Michael B. Cooper

Fasting and postprandial determinants for the occurrence of small dense LDL species in non-insulin-dependent diabetic patients with and without hypertriglyceridaemia: the involvement of insulin, insulin precursor species and insulin resistance

We have studied low density lipoprotein (LDL) subclass distribution in a group of male patients with non-insulin-dependent diabetes mellitus (NIDDM) and investigated its relationships to fasting and postprandial triglyceride (TG)-rich lipoproteins, insulin resistance, lipoprotein lipase (EC 3.1.1.3; LPL), hepatic lipase (EC 3.1.1.34; HL), lecithin:cholesterol acyl transferase (EC 2.3.1.43; LCAT) and cholesteryl ester transfer protein (CETP) activities. LDL was subfractionated by density gradient ultracentrifugation. Postprandial lipoproteins were measured after an oral fat load using retinyl palmitate as a marker for intestinal TG-rich lipoproteins. Hypertriglyceridaemic NIDDMs (HTG) had a preponderance of small dense LDL particles present in the plasma and reduced amounts of large buoyant species when compared to normotriglyceridaemic patients (NTG) and controls. Both groups of diabetics were more insulin resistant than the controls (P < 0.05) and had raised concentrations of proinsulin (P < 0.05), although insulin content did not differ significantly. 32-33 split proinsulin (SPI) was the major insulin-like molecule present in HTG and was present in significantly higher amounts in these patients (P < 0.05) than either NTG or control subjects and correlated significantly with the presence of small dense LDL particles. After a test meal, the postprandial chylomicron response was greater in HTG than either NTG diabetics or controls (P < 0.05). Chylomicron remnants were present to a greater extent in HTG than in NTG and controls (P < 0.05), although in this case NTG also contained more chylomicron remnants than control subjects (P < 0.05). There was no difference in the LPL activity, CETP and LCAT between diabetics and controls, whereas an increase in hepatic lipase activity was seen in the HTG diabetics (P < 0.05). Both CETP and LCAT activities increased postprandially. Multivariate analysis showed that TG, HDL content and HL activity were the most important determinants of small dense LDL concentration in the fasting state (R2 = 67%). Postprandially, chylomicron remnant clearance, HL and insulin resistance were the major determinants (R2 = 61%) of LDL-III.

The effect of superoxide generation on the ability of mitochondria to take up and retain Ca2

When heart or liver mitochondria are exposed to superoxide radicals generated from xanthine + xanthine oxidase their ability to take up and to retain Ca2+ is impaired. The rate of oxidation of pyruvate + malate as substrates is diminished and the appearance of thiol groups when the mitochondria are supplied with these substrates is abolished. These inhibitory effects are offset if respiration is supported by succinate in presence of rotenone provided that a substrate (β‐hydroxybutyrate) is provided to maintain the reduction of NADH. The data agree with the thesis that a generation of thiol groups is essential to maintain membrane integrity and that the generation depends on provision of reduced NAD(P)H.

Antioxidant enzyme activities in heart and skeletal muscle of rats of different ages

The activity of antioxidant enzymes was measured in cardiac and skeletal muscle in rats aged either 4, 15, or 27 months. Generally, regardless of age, heart contains a greater content of these enzymes than skeletal muscle. Whereas skeletal muscle showed age-dependent increases in glutathione peroxidase, glutathione reductase, and catalase activities, heart tissue showed increases in only the glutathione peroxidase activity. Neither tissue showed any significant age-dependent change in cytosolic or mitochondrial superoxide dismutase content or in cytochrome oxidase.

Carnitine and acetylcarnitine in red blood cells

Carnitine and acetylcarnitine were found to be present in human erythrocytes. Their presence was not as a factor of leucocyte contamination. Carnitine is present within the erythrocyte at a level comparable to that of the plasma, whilst acetylcarnitine is more concentrated within the cell. Red blood cell carnitine and acetylcarnitine do not freely exchange with plasma but intra-erythrocyte acetylcarnitine has a significant relationship to the plasma levels.

The effect of age on the activity of enzymes of peroxide metabolism in rat brain

The activity of some enzymes associated with peroxide metabolism and cytochrome oxidase activity was measured in cortex, striatum, hypothalamus, and hippocampus from brains of rats aged either 4, 15, or 27 months. Cytochrome oxidase activity was greatest in the cortex, but no significant age-related changes in the activity of cytochrome oxidase, superoxide dismutase, or glutathione peroxidase were found in any of the brain areas. In contrast, glutathione reductase activity increased as a function of age in all regions. In general, the activity of catalase fell on maturation of the animal to adulthood and then showed a trend to increase with age.

