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Background: Erythropoietic protoporphyria is a rare photodermatosis of childhood, and the diagnosis can be delayed. A deficient ferrochelatase enzyme leads to accumulation of protoporphyrins in the dermis, causing phototoxic burning., Objective: To report three cases with great variability in severity of symptoms and age at diagnosis. We discuss clinical and biochemical findings, mutation analysis, and therapeutic options., Methods: We report three cases with different degrees of photosensitivity, laboratory results, psychosocial impact, and preventive and therapeutic treatments., Results: The diagnosis of erythropoietic protoporphyria was confirmed by both typical elevation of plasma porphyrins and the discovery of a mutated FECH gene., Conclusion: Erythropoietic protoporphyria should be suspected in any cases of childhood photosensitivity. Systemic complications are unusual. Mutation analysis confirms the diagnosis. Photoprotection is the cornerstone of treatment.

Objective: To investigate the demographic, clinical, biochemical and genotypic features of patients with erythropoietic protoporphyria (EPP) in a Swedish cohort., Design: Cross-sectional questionnaire, biochemical and genetic study., Setting: Sweden., Subjects: Fifty-one Swedish individuals known in 2008 to have EPP confirmed by molecular diagnosis. There were no exclusion criteria; all patients were included in the demographic and genetic study. A total of 92% participants completed the questionnaire study and 82% the biochemical study., Results: The prevalence of EPP was 1 : 180,000. Nine novel ferrochelatase gene mutations were found. The most commonly reported age at onset of symptoms was the first year of life and the mean age at diagnosis was 22 years. Painful photosensitivity was the main symptom. Exogenous factors other than sunlight were frequently reported to cause cutaneous symptoms. One in five patients reported a positive effect of beta-carotene therapy. A marked impact of EPP on quality of life was reported. Women had a significantly lower mean erythrocyte protoporphyrin concentration than men. Of all participants, 84% had insufficient vitamin D concentrations, 44% had below normal serum ferritin or transferrin saturation levels and red cell abnormalities were common., Conclusions: The notably delayed diagnosis suggests the need for an increased awareness of EPP. Disturbed erythropoiesis, biochemical signs of iron deficiency and low vitamin D levels are frequent findings in this disease. New and better treatments are needed as current treatment options for symptom amelioration are limited. Vitamin D supplementation should be considered., (© 2010 The Association for the Publication of the Journal of Internal Medicine.)

Erythropoietic protoporphyria (EPP) is a rare hereditary disorder due to a partial deficiency of ferrochelatase (FECH). The genotype of EPP patients features a mutation on one allele of the FECH gene and a common hypomorphic FECH IVS3-48c on the other allele (M/c). The resulting enzyme activity in patients is ∼35% of that in normal individuals. Ferrochelatase deficiency results in the accumulation of protoporphyrin in the skin, which is responsible for the clinical symptom of cutaneous photosensitivity in patients. In this study, we report the identification of a novel FECH mutation delT23 in an 11-member EPP family of Jewish origin. Two EPP siblings shared an identical genotype of delT23/IVS3-48c (M/c). They were both photosensitive and showed highly increased erythrocyte protoporphyrin. The genotype of the patients' mother, who did not present with any EPP clinical symptoms, was delT23/IVS3-48t (M/t). The patients' father, an offspring of consanguineous parents, was homozygous IVS3-48 c/c. He exhibited a mild photosensitivity, and an increase of 4-fold in erythrocyte protoporphyrin. His FECH mRNA amount was 71% of that of genotype t/t. It is the first reported case of an individual with c/c genotype who exhibits both biochemical and clinical indications of EPP. These results suggest that IVS3-48c is a functional variant of ferrochelatase. The clinical symptoms and biochemical abnormalities in the patients' father could be the result of an interaction between genetic and environmental factors. In addition, the frequency of IVS3-48c in the Ashkenazi Jewish population was estimated at 8%, which is similar to that in the European populations.

