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Biomedical subjects

B Middleton

Publications and source records attributed to B Middleton.

At least 91 records · Page 5Linked to original sources
PubMed1986

Diurnal variation in the fraction of 3-hydroxy-3-methylglutaryl-coenzyme A reductase in the active form in the mammary gland of the lactating rat.

'Expressed' and 'total' activities of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase) were measured in freeze-clamped samples of mammary glands from lactating rats at intervals throughout the 24 h light/dark cycle. 'Expressed' activities were measured in microsomal fractions isolated and assayed in the presence of 100 mM-KF. 'Total' activities were determined in microsomal preparations from the same homogenates but washed free of KF and incubated with exogenously added sheep liver phosphoprotein phosphatase before assay. Both 'expressed' and 'total' activities of HMG-CoA reductase underwent a diurnal cycle, which had a major peak 6 h into the light phase and a nadir 15 h later, i.e. 9 h into the dark period. Both activities showed a secondary peak of activity (around 68% of the maximum activity) at the time of changeover from dark to light, with a trough in the value of the 'expressed' activity that was close to the nadir value. 'Expressed' activity was lower than 'total' at all time points, indicating the presence of enzyme molecules inactivated by covalent phosphorylation. Nevertheless the 'expressed'/'total' activity ratio was comparatively constant and varied only between 43% and 75%. Immunotitration of enzyme activity, with antiserum raised in sheep against purified rat liver HMG-CoA reductase, confirmed the presence of both active and inactive forms of the enzyme and indicated that at the peak and nadir the variation in 'expressed' HMG-CoA reductase activity resulted from changes in the total number of enzyme molecules rather than from covalent modification. The sample obtained after 3 h of the light phase exhibited an anomalously low 'total' HMG-CoA reductase activity, which could be increased when Cl- replaced F- in the homogenization medium. The result suggests that at that time the activity of the enzyme could be regulated by mechanisms other than covalent phosphorylation or degradation.

Animals↗
PubMed1986

A study of the metabolism of [U-14C]3-methyl-2-oxopentanoate by rat liver mitochondria using h.p.l.c. with continuous on-line monitoring of radioactive intact acyl-coenzyme A intermediates.

A reverse-phase h.p.l.c. system for the resolution of the acyl-CoA intermediates of the degradation of 3-methyl-2-oxopentanoate is described. The validation of a method for the measurement of radioactive intermediates produced by the incubation of [U-14C]3-methyl-2-oxopentanoate with rat liver mitochondrial fractions is described. The absence of bicarbonate caused the accumulation of [14C]propionyl-CoA. The accumulation of [14C]2-methylbutyryl-CoA was observed in incubations with mitochondrial fractions derived from riboflavin-deficient animals. Studies of the accumulation of labelled intermediates with time suggest that there is regulation of the pathway of isoleucine degradation at methylmalonyl-CoA mutase, as suggested for valine [Corkey, Martin-Requero, Walajtys-Rode, Williams & Williamson (1982) J. Biol. Chem. 257, 9668-9676]. These studies demonstrate that h.p.l.c. with on-line continuous radiochemical detection is a powerful method for the investigation of the control of intermediary metabolism.

Acyl Coenzyme A↗
PubMed1986

3-Ketothiolase deficiency.

Two patients have been studied in whom the activity of the short chain-length-specific mitochondrial 3-ketothiolase was found to be deficient. Use of a range of 3-ketoacyl-CoA substrates showed that the other 3-ketothiolase isoenzymes were normal in each case. Both patients had episodic ketosis and metabolic acidosis. One patient had substantial evidence of damage to the central nervous system and two siblings who had died of the disease. The organic aciduria was characterized by the excretion of 2-methyl-3-hydroxybutyric acid and tiglyglycine. In one patient the organic aciduria was very subtle and was masked during the presence of ketosis, but it was clarified by an isoleucine load after recovery from ketosis.

3-Hydroxybutyric Acid↗
PubMed1986

Dietary vitamin A and cancer--a multisite case-control study.

