Enzymic Cleavage Of In Vivo Formed Maillard-Type Compounds Involved In Haemostasis

Desoxyfructose derivatives of serotonin (Mester et al.,1975), of haemoglobin (Flückiger and Winterhalter, 1976), of poly-L-lysine (Mester et al., 1975) and of lysine rich histones (Kertesz-Crisba, 1977) are easily formed in vivo by a simple Maillard-type chemical reaction. Some of these compounds interfere with platelet functions (Mester et al.,1976) or contribute to the thickening of the basal membrane of blood vessels (Cerami et al., 1979).While the chemical synthesis of Maillard-type compounds proceeds readily even in vivo, the chemical cleavage of them needs sever conditions which certainly do not exist in vivo (Gottschalk, 1952). However, a slow liberation of serotonin from desoxyfructo-serotonin is observed in vivo, suggesting the existence of an enzyme system for the cleavage of Maillard-type sugar-amine derivatives. In vitro, using a sheep liver microsomal preparation rich in Cytochrome P450 enzyme, the liberation of serotonin is in linear correlation with the enzyme concentration. The cleavage of desoxyfructo-serotonin is activated by NADPH having its optimum at pH=7.4, excluding definitely the occurence of a chemical hydrolysis.Factors interfering with the enzyme system involved in the cleavage of Maillard-type compounds, may also interfere with haemostasis.

4,4′-dimethylcholesta-7,9,14-trienol is an intermediate in the demethylation of dihydroagnosterol

1. 4,4′-Dimethylcholesta-7,9,14-trienol is an intermediate in the metabolism of dihydroagnosterol to cholesterol by rat liver homogenate. 2. This triene is reduced by a rat liver microsomal preparation in the presence of NADPH to give 4,4′-dimethylcholesta-7,9-dienol under anaerobic conditions. 3. Reduction of the triene in the presence of [4-3H2]NADPH resulted in the incorporation of 3H into the product. 4. Under aerobic conditions the triene is converted into cholesterol by a rat liver homogenate.

Binding of diethylstilboestrol to deoxyribonucleic acid by rat liver microsomal fractions in vitro and in mouse foetal cells in culture

Diethylstillboestrol, a synthetic and carcinogenic hormone, binds to DNA as a result of incubation with a liver microsomal preparation in vitro and on incubation with primary mouse foetal cells in culture. Enzymic digestion of DNA samples thus prepared gives several covalent deoxyribonucleoside-diethylstilboestrol products from the microsomal system. One of these is produced in small but significant yield in the tissue-culture system.

Cyclic metabolic pathway of a butylated hydroxytoluene by rat liver microsomal fractions

A cyclic metabolic pathway was obtained when 3,5-di-t-butyl-4-hydroxytoluene (BHT) was incubated with a rat liver microsomal preparation. The pathway is as follows: BHT → 4-hydroperoxy-4-methyl-2,6-di-t-butylcyclohexa-2,5-dienone (BHT-OOH) → 4-hydroxy-4-methyl-2,6-di-t-butylcyclohexa-2,5-dienone (BHT-30OH) → BHT. This metabolic pathway suggests that antioxidants such as BHT owe their high efficacy, at least in part, to their metabolic regeneration.

Formation of bilirubin glucoside

1. Rat liver microsomal preparation can effect the transglucosylation from UDP-glucose to bilirubin in the presence of Mg2+. 2. Other nucleotides, namely CDP-glucose, ADP-glucose and GDP-glucose, were not active as glucosyl donors. 3. Only trace amounts of galactose, galacturonic acid and N-acetylglucosamine were conjugated to bilirubin when their respective UDP derivatives were used in the reaction mixture. 4. The azobilirubin glucosides produced by coupling with p-diazobenzenesulphonic acid and diazotized ethyl anthranilic acid were separable from the corresponding azobilirubin glucuronides by t.l.c. 5. The glucoside was, however, hydrolysed by both β-glucosidase and various preparations of β-glucuronidase; azobilirubin and glucose were liberated in the process. 6. Kinetic studies showed that the effects of pH and Mg2+ on the two conjugating systems were similar. 7. The specific activities of hepatic bilirubin UDP-glucosyltransferase, expressed as μg of bilirubin ‘equivalents’ conjugated/h per mg of protein, are respectively 1.7 and 2.4 for male and female rats. 8. The Km values for bilirubin and UDP-glucose are 5.7×10-5m and 1.6×10-3m respectively. 9. The glucoside and glucuronide conjugations of bilirubin are discussed in relation to the availability of the conjugating agents and aglycone in the liver.