Objectives: To improve the traditional fresh blood film method to a high-throughput analysis of the presence of circulating fluorescent red cells (fluorocytes) in erythropoietic protoporphyria (EPP) using an automated imaging system., Methods: Based on the autofluorescence of protoporphyrin, we used an automatic image acquisition platform for examining fluorocytes in peripheral blood with minimal sample preparation. The image acquisition is easy-to-use under automated operations of excitation, focusing, detection and data analysis. Quality image and semi-quantitative fluorescence measurement of fluorocytes can be generated in a single step. For high-throughput analysis, the platform can image more than 200 96-well micro-plates, i.e. 19200 samples, in approximately 10 hours. Importantly, the reagent cost of analysis is negligible., Results: In this pilot study, three EPP patients were diagnosed and 4000 normal individuals were screened for EPP by this method. Our results showed that the method can distinguish the overt case and asymptomatic carriers. It gives reliable evidence for rapid EPP screening., Conclusion: This automated imaging system provides multiple advantages that improve the traditional fresh blood film method as a more effective diagnostic tool and facilitates population screening for EPP. As fluorocytes are present in the umbilical cord blood of EPP patients, this high-throughput method can be potentially used for newborn screening of EPP.

Ferrochelatase is the terminal enzyme in the heme biosynthesis pathway. Under anaerobic conditions it catalyzes the insertion of ferrous iron into the protoporphyrin IX ring to form protoheme. In the absence of iron and under aerobic conditions, the enzyme will use zinc or mercury as a substitute. The assay described in this unit uses zinc under aerobic conditions and the artificial substrate mesoporphyrin to analyze the formation of zinc-mesoporphyrin by HPLC with UV or fluorescence detection.

Ferrochelatase, estimated as zinc chelatase, was measured in the lymphocytes of 30 patients with erythropoietic protoporphyria (EPP), in 35 first- or second-degree relatives of patients with EPP, and in 50 healthy controls. In 30 EPP patients the zinc chelatase level (mean +/- standard deviation, SD) was 0.45 +/- 0.10 nmol of zinc protoporphyrin per hour per milligram of protein, in 14 EPP carriers the zinc chelatase level (mean +/- SD) was 0.42 +/- 0.09 and in 50 healthy controls the zinc chelatase level (mean +/- SD) was 0.84 +/- 0.27. All patients with EPP were also demonstrated to have an elevated protoporphyrin level in their red blood cells: the erythrocyte protoporphyrin levels were as follows EPP patients (mean +/- SD) 1300 +/- 758 nmol protoporphyrin/dl, EPP carriers (mean +/- SD) 60 +/- 24, and healthy controls (mean +/- SD) 50 +/- 25 (P < 0.001 for EPP patients compared to controls and EPP carriers). The families of 12 out of 15 EPP patients were examined with respect to the mode of inheritance of the disorder. Of 35 relatives, 14 were carriers of EPP, as characterized by reduced zinc chelatase activity in lymphocytes and by a normal protoporphyrin level in red blood cells. None of the 14 EPP carriers had presented with clinical symptoms of EPP. The mod of inheritance was autosomal dominant in seven of the 12 examined families, and autosomal recessive in two. In two families only one parent could be investigated, but we nevertheless concluded that the inheritance was autosomal dominant. Inheritance in one EPP family could not be elucidated as both parents showed normal zinc chelatase levels and did not demonstrate abnormal erythrocyte protoporphyrin levels.

Three blonde d'Aquitaine calves (one male and two females) about four months old, exhibited skin lesions just after birth, the site and nature of which suggested photosensitisation. Their porphyrin metabolism indicated a marked decrease in the activity of lymphocytic ferrochelatase, leading to a diagnosis of congenital erythrocytic protoporphyria. The associated nervous disorders of the 'recurrent epileptiform seizure' type are discussed in the light of complementary histological and biochemical tests.

Rapid, sensitive and specific high-performance liquid chromatographic assays are described for protoporphyrinogen oxidase and ferrochelatase in human leucocytes. The enzyme reaction products were separated and quantitated by reversed-phase high-performance liquid chromatography with fluorescence detection. The optimal pH for the protoporphyrinogen oxidase assay was 8.6 and the Michaelis constant for protoporphyrinogen IX was 9.78 +/- 0.96 microM (mean +/- S.D.). The mean (+/- S.D.) activity of protoporphyrinogen oxidase in fourteen apparently healthy subjects was 0.146 +/- 0.023 nmol protoporphyrin IX per min per mg protein. In one patient with variegate porphyria, the activity was 0.028 nmol protoporphyrin IX per min per mg protein. The optimal pH for ferrochelatase was 7.4 and with protoporphyrin and Zn2+ as substrates, the Michaelis constants were 1.49 and 8.33 microM, respectively. The mean activity of ferrochelatase in ten control subjects was 0.24 nM Zn-protoporphyrin or 2.05 nM Zn-mesoporphyrin formed per h per mg protein.

