Biochemical and genetic characterization of four cases of hereditary coproporphyria in Spain.

We report a biochemical and genetic characterization of four cases of hereditary coproporphyria (HCP) in Spain. All patients showed a typical HCP porphyrin excretion pattern with a high concentration of coproporphyrins in feces and inverted I:III isomer ratio. The porphyrin precursors in urine were found elevated in two patients who showed acute symptoms. The analysis of the CPO gene showed that three cases harboured novel mutations: V135A (404T>C; exon 1); L214R (641T>G; exon 2); and P249R (746C>G; exon 3) and in the fourth, a previously described R426X mutation in exon 6.

Comparison of two assay methods for activities of uroporphyrinogen decarboxylase and coproporphyrinogen oxidase.

Uroporphyrinogen decarboxylase (UROD) and coproporphyrinogen oxidase (copro'gen oxidase) are two of the least well understood enzymes in the heme biosynthetic pathway. In the fifth step of the pathway, UROD converts uroporphyrinogen III to coproporphyrinogen III by the decarboxylation of the four acetic acid side chains. Copro'gen oxidase then converts coproporphyrinogen III to protoporphyrinogen IX via two sequential oxidative decarboxylations. Studies of these two enzymes are important to increase our understanding of their mechanisms. Assay comparisons of UROD and copro'gen oxidase from chicken blood hemolysates (CBH), using a newly developed micro-assay, showed that the specific activity of both enzymes is increased in the micro-assay relative to the large-scale assay. The micro-assay has distinct advantages in terms of cost, labor intensity, amount of enzyme required, and sensitivity.

Multiple chemical sensitivity syndrome and porphyria. A note of caution and concern.

Growing numbers of patients suffering from many symptoms believe that they have a condition called multiple chemical sensitivity syndrome (MCSS). It has been suggested that this syndrome can be triggered by exposure to any of a large and usually incompletely defined number of natural and synthetic chemical substances. Major medical organizations, including the National Research Council and the American Medical Association, have not recognized MCSS as a clinical syndrome because of a lack of valid, well-controlled studies defining it and establishing pathogenesis or origin. Lately, some have proposed that many patients with MCSS suffer from hereditary coproporphyria. However, this purported association is based chiefly on results from a single reference laboratory of a fundamentally flawed assay for erythrocyte coproporphyrinogen oxidase. Although patients with MCSS may, at times, have modest increases in urinary coproporphyrin excretion, this is a common finding found in many asymptomatic subjects or patients with diverse other conditions (eg, diabetes mellitus, heavy alcohol use, liver disease, and many kinds of anemia). Such secondary coproporphyrinuria does not indicate the existence of coproporphyria. To our knowledge, there is no scientifically valid evidence to support an association between MCSS and coproporphyria, nor is there any unifying hypothesis for rationally linking these 2 disorders.

Effect of covalent modification on coproporphyrinogen oxidase from chicken red blood cells.

The biosynthesis of heme is a complex multi-step pathway requiring the efforts of eight enzymes. The initial enzymes in the heme biosynthetic pathway have been well characterized in relation to their mechanisms. Coproporphyrinogen oxidase (Copro'gen oxidase) is one of the last three enzymes in the pathway and is one of the least well understood. Copro'gen oxidase converts coproporphyrinogen III to protoporphyrinogen IX via oxidative decarboxylation of the 3- and 8-propionic side chain moieties. To further our understanding of the recognition and binding of substrate, Copro'gen oxidase was partially purified from chicken red blood cell hemolysates then incubated with covalent modifiers of specific amino acids. Incubation with tetranitromethane, p-hydroxyphenylglyoxal, N-acetylimidazole, or trinitrobenzenesulfonic acid resulted in substantial reduction of Copro'gen oxidase activity implying the presence of critical tyrosine, arginine and lysine residues. We conclude that these amino acids play important roles in the enzymic mechanism (for both binding and catalysis) of Copro'gen oxidase.

Acute hereditary coproporphyria induced by the androgenic/anabolic steroid methandrostenolone (Dianabol).

Acute attacks of porphyria can be induced by certain drugs. We report a case of acute coproporphyria induced by methandrostenolone. This is the first report of acute porphyria induced by an androgenic, anabolic steroid.

Childhood porphyrias: implications and treatments.

In describing the clinical, biochemical, and family findings in five children with porphyria, we examine initial treatments and, where appropriate, the effectiveness of long-term therapy. We note that porphyria diagnosis, particularly in childhood, relies heavily on specialist laboratory investigations. Because disease expression in some porphyrias requires exposure to precipitating factors, it may be prevented or delayed by their avoidance.

Inhibition of human lymphocyte coproporphyrinogen oxidase activity by metals, bilirubin and haemin.

