Biosynthesis of mercapturic acids from allyl alcohol, allyl esters and acrolein

1. 3-Hydroxypropylmercapturic acid, i.e. N-acetyl-S-(3-hydroxypropyl)-l-cysteine, was isolated, as its dicyclohexylammonium salt, from the urine of rats after the subcutaneous injection of each of the following compounds: allyl alcohol, allyl formate, allyl propionate, allyl nitrate, acrolein and S-(3-hydroxypropyl)-l-cysteine. 2. Allylmercapturic acid, i.e. N-acetyl-S-allyl-l-cysteine, was isolated from the urine of rats after the subcutaneous injection of each of the following compounds: triallyl phosphate, sodium allyl sulphate and allyl nitrate. The sulphoxide of allylmercapturic acid was detected in the urine excreted by these rats. 3. 3-Hydroxypropylmercapturic acid was identified by g.l.c. as a metabolite of allyl acetate, allyl stearate, allyl benzoate, diallyl phthalate, allyl nitrite, triallyl phosphate and sodium allyl sulphate. 4. S-(3-Hydroxypropyl)-l-cysteine was detected in the bile of a rat dosed with allyl acetate.     

Conversion of thyroxine into tri-iodothyronine by rat liver homogenate.

By using a highly specific radioimmunoassay the formation of tri-iodothyronine by the deiodination of thyroxine was studied in rat liver homogenate. Several observations suggest that the reaction observed is enzymic in nature. Pre-heating the homogenate for 30 min at 56 degrees C completely abolished conversion of thyroxine into tri-iodothyronine; the component of rat liver homogenate responsible could be saturated with substrate; iodotyrosines displayed competitive activity. Between 0 degrees and 37 degrees C, the tri-iodothyronine-production rate was positively correlated with incubation temperature. The addition of NAD+ enhanced conversion into tri-iodothyronine, which suggests that an oxidative mechanism is involved. 5-Propyl-2-thiouracil and 6-propyl-2-thiouracil, both known to prevent deiodination in vivo, greatly decreased the deiodiantion activity of rat liver homogenate.     

Inhibition of rat liver glutathione S-transferase isoenzymes by acrolein

The in vitro effect of the toxin and teratogen, acrolein, on the fetal rat liver glutathione S-transferase isoenzyme, YcYfetus, was investigated and compared with acrolein's effect on some of the adult rat liver glutathione S-transferase isoenzymes. Acrolein was found to inhibit all the isoenzymes investigated and double-reciprocal plots suggest that inhibition is either noncompetitive or mixed-type noncompetitive. It is therefore attractive to suggest that should a similar situation arise in vivo, it may provide one mechanism for the teratogenicity of acrolein.     

Conversion of proinsulin into insulin by cathepsins B and L from rat liver lysosomes

Conversion of proinsulin and intermediate forms of proinsulin into insulin were studied with rat liver cell fractions and purified lysosomal proteinases by using the technique of polyacrylamide disc-electrophoresis. Both substrates were degraded very rapidly by homogenates and crude lysosomal fractions to split products not detectable on disc-electropherograms. Neither breakdown nor conversion were detected with the cytosol and the microsomal fraction. With partially purified lysosomal fractions (mol. wt. approx. 25 000) or with highly purified cathepsin L or cathepsin B (B1) proinsulin was converted into products migrating like the intermediate forms and insulin, and the intermediates were converted into products migrating like insulin and deoctapeptide-insulin in disc-electropherograms. The mechanism of conversion seems to be different for both enzymes. The results force us to conclude that lysosomal cathepsins, especially cathepsins L and B might be involved in the process of conversion of proinsulin into insulin and perhaps also of other precursors into biologically active proteins in vivo.     

Conversion of furfural into furfuryl alcohol by saccharomyces cervisiae 354

The influence of aeration, stirring conditions, and the addition of furfural on the yield and productivity of furfural – furfuryl alcohol bioconversion by the yeast strain Saccharomyces cerevisiae 354 was investigated. The formation of furfuryl alcohol increases up to 32 hours of incubation corresponding to the addition of furfural, while the cell growth essentially ceased at 20 hours. The conversion of furfural into furfuryl alcohol under anaerobic and low aeration conditions was 70% and the productivity 0.5g · 1^?1 · h^?1, when the final concentrationof of furfural amounted to 35 g · 1^?1.     

Conversion of green note aldehydes into alcohols by yeast alcohol dehydrogenase

Green note aldehydes were successfully reduced into their corresponding alcohol by commercial yeast alcohol dehydrogenase. Among different yeasts tested for their ability to convert (Z)-3-hexenal into (Z)-3-hexenol, Pichia anomala gave the best results. Conversion yields higher than 90% were also obtained by directly conducting the reaction in the medium where (Z)-3-hexenal is produced by the action of lipoxygenase and hydroperoxide lyase on linolenic acid.     