The relationship between an index of dietary vitamin A and cancer risk at 25 sites was assessed in retrospective case-control studies. Common control groups for males and females were used in all analyses. Relative risk estimates were derived from multiple logistic regression analyses that controlled for age, alcohol consumption, and smoking exposure. We found that among males, dietary vitamin A is associated with lower risk for cancers of the tongue, floor and other mouth, pharynx, larynx, esophagus, and lung but higher risk for Hodgkin's disease and leukemia. Among females, we found that dietary vitamin A had less effect on risk generally but was associated with lower risk for bladder cancer. These findings are consistent with previous research that showed dietary vitamin A to be associated with decreased risk of squamous epithelial cancers. The association of dietary vitamin A and increased risk of Hodgkin's disease and leukemia among males in addition to the disparity in effect of dietary vitamin A on risk between males and females are areas worthy of further research.

Adult↗
PubMed1985

Factors associated with the elderly falling in intermediate care facilities.

In intermediate care facilities, the records of 41 patients who had fallen and 36 controls were reviewed retrospectively, and the two groups compared for demographics, diagnoses, blood pressures over the prior two months, and prescribed medication. Seven of the sample had a recent weight loss recorded; all seven were in the group that fell. The mean number of medications prescribed for the group of fallers was significantly greater than the mean prescribed for the control group. The mean number of medication changes during the two weeks prior to the fall was significantly greater than the mean number of medication changes during the two weeks prior to data collection for the control group. Our data suggest that caution should be exercised in multiple drug prescribing for patients in intermediate care facilities and that recent weight loss and medication changes may be risk factors for falling.

Accident Prevention↗
PubMed1984

A mevalonate requirement for maintenance of fatty acid and protein synthesis during hormonally stimulated development of mammary gland in vitro.

The effect of compactin on hormonally induced lipogenesis and protein synthesis was studied in vitro in explants of mammary gland from mid-pregnant rabbits. Compactin blocks mevalonate synthesis by the specific inhibition of 3-hydroxy-3-methylglutaryl-CoA reductase, and in this system, culture with 10 microM compactin for 24, 48, and 72 h inhibited incorporation of [1-14C]acetate (but not [2-14C]mevalonate) into sterol by 98, 95, and 86%, respectively. Removal of compactin prior to assay rapidly reversed this effect and was associated with increased tissue 3-hydroxy-3-methylglutaryl-CoA reductase activity. Fatty acid synthesis (measured by incorporation of [1-14C]acetate or [4,5-3H]leucine) and protein synthesis (measured by incorporation of [4,5-3H]leucine) were both inhibited by around 50% after culture with compactin. This inhibition was not rapidly reversed by removal of compactin prior to assay, but it was prevented by inclusion of 1 mM mevalonolactone in the culture medium. After removal of compactin and continued culture in its absence for 24 h with hormones, the normal tissue capacity for fatty acid and protein synthesis was restored, indicating no permanent cell damage. The results suggest a specific requirement for mevalonate (or derived products) for the hormonal maintenance of the increased fatty acid and protein synthesis characteristic of the development of the mammary gland.

Acetates↗
PubMed1984

Biochemical investigations on a patient with a defect in cytosolic acetoacetyl-CoA thiolase, associated with mental retardation.

A severely mentally retarded boy with two normal siblings was persistently found to excrete elevated amounts of 3-hydroxybutyrate and acetoacetate. Enzyme analysis in cultured fibroblasts revealed a probable deficiency in cytosolic acetoacetyl-CoA thiolase which was about half the control activity with normal mitochondrial thiolase activities. Treatment with reduced dietary fat was initiated and a rapid reduction of the ketosis to biochemical normality was demonstrated. Shortly after initiating dietary treatment he presented with severe gastrointestinal problems and the histological features of colitis cystica superficialis. This appeared to respond to intravenous hydrocortisone therapy with an apparent complete recovery.

Acetyl-CoA C-Acetyltransferase↗
PubMed1984

The effects of 3,5,5-trimethylcyclohexanol on hepatic cholesterol synthesis, bile flow and biliary lipid secretion in the rat.