Coproporphyrinogen III oxidase activity in human lymphocytes was found to be inhibited by cadmium and mercury but not by lead. The organo-metal compounds tributyltin and methylmercury were effective inhibitors of this haem biosynthetic pathway enzyme. Haemin (the ultimate product of the pathway) and bilirubin (a product of haem catabolism) were also shown to be inhibitory. Kinetic studies performed under initial velocity conditions showed that bilirubin was a non-competitive inhibitor and that one bilirubin molecule was bound to both the enzyme and enzyme substrate complex. The analysis also showed haemin to be a non-competitive inhibitor in which two haemin molecules bind to the enzyme whereas the enzyme substrate complex accepts only one haemin molecule. The possible physiological significance of the inhibition of coproporphyrinogen III oxidase activity by haemin and bilirubin is discussed.

An unusual case of variegate porphyria with possible homozygous inheritance.

We report an unusual case of variegate porphyria in a young girl with epilepsy, mental retardation and premature adrenarche. Symptoms of porphyria commenced about the age of 12 years and death occurred about 18 months later. The patient had very low protoporphyrinogen oxidase activity in her cultured fibroblasts. Both parents had half the normal activity of this enzyme in lymphocytes and are heterozygous for the abnormal gene for variegate porphyria. Therefore, it is possible that the patient was a homozygous variant. Anticonvulsant therapy and low hepatic 5 alpha reductase activity were probably other contributing factors to the severity of the condition in this patient.

Accurate and specific HPLC assay of coproporphyrinogen III oxidase activity in human peripheral leucocytes.

An HPLC method is described for the assay of coproporphyrinogen III oxidase in human leucocytes. The optimal pH for the assay was 6.5-7.0 and the Km for coproporphyrinogen III was 0.12 +/- 0.021 mumol/l. The mean activity in 28 apparently health subjects was 0.249 (2 SD range 0.130-0.368) nmol/h per mg protein. In two patients with hereditary coproporphyria, the activities were 0.029 and 0.078 nmol/h per mg protein.

Coproporphyrinogen oxidase activity and porphyrin concentrations in peripheral red blood cells in hereditary sideroblastic anaemia.

The activity of coproporphyrinogen oxidase and the concentrations of coproporphyrin and protoporphyrin (measured by HPLC) in peripheral red blood cells were established in 2 families with different types of hereditary sideroblastic anaemia. 2 males and 4 females were members of a family with an X-chromosome-linked and pyridoxine-responsive HSA, and 3 females were members of another family where the mode of inheritance is not clear and where pyridoxine did not produce a haematological response. Coproporphyrinogen oxidase activity was normal in 8 of 9 patients and slightly decreased only in 1 patient. All patients had normal red cell coproporphyrin concentrations, but red cell protoporphyrin concentration was decreased in 4 patients. These findings indicate that in vivo haem synthesis was not impaired at the step of coproporphyrinogen oxidase, hence enzymatic defects in earlier steps of haem synthesis are more evident. Earlier suggestions of impaired haem synthesis at this level, based on observed increased concentrations of coproporphyrin in peripheral red blood cells might be explained by the use of unspecific methods.

Harderoporphyria: a variant hereditary coproporphyria.

Three siblings with intense jaundice and hemolytic anemia at birth were found to excrete a high level of coproporphyrin in their urine and feces; the pattern of fecal porphyrin excretion was atypical for hereditary coproporphyria because the major porphyrin was harderoporphyrin (greater than 60%; normal value is less than 20%). The lymphocyte coproporphyrinogen III oxidase activity of each patient was 10% of control values, which suggests a homozygous state. Both parents showed only mild abnormalities in porphyrin excretion and lymphocyte coproporphyrinogen III oxidase activity decreased to 50% of normal values, as is expected in heterozygous cases of hereditary coproporphyria. Kinetic parameters of coproporphyrinogen III oxidase from these patients were clearly modified, with a Michaelis constant 15-20-fold higher than normal values when using coproporphyrinogen or harderoporphyrinogen as substrates. Maximal velocity was half the normal value, and we also observed a marked sensitivity to thermal denaturation. The possibility that a mutation affecting the enzyme on the active center which is specifically involved in the second decarboxylation (from harderoporphyrinogen to protoporphyrinogen) was eliminated by experiments on rat liver that showed that coproporphyrinogen and harderoporphyrinogen were metabolized at the same active center. The pattern of porphyrin excretion and the coproporphyrinogen oxidase from the three patients exhibited abnormalities that were different from the abnormalities found in another recently described homozygous case of hereditary coproporphyria. We suggest naming this variant of coproporphyrinogen oxidase defect "harderoporphyria."

Abnormal haem biosynthesis in chronic alcoholics.

