Iron loading of cultured hepatocytes. Effect of iron on 5-aminolaevulinate synthase is independent of lipid peroxidation.

Cultured chick embryo hepatocytes were iron-loaded with ferric nitrilotriacetate. Iron-loading was confirmed by both quantitative cellular iron determinations and ultrastructural studies. With iron-loading, lipid peroxidation, as detected by malonaldehyde released into the medium, occurred at a linear rate for 12h, after which time the rate of malonaldehyde production decreased. No cell toxicity, as detected by lactate dehydrogenase release, was noted. The amount of malonaldehyde recovered in the medium after 18h of exposure to iron represented 24-33% of the total malonaldehyde that could be produced by incubating lysed cells with iron and ascorbate. Cellular glutathione was not affected by iron-stimulated lipid peroxidation, but was increased by allylisopropylacetamide. Although iron-loading by itself had no effect on activity of 5-aminolaevulinate synthase, the first and rate-limiting step in haem synthesis, iron-loading in the presence of the porphyrogenic drug allylisopropylacetamide increased levels of 5-aminolaevulinate synthase 6-fold over levels induced by the drug alone. The antioxidant, butylated hydroxytoluene, totally inhibited iron-stimulated lipid peroxidation, but did not interfere with the effect of iron-loading to potentiate an increase in 5-aminolaevulinate synthase. After 18h of exposure to iron, followed by a change to fresh medium, the iron remaining within the cells did not stimulate further lipid peroxidation over the following 18h, but did potentiate an increase in 5-aminolaevulinate synthase on exposure to allylisopropylacetamide. It therefore appears that lipid peroxidation is not the mechanism by which iron potentiates induction of hepatic 5-aminolaevulinate synthase.

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.

Decreased activity of uroporphyrinogen decarboxylase caused by 2,4,5,3',4'-pentabromobiphenyl in chick embryo hepatocyte cultures. Difference in activity in intact or homogenized cells.

Uroporphyrinogen decarboxylase activity was investigated in cultures of chick embryo liver by two different methods: (1) analysis of porphyrin composition following incubation of intact cells with delta-aminolevulinic acid; and (2) a more conventional direct enzymic assay of cell homogenates. Activity was detectibly decreased following exposure of cells to 100 ng/ml 2,4,5,3',4'-pentabromobiphenyl using the first method, but not the second. This decrease in activity was reversed by homogenizing the cells treated with 100 ng/ml pentabromobiphenyl. It is concluded that the direct homogenate assay of the enzyme may miss or underestimate decreases in its in vivo activity.

Increases in cytochrome p-450 in cultured hepatocytes mediated by 3- and 4-carbon alcohols.

The amount of cytochrome P-450 was increased to different extents after treatment of cultured chick embryo hepatocytes with n-propanol, isopropanol, n-butanol, or isobutanol. These increases were associated with increases in benzphetamine demethylase activity, a cytochrome P-450-catalyzed oxidation, and glucuronidation of phenol red, catalyzed by UDP-glucuronyl transferase. The responses were similar to those obtained with ethanol or propylisopropylacetamide, which the phenobarbital-like inducers. Pretreatment of cells with cycloheximide prevented the increases in both cytochrome P-450 and glucuronidation of phenol red, indicating that protein synthesis was required for these responses.

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.

Formation of cobalt protoporphyrin by chicken hepatocytes in culture. Relationship to decrease of 5-aminolaevulinate synthase caused by cobalt.

Cobalt protoporphyrin generated from 5-amino[4-14C]laevulinate by homogenates or primary cultures of chick embryo liver exposed to CoCl2 was found to be radioactivity unextractable by acid/acetone, when extra protein was added. The activity of ferrochelatase was required for formation of cobalt protoporphyrin since inhibition of ferrochelatase with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (in the presence of cycloheximide) inhibited formation of cobalt protoporphyrin and resulted in accumulation of protoporphyrin. Cobalt protoporphyrin was detected spectrophotometrically in hepatocyte cultures exposed to the combination of 2-allyl-2-isopropylacetamide and CoCl2: (1) as the pyridine haemochrome of the protein pellet remaining after acid-acetone extraction of the cells, or (2) as the material extracted from the protein pellet with acetic acid-pyridine-chloroform. The amount of cobalt protoporphyrin increased with increasing CoCl2 concentration as cellular haem declined. The decrease in haem was about equal to the amount of cobalt protoporphyrin that accumulated. 2-Allyl-2-isopropylacetamide and polychlorinated biphenyls were both powerful inducers of 5-aminolaevulinate synthase. The former led to protoporphyrin accumulation, whereas with the latter, uroporphyrin accumulated, probably due to a concomitant decrease in activity of uroporphyrinogen decarboxylase. The decrease in activity of 5-aminolaevulinate synthase produced by administration of CoCl2 was greater after treatment with 2-allyl-2-isopropylacetamide than after treatment with allylisopropylacetamide and 3,4,3',4'-tetrachlorobiphenyl. We conclude: (a) that cobalt protoporphyrin is readily formed in cultured hepatocytes, and (b) that its formation accounts for the action of cobalt on 5-aminolaevulinate synthase.

