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J F Healey

Publications and source records attributed to J F Healey.

At least 37 records · Page 2Linked to original sources
PubMed1988

Regulation of hepatic haem metabolism. Disparate mechanisms of induction of haem oxygenase by drugs and metals.

We studied drug- and metal-mediated increases in activity of haem oxygenase, the rate-controlling enzyme for haem breakdown, in chick-embryo hepatocytes in ovo and in primary culture. Phenobarbitone and phenobarbitone-like drugs (glutethimide, mephenytoin), which are known to increase concentrations of an isoform of cytochrome P-450 in chick-embryo hepatocytes, were found to increase activities of haem oxygenase as well. In contrast, 20-methylcholanthrene, which increases the concentration of a different isoform of cytochrome P-450, had no effect on activity of haem oxygenase. Inhibitors of haem synthesis, 4,6-dioxoheptanoic acid or desferrioxamine, prevented drug-mediated induction of both cytochrome P-450 and haem oxygenase in embryo hepatocytes in ovo or in culture. Addition of haem restored induction of both enzymes. These results are interpreted to indicate that phenobarbitone and its congeners induce haem oxygenase by increasing hepatic haem formation. In contrast, increases in haem oxygenase activity by metals such as cobalt, cadmium and iron were not dependent on increased haem synthesis and were not inhibited by 4,6-dioxoheptanoic acid. We conclude that (1) induction of hepatic haem oxygenase activity by phenobarbitone-type drugs is due to increased haem formation, and (2) induction of haem oxygenase by drugs and metals occurs by different mechanisms.

Animals↗
PubMed1987

Haem synthesis from exogenous 5-aminolaevulinate in cultured chick-embryo hepatocytes. Effects of inducers of cytochromes P-450.

The effects of inducers of cytochrome P-450 on haem biosynthesis from 5-aminolaevulinate were examined by using cultured chick-embryo hepatocytes. Cultures treated with either 2-propyl-2-isopropylacetamide or 3-methylcholanthrene contained increased amounts of cytochrome P-450 and haem. After treatment for 3 h with 5-amino[4-14C]laevulinate, the relative amounts of radioactivity accumulating as haem corresponded to the relative amounts of total cellular haem, but not to increases in the amounts of cytochrome P-450. Treatment with 5-aminolaevulinate did not alter cellular haem or cytochrome P-450 concentrations in either control or drug-treated cultures. The mechanism of the enhanced accumulation of radioactivity in haem was investigated. Although 2-propyl-2-isopropylacetamide enhanced the uptake of 5-aminolaevulinate and increased the cellular concentration of porphobilinogen 1.5-fold, these changes did not account for the increases in haem radioactivity. The inducing drugs had no effect on the rates of degradation of radioactive haem, but appeared to enhance conversion of protoporphyrin into haem. This latter effect was shown by: (1) a decreased accumulation of protoporphyrin from 5-aminolaevulinate in cells treated with inducers, and (2) complete prevention of this decrease if the iron chelator desferrioxamine was present. We conclude that inducers of cytochrome P-450 may increase haem synthesis not only by increasing activity of 5-aminolaevulinate synthase, but also by increasing conversion of protoporphyrin into haem.

Allylisopropylacetamide↗
PubMed1986

High-performance liquid chromatographic separation and quantitation of tetrapyrroles from biological materials.

We describe a rapid, reverse-phase HPLC procedure for separating and quantifying tetrapyrroles of biological interest. This procedure uses a 5-micron C18 column and the mobile phase is ammonium phosphate (pH 3.5) with a methanol gradient that is increased from 61 to 100%. Detection is by absorbance at 405 nm or by fluorescence. Porphyrins, heme, and the heme breakdown products, biliverdin and bilirubin, can be separated from a single injection in 25 min. Injections can be made every 40 min. Limits of detection are about 0.1 pmol for porphyrins, 5 pmol for heme, and 10 pmol for biliverdin and bilirubin. We present examples of the use of the system for separating tetrapyrroles formed by primary cultures of chick embryo hepatocytes and homogenates of rat liver.

Animals↗
PubMed1986

Hepatic microsomal function in rats with chronic dietary iron overload.

