Herbert L. Bonkowsky

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.

Hormonal requirements for the induction of cytochrome P450 in hepatocytes cultured in a serum-free medium

Abstract Drug mediated induction of cytochrome P450 was studied in cultures of hepatocytes that had never been cultured in the presence of serum. Propylisopropylacetamide induced a five-fold increase in cytochrome P450, approximating in ovo induced levels, when triiodothyronine and/or dexamethasone were included in the culture medium. Insulin was apparently not required for this induction. Cytochrome P450, free of cytochrome oxidase, could be fully recovered from cell homogenates in a 8700g supernatant, by use of a buffer containing 0.2% Emulgen.

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.

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.

Hepatic cytochrome P-450 in chronically hypoxemic rats

Abstract The basal level of hepatic cytochrome P-450 and its inducibility by phenobarbital pretreatment have been found to be enhanced by chronic hypoxemia. Pentobarbital sleeping times were decreased in parallel to changes in levels of cytochrome P-450. The increase in level of hepatic cytochrome P-450 in chronically hypoxemic rats occurred despite the increased levels of hepatic heme oxygenase which previously were associated with the chronic hemoglobinemia of the hypoxemic state. Chronically hypoxemic rats may provide a useful model for study of control of hepatic heme and hemoprotein metabolism.

A rapid, simple method for obtaining radiochemically pure hepatic heme

Abstract Radioactivity-labelled heme has usually been isolated from liver to which unlabelled carrier has been added by long, laborious techniques involving organis solvent extraction followed by crystallization. We have devised a simpler, rapid method for obtaining radiochemically-pure heme synthesized in vivo in rat liver from δ-amino[4- 14 C]levulinate, by modifying our previous procedure (Bonkowsky et al. (1975) J. Clin. Invest. 56, 1139–1148). This method, in which the heme is extracted into ethyl acetate/glacial acetic acid and in which porphyrins are removed from the heme-containing organic phase with HCl washes, does not require addition of carrier heme. The new method gives better heme recoveries than and heme specific activities identical to, those obtained using the crystallization method. In this new method heme must by synthesized from δ-amino[4- 14 C]levulinate; it not satisfactory to use [2- 14 C]-glycine substrate because non-heme counts are isolated in the heme fraction.

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 δ‐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.

Assay of δ-aminolevulinic acid synthetase in homogenates of mouse, rat, and human liver: Species differences in requirement for an exogenous succinyl-CoA-generating system

Conditions required for optimal assay of low levels of activity of hepatic delta-aminolevulinic acid synthetase have been studied, comparing dilute homogenates of mouse, rat, and human livers. The assay method used was a modification of that described by Ebert et al. (Biochim, Biophys, Acta (1970) 208, 236-250), and livers were studied from both untreated animal and human subjects and subjects pretreated with porphyrinogenic compounds. In homogenates of mouse and human but not rat liver, maximal rates of delta-aminolevulinic acid formation required addition to the incubation mixture of an exogenous system for succinyl-CoA generation. The requirement for this generating system was increased if livers from pretreated subjects were frozen and stored prior to assay, suggesting that the endogenous capacity for succinyl-CoA generation was more labile than delta-aminolevulinic acid synthetase under these conditions. Of the metabolic inhibitors tested (F-, malonate, and arsenite), only F- (100 mM final concentration) enhanced activity. Increasing the permeability of mitochondria by quick freeze-thawing of fresh homogenates just before assay did not increase the rate of delta-aminolevulinic acid formation.

KINETICS OF HEPATIC HEME TURNOVER IN RATS GIVEN ALLYLISOPROPYLACETAMIDE: A NEW MODEL FOR LIVER HEME CATABOLISM

Kinetic analysis of hepatic heme turnover, for 49h after labelling with 14 C-δ-aminolevulinate (ALA), revealed the existence of two heme pools. A single injection of allylisopropylacetamide, 4h after 14 C-ALA, led to production of “green pigments” and increased heme turnover. This increased turnover was due to an increase in the size of the more rapidly turning-over pool, not to an increase in the rate constants for turnover from either pool. Green pigments also underwent rapid biphasic turnover.

COINCIDENT INDUCTION OF CYTOCHROME P450 AND PHENOL RED CONJUGATION IN CULTURED CHICK EMBRYO HEPATOCYTES: EFFECTS OF COBALTOUS AND MANGANOUS IONS

Cytochrome P450 and conjugation of phenol red are inducible in cultures of chick embryo hepatocytes. Cobaltous and manganous ions cause decreases in P450, which is partially restored in the intact cells by hemin.