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.     

Conversion of proalbumin into serum albumin in the secretory vesicles of rat liver

The conversion site of proalbumin into serum albumin was investigated in the subcellular fractions of rat liver labeled with [³H] leucine in vivo. In the cisternae-rich fraction of the Golgi complex as well as in the microsomal fraction most of the labeled albumin was detected as proalbumin, while in the secretory vesicles, which were obtained in increased amount by oral administration of ethanol, more than 70% of the labeled albumin was found as serum type, indicating that conversion of proalbumin into serum albumin occurs within the secretory vesicles in rat liver. Little accumulation of albumin was observed in colchicine-treated rats.     

Oxidative demethylation of t-butyl alcohol by rat liver microsomes

Tertiary butyl alcohol has often been used experimentally as a “non-metabolizable” alcohol. In this report, evidence is presented that t-butanol serves as a substrate for rat liver microsomes and that it is oxidatively demethylated to yield formaldehyde. The apparent K_m for t-butanol is 30 mM while V_max is about 5.5 nmol per min per mg microsomal protein. Formaldehyde production is stimulated by azide, which prevents destruction of H₂O₂ by catalase. Hydroxyl radical scavenging agents, such as benzoate, mannitol, and 2-keto-4-thiomethylbutyrate, suppress formaldehyde production. Therefore, the microsomal reaction pathway appears to involve the interaction of t-butanol with hydroxyl radicals generated from H₂O₂ by the microsomes. Formaldehyde is also produced when t-butanol is incubated with model hydroxyl radical-generating systems such as the iron-EDTA-stimulated oxidation of xanthine by xanthine oxidase or the iron-EDTA-catalyzed autoxidation of ascorbate. These results indicate that t-butanol cannot be used to distinguish metabolically-linked from non-metabolically-linked actions of ethanol.     

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.     

Destruction of liver haem by norethindrone. Conversion into green pigments

1. Factors affecting the norethindrone-mediated conversion of hepatic haem into green pigments have been studied in the rat. Concentrations of haem and green pigments were estimated spectrophotometrically after esterification and separation by silica gel high-pressure liquid chromatography (h.p.l.c.). 2. Accumulation of green pigments in the liver was dependent on the dose of steroid and the time after dosing, maximum values being reached after 4–8h. Phenobarbitone pretreatment of rats resulted in an 8-fold increase in the concentration of green pigments at these times. 3. In microsomal systems in vitro, the formation of green pigments in the presence of NADPH and norethindrone was also dependent on the concentration of steroid and incubation times. Reaction rates very rapidly became non-linear with time, consistent with the self-catalysed destruction of the form(s) of cytochrome P-450 responsible for the metabolic activation of norethindrone. Microsomal mixtures incubated for a short period of time (1min) with norethindrone gave only one green-pigment peak after h.p.l.c. Longer incubation times gave four or five additional green pigments. Results suggested that multiple green pigments may arise by metabolic transformation of a single precursor. 4. When liver haem was prelabelled with ¹⁴C by using 5-amino[4-¹⁴C]laevulinic acid, subsequent dosing with norethindrone in vivo gave rise to three major ¹⁴C-labelled-green-pigment peaks on h.p.l.c. None of these components had the same retention times as the green pigments produced by microsomal fractions in vitro. 5. When liver haem was prelabelled with ⁵⁹Fe by using ⁵⁹FeCl₃, norethindrone administration resulted in the detection of ⁵⁹Fe-labelled green pigments if subsequent esterification was carried out under neutral conditions with trimethyloxonium tetrafluoroborate, but not when carried out under acidic conditions with methanol/H₂SO₄. These results suggested that green pigments normally contain chelated iron and that metal-free green pigments are not produced by the liver.     

Developmental changes in rat liver alcohol dehydrogenase

Hepatic alcohol dehydrogenase activity and mass content change coordinately during development in male rats. Enzyme activity and mass content increase continuously after birth to 100 and 80% of maximal values within 6 weeks (2.6 ± 0.4 μmole/min/g liver and 92 ± 20 μg/g liver), respectively. When expressed per milligram of soluble proteins, both parameters peak at 3 weeks (0.052 ± 0.002 μmole/min/mg protein and 2.0 ± 0.4 μg/mg protein) and then decrease gradually to plateau levels. These decreases probably arise from a “surge” in soluble liver protein levels that occurs after weaning. Similar developmental patterns also occur in female rats. These findings are the first quantitative measurements of this enzyme in developing animals.