We determined whether alterations in hepatic microsomal function occur in association with iron-induced lipid peroxidation in vivo in rats with chronic dietary iron overload. In rats fed a 2.0% carbonyl iron diet for a period of 20 wk, there was no significant microsomal conjugated diene formation (evidence of microsomal lipid peroxidation) or difference in cytochrome P450 concentration found at mean (+/- SEM) hepatic iron concentrations of 1210 +/- 92 micrograms/g liver (wet wt) or 2730 +/- 100 micrograms/g. At a hepatic iron concentration of 4090 +/- 245 micrograms/g, however, there was significant conjugated diene formation (p less than 0.001) and a 56% decrease in the cytochrome P450 concentration (p less than 0.001). In rats fed a 2.5% carbonyl iron diet for 10 wk, achieving a liver iron concentration of 4820 +/- 420 micrograms/g, there was significant microsomal conjugated diene formation (p less than 0.001), a 35% reduction in cytochrome P450 (p less than 0.005), and a 16% reduction in aminopyrine demethylase activity (p less than 0.025), but only an 8% reduction in glucose-6-phosphatase activity (p = not significant). Finally, in rats fed a 3.0% iron-supplemented diet for 7 wk, achieving a liver iron concentration of 2730 +/- 205 micrograms/g, there was a 23% reduction in cytochrome P450 (p less than 0.025), a 28% reduction in cytochrome b5 (p less than 0.001), and a 47% increase in heme oxygenase activity (p less than 0.025) (heme oxygenase activity measured in this group only). We conclude that oral iron loading can produce microsomal lipid peroxidation in vivo that is associated with selective decreases in microsomal hemoprotein concentrations and cytochrome P450-dependent enzymes.

Aminopyrine N-Demethylase↗
PubMed1986

Hepatic heme and drug metabolism in rats with chronic mountain sickness.

Rats chronically exposed to hypobaric conditions develop pulmonary hypertension, right heart failure, hemoglobinemia, and in preliminary studies were recently found to have increased hepatic cytochrome P-450 content and activity of heme oxygenase, the rate-limiting enzyme for heme breakdown. To further delineate effects of chronic hypoxic, hypobaric exposure, on hepatic physiology and biochemistry, we have studied heme and drug metabolism in male Sprague-Dawley rats exposed to hypoxic conditions for 4-5 wk. Hypoxia, produced by exposure of rats to room air under hypobaric conditions (approximately 380 Torr), caused marked polycythemia [hematocrit (Hct) 70% vs. control Hct 43%], plasma hemoglobinemia, depletion of plasma haptoglobin, and decreased hemopexin concentrations. It also led to significant (20-30%) increases in concentrations of total hepatic heme and microsomal cytochrome P-450 and increased activities of heme oxygenase. In contrast, activity of 5-aminolevulinate synthase, the rate-limiting enzyme of hepatic heme synthesis, was significantly decreased in hypoxic rats and was not as inducible as in control normoxic rats. Hypoxia did not alter the rest of the heme synthetic pathway, as shown by a normal rate of conversion of 5-aminolevulinate to heme. Hypoxic exposure had no effect on the concentration of hepatic cytochrome-b5 but decreased activity of NADPH-cytochrome c reductase. Rates of metabolism of aminopyrine, benzphetamine, ethoxyresorufin, and warfarin were similar in hepatic microsomes obtained from hypoxic and normoxic rats. Thus the oxygen-requiring processes of hepatic heme and drug metabolism were well maintained despite chronic profound hypoxia sufficient to cause cardiopulmonary complications.

5-Aminolevulinate Synthetase↗
PubMed1985

Conversion of 5-aminolaevulinate into haem by homogenates of human liver. Comparison with rat and chick-embryo liver homogenates.