Lead exposure is a well-known cause of increases in urinary coproporphyrin and erythrocyte zinc-protoporphyrin, so these compounds are often used to monitor occupational lead exposure. The increased concentrations are usually assumed to result from lead inhibition of two of the mitochondrial enzymes of heme biosynthesis, coproporphyrinogen oxidase (EC 1.3.3.3) and ferrochelatase (EC 4.99.1.1). We studied 88 subjects in whom the degree of occupational lead exposure was established by measuring erythrocyte lead and protoporphyrin. Assay of lymphocyte coproporphyrinogen oxidase and ferrochelatase activities showed that these enzymes were unaffected by lead exposure, as was a related enzyme, lymphocyte NADH-ferricyanide reductase. We propose alternative explanations for the increased concentrations of coproporphyrin and zinc-protoporphyrin seen in lead exposure.

Erythropoietic protoporphyria (EPP) is associated with a deficiency of protohaem ferrolyase. We have used a novel assay for this enzyme based on its ability to utilize zinc as a substrate to investigate the inheritance of EPP in nine affected families. Zinc chelatase activity was markedly reduced in peripheral blood mononuclear cells from 14 EPP patients (mean, 3.3 nmol Zn protohaem/h/mg protein; range, 0.3-8.0) when compared with 41 controls (16.8 +/- 3.6) p less than 0.01. In three families with parent-to-child transmission of disease, the asymptomatic parent had an enzymatic activity within the normal range. In three pedigrees where the parents were asymptomatic, enzymatic activities were below the 95% confidence limits in both. Zinc chelatase activity was below the mean control value in 17 of the 18 parents in nine affected pedigrees, and six of seven asymptomatic offspring of patients with protoporphyria. The findings suggest that EPP is not transmitted as a simple dominant trait and that inheritance of more than one gene may be required for disease expression.

Effects of erythropoietin treatment on haem synthesis in peripheral blood were evaluated in 11 patients on haemodialysis. After 2 weeks of erythropoietin, mean (SEM) uroporphyrinogen-l synthase activity increased significantly from 88 (10) to 116 (9) pmol/h per mg protein. Haem synthase activity, thought to be the rate-limiting step in erythroid haem synthesis, also showed a significant increase from 4.5 (0.8) to 8.4 (1.8) pmol/h per 10(6) reticulocytes. 4 patients, who showed only a partial response to erythropoietin, had significantly higher serum aluminium concentrations than the 7 who responded fully (225 [44] vs 55 [23] micrograms/l); erythrocyte protoporphyrin concentrations in partial responders were also much higher than in responders (973 [120] vs 388 [29] nmol/l). Aluminium intoxication may cause resistance to erythropoietin by interference with haem synthesis, with accumulation of protoporphyrin.

Elevation of zinc protoporphyrin (ZPP) levels in the blood has served as an indicator of lead poisoning and iron deficiency anemia for many years. We have discovered that sublethal doses of whole body irradiation with x-rays also elevates ZPP 2-3-fold over normal levels. The ZPP level does not begin to increase until days 12-14 postirradiation and peaks between days 18 and 20 before returning to normal levels between days 28 and 35. Increasing the radiation dose delays the onset of the rise in ZPP, but does not affect the magnitude of the elevation. At lethal doses, ZPP elevation is not observed. Neither of the two previously described mechanisms that cause elevations of ZPP, namely iron deficiency and inhibition of ferrochelatase, are responsible for the radiation-induced elevation of ZPP. The elevation of ZPP appears to be correlated with the recovery of the hematopoietic system from radiation injury.

