Characterization of sulphatide-containing lipoproteins in rat brain

—(1) Water-soluble [³⁵S]sulphatide is found in the 105,000 g supernatant (SN) of rat brain after intraperitoneal injection of Na₂³⁵SO₄. This labelled sulphatide has a density between those of free lipid and free protein. (2) Fractionation of SN by preparative acrylamide gel electrophoresis indicates that the [³⁵S]sulphatide is not distributed among all SN proteins, but is associated with certain specific proteins. One of the isolated [³⁵S]sulphatide-containing proteins appears homogeneous by analytical acrylamide gel electrophoresis at several pH values. (3) Comparison of the turnover of [³⁵S]sulphatide in microsomes, SN, and myelin indicates that these three subcellular fractions behave as distinct metabolic pools, which meet the requirements for a precursor-product relationship between microsomes and SN and between SN and myelin. (4) These results suggest that sulphatide, synthesized in the microsomes, is transported to the myelin membrane as water-soluble sulphatide containing Iipoproteins in SN.     

Studies of water soluble lipoproteins in rat brain

—The occurrencc of water soluble lipoprotein in the 105,000 g supernatant of rat brain homogenate has been demonstrated. The lipoproteins of brain differed from those of the serum with respect to lipid composition, density and reaction to serum lipoprotein antibodies. Acrylamide gel electrophoresis resolved the brain supernatant into four zones which stained with lipid dye. It was possible to demonstrate that these zones also contained protein by double staining of the gel for lipid and protein. Two of the zones differed in their mobility from serum lipoprotein. Preparative acrylamide gel electrophoresis isolated protein fractions in supematant which contained both lipid and protein and permitted preliminary identification of the lipoprotein bands amongst all the protein bands resolved by acrylamide gel electrophoresis.     

Synthesis of thiamine triphosphate in rat brain in vivo

Thiamine metabolism in vivo was studied by intracerebroventricular injection of labeled thiamine in rat brain. Labeled thiamine was found to be rapidly converted to the phosphorylated thiamine esters. The distribution of the radioactive thiamine compounds was reached to steady state at 3 hr after injection: thiamine, thiamine monophosphate, thiamine pyrophosphate, and thiamine triphosphate were 8–12%, 12–14%, 72–74%, and 2–3%, respectively, in cerebral cortex. The presence of labeled thiamine triphosphate in the brain was further confirmed by the treatment with thiamine triphosphatase which had an absolute substrate specificity for thiamine triphosphate. These results suggest that thiamine triphosphate is synthesized in vivo in rat brain.     

Synthesis of glutamate and aspartate in rat brain synaptosomes

ATP and glutamine are the sources of endogenous ammonia in rat brain synaptosomes. The amount of endogenous ammonia formed from exogenous ATP is not sufficient to assure the maximum rate of aspartate and glutamate accumulation in the synaptosomes utilizing pyruvate + malate. Addition of exogenous NH4+ or depolarization of synaptosome plasma membranes with high K+ concentration led to a twofold increase in the rate of accumulation of these amino acids. This indicates that both exogenous and endogenous NH4+ is involved in the synthesis of aspartate and glutamate in nerve terminals. Accumulation of glutamate was stimulated by aminooxyacetate and inhibited by haloperidol which indicates that NH4+ is bound in the reaction catalysed by glutamate dehydrogenase. Endogenous oxaloacetate derived from pyruvate metabolism was the substrate for synthesis of aspartate. Additive inhibition of aspartate accumulation by fluorocitrate and (-) hydroxyacetate shows that, in addition to the tricarboxylic acid cycle, the reaction catalysed by ATP-citrate lyase serves in the synaptosomes as another source of oxaloacetate.     

Synthesis of taurine in rat liver and brain in vivo

The in vivo formation of taurine and the analysis of labeled taurine precursors was examined in rat brain and liver at different times after an intracisternal injection of [³⁵S]cysteine and an intraperitoneal injection of [³H]cysteine, simultaneously administered. The distribution pattern of radioactivity was similar in liver and brain. Most of the labeling in both organs (85% in brain and 80% in liver) was recovered in glutathione (oxidized and reduced), cysteic acid, cysteine sulfinic acid, hypotaurine, cystathionine, and a mixed disulfide of cysteine and glutathione. The relative rates of labeling of cysteine sulfinic acid and taurine in liver and brain suggest than in vivo, liver possesses a higher capacity for taurine synthesis than brain. A small amount of [³H]taurine was detected in brain after intraperitoneal injection of [³H]cysteine. The time of appearance of this [³H]taurine as well as the fact that it occurs when [³H]cysteine is not detectable in brain or plasma suggests that it was probably not synthesized in brain from labeled precursors but formed elsewhere and transported into the brain through an exchange process.     