To assess whether the synthesis of haem can be studied in small amounts of human liver, we measured kinetics of the conversion of 5-aminolaevulinate into haem and haem precursors in homogenates of human livers. We used methods previously developed in our laboratory for studies of rat and chick-embryo livers [Healey, Bonkowsky, Sinclair & Sinclair (1981) Biochem. J. 198, 595-604]. The maximal rate at which homogenates of human livers converted 5-aminolaevulinate into protoporphyrin was only 26% of that for rat, and 58% of that for chick embryo. In the absence of added Fe2+, homogenates of fresh human liver resembled those of chick embryos in that protoporphyrin and haem accumulated in similar amounts, whereas fresh rat liver homogenate accumulated about twice as much haem as protoporphyrin. However, when Fe2+ (0.25 mM) was added to human liver homogenates, mainly haem accumulated, indicating that the supply of reduced iron limited the activity of haem synthase, the final enzyme in the haem-biosynthesis pathway. Addition of the potent iron chelator desferrioxamine after 30 min of incubation with 5-amino[14C]laevulinate stopped further haem synthesis without affecting synthesis of protoporphyrin. Thus the prelabelled haem was stable after addition of desferrioxamine. Since the conversion of 5-amino[14C]laevulinate into haem and protoporphyrin was carried out at pH 7.4, whereas the pH optimum for rat or bovine hepatic 5-aminolaevulinate dehydratase is about 6.3, we determined kinetic parameters of the human hepatic dehydrase at both pH values. The Vmax was the same at both pH values, whereas the Km was slightly higher at the lower pH. Our results indicate that the synthesis of porphyrins and haem from 5-aminolaevulinate can be studied with the small amounts of human liver obtainable by percutaneous needle biopsy. We discuss the implications of our results in relation to use of rat or chick-embryo livers as experimental models for the biochemical features of human acute porphyria.

Aminolevulinic Acid↗
PubMed1984

Formation of cytochrome P-450 containing haem or cobalt-protoporphyrin in liver homogenates of rats treated with phenobarbital and allylisopropylacetamide.

The potent porphyrogen allylisopropylacetamide and related compounds decrease hepatic concentrations of cytochrome P-450. This decrease occurs particularly in phenobarbital-induced cytochrome P-450 and is caused by suicidal breakdown of the haem of cytochrome P-450. Quantitative rocket immunoelectrophoresis showed that the protein moiety of the major phenobarbital-inducible form of hepatic cytochrome P-450 was not diminished up to 1 h, but was markedly decreased (to 43% of that of the phenobarbital-treated control) at 20 h after allylisopropylacetamide treatment. In contrast, the concentration of total cytochrome P-450, measured spectrophotometrically, decreased to 30-40% of the control at both 1 and 20 h after allylisopropylacetamide. Cytochrome P-450-dependent demethylations of ethylmorphine and benzphetamine decreased to a similar extent. When liver homogenates from rats treated with allylisopropylacetamide 1 h before being killed were incubated with haem, functional holocytochrome P-450 could be reconstituted from the apoprotein. Incubation with haem increased spectrophotometrically measurable cytochrome P-450 to 69%, ethylmorphine demethylase to 64% and benzphetamine demethylase to 93% of the activities in rats treated with phenobarbital alone. At 20 h after allylisopropylacetamide treatment, however, little or no reconstitution of cytochrome P-450 occurred after incubation with haem. When liver homogenates were incubated with cobalt and protoporphyrin, and microsomal proteins were then subjected to polyacrylamide-gel electrophoresis, cobalt-protoporphyrin was found specifically associated with proteins of Mr 50 000-53 000. When homogenates from rats given allylisopropylacetamide for 1 h or 20 h were compared, it was found that the extent of this association was higher in livers from the rats containing more apocytochrome P-450, suggesting that cobalt-protoporphyrin had associated with the apocytochrome. The data provide insight into the association of haem with the protein moiety of cytochrome P-450 and factors affecting breakdown of this protein.

Acetamides↗
PubMed1983

Iron and the liver: acute effects of iron-loading on hepatic heme synthesis of rats. Role of decreased activity of 5-aminolevulinate dehydrase.

Acute iron loading of rats, by intraperitoneal administration of iron-dextran (500 mg Fe/kg body wt 18-20 h before killing) decreased by 30% the rate of conversion of 5-amino-[14C]levulinate ([14C]ALA) into heme as measured with a recently described procedure for liver homogenates (1981. Biochem. J. 198: 595-604). The decrease in conversion of labeled ALA into heme caused by iron loading was shown to be due to a 70-80% decrease in activity of ALA dehydrase. The decrease in activity of ALA dehydrase caused by iron loading was not associated with a decrease in hepatic concentrations of GSH, nor could it be reversed by addition of dithiothreitol, Zn2+ or chelators of Fe2+ and Fe3+. Addition of FeSO4, ferric citrate, or ferritin to homogenates of control liver had no effect of activity of ALA dehydrase. The decrease in activity of ALA dehydrase, caused by iron-dextran, was mirrored by a reciprocal increase in ALA synthase. Iron-dextran potentiated the induction of ALA synthase by allylisopropylacetamide. However, this potentiation could be dissociated from the decrease in ALA dehydrase caused by iron loading.