The activities of six of the enzymes of haem biosynthesis have been examined in eleven chronic alcoholics admitted to hospital for alcohol withdrawal. The mitochondrial enzymes delta-aminolaevulinic acid (ALA) synthase, coproporphyrinogen oxidase and ferrochelatase were monitored in peripheral leucocytes and the cytosolic enzymes ALA dehydratase, uroporphyrinogen-1-synthase and uroporphyrinogen decarboxylase in peripheral erythrocytes. Compared with control subjects the activity of the initial and rate controlling enzyme of the pathway, ALA synthase, was increased (P less than 0.01) and the activities of ALA dehydratase and uroporphyrinogen decarboxylase depressed (P less than 0.01, P less than 0.02 respectively) on the day after admission but all returned to normal by the tenth to twentieth days after alcohol withdrawal. This stimulation of ALA synthase and inhibition of uroporphyrinogen decarboxylase explains the mechanism by which chronic alcohol ingestion may precipitate cutaneous hepatic porphyria. Two of the alcoholics were anaemic without evidence of haematinic deficiency and this was associated with depressed ferrochelatase activity and iron and porphyrin accumulation. The anaemia and related biochemical abnormalities in these two subjects were all corrected with alcohol withdrawal. It is proposed that inhibition of ferrochelatase activity is the biochemical basis of alcohol related sideroblastic anaemia.

Acute ethanol ingestion and haem biosynthesis in healthy subjects.

The effects of acute ethanol ingestion on the activities of the enzymes of haem biosynthesis in peripheral blood cells have been monitored in eight healthy subjects. The mitochondrial enzymes delta-aminolaevulinic acid (ALA) synthase, coproporphyrinogen oxidase and ferrochelatase were measured in leucocytes and the cytosolic enzymes ALA dehydratase, porphobilinogen (PBG) deaminase and uroporphyrinogen decarboxylase in erythrocytes. Ingestion of 1 . 316 mol ethanol resulted in increased activity of the rate-controlling enzymes ALA synthase and PBG deaminase and decreased activity of the other four enzymes. There was also increased urinary excretion of coproporphyrin. These observations may be relevant to the biochemical mechanisms involved in the ethanol-related conditions, sideroblastic anaemia, cutaneous hepatic porphyria and hepatic siderosis.

Haem biosynthesis in rheumatoid disease.

The activities in blood of six enzymes of the haem biosynthetic pathway have been determined in 12 patients with rheumatoid disease, six of whom were anaemic. The porphyrin and porphyrin-precursor intermediary products of haem biosynthesis were also determined in blood, urine and faeces. No significant differences were found between anaemic and non-anaemic subjects. Failure of delta-amino-laevulinate synthase activity to increase in response to anaemia may be the nature of the marrow unresponsiveness suggested as one factor in the causation of the anaemia. Normal ferrochelatase activity and normal concentrations of free protoporphyrin support the view that iron is effectively unavailable although present in normal amounts. Coproporphyrinogen oxidase activity was significantly depressed.

Hereditary coproporphyria and epilepsy.

A 9-year-old boy with mental deterioration and epilepsy suffered an acute attack of hereditary coproporphyria associated with worsening of seizure control. Leucocyte coproporphyrinogen oxidase activity was undetectable in the patient during this attack, and was reduced in his mother, a latent case. The complex relationship between porphyria, epilepsy, and anticonvulsant drugs is discussed.

Hereditary coproporphyria. Demonstration of the abnormalities in haem biosynthesis in peripheral blood.

Hereditary coproporphyria is biochemically distinct from the other porphyrias and is characterized by excessive excretion of coproporphyrin in faeces and usually in urine. The laboratory findings in 28 patients with this disease are presented and the clinical details of eight patients who have been in attack summarised. The remaining 20 patients were latent for the disease. In all patients studied the activity of delta-aminolaevulinic acid synthase was raised and coproporphyrinogen oxidase depressed in the leucocyte. This indicates the partial enzyme block in the haem biosynthetic pathway in this disease. The activities of the other enzymes in the pathway, leucocyte ferrochelatase and erythrocyte delta-aminolaevulinic acid dehydratase, porphobilinogen deaminase and uroporphyrinogen decarboxylase showed no consistent change. On review of 111 cases, 35 per cent presented in acute attack: 80 per cent had abdominal pain, 34 per cent vomiting, 29 per cent solar sensitivity, 23 per cent neurological involvement, 23 per cent psychiatric symptoms and 20 per cent severe constipation. Only two fatalities have been published, both from respiratory failure. There was a female preponderance of cases in attack of 2-5:1 and in the latent cases of 1-5:1 suggesting hormonal provocation in the uncovering of the disease. Drugs were implicated as precipitating 54 per cent of acute attacks and in 34 per cent of cases, these were barbiturates. This study demonstrates the reduction in activity of coproporphyrinogen oxidase in the haem biosynthetic pathway and the elevation of delta-aminolaevulinic acid synthase in the peripheral blood. These features, together with the typical abnormal porphyrin excretion pattern, appear to be diagnostic of hereditary coproporphyria whether in attack, remission, or in the latent form.