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Biomedical subjects

J R Bloomer

Publications and source records attributed to J R Bloomer.

At least 55 records · Page 3Linked to original sources
PubMed1986

Fulminant Wilson's disease treated with postdilution hemofiltration and orthotopic liver transplantation.

A 22-yr-old woman presented with fulminant Wilson's disease. The diagnosis was suspected clinically and was later confirmed with chemical and pathologic studies. She presented with acute hepatic failure, hemolysis, and acute anuric renal failure. Postdilution hemofiltration and continuous arteriovenous hemofiltration with oral D-penicillamine allowed removal of a total of 95,700 micrograms of copper; 78,665 micrograms of the total were removed via postdilution hemofiltration alone. On the 57th day, the patient received successful liver and renal transplants. We found that the determination of serum copper was instrumental in the diagnosis of fulminant Wilson's disease, that postdilution hemofiltration allowed a rapid removal of copper in the presence of renal failure, and that, finally, orthotopic liver transplantation should be performed early in the clinical course of these patients. This patient is the longest survivor of this serious condition.

Acute Disease↗
PubMed1983

Enzymatic formation of zinc-protoporphyrin by rat liver and its potential effect on hepatic heme metabolism.

The mitochondrial enzyme heme synthase (ferrochelatase) catalyzes the formation of heme from ferrous iron and protoporphyrin. Using a fluorometric assay, the enzymatic formation of zinc-protoporphyrin in rat liver tissue was compared with heme formation. With sonicated liver homogenate, the rate of zinc-protoporphyrin formation was similar to that of heme when each metal was present alone in the reaction mixture. When combined in the reaction mixture, the two metals competed for the enzyme. With intact mitochondria, zinc was a better substrate, as 90% of the product was zinc-protoporphyrin in the presence of 50 microM zinc and 50 microM iron. The effect of zinc-protoporphyrin on the induction of delta-aminolevulinic acid synthase, the rate-limiting enzyme in hepatic heme biosynthesis, was also examined in the rat. Intraperitoneal injection of allylisopropylacetamide (350 mumol) induced activity of the enzyme fourfold. Concomitant administration of zinc-protoporphyrin (4 mumol) suppressed the induction by 52%, nearly as effectively as the same amount of heme. These findings indicate that the formation of zinc-protoporphyrin in liver may (a) inhibit the synthesis of heme and (b) exert negative feedback regulation on delta-aminolevulinic acid synthase. This combination would reduce the hepatic heme level.

5-Aminolevulinate Synthetase↗
PubMed1982

Protoporphyria.

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Adolescent↗
PubMed1982

A radiochemical assay for heme synthase activity.

A radiochemical assay for heme synthase (ferrochelatase) activity is described in this report. The principle of the assay is to measure the incorporation of 59Fe, added to the reaction flask with a known amount of iron, into heme. Iron is maintained in the ferrous state, and oxygen is excluded from the flask during the incubation of the substrates with enzyme. Labeled heme product is extracted by solvent partitioning and counted in a gamma scintillation spectrometer. The assay can detect picomole amounts of product and is most useful when low levels of heme synthase activity are present.

Animals↗
PubMed1982

Evidence that hepatic crystalline deposits in a patient with protoporphyria are composed of protoporphyrin.

The livers of patients with protoporphyria and hepatic disease typically contain pigment crystals which may be a factor in the pathogenesis of liver injury. We isolated and characterized these crystals from a patient who developed hepatic failure and underwent liver transplantation. The prophyrin concentration in the liver was 516 micrograms/g wet weight (normal is a trace amount), over 99% of which was protoporphyrin. Through a discontinuous sucrose density gradient, followed by tissue digestion with deoxycholate, pigment crystals were obtained that had ultrastructural features like those in the whole liver section. After the crystals were dissolved in perchloric acid-methanol, their fluorescence emission and excitation spectra were identical to those of an extract of whole liver tissue and to a protoporphyrin standard. This provides evidence that the pigment crystals in livers of patients with protoporphyria and hepatic disease are composed of protoporphyrin.

Crystallization↗
PubMed1982

Bovine protoporphyria: documentation of autosomal recessive inheritance and comparison with the human disease through measurement of heme synthase activity.

