Anthony Heape

Phospholipid and fatty acid composition of erythrocytes in type I and type II diabetes.

Different data have been reported concerning modifications of the erythrocyte lipid composition in the different types of diabetes. The heterogeneity of diabetes could be a cause for such differences. Ten type I and ten type II diabetics were carefully selected. The patients were poorly controlled (the mean glycosylated hemoglobin was 12.8% +/- 0.7%); their mean age was 54 +/- 5 years, with a mean duration of diabetes of 18 +/- 4 years. One half of them had severe diabetic complications (nephropathy, retinopathy, and/or polyneuropathy). The diabetics were compared with ten controls. The phospholipid composition was determined by HPTLC analysis, and the fatty acid moieties of the total phospholipids were measured by gas liquid chromatography associated with mass spectrometry. Under well-defined experimental conditions, these results demonstrated a slight, but significant (P less than .05), increase in the phosphatidylethanolamine (PE)/phosphatidylserine (PS) ratio using a Ninhydrin quantitation method; there was also an increase in two minor lipids content (phosphatidylinositol, phosphatidic acid) and the appearance of a lysolipid (lysoPE) in the patients. Whatever the type of diabetes, the red blood cells of diabetics showed no significant differences in their fatty acid contents.

Decrease of lipid extractability of chloroform-methanol upon water addition to human erythrocytes☆

The yield of lipids extractable by chloroform-methanol 2:1 from human erythrocytes decreases as a function of the relative amount of water added to--or present in--the erythrocyte pellet prior to the lipid extraction. Only slight modifications are observed as long as the volume of water does not exceed that of the red blood cell pellet. As the volume of added water increases, the phosphatidylserine recovery drops dramatically and tends to zero while the yield of the other phospholipids remains unchanged. This phenomenon is not observed when the lipids are extracted by a mixture of isopropanol-chloroform.

A developmental study of fatty acyl group contents in the periphéral nervous system of normal and trembler mice

We quantitated the content of the fatty acyl groups of sciatic nerves from normal and Trembler mice between the postnatal ages of 5 and 60 d. Palmitoyl, stearoyl, and unsaturated 18-carbon groups increased normally in Trembler nerves during the first 9 d, after which their levels were notably lower than those observed for the normal littermates. In good agreement, the synthesis of palmitic acid by the fatty acid synthetase was normal in the PNS of 9-d-old Trembler mice. Myelin-specific saturated very long chain fatty acyl groups (VLCFAG) were deficient at all ages studied, the deficiency increasing from about 2.5-fold at 5 d, to 26-fold at 60 d, compared to the maximal 3- to 4-fold decrease observed for the more ubiquitous shorter chains. The results presented in this paper suggest that the highly abnormal VLCFAG content of the mutant nerves cannot be accounted for by an abnormal fatty acid synthetase activity. For the fatty acyl chains with 18 carbon atoms, however, the deficiency observed after 9 d of age can be almost entirely explained by the diminished levels of palmitic acid. The relatively late occurrence of the palmitic acid deficiency compared with that of the VLCFAG and the normal palmitate synthetase activity observed in young mutant mice indicates that the former is an indirect consequence of the Trembler mutation.

Increased acylation of lysophosphatidylcholine in microsomes of trembler mouse peripheral nerves

We report here the presence of a Stearoyl-CoA: [1-palmitoyl]-glycerophosphorylcholine (LPC) transacylase activity in the microsomal fraction of normal and Trembler mouse sciatic nerves. Under the experimental conditions studied as a function of incubation time, protein concentration, acyl-CoA and LPC concentrations, the transacylase specific activity was 2-3 times higher in the microsomes of the mutants nerves than in those of the control. The addition of 5 mM ATP-Mg to the incubation medium, in the absence of bovine serum albumin, leads to a 90% decrease of the stearoyl-CoA thioesterase activity, but increases the transacylation by only 10-20%. This may be due to the low value (10 microM) of the apparent K(m) for C(18)-CoA observed for the mutants transacylase. In microsomes from control nerves, transacylation requires exogenous LPC, whereas in Trembler mouse sciatic nerve microsomes, the transacylase can use endogenous LPC.