5-Aminolevulinate Synthetase↗
PubMed1982

Decrease in hepatic cytochrome P-450 by cobalt. Evidence for a role of cobalt protoporphyrin.

Exposure of cultured chick-embryo hepatocytes to increasing concentrations of CoCl2 in the presence of allylisopropylacetamide results in formation of cobalt protoporphyrin, with a reciprocal decrease in haem and cytochrome P-450. Treatment of rats with CoCl2 (84 mumol/kg) and 5-aminolaevulinate (0.2 mmol/kg) also results in formation of cobalt protoporphyrin and a decrease in cytochrome P-450 in the liver. Hepatic microsomal fractions from rats treated with phenobarbital, CoCl2 and 5-aminolaevulinate were analysed by polyacrylamide gel electrophoresis. Cobalt protoporphyrin was associated mainly with proteins of 50000-53000 mol.wt. The results suggest that the formation of cobalt protoporphyrin occurred at the expense of the synthesis of haem, leading to a decrease in cytochrome P-450. Furthermore, the cobalt protoporphyrin that was formed may itself have been incorporated into apocytochrome P-450.

Animals↗
PubMed1981

Conversion of 5-aminolaevulinate into haem by liver homogenates. Comparison of rat and chick embryo.

1. We have studied the kinetics of the conversion of 5-aminolaevulinate into haem and haem precursors in homogenates of livers of rats and chick embryos. Homogenates of fresh liver from both species efficiently convert 5-aminolaevulinate into haem. After frozen storage for 1 year, homogenates of rat, but not chick, liver have decreased rates of formation of haem with accumulation of more protoporphyrin. The rate of haem formation after storage is restored by addition of Fe2+ and menadione. 2. At all initial concentrations of 5-aminolaevulinate tested (2 microM-1 mM), homogenates of rat liver accumulate less protoporphyrin than haem. In contrast, homogenates of chick embryo liver accumulate more protoporphyrin than haem at concentration of 5-aminolaevulinate greater than 10 microM. Conversion of protoporphyrin into haem by homogenates of fresh or frozen chick embryo liver is not increased by addition of Fe2+. 3. Homogenates of liver from both species accumulate porphobilinogen; the kinetic parameters for this process reflect those of 5-aminolaevulinate dehydratase. 4. The results show that the rate-limiting enzyme for the hepatic conversion of 5-aminolaevulinate into protoporphyrin is porphobilinogen deaminase. In addition, chick liver, compared with rat liver, has only about one-fifth the activity of ferrochelatase, the final enzyme of the haem biosynthetic pathway, which inserts Fe2+ into protoporphyrin to form haem. 5. Comparison of these results with previous studies indicates that the homogenate system described here provides physiologically and clinically relevant information for study of hepatic haem synthesis and its control.

Aminolevulinic Acid↗
PubMed1981

Iron and the liver. Acute and long-term effects of iron-loading on hepatic haem metabolism.

We have determined the dose-response curves (100-900 mg of Fe/kg body wt.) and the time course over 84 days for the effects of a single injection of iron-dextran on rat hepatic 5-aminolaevulinate synthetase, cytochrome P-450, iron content, and GSH (reduced glutathione). Porphyrins in liver and urine have also been measured. (1) At 2 days after treatment, a dose of 500 mg of Fe/kg produced a 20-fold increase in iron concentration, which was maintained for 14 days. Total hepatic iron remained constant over 63 days, falling slightly by 84 days. (2) The activity of 5-aminolaevulinate synthetase was maximally increased (6-fold) 12-24 h after iron treatment. By 48 h the activity fell to less than twice the control value and thereafter remained slightly above the control value (1.1-1.5-fold) until 84 days after iron treatment. Liver GSH concentrations were unaffected by iron. Porphyrins in liver and urine were either unchanged or decreased. (3) Hepatic cytochrome P-450 decreased after iron treatment to a minimum (63% of control) at 48 h after iron administration and gradually returned to the control value by 28 days. (4) Iron-dextran potentiated 2 allyl-2-isopropyl-acetamide-induced synthesis of hepatic 5-aminolaevulinate. Potentiation occurred if the drug was given at the same time or 36 h after iron administration, but did not occur if the drug was given 14 or 64 days after iron administration. (5) The results are discussed in relation to proposed mechanisms for the effects of iron on hepatic haem metabolism.