We describe a fluorometric assay for heme synthetase, the enzyme that is genetically deficient in erythropoietic protoporphyria. The method, which can readily detect activity in 1 microliter of packed human lymphocytes, is based on the formation of zinc protoheme from protoporphyrin IX. That zinc chelatase and ferrochelatase activities reside in the same enzyme was shown by the competitive action of ferrous ions and the inhibitory effects of N-methyl protoporphyrin (a specific inhibitor of heme synthetase) on zinc chelatase. The Km for zinc was 11 micrograms and that for protoporphyrin IX was 6 microM. The Ki fro ferrous ions was 14 microM. Zinc chelatase was reduced to 15.3% of the mean control activity in lymphocytes obtained from patients with protoporphyria, thus confirming the defect of heme biosynthesis in this disorder. The assay should prove to be useful for determining heme synthetase in tissues with low specific activity and to investigate further the enzymatic defect in protoporphyria.

The aim of this study was to investigate a possible relationship between exposure to sulfides and disturbances of the synthesis of heme and the erythrocytes. Eighteen workers exposed to sulfides at a pulp and paper plant were examined and compared with individually matched referents from a thermomechanical pulp plant without such exposure. The exposure levels of methylmercaptan, dimethylsulfide, and dimethyldisulfide were low. However, five subjects were exposed to high levels of short duration, and their data were analyzed separately. The activity of the enzymes delta-aminolevulinic acid synthase and heme synthase in reticulocytes, characteristics of the erythrocytes, and the iron status were analyzed. A minor decrease, not statistically significant, was observed for the enzymes among the five highly exposed subjects. However, the concentrations of iron and transferrin were elevated and the concentration of ferritin was low in comparison to the corresponding levels of the referents. This combination will not occur spontaneously. A previous study indicated that sulfides may inhibit heme synthesis, and the present study suggests that they may also disturb iron metabolism.

Experimental sideroblastic anaemia was produced in normal and in iron loaded guinea pigs by intraperitoneal (i.p.) administration of isoniazid and cycloserine. Subsequently, the activities of delta-aminolaevulinic acid synthase (ALA-S) and of haem synthase in peripheral red blood cells were measured and in particular the relationships of enzyme activities to the iron status were examined. The ALA-S activity showed a similar decrease in all animals with sideroblastic anaemia. The haem synthase activity was increased probably due to secondary induction, but it was significantly less increased in animals with the highest values for iron status. This finding indicates that mitochondrial iron accumulation may have limited the compensatory increase of haem synthase activity. It is likely that also in human sideroblastic anaemia mitochondrial iron overload may have a secondary limiting effect on the haem synthase activity in erythroid cells.

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.

In 130 pregnant women (41 without complications, 40 with prematurity, 49 with EPH-Gestosis) Serumferritin, ferrochelatase (FCH), delta-aminolaevulinic-aciddehydratase (D-ALA-D), porphobilinogendeaminase (PBG-D) as well as hematologic routine parameters were measured. Regarding serumferritin, both uncomplicated and cases with pathologic conditions revealed a significant decrease in group II (28th week of pregnancy) as compared to group I (12th to 28th week of pregnancy). Women with EPH-Gestosis showed the lowest values. Activities of hemesynthesizing enzymes did not show any significant differences, neither between the two groups nor between uncomplicated and pathologic cases. Compared to healthy, non pregnant women; in pregnant women we found a significant increase in D-ALA-D and PBG-D, but a significant decrease in FCH. Enzyme pattern in pregnancy reveals an increased synthesis of porphobilinogen and an increased conversion of porphobilinogen to porphyrin. The low activity of FCH we measured in our study could be a reason for the elevation of free protoporphyrin in the erythrocytes.

We studied the porphyrin metabolism of a 7-year-old Japanese boy with erythropoietic protoporphyria (EPP) and his family members. Leukocyte ferrochelatase activity was markedly decreased in this patient, being approximately 12% of the mean value of normal controls (4 aged-matched healthy boys). In contrast, leukocyte delta-aminolevulinic acid (ALA) synthase activity was normal. The free protoporphyrin content of erythrocytes was greatly increased (4.3 mg/100 ml RBC), while erythrocyte ALA dehydratase and porphobilinogen (PBG) deaminase activities were 1.7- and 2.2-fold of respective control values. A survey of his family revealed that 12 of 19 members probably had manifest EPP or were EPP carriers. These results suggest that, in EPP, there might be an inherited impairement of ferrochelatase activity which gives rise to an elevation of erythroblast ALA dehydratase and PBG deaminase activities to compensate for a resultant decrease in heme production.