Protoporphyria is an autosomal dominant disease in man in which protoporphyrin accumulated because of a defect in heme synthase (ferrochelatase) activity. A disease has been described in cattle that has the same manifestations as does the human disease. We measured heme synthase activity in sonicates of cultured skin fibroblasts and whole liver homogenates from animals with protoporphyria, their unaffected parents, and normal cattle in order to examine the mode of inheritance and compare it with human protoporphyria. The mean activity (+/- SEM) in fibroblasts from the three groups was 2.0 +/- 0.4, 47 +/- 12, and 149 +/- 10 pmol heme formed/mg protein per hr, respectively, consistent with autosomal recessive inheritance. Similarly, the levels of heme synthase activity in livers of the parents were intermediate to those of normal animals and of animals with protoporphyria. When compared with normal human fibroblasts and liver, the specific activity of heme synthase in normal bovine tissue was significantly higher. These studies indicate that manifestations of protoporphyria do not occur in cattle unless the animal is homozygous for the gene defect, whereas in humans, the heterozygous condition is sufficient. This is probably because the specific activity of heme synthase in cells of heterozygous animals is not reduced to a level that significantly alters heme metabolism.

Animals↗
PubMed1981

Enzyme defects in the porphyrias and their relevance to the biochemical abnormalities in these disorders.

Defects in enzymes of the heme biosynthesis pathway underlie the biochemical abnormalities which occur in the porphyrias. Porphyrins and porphyrin precursors are accumulated and excreted in excessive amounts because of the enzyme defects. This is illustrated by studies in protoporphyria and variegate porphyria, disorders in which the biochemical abnormalities indicate a defect(s) in the terminal part of the heme biosynthesis pathway. The activity of heme synthease (ferrochelatase), which catalyzes the chelation of ferrous iron to protoporphyrin, is deficient in tissues of patients with protoporphyria. This causes protoporphyrin to be accumulated and excreted excessively. In variegate porphyria protoporphyrinogen oxidase, which catalyzes the oxidation of protoporphyrinogen to protoporphyrin, appears to be defective. As a result, protoporphyrinogen may be excreted in increased amounts in bile, where it is subsequently auto-oxidized to protoporphyrin. The following questions have arisen as a result of the demonstrations of enzyme defects in tissues of patients with porphyria: (1) Will different defects in the same enzyme be found among patients who fulfill the clinical and biochemical criteria for diagnosis of a specific porphyria? That is, does genetic heterogeneity exist in each of the porphyrias? (2) Why do some patients with an enzyme defect not have biochemical abnormalities? (3) Why is one type of tissue, usually the liver, the major site of expression of the biochemical abnormality, when the enzyme defect can be demonstrated in all tissues?

Fibroblasts↗
PubMed1980

The enzymatic defect in variegate prophyria. Studies with human cultured skin fibroblasts.

Fecal protoporphyrin is increased in patients with variegate porphyria, even during clinical remission, suggesting an enzymatic defect in the terminal portion of the heme biosynthetic pathway. We measured the activities of protoporphyrinogen oxidase, which catalyzes the oxidation of protoporphyrinogen to protoporphyrin, and heme synthase, which catalyzes the chelation of iron to protoporphyrins, in cultured skin fibroblasts from five normal controls and five patients with variegate porphyria. Heme synthase activity was shown to be normal in variegate porphyria cells by direct assay in cell sonicates and indirect assay in intact cells. Protoporphyrinogen oxidase activity, however, was reduced to 43 per cent of normal in sonicates of variegate porphyria cells (0.90 +/- 0.13 vs. 2.12 +/- 0.25 nmol of protoporphyrin per milligram of protein per hour [mean +/- S.E.M.] [P less than 0.005]). We conclude that protoporphyrinogen oxidase activity is deficient in variegate porphyria. Fecal protoporphyrin may increase because an excess amount of protoporphyrinogen is excreted into bile and subsequently auto-oxidized to protoporphyrin.

Adult↗
PubMed1980

A fluorometric assay for measurement of protoporphyrinogen oxidase activity in mammalian tissue.

The oxidation of protoporphyrinogen to protoporphyrin, which is catalyzed by the enzyme protoporphyrinogen oxidase, is an important step in heme biosynthesis. We describe a fluorometric assay for protoporphyrinogen oxidase activity in mammalian tissues which measures the conversion of the non-fluorescent protoporphyrinogen to the fluorescent protoporphyrin. Using these assay conditions, the mean level of protoporphyrinogen oxidase activity in sonicated rat liver mitochondria was 12.3, and in sonicated human cultured skin fibroblasts was 1.97 nmol protoporphyrin/mg protein/h.

Animals↗
PubMed1980

Effect of an exogenous succinyl-CoA-generating system on the measurement of delta-aminolevulinic acid synthase activity in rat liver tissue by a radiochemical assay.