The chemical trimethylcyclohexanol (TMC) is closely related to menthol, the major component of a terpene preparation with known choleretic and cholelitholytic activity. Its effects on hepatic cholesterol synthesis and bile secretion were examined in the rat. In both acute and long-term dosing experiments TMC significantly inhibited hepatic S-3-hydroxy-3-methylglutaryl coenzyme A (HMGCoA) reductase. TMC was a potent choleretic, with detectable effects on bile flow at low doses, which also reduced coupling of cholesterol secretion to bile salt secretion. Single large doses tended to lower biliary cholesterol output and caused significant reduction in cholesterol saturation index after biliary diversion for 1 h. TMC and its widely prescribed ester cyclandelate, which is rapidly degraded to TMC after ingestion, should be investigated further as potential cholelitholytic treatments in man.

Animals↗
PubMed1983

In vivo inhibition of hepatic lipogenesis in the rat by cyclandelate (3,3',5-trimethylcyclohexanylmandelate).

Rates of hepatic lipogenesis were measured in vivo in rats by incorporation into lipids of [3H] from injected [3H]H2O 17 hr after a single oral dose of cyclandelate (3,3',5-trimethylcyclohexanylmandelate, a vasoactive substance). Cyclandelate administration resulted in a significant inhibition (40-60%) of both sterol and fatty acid synthesis in the livers which was independent of the 3.2-fold diurnal variation in the rates of hepatic sterol and fatty acid synthesis. The inhibition of accumulation of newly synthesized fatty acid in intestine also reached statistical significance. The accumulation of newly synthesized sterol was significantly depressed in serum but did not result in any change in the concentration of serum total cholesterol. These results are interpreted in terms of the inhibitory effect of cyclandelate on hepatic 3-hydroxy-3-methylglutaryl-CoA reductase previously reported by us (Biochem. Pharmac. 32, 649, 1983).

Animals↗
PubMed1983

The synthesis and characterisation of 2-methylacetoacetyl coenzyme A and its use in the identification of the site of the defect in 2-methylacetoacetic and 2-methyl-3-hydroxybutyric aciduria.

(1) 2-Methylacetoacetyl coenzyme A was prepared, purified by ion exchange chromatography as judged by TLC and HPLC and a number of its properties characterised. (2) An assay of 3-oxothiolase in homogenates of cultured human fibroblasts using 2-methylacetoacetyl CoA as substrate is described. (3) This enzyme activity was shown to be absent in fibroblasts from two patients with 2-methylacetoacetic and 2-methyl-3-hydroxybutyric aciduria. (4) These patients also showed decreased activity (42% normal) with acetoacetyl CoA, and indicated that the defective thiolase could also utilize this substrate in normals. The residual activity with acetoacetyl CoA in the patients' fibroblasts resembled the cytosolic acetoacetyl CoA-specific thiolase in properties. We suggest that the enzyme defective in the patients was the mitochondrial acetoacetyl CoA thiolase involved in ketone body utilization in extrahepatic tissues.

Acetoacetates↗
PubMed1983

The inhibition of hepatic S-3-hydroxy-3-methylglutaryl-CoA reductase by 3,3,5-trimethylcyclohexanol and its mandelic acid ester, cyclandelate.

Rat hepatic HMGCoA reductase was found to be at least 50% inhibited 17 hr after administration of a single oral dose of 3,3,5-trimethylcyclohexanyl mandelate (cyclandelate), a vasoactive substance. This inhibition was also found in rats given the 3,3,5-trimethylcyclohexanol component but only slight inhibition was seen after an equimolar dose of mandelate. The inhibition of HMGCoA was observed both around the high point and near the low point of the diurnal activity cycle. The effect did not persist to 41 hr after treatment. There was no direct inhibition of HMGCoA reductase by trimethylcyclohexanol when added to the assay system in vitro. The in vivo effect of these inhibitors was specific for HMGCoA reductase. There was no change, neither elevation nor depression, of the amount of microsomal membrane components cytochromes b5 and P-450, not was the activity of another microsomal enzyme, arylesterase, affected by dosing with cyclandelate or trimethylcyclohexanol.

Administration, Oral↗
PubMed1982

The existence of an inner-membrane-bound, long acyl-chain-specific 3-hydroxyacyl-CoA dehydrogenase in mammalian mitochondria.