We describe a new rapid, sensitive high-performance liquid-chromatographic (HPLC) method for determining ferrochelatase (EC 4.99.1.1) activity in lymphocytes. Zinc and mesoporphyrin are incubated aerobically with sonicated lymphocytes and the zinc mesoporphyrin formed is extracted with dimethyl sulfoxide-methanol-EDTA for quantification by HPLC. Incubation conditions, including the concentration of the palmitic acid activator, were optimized. The Michaelis constant (Km) was 2.1 mumol/L for mesoporphyrin, 22.2 mumol/L for zinc. The mean ferrochelatase activity (expressed as zinc mesoporphyrin formed per hour per milligram of lymphocyte protein) for a reference population was 3.25 (SD 0.43) nmol.h-1.mg-1. For three patients with erythrohepatic protoporphyria (EHP), activities were 1.11, 1.30, and 1.35. Neutrophils contain negligible ferrochelatase activity.

The molecular weight of protoheme ferro-lyase [EC 4.99.1.1], a mitochondrial enzyme, was estimated by the radiation inactivation method. Irradiation in vacuo caused less inactivation than that in air. The approximate molecular weight determined by this method was in the range from 250,000 to 320,000. The significance of the location of the enzyme in connection with its role in heme synthesis is discussed on the basis of Singer's membrane theory.

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.

The activity of heme synthetase (ferrochelatase), the enzyme(s) which catalyzes the formation of heme from iron and protoporphyrin IX, was studied in the various fractions of a cell-free reticulocyte system which synthesizes hemoglobin. The ribosomal fraction contained heme synthetase activity and its characteristics were similar to that described in avian erythrocytes, human and rat liver, and rabbit reticulocytes. It has a pH optimum of 7.5, required sulfhydryl reagents, was denatured by heat and was unstable on freezing. Heme synthetase is a mitochondrial enzyme. Fragments of mitochondrial membrane were identified in the ribosome fraction by both electron microscopy and the presence of cytochrome oxidase activity.

After reviewing of the sources of lead poisoning in birds, the diagnostic value of several clinicopathologic parameters for the diagnosis of plumbism in birds is discussed. These parameters include venous lead concentration, plasma delta-aminolaevulinic acid dehydratase concentration (ALA-D), aminolaevulinic-acid (ALA) excretion in urine, free erythrocytic protoporphyrin (FEPP) and zinc protoporphyrin (ZPP) concentrations in venous blood, and the percentage of fluorocytes, reticulocytes, and erythrocytes with basophilic stippling in peripheral blood. The CaNa2 EDTA lead mobilization test is also mentioned.

Ferrochelatase (EC 4.99.1.1) was purified 2000-fold to apparent homogeneity from isolated chicken erythrocyte mitochondria. The purified enzyme yields a single band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis with an apparent Mr of 42 000. The enzyme utilizes proto-, meso- and deutero-porphyrin with Km values of 37, 51 and 80 microM respectively. The disubstituted porphyrins 2,4-bisglycol deutero-porphyrin and 2,4-disulphonic deuteroporphyrin were not substrates. Mn2+, Hg2+, Pb2+ and Co2+ were strong inhibitors of the purified enzyme. Palmitic acid and oleic acid stimulated activity, whereas linoleic acid inhibited and phospholipids had variable effects. Chicken ferrochelatase was inhibited by N-ethylmaleimide and iodoacetamide. Inhibition by iodoacetamide was pseudo-first-order, but inhibition by N-ethylmaleimide appeared to be biphasic in nature with an initial high rate followed by a much lower rate of inactivation. The characteristics of the chicken erythrocyte enzyme are compared with those previously reported for mammalian liver ferrochelatase.

