Search PubMed⌕ Search

Biomedical subjects

B Middleton

Publications and source records attributed to B Middleton.

At least 109 records · Page 6Linked to original sources
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↗
PubMed1982

Studies on a stable, mild diabetes induced by streptozotocin in rats.

A stable, mild diabetes in rats maintained on a normal laboratory diet was induced by a single i.p. injection of streptozotocin. Irreversible damage of the pancreatic islet B cells was complete by 3 days after treatment and plasma immunoreactive insulin was undetectable throughout the remaining 12-week period of investigation. The diabetes was characterized by hyperglycaemia of over 30 mM and a constant elevation of plasma alanine and branch-chain amino acids throughout the 12 weeks. In contrast to severe diabetes, plasma free fatty acids rose only gradually from normal values to reach 1.5 mM by week 12, ketone bodies were only slightly elevated (0.7 mM maximum) and liver glycogen was maintained throughout at around 30% of the normal, fed value. Starvation for up to 40 h caused only slight changes (in contrast with non-diabetic animals) and in particular no changes in free fatty acid or ketone bodies were found. These metabolic results are discussed in relation to the mechanisms thought to control those processes.

Amino Acids↗
PubMed1981

The control of sterol synthesis during development of rabbit mammary gland.

A 9-fold increase in the rate of conversion of [14C]-acetate to nonsaponifiable lipids was seen on culturing explants of pregnant rabbit mammary gland in the presence of insulin, prolactin, and cortisol. Stimulation of [3H]mevalonate into nonsaponifiable lipid was 3-fold under the same conditions. With both substrates activities were maximal after 24 h in culture. Incorporation of acetate into saponifiable lipid was stimulated 20-fold after 24 h and almost 50-fold after 48 h in the presence of hormones. Radioactivity from [3H]mevalonate was incorporated into saponifiable lipid after 48 h in culture with hormones suggesting an active trans-methyl-glutaconyl shunt at this time. Changes in the rate of acetate conversion to nonsaponifiable lipid correlated well with the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) in the glands of mid-pregnant and mid-lactating rabbits. Enzyme activity rose from 3.3 pmol/min/mg of 100,000 X g pellet protein in the mid-pregnant gland to an apparent value of 15.6 pmol/min/mg of 100,000 X g pellet protein in the lactating gland. When corrected for the very high content of milk protein in this 100,000 X g pellet, the HMG-CoA reductase activity was 36-fold greater in lactating compared to pregnant mammary gland. Hepatic reductase in both pregnant and lactating animals was approximately 25 pmol/min/mg of microsomal protein. This increase in capacity for sterol synthesis in mammary gland during the transition from pregnancy to lactation suggests that this tissue could synthesize a major proportion of milk sterol.

Acetates↗
PubMed1980

Elevation of serum high density lipoprotein cholesterol by rowachol, a proprietary mixture of six pure monoterpenes.

Rowachol, a proprietary choleretic containing 6 pure monoterpenes markedly elevates serum HDL cholesterol (SHDL-C) concentrations in man. The concentration of SHDL-C showed a progressive increase in 16 patients treated with 6-9 capsules of Rowachol daily for periods of 2-28 weeks. There was no accompanying significant change in the concentrations of serum total cholesterol or triglyceride. In view of the significant inverse relationship between SHDL-C concentration and the risk of developing ischaemic heart disease, it is suggested that Rowachol and possibly other terpenes merit further investigation as possible therapeutic agents in the prevention and treatment of atheroma.

Adolescent↗
PubMed1977

Elimination of glucose interference and improved precision in a continuous-flow analysis for inulin.

A modified fermentation procedure based on one described by Homer Smith in his textbook was used to eliminate glucose interference in the automated detrmination of inulin. The manifold of Fjeldbo and Stamey [J. Lab. Clin. Med. 72, 353 (1968)] was modified to enable us to insert the fermentation procedure without sacrificing precision in continuous-flow analysis for inulin. Analytical recoveries of inulin from plasma and urine with the modified manifold were 100.1 +/- 0.8% (SD) and 99.9 +/- 1.6%. Corresponding recoveries of inulin from specimens of plasma and urine containing 5 g of glucose per liter and treated with yeast were 99.6 +/- 1.5% and 100.0 +/- 1.6%.

Blood Glucose↗
PubMed1974

An enzyme-bound intermediate in the biosynthesis of 3-hydroxy-3-methylglutaryl-coenzyme A.