Studies on the mechanism and regulation of C-4 demethylation in cholesterol biosynthesis. The role of adenosine 3′:5′-cyclic monophosphate

1. An assay for demethylation has been developed based on the release of tritium from 4,4-dimethyl[3α-3H]cholest-7-en-3β-ol (II). 2. The maximum release of 3H from 3α-3H-labelled compound (II) in a rat liver microsomal preparation occurs in the presence of NADPH and NAD+ under aerobic conditions. 3. Incubation of 3α-3H-labelled compound (II) with NADPH under aerobic conditions leads to the formation of a 3α-3H-labelled C-4 carboxylic acid. This compound undergoes dehydrogenation on subsequent anaerobic incubation with NAD+. 4. The 3H released from the steroid was located in [4-3H]nicotinamide and the medium. Incubation with synthetic [4-3H2]NADH gave a similar result. 5. In the presence of glutamate dehydrogenase and α-oxoglutarate part of the 3H released from the steroid was transferred to glutamate. 6. A series of 3-oxo steroids were reduced equally well by [4-3H2]NADH and [4-3H2]NADPH. The reduction of 5α-cholest-7-en-3-one was shown to use the 4B H atom from the nucleotide. 7. 3′:5′-Cyclic AMP was shown to be a competitive inhibitor of the 3β-hydroxy dehydrogenase enzyme in the demethylation reaction.

Metabolic hydroxylations of trans-stilbene

1. A study was made of the hydroxylation of trans-stilbene in rabbits, guinea pigs and mice, as well as by rabbit liver microsomes. 2. In the rabbit in vivo, trans-stilbene is converted into 4-hydroxy-,4,4′-dihydroxy-,3-hydroxy-4-methoxy-and 4-hydroxy-3-methoxy-stilbene, and hydroxylation plays a more significant role in the metabolism of trans-stilbene than has previously been reported. 3. Investigation of the hydroxylation of 4-hydroxystilbene in the rabbit in vivo demonstrated its ready conversion into 4,4′-dihydroxystilbene and established its intermediacy in the formation of this compound and the methylated analogues of 3,4-dihydroxystilbene. 4. Hydroxylation of trans-stilbene in the guinea pig was found to follow a pattern similar, both qualitatively and quantitatively, to that in the rabbit. 5. Studies in the mouse revealed only limited yields of 4,4′-dihydroxystilbene. 6. Studies of the hydroxylation of trans-stilbene and 4-hydroxystilbene by rabbit liver microsomes located two of the reactions that occur with these compounds in vivo. 7. Work with a solubilized liver-microsomal preparation provided evidence that ‘stilbene hydroxylase’ activity is not completely lost on solubilization, thus allowing for future microsomal enzyme-isolation studies.

Drug-metabolizing activity of the human placenta

The drug-metabolizing activity of a 9000 × g supernatant fraction prepared from human placentae obtained immediately after birth was investigated. For comparative purposes a similar rat liver microsomal preparation was used. The metabolism of pentobarbital by oxidation, and of amphetamine by deamination or hydroxylation, proceeded at similar rates in the two tissues. The rate of metabolism of meperidine in the human placental preparation was about two-thirds of that found with the rat liver enzyme. The rate of metabolism of aminopyrine, as determined by measuring the formation of 4-aminoantipyrine or the production of formaldyhyde, was insignificant in the placental preparation, but the metabolism of 4-aminoantipyrine occurred much more rapidly in this tissue than in rat liver. The nitroreductase activity of the human placenta was low; a sixfold activation was achieved by adding a source of glucose 6-phosphate dehydrogenase but the activity was still only one-third of that found in rat liver. The cofactor requirements for the drug-metabolizing enzymes studied were found to be similar in the two tissues. The significance of these findings with respect to the effects of drugs on the mother and the fetus is discussed.