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.

The diagnosis of acute intermittent porphyria. Usefulness and limitations of the erythrocyte uroporphyrinogen I synthase assay.

In acute intermittent porphyria (AIP) the inherited metabolic defect residues in a partial enzyme deficiency at the uroporphyrinogen I synthase (URO-S) step of heme biosynthesis. Assay of this enzyme in erythrocytes is increasingly used for diagnosis of the genetic defect. Erythrocyte URO-S activity was measured by three laboratories in members of 14 AIP kindreds and found helpful for diagnosing the asymptomatic carrier state, since activity of the enzyme was usually distributed bimodally. However, a definite diagnosis of the carrier state was not always possible with this assay: approximately 7% of a total of 340 determinations were inconclusive. Repeat enzyme assays, enzyme assays by more than one laboratory, quantitative urine porphobilinogen determinations, and studies of other family members all aided in arriving at a firm diagnosis for most of those in whom a single erythrocyte URO-S measurement was inconclusive. Because the enzyme is unstable, blood specimens should be stored frozen if immediate assay is not possible.

Stimulation of hemoglobin synthesis in murine erythro-leukemia cells by low molecular weight ketones, aldehydes, acids, alcohols and ethers.

The effects of short chain (C1-C5) aldehydes, ketones, acids alcohols and ethers on murine erythroleukemia (MEL) cells were examined to determine which particular chemical moieties and some of their combinations stimulated hemoglobin synthesis in these cells. The C4 series of compounds was active at lower concentrations than homologs of shorter chain lengths. Within an homologous series the potency and efficacy of the alcohol was always less then that of the acid and aldehyde compounds. Though hepanoic acid was found to be an inducer of hemoglobin synthesis in MEL cells, the 4,6-dioxoheptanoic acid analog is a potent inhibitor of hemoglobin synthesis. Analysis of porphyrin content of MEL cells incubated with the inducers 2-butanone, 2-methoxyethanol, acetone and methanol, showed that increased hemoglobin synthesis was always accompanied by the accumulation of porphyrins, most of which was protoporphyrin. These studies suggest that low molecular ketones, aldehydes, acids, ethers and alcohol can correct the defect in erythroid differentiation exhibited by MEL cells and they further suggest that the physiological trigger for inducing hemoglobin synthesis in these cells is less discriminating than previously recognized.

Intrahepatic hematoma after aspiration liver biopsy. A prospective randomized trial using two different needles.

The purpose of this study was twofold: (1) to determine the incidence of intrahepatic hematoma after liver biopsy by the aspiration technique and (2) to compare the incidence of hematoma after biopsy with a needle of 1.6 mm vs 1.9 mm OD. Fifty-one patients were randomly assigned to be biopsied with the smaller needle and 46 with the larger. Hematomas, diagnosed by development of characteristic transient defects on liver scans done shortly after biopsy, occurred in two (4%) of those biopsied with the smaller needle and in none of those biopsied with the larger. This difference is not statistically significant. Neither patient who developed a hematoma had changes in vital signs, CBC, or serum alkaline phosphatase. One had a fivefold rise in SGOT; n the other, the SGOT remained normal. If these results are considered with those of other prospective trials of similar design, the overall incidence of intrahepatic hematoma after aspiration biopsy may be estimated to be 2.3% when needles of 1.6-2.0 mm (OD) are used. Many of these go undetected because, justifiably, scans are not routinely done shortly after biopsies.

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.

Iron increases collagenase production by rabbit synovial fibroblasts.

Ionic iron, as the chelate FeNTA, was taken up by rabbit synovial fibroblasts in monolayer culture. Uptake was accompanied by increased production of latent collagenase and PGE2. Concomitant addition of desferrioxamine, a specific chelator of Fe+++, prevented iron uptake and induction of collagenase and PGE2. Collagenase induced by iron may have a role in the pathogenesis of certain disease states associated with abnormal iron deposition.

Seizure management in acute hepatic porphyria: risks of valproate and clonazepam.

Seizures may occur in acute intermittent porphyria or other hepatic porphyrias. Management is difficult, because barbiturates and hydantoins exacerbate the porphyric state. We studied one patient with major motor seizures and acute intermittent porphyria. The seizure disorder was exacerbated by phenytoin and did not respond to a high-carbohydrate diet or to intravenous hematin. Clonazepam was ineffective in treating the seizures and, in high doses, seemed to exacerbate the porphyria. Both clonazepam and valproate were porphyrinogenic in experimental test systems. Because both drugs may exacerbate the acute hepatic porphyrias, bromide remains the drug of choice to treat these seizures.