L-3-Hydroxyacyl-CoA dehydrogenase was measured in mammalian tissues with long-chain (C16) and short-chain (C4) 3-ketoacyl-CoA substrates. The majority of the activity was mitochondrial. Activity with C16 substrate was enriched in the inner membrane fraction while the matrix showed enrichment of activity with C4 substrate. Gel-filtration in the presence of nonionic and ionic detergents separated two peaks of activity with C16 substrate from liver, heart, kidney and brown adipose tissue mitochondria. The highest molecular weight activity was associated with membrane fragments and accounted for 18-32% of the total activity with C16 substrate. This enzyme showed preference for long-chain substrate, the C16/C4 activity ratio being 6-15 for the different mitochondria studied. The other enzyme had a molecular weight of 71 000 and showed highest activity with C4 substrate (C16/C4 activity ratio of 0.2). The substrate specificity of the membrane-associated enzyme was not altered by extraction with detergent or lipid solvents. The membrane-associated enzyme could be partially purified by treatment with high concentrations of KCl and EDTA, when it showed a molecular weight of 186 000 while retaining its high C16/C4 activity ratio. Protease treatment caused loss of C4 activity from the high-molecular-weight enzyme without affecting its C16 activity. We conclude that an inner membrane-associated, long-chain-specific 3-ketoacyl-CoA dehydrogenase coexists in mitochondria with the better-documented matrix enzyme which shows preference for shorter-chain-length substrates.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1982

Inhibition of hepatic S-3-hydroxy-3-methylglutaryl-CoA reductase and in vivo rates of lipogenesis by a mixture of pure cyclic monoterpenes.

A proprietary mixture of pure cyclic monoterpenes (Rowachol) inhibited hepatic HMGCoA reductase by 50-60% when measured 17 hr after the oral administration of a single dose to rats. The extent of this inhibition was independent of the normal activity range of HMGCoA reductase within its diurnal cycle and the same inhibition (65%) was found in 24 hr starved animals where the control reductase activity was less than 20% that of normal fed rats. De novo sterol and fatty acid synthesis in intact, fed rats was measured by incorporation of 3H from injected H2O. In rats treated with Rowachol the rate of sterol synthesis in vivo was inhibited 52% in liver and 44% in testis with no significant effects in other tissues. The synthesis of non sterol (isoprenoid) compounds in testis was unaffected and the inhibition of sterol synthesis in this tissue probably reflects decreased acquisition of newly synthesized material from liver rather than any effect on the endogenous process. In the same animals the rate of fatty acid synthesis was inhibited 55% in liver. These effects were associated with a significant depletion of liver glycogen which may account for the reduction in rate of fatty acid synthesis. We conclude that the reported cholelitholytic action of monoterpenes is associated with the physiological inhibition of hepatic sterol synthesis mediated by decrease HMGCoA reductase activity.

Animals↗
PubMed1982

The mechanism of cyclic monoterpene inhibition of hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase in vivo in the rat.

Seventeen hours after a single oral dose of the cyclic monoterpenes cineole or menthol, rat liver 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity was inhibited by up to 70%. The transient nature of this effect (no inhibition 41 h after dosing) was compatible with the rapid metabolism and excretion of these terpenes. Neither menthol, and its major metabolite, menthylglucuronide, nor cineole acted as direct inhibitors of HMG-CoA reductase activity in vitro, although menthol was found to bind to liver microsomes Ks approximately 0.1 mM). Unlike the short term effects of dietary cholesterol, terpene administration did not affect HMG-CoA reductase activity by modulation of the lipid microenvironment of the enzyme. Thus, following menthol or cineole treatment, we found no deviations from the normal kinetic responses to changes in temperature or in concentration of HMG-CoA. Furthermore, the inhibitory effect was still seen after solubilization of the enzyme from microsomes. The loss of HMG-CoA reductase activity was not associated with increased phosphorylation of the enzyme. Immunotitration of HMG-CoA reductase from terpene-treated rats showed that activity loss was due to less enzyme molecules (together with some possibly "cripple" enzyme), indicating that rates of enzyme synthesis or degradation had been altered. Since menthol inhibition of reductase was still observed in rats deprived of foods, we conclude that the effect is not mediated by those hormones whose concentration is changed during fasting (insulin, glucagon, and adrenaline).

Animals↗