latent disease who have normal urinary delta-aminolevulinic acid and porphobilinogen excretion. It also appears to distinguish other types of porphyria from acute intermittent porphyria. It must also be recognized that some red blood cells URO-S determinations will yield indeterminate results; therefore, repeat assays, including examination of kinship, will improve discrimination and confidence in the final diagnosis.

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.

Erythrocyte porphobilinogen deaminase activity in liver disease.

The activities of erythrocyte porphobilinogen deaminase were studied in patients with various liver diseases and in control groups. The lowest enzyme activities were found in patients with acute intermittent porphyria, and the highest ones in those with increased hemopoietic activity. Patients with liver cirrhosis or chronic active hepatitis had porphobilinogen deaminase activities that were significantly higher than in normal subjects and did not depend on disease activity. In patients with acute hepatitis, porphobilinogen deaminase activities varied depending on the phase of disease, being normal at onset and after 3-4 mo, and elevated to the values observed in chronic liver disease between 2 and 4 wk of hospitalization. The differences in porphobilinogen deaminase activities between patients with liver disease and controls did not relate to red cell age as determined by density gradient centrifugation. Therefore, although the mechanism responsible for the increase in porphobilinogen deaminase activities in liver disease is not clear, the results of this study suggest that it is independent of the presence of immature red cells in the circulation.

Red blood cell porphobilinogen deaminase in the evaluation of acute intermittent porphyria.

We measured the activity of the enzyme porphobilinogen deaminase in red blood cells of 222 persons. Ninety-seven of 107 patients with acute intermittent porphyria had enzyme activity below the normal range, whereas 55 of 56 patients with other types of porphyria had normal activity. This underscores the utility of this test in confirming the diagnosis of acute intermittent porphyria. Measurement of enzyme activity in 41 families with acute intermittent porphyria demonstrated that deficient activity is inherited as an autosomal dominant trait. Many latent carriers of the genetic defect were identified by family studies, permitting appropriate precautions to avoid potentially lethal porphyric attacks.

Porphobilinogen deaminase and the synthesis of porphyrin isomers in the Dubin-Johnson syndrome.

Porphyrin synthesis was studied in a family of 9.3 of whom had the Dubin-Johnson syndrome (DJS). Subjects with DJS had modest increases in urinary porphyrin concentration with a marked increase in the series I isomer of coproporphyrin (tetracarboxylic) as well as in the octa-, hepta-, hexa- and penta-carboxyl porphyrins. Activity of erythrocyte porphobilinogen deaminase (PBG-D) was also increased. Non-expressing carriers in the family had normal levels of urinary porphyrins but modest increases in both series I isomer accumulation and PBG-D activity. These results may provide a rationale for the altered synthesis of porphyrin in DJS.

Increased activity of porphobilinogen deaminase in erythrocytes during attacks of acute intermittent porphyria.

The activity of porphobilinogen deaminase was determined in 25 patients with acute intermittent porphyria during and after fully developed attacks of porphyria. It was found that in most cases (in 20 of 25) it was higher than 24.3 nmoles/ml erythrocytes/hour, a value considered as characteristic for acute intermittent porphyria, and that it decreased during convalescence and remission. In a proportion of these cases the decrease in the activity of the enzyme was parallelled by decreasing urinary excretion of porphobilinogen. A normal activity of porphobilinogen deaminase during an attack of porphyria can be a source of error in the differential diagnosis of porphyria.

Variant of acute intermittent porphyria with normal erythrocyte uroporphyrinogen-I-synthase activity.

A kindred in which several members have otherwise typical acute intermittent porphyria but normal erythrocyte uroporphyrinogen-I-synthase activity has been described from Finland. We studied two porphyric members of this kindred, two patients with typical acute intermittent porphyria, and two healthy controls using the delta-aminolaevulinic acid loading test and by measuring the erythrocyte enzymes of haem biosynthesis. The excretion pattern of haem precursors after the delta-aminolaevulinic loading test in the members of the kindred studied, was similar to that in typical acute intermittent porphyria suggesting an identical enzyme defect in the liver. The activity of all red cell enzymes studied was normal in the members of the kindred. The results suggest that porphyria in the kindred studied is a variant of acute intermittent porphyria, where the uroporphyrinogen-I-synthase defect is manifested in the liver but not in red cells.