Calcium and magnesium ion losses in response to stimulants of efflux applied to heart, liver and kidney mitochondria

Abstract The efflux of Ca 2+ from previously Ca 2+ -loaded heart, liver or kidney mitochondria is accompanied by an approximately equal loss of endogenous Mg 2+ irrespective of the agent applied to stimulate efflux which may be Na + , a mercurial, a long chain fatty derivative or thyroxin. The proportion of Mg 2+ (and of the accompanying adenine nucleotide) in relation to the Ca 2+ is diminished if Mg 2+ is added to the medium. The similarity between data from different mitochondria and with different agents accords with the ion losses involving a common factor such as generation within the membrane of lysophospholipid by Ca 2+ in transit. It is shown that lysophospholecithin stimulates Ca 2+ efflux with a hyperbolic concentration dependence.

Inhibition of Ca2+ stimulated ion losses from mitochondria by inhibitors of calmodulin.

The efflux of Ca 2+ from heart liver and kidney mitochondria previously loaded with Ca 2+ , has been shown to be accompanied by correlated losses of other components such as Mg 2+ and adenine nucleotides. The presence of the inhibitors of calmodulin, trifluoroperazine and dinucaine (probably acting by inhibiting Ca 2+ stimulation of endogenous phospholipase A 2 ), is now shown not only to slow efflux of Ca 2+ ; but also to inhibit losses of Mg 2+ and adenine nucleotides. In the presence of the calmodulin inhibitors, the efflux of Ca 2+ tends to resemble the efflux of Sr 2+ from Sr 2+ loaded mitochondria. Since Sr 2+ does not activate phospholipase A 2 or calmodulin, all 3 results point to the involvement of calmodulin in the Ca 2+ mediated increase of mitochondrial permeability.

Citrate interference with the determination of acetylcarnitine: a method for its elimination.

Citrate can interfere with the determination of acetylcarnitine when measured by a radioisotopic method. In this report we present a modified procedure designed to avoid this effect. Recovery of added acetylcarnitine was approximately 95%. Acetylcarnitine in normal human plasma was found to range from 0.8 to 19.9 nmol/ml with a mean of 6.3; that of human skeletal muscle ranged from 0.1 to 1.6 nmol/mg protein with a mean of 1.08. The results confirm that exercise gives rise to increased acetylcarnitine content of plasma.

Cytochrome P-450 associated with free polysome fractions

It has been suggested that constitutive membrane proteins are synthesised on rough endoplasmic reticulum with subsequent translocation, where appropriate, to smooth membranes, [l-4]. However, alternative models may be possible and indeed evidence has been presented suggesting that some membrane proteins may be inserted from a cytoplasmic pool [5-S]. A recent report [9] described the presence of cytochrome P-4.50, a component of the microsomal mixed function oxidase both of completed molecules and in nascent forms, in the free polysome fractions derived from rabbit liver. These molecules were clsimed to be associated with a class of free heavy polysomes. We have re-evaluated the experimental basis of this claim, working mainly with rat liver. We conclude that cytochrome P-450 in fractions of free hepatic polysomes is associated with membranous contamination and is not present on the ribosomes.

The biosynthesis of cytochrome P450 by rough endoplasmic reticulum in vitro: A significant proportion of newly-biosynthesised cytochrome P450 is resistant to proteolytic digestion in intact vesicles

Membrane proteins synthesised on polysomes of the rough endoplasmic reticulum are thought to be directly inserted into the membrane at the site of synthesis [l-6]. However, we have recently demonstrated that a part of the newly-biosynthesised cytochrome P4.50, a major membrane protein, can be found inside vesicles derived from the rough endoplasmic reticulum after protein synthesis in vitro [7,8]. Newly-biosynthesised cytochrome P450 has also been located inside vesciles derived from rough and smooth endoplasmic reticulum after incorporation of radiolabelled amino acids in situ by perfusion of the liver [8]. In these studies intravesicular content is defined as the material released from reticular fractions by treatment with a low concentration of deoxycholate (0.05% w/v). Under the experimental conditions employed in these studies this level of detergent does not solubilise any of the spectrallydetectable cytochrome P450 [7-91. However, the possibility cannot be entirely eliminated that the newly-biosynthesised intravesicular cytochrome P450 is a minor component which becomes highly radiolabelled during protein synthesis and is released from the membrane by low concentrations of deoxycholate. Intact microsomal vesicles are impermeable to macromolecules and thus treatment with proteases affects only those susceptible bonds which are present in proteins exposed on the outer membrane surface [lo]. Treatment of vesicles with deoxycholate (0.05%