Rat liver tissue was used to examine the effect of an exogenous succinyl-CoA-generating system on the radiochemical assay for delta-aminolevulinic acid synthase (succinyl-CoA:glycine C-succinyltransferase (decarboxylating), EC 2.3.1.37) activity developed by Ebert et al. (Ebert, P.S., Tschudy, D.P., Choudry, J.N. and Chirigos, M.A. (1970) Biochim. Biophys. Acta 208, 236--250). In the absence of exogenous succinate thiokinase, 34--62% (average 55%) of the radioactivity in the final column eluate could be attributed to delta-amino-[4-14C]levulinic acid, as assessed by conversion of delta-aminolevulinic acid in the eluate to a pyrrole. The addition of succinate thiokinase markedly enhanced the formation of the contaminant(s), as succinyl-CoA was metabolized to a compound or compounds that eluted chromatographically with delta-amino-levulinic acid. This effect was abolished by 10 mM EDTA, probably because the generation of succinyl-CoA was suppressed due to the chelation of Mg2+. These observations indicate that this radiochemical assay should be carefully examined for each set of assay conditions employed.

5-Aminolevulinate Synthetase↗
PubMed1980

Benign recurrent intrahepatic cholestasis: studies of bilirubin kinetics, bile acids, and cholangiography.

Three patients with benign recurrent intrahepatic cholestasis are described. They had had between five and 16 attacks of cholestasis. Between attacks the liver function tests, including serum bile acids, were normal. Serial serum bilirubin and bile acid estimations during the cholestasis in one patient revealed a consistent discrepancy between the serum bilirubin and bile acid concentrations during three consecutive attacks. In the other two patients the serum concentrations of bile acids and bilirubin varied in parallel. Analysis of the individual serum bile acids did not reveal high concentrations of any 'toxic' bile acid. In one patient, plasma bromsulphthalein (BSP) curves were obtained during both remission and cholestatic periods. The 45 minute retention was slightly increased (10.8%) during remission. During the cholestasis, the 45 minute retention (25%) and the fractional extraction coefficient (Ke=0.069 min-1) were markedly abnormal. The hepatic clearance of unconjugated radiobilirubin was normal at all times in this patient, although during cholestasis, conjugated bilirubin reflexed from the liver to the plasma and was then cleared slowly with a half life of approximately 12 hours. Treatment with corticosteroids, cholestyramine, and phenobarbitone was unsatisfactory.

Adult↗
PubMed1980

Characterization of deficient heme synthase activity in protoporphyria with cultured skin fibroblasts.

Heme synthase (ferrochelatase) activity, as determined by the chelation of ferrous iron to protoporphyrin or deuteroporphyrin, is reduced to 10-25% of normal in tissues of patients with protoporphyria. With cultured skin fibroblasts from seven patients with protoporphyria and six normal individuals, the present studies examined the enzymatic defect.Heme synthase activity in normal and protoporphyria fibroblasts had the same pH optimum, showed similar inhibition by divalent metals, and had the highest specific activity in the mitochondrial-enriched fraction. The ultrastructural features and other biochemical parameters of mitochondria were normal in protoporphyria cells, excluding a general mitochondrial defect. Measurement of the rate of deuteroheme formation at different concentrations of substrate demonstrated a significant reduction in the apparent K(m) for deuteroporphyrin in detergent-treated sonicates of protoporphyria fibroblasts compared to normal (7.5 +/- 0.9 muM, mean +/- SEM, vs. 17.4 +/- 1.8), as well as a decrease in the velocity of reaction (mean level was 21% of normal). Studies with intact cells, in which heme synthase activity was estimated indirectly, also indicated that the apparent K(m) for porphyrin substrate was significantly lower in protoporphyria lines. These data show that heme synthase in protoporphyria fibroblasts has markedly reduced catalytic activity despite an increased affinity for porphyrin substrate. This could be caused by either a change in the enzyme protein, or an alteration of its micro-environment.

Cells, Cultured↗
PubMed1979

Enzymatic fluorometry for estimating serum total bile acid concentration.

Fasting serum total bile acid (SBA) levels were estimated by enzymatic fluorometry (EF) in 36 subjects without liver disease, 28 with hepatic lesions and impaired hepatic function, and 79 with hepatic lesions and normal function. Fasting and postprandial EF-SBA levels were compared in nine normal subjects and nine patients with cholestasis, and SBA assays by EF and gas-liquid chromatography (GLC) were compared in 28 patients with hepatic lesions and impaired function. Levels of SBA were below 9 mumole/L in all but two of the 36 subjects without liver disease, and above that level in all 28 with impaired hepatic function and 17 (24%) of the 70 with hepatic lesions and normal liver function. In most subjects, EF detected notable postprandial rises in SBA. Enzymatic fluorometric and GLC-SBA values were closely correlated (coefficient of correlation, 0.869).

3-Hydroxysteroid Dehydrogenases↗