1. Purified 3-hydroxy-3-methylglutaryl-CoA synthase from baker's yeast (free from acetoacetyl-CoA thiolase activity) catalysed an exchange of acetyl moiety between 3'-dephospho-CoA and CoA. The exchange rate was comparable with the overall velocity of synthesis of 3-hydroxy-3-methylglutaryl-CoA. 2. Acetyl-CoA reacted with the synthase, giving a rapid ;burst' release of CoA proportional in amount to the quantity of enzyme present. The ;burst' of CoA was released from acetyl-CoA, propionyl-CoA and succinyl-CoA (3-carboxypropionyl-CoA) but not from acetoacetyl-CoA, hexanoyl-CoA, dl-3-hydroxy-3-methylglutaryl-CoA, or other derivatives of glutaryl-CoA. 3. Incubation of 3-hydroxy-3-methylglutaryl-CoA synthase with [1-(14)C]acetyl-CoA yielded protein-bound acetyl groups. The K(eq.) for the acetylation was 1.2 at pH7.0 and 4 degrees C. Acetyl-labelled synthase was isolated free from [1-(14)C]acetyl-CoA by rapid gel filtration at pH6.1. The [1-(14)C]acetyl group was removed from the protein by treatment with hydroxylamine, CoA or acetoacetyl-CoA but not by acid. When CoA or acetoacetyl-CoA was present the radioactive product was [1-(14)C]acetyl-CoA or 3-hydroxy-3-methyl-[(14)C]glutaryl-CoA respectively. 4. The isolated [1-(14)C]acetyl-enzyme was slowly hydrolysed at pH6.1 and 4 degrees C with a first-order rate constant of 0.005min(-1). This rate could be stimulated either by raising the pH to 7.0 or by the addition of desulpho-CoA. 5. These properties are interpreted in terms of a mechanism in which 3-hydroxy-3-methyl-glutaryl-CoA synthase is acetylated by acetyl-CoA to give a stable acetyl-enzyme, which then condenses with acetoacetyl-CoA yielding a covalent derivative between 3-hydroxy-3-methylglutaryl-CoA and the enzyme which is then rapidly hydrolysed to free enzyme and product.

Acetyl Coenzyme A↗
PubMed1974

The kinetic mechanism and properties of the cytoplasmic acetoacetyl-coenzyme A thiolase from rat liver.

1. Cytoplasmic acetoacetyl-CoA thiolase was highly purified in good yield from rat liver extracts. 2. Mg(2+) inhibits the rate of acetoacetyl-CoA thiolysis but not the rate of synthesis of acetoacetyl-CoA. Measurement of the velocity of thiolysis at varying Mg(2+) but fixed acetoacetyl-CoA concentrations gave evidence that the keto form of acetoacetyl-CoA is the true substrate. 3. Linear reciprocal plots of velocity of acetoacetyl-CoA synthesis against acetyl-CoA concentration in the presence or absence of desulpho-CoA (a competitive inhibitor) indicate that the kinetic mechanism is of the Ping Pong (Cleland, 1963) type involving an acetyl-enzyme covalent intermediate. In the presence of CoA the reciprocal plots are non-linear, becoming second order in acetyl-CoA (the Hill plot shows a slope of 1.7), but here this does not imply co-operative phenomena. 4. In the direction of acetoacetyl-CoA thiolysis CoA is a substrate inhibitor, competing with acetoacetyl-CoA, with a K(i) of 67mum. Linear reciprocal plots of initial velocity against concentration of mixtures of acetoacetyl-CoA plus CoA confirmed the Ping Pong mechanism for acetoacetyl-CoA thiolysis. This method of investigation also enabled the determination of all the kinetic constants without complication by substrate inhibition. When saturated with substrate the rate of acetoacetyl-CoA synthesis is 0.055 times the rate of acetoacetyl-CoA thiolysis. 5. Acetoacetyl-CoA thiolase was extremely susceptible to inhibition by an excess of iodoacetamide, but this inhibition was completely abolished after preincubation of the enzyme with a molar excess of acetoacetyl-CoA. This result was in keeping with the existence of an acetyl-enzyme. Acetyl-CoA, in whose presence the overall reaction could proceed, gave poor protection, presumably because of the continuous turnover of acetyl-enzyme in this case. 6. The kinetic mechanism of cytoplasmic thiolase is discussed in terms of its proposed role in steroid biosynthesis.

Acetyl-CoA C-Acetyltransferase↗