5-Aminolevulinate Synthetase↗
PubMed1980

Metabolism of hepatic haem and 'green pigments' in rats given 2-allyl-2-isopropylacetamide and ferric citrate. A new model for hepatic haem turnover.

We have studied effects of single doses of 2-allyl-2-isopropylacetamide and ferric citrate on hepatic haem turnover in rats. Haem was pre-labelled by intraperitoneal administration of 5-amino-[4-(14)C]laevulinate 4h before other treatments. Computer-assisted analysis of the haem decay curve showed that at least two exponential components were involved implying two haem pools. In control rats the size of the rapidly-turning-over pool was 38% of the total. Treatment with 2-allyl-2-isopropylacetamide alone resulted in a 2.3-fold increase in the fractional size of this pool. Treatment with ferric citrate alone increased the size of this pool 1.8-fold; treatment with both agents together had no measurable effect beyond that produced by 2-allyl-2-isopropylacetamide. The apparent rate constant for disappearance of labelled haem from the first pool was not affected by treatment with 2-allyl-2-isopropylacetamide or ferric citrate, indicating that these treatments affect hepatic haem turnover primarily by altering the distribution of haem synthesized in the liver. The increased haem degradation after treatment with 2-allyl-2-isopropylacetamide alone was associated with the accumulation of ;green pigments' in the liver. These pigments were detectable 1.5h after drug treatment; their total amount increased steadily for 28h and then declined. Despite this increase in amount, radioactivity of the ;green pigment' fraction fell rapidly in a biphasic fashion. Some of the radioactivity that initially was found in the ;green pigment' fraction was later found in an aqueous fraction, not extractable by acidic ethyl acetate. Rats given ferric citrate together with 2-allyl-2-isopropylacetamide accumulated less ;green pigments'.

Acetamides↗
PubMed1979

Iron and the liver: subcellular distribution of iron and decreased microsomal cytochrome P-450 in livers of iron-loaded rats.

To understand better the intracellular iron distribution and metabolic consequences of chronic hepatic iron overload, rats were given large doses of iron dextran or ferric citrate intraperitoneally. They accumulated large quantities of iron within Kupffer cells and hepatocytes. The relative subcellular iron distributions were similar in controls and iron-loaded rats, despite a ten- to 20-fold difference in hepatic iron concentration. Electron microscopy of whole liver and subcellular particulate fractions suggested that iron was present in highest concentration in lysosomes, which were rendered more labile by its presence. Nevertheless, quantitative iron determinations on all subcellular fractions, obtained by two preparative methods, showed that most of the iron was present in the "soluble" fraction. The amount of iron in the "microsomal" fraction varied, depending on the techniques used for preparation of this fraction. Cytochrome P-450 and total heme concentrations were decreased 40% to 50% in microsomes isolated from iron-loaded livers.

Animals↗
PubMedDate not supplied

Hepatic heme synthesis in a new model of experimental hemochromatosis: studies in rats fed finely divided elemental iron.

Rats fed chow containing finely divided elemental iron (from carbonyl-iron) develop hepatic iron overload resembling human hereditary hemochromatosis in that deposition of iron is primarily in periportal hepatocytes and with hepatic iron concentrations sufficiently high to be associated in the human disease with hepatic fibrosis or cirrhosis. In recent studies using this model, we reported changes in hepatic hemoproteins and heme oxygenase, the rate-controlling enzyme of heme breakdown. We now report effects of iron-loading on three enzymes of heme synthesis: 5-aminolevulinate synthase; the first and rate-controlling enzyme of the pathway, 5-aminolevulinate dehydrase (or porphobilinogen synthase), and uroporphyrinogen decarboxylase, the activity of which is decreased in porphyria cutanea tarda, a liver disease in which iron is known to play an important but still poorly understood role. Of the three enzymes, only activity of the dehydrase was altered by iron-loading: it was decreased significantly as early as 1 week after starting iron feeding, and with marked iron overload was 30 to 32% of control values. The degree of decrease was inversely related (r = -0.77 to -0.88) to the degree of iron overload and was partially reversed within 1 to 3 days when feeding of the iron-supplemented diet was stopped. The decrease in dehydrase activity was not attributable to lack of reduced glutathione or other disulfide-reducing agents or to zinc deficiency; nor was evidence found for inhibition by iron compounds or other possible inhibitors present in iron-loaded livers.(ABSTRACT TRUNCATED AT 250 WORDS)

5-Aminolevulinate Synthetase↗