High uptake and rapid metabolism of palmitate in peripheral nerves of normal and trembler mice in vivo: similarities and differences

In vivo, short-term palmitate metabolism was studied in normal and Trembler mouse sciatic nerves, after intraneural injection of 6.34 pmol of [(3)H]palmitate. After 30 min of in vivo incubation, all the [(3)H]palmitate available for short-term lipid metabolism (90% of the injected substrate) had already been used up in both normal and Trembler sciatic nerves. Despite the normal metabolic level of the [(3)H]palmitate, the distribution of the label between the various polar lipids was abnormal in the mutants nerves: sphingomyelin and phosphatidylcholine were more labelled than in normal nerves, while the other lipids, and particularly the cerebrosides, were less labelled. The labelled fatty acid distribution in four polar lipids, purified by thin-layer chromatography, was also abnormal, correlating with a decreased overall [(3)H]palmitate elongation (after 2 h, normal = 44%, Trembler = 24%), and a severely decreased synthesis (5- to 10-fold) of saturated very long-chain fatty acids. In normal nerves, saturated very long-chain fatty acids represented about 8% of the total labelled fatty acids and they were preferentially incorporated into the cerebrosides. The rapid depletion of the [(3)H]palmitate, the high level of its incorporation into polar lipids of both normal and Trembler mouse sciatic nerves and the excellent reproducibility of the results, have led us to propose a novel pulsed wave method for the in vivo study of intracellular lipid transport phenomena implicated in peripheral nerve myelin assembly.

Po, MBP, histone, and DNA levels in sciatic nerve

We studied the quantitative changes in proteins (total, Po, MBP, and histones) and DNA from sciatic nerves of normal and Trembler mice during postnatal development. Polyacrylamide gel electrophoresis and immunoblotting procedures allowed an accurate characterization of Po, MBP, and histones, as well as the comparison of their respective amounts from d 2 to d 120 after birth. It was found that 1. The immunoblotting procedure ascertains the presence of Po in the sciatic nerve of Trembler. In the 2-d-old mice, Po is detected in essentially similar amounts in Trembler and normal PNS, whereas its level in adult mutant sciatic nerves is never greater than 20% of the control. The sharp increase in Po levels observed during the third week in the normal nerves is not observed in those of the mutant; 2. MBP species are at most 4% of the control in the 10- to 12-d-old Trembler mice, whereas they were not detectable in adult nerves. The distribution of the different MBP species is the same in both mutant and control mice; 3. In normal mice, Po and MBP accumulate at similar rates, but the 14 kDa MBP accumulates faster than the 18.5 kDa MBP; and 4. Histone and DNA contents decrease 3- to 5-fold in normal nerves, whereas they remain constant, or increase slightly, in the mutant.

Technique for injection into the sciatic nerve of the mouse for quantitative in vivo metabolic studies

In this paper we describe a technique for intraneural injections, applicable to mouse peripheral nerves, which, compared with previous techniques, reduces trauma to the nerves and increases the level and reduces the variability of label recovery. Our technique employs glass needles (tip diameter, 50 micron) linked to a peristaltic pump by polyethylene tubing to inject small volumes (in the microliter range) of radiolabeled substrate solutions into mouse sciatic nerves, and allows the recovery of 20.9 +/- 1.9% (mean +/- standard deviation) and 30.5 +/- 4.8% of the injected radioactivity for 2 microliter [3H]acetate and 0.5 microliter of [3H]stearate, respectively.

A comparison of substrate-dependent fatty acyl-CoA synthetase activities and ATP hydrolysis in membrane pellets from normal and trembler mouse sciatic nerves.

Acyl-CoA synthetase activity was investigated in membrane fractions from normal and Trembler mouse sciatic nerves. Stearoyl-CoA synthesis in the Trembler assays was always about twice that observed in the control experiments under various conditions of CoA and ATP concentrations. In experiments using varying concentrations of fatty acid, the activation of stearate was very low in both systems (normal and Trembler), up to a substrate concentration of 20 mM, suggesting that fatty acid micelles are required for full activity. ATP, at concentrations of up to 1 mM, was totally (Trembler) or partially (control) hydrolyzed within 15 min, resulting in an apparently lower ligase activity in the mutant than in the control when an ATP-regenerating system was not added. The activation of saturated fatty acids decreased with increasing chain length. However, lignoceroyl-CoA formation was detected in the mutant but not in the control.

Evidence for an Intermembrane Transport of Lipids to Peripheral Nerve Myelin in vivo by a Novel Pulsed Wave Procedure

Three principal methods have been employed for the study of intracellular transport in nervous tissues: autoradiography associated with electron microscopy, time-staggered double-isotope labelling and isotopic pulse-chase experiments. Although the autoradiographical (Gould and Dawson, 1976; Gould et al., 1987; Kumara-Siri and Gould, 1980; Rawlins, 1973) and the double-labelling methods (Konat et al., 1985; Pereyra et al., 1983; Shimomura et al., 1984) can provide useful complementary information, only the isotopic pulse-chase experiments (Benjamins and Iwata, 1979; Burkart et al., 1982; Konat, 1981; Konat et al., 1985), associated with the use of intracellular transport inhibitors, would be able to demonstrate unambiguously the existence of intermembrane transfer events implicated in membrane biogenesis. No in vivo studies of peripheral nerves have been carried out using this method, most probably because of the difficulty in controlling the experimental conditions.