1. Analysis of reticulocytes for delta-amino-laevulinic acid synthase (AmLev synthase, EC 2.3.1.37) and haem synthase (EC 4.99.1.1) activity in 17 workers in pulp production with low-level hydrogen sulphide and methylmercaptan exposure showed decreased activities in eight and six cases respectively. 2. Erythrocyte protoporphyrin concentration was below the control range in seven cases. 3. Low AmLev synthase and haem synthase activities were found in one patient with hydrogen sulphide intoxication 1 week after the event. The activities had returned to the control levels 2 months later, though erythrocyte protoporphyrin remained abnormally low. 4. In vitro, hydrogen sulphide inhibited haem synthase with an apparent Ki of 3.4 mmol/l. Sulphide anion, on the other hand, inhibited AmLev synthase activity 85% at 10 mmol/l concentration. Thiosulphate anion inhibited AmLev synthase activity 18% (Ki 27 mmol/l) and haem synthase activity 43% at 10 mmol/l concentration. Selenite inhibited AmLev synthase (Ki 5.1 mmol/l) and haem synthase (Ki 9.0 mmol/l). 5. The assay of AmLev synthase and haem synthase could be a valuable addition to the assessment of workers' health in industries generating hydrogen sulphide or/and methylmercaptan, although the mechanism of the toxic effect remains speculative.

In 44 patients with rheumatoid arthritis and 30 control persons, the activities of the following three hemesynthesizing enzymes were determined: delta-aminolevulinic acid dehydratase (D-ALA-D), porphobilinogen deaminase (PBG-D), and ferrochelatase (FCH). Compared with the control persons in patients with rheumatoid arthritis, statistically significant decreased activities of FCH were determined, whereas no differences between the two groups tested occurred with D-ALA-D and PBG-D.

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.

Heme metabolism and in vitro erythropoietic growth (CFU-E, BFU-E) were examined in bone marrow cells taken from two siblings with apparent familial hypochromic microcytic anemia. Bone marrow cells from both patients grew adequate numbers of CFU-E and BFU-E colonies in culture in the presence of erythropoietin. In addition, small numbers of endogenous CFU-E were seen in 7-day cultures. Assays on bone marrow cells taken from both patients revealed that baseline delta-aminolevulinic synthase activity was considerably reduced, but increased six to seven fold (to normal levels) when patients' cells were exposed to pyridoxal phosphate (PLP). In both cases, ferrochelatase and delta-aminolevulinic acid dehydratase activities were normal. Bone marrow heme oxygenase showed no significant differences in activities between normals and patients values in the absence or presence of PLP. In contrast, heme synthesis by patients' bone marrow was less than that of normals. This study demonstrates that bone marrow cells from patients with this rare disorder have some disturbances in heme metabolism, whereas erythropoiesis appeared to be normal when cultured with adequate nutrients in vitro.

Anemia is a constant complication of uremia. As it has been suggested that uremic toxins (middle molecules) play an important role in the mechanism of anemia, we studied the activities of three heme-synthesizing enzymes: delta-aminolevulinic acid dehydratase, porphobilinogen deaminase and ferrochelatase. In 26 patients on regular dialysis therapy, all three enzymes had significantly lower values than in normal control subjects. From our results, it can be assumed that the decreased heme biosynthesis in chronic uremic patients might be caused by a lack of erythropoietin or by uremic toxins inhibiting erythropoietin and/or heme-synthesizing enzymes.

Protoporphyrinogen oxidase activity and ferrochelatase activity were measured in leucocytes from patients with porphyria variegata. The mean activity of protoporphyrinogen oxidase (PPO) in porphyria variegata (PV) was about 50% of normal (P less than 0.05). The mean activity of ferrochelatase with 59Fe2+ sulphate and protoporphyrin as substrates (in the presence of ascorbic acid) was reduced by 40% (P less than 0.009). The mean activity of ferrochelatase with 59Fe3+ chloride and protoporphyrin as substrates (in the presence of reduced glutathione) was increased by 65% (P less than 0.005). Both are statistically highly significant. The findings are interpreted as follows: (a) The occurrence of a low level of protoporphyrinogen oxidase in PV is confirmed. (b) The findings indicate a concurrent structural change in ferrochelatase (this may be structurally related to (a) but no evidence of this is at present available).

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.