Disc electrophoresis of rat plasma lipoproteins

The disc electrophoresis of lipoproteins of unfractionated rat plasma is described. The plasma was prestained with Sudan black B and electrophoretically separated at polyacrylamide gel concentrations of 7.5, 5, and 3.75%. At least four lipoprotein components were observed, and an additional 2-3 components in the main gel and 2-5 components in the spacer gel possibly were present. Densitometry of the resolved gel patterns indicated good reproducibility. Thin-layer chromatography of lipids extracted from the Sudan black B-binding components confirmed the lipoprotein nature of these components of rat plasma. A comparison of the disc electrophoretic patterns of human serum and rat plasma suggested that the low-density lipoprotein components of rat plasma are smaller in size than those of human serum.     

Distribution of oleoyl-estrone in rat plasma lipoproteins.

Pooled adult normal rat plasma was used for the separation of lipoprotein fractions: VLDL, LDL and HDL, from which a total lipids extract was obtained. The presence of fragments with the MW of estrone and oleoyl-estrone in the lipoprotein fractions was analyzed by HPLC-MS. The results show that oleoyl-estrone is the major estrone component in lipoproteins; this molecular species was present in all three lipoprotein lipid extracts. The lipoprotein fractions were used for the analysis of protein and lipid classes: triacylglycerols, total and esterified cholesterol and phospholipids as well as acyl-estrone. About half of the total acyl-estrone was in the HDL fraction and only about 10% in the VLDL fraction. HDLs contained about one molecule in 50 particles, LDLs one molecule per particle and VLDLs 15 molecules per particle, i.e. given their size, the larger lipoproteins contained more oleoyl-estrone than the HDLs. The distribution of this hormone suggests that oleoyl-estrone is lost with other lipids as the lipoproteins shrink. The results presented show that oleoyl-estrone is a molecule found naturally in rat lipoproteins in low concentrations - the lowest in HDLs - that are consistent with its postulated role in the control of body weight.     

Synthesis and binding of 3H-oxymorphazone to rat brain membranes

Oxymorphazone is a 14-hydroxydihydromorphinone derivative which contains a C-6 hydrazone group and hence could serve as an irreversible label for opioid receptors. 3H-oxymorphazone was synthesized by the reaction of 3H-oxymorphone with excess hydrazine. A specific radioactivity of 640 GBq/mmol (17,3 Ci/mmol) was achieved. Both the unlabelled compound and the tritiated ligand show high affinity to mu and kappa opiate receptor subtypes in rat brain membranes. Two binding sites were detected by equilibrium binding studies, with apparent Kd values of 0.62 nM and 28 nM. About 20% of the H-oxymorphazone specific binding is irreversible after reaction at 1 nM ligand concentration, and this can be enhanced by a higher concentration of tritiated ligand. No azine formation was detected. Preincubation of the membranes with unlabelled oxymorphazone resulted in an irreversible blockade of the high affinity 3H-naloxone binding sites.     

Effect of portacaval anastomosis on rat lipoproteins

The distribution of cholesterol (C), triglycerides (TG), phospholipids (PL) and protein in the different lipoproteins was studied in male Wistar rats under 2 conditions: control and 2 months after portacaval anastomosis (PCA). PCA decreased the levels of cholesterol and the other components in chylomicrons (-90%), very low density lipoproteins (-65 to -78%), LDL2 (1.040 less than d less than 1.063 g/ml; -51 to -61%) and HDL (1.063 less than d less than 1.21 g/ml), whereas no change was observed in LDL1 (1.006 less than d less than 1.040 g/ml). Apoprotein C contents were decreased in all lipoproteins. The relative proportions of C, TG, PL and proteins in lipoproteins were essentially unchanged by the shunt, suggesting a reduced number of lipoprotein particles in plasma after PCA. It was concluded that PCA reduced the levels of all lipoproteins secreted by liver and/or the intestine without modifying those of intraplasmatic origin (LDL1).     

Interactions between model triacylglycerol-rich lipoproteins and high-density lipoproteins in rat, rabbit and man

There are inverse relationships between HDL cholesterol and plasma triacylglycerol concentrations in normal and in hypertriglyceridemic individuals. To investigate the interactions between triacylglycerol-rich lipid particles and HDL, a lipid emulsion model of the triacylglycerol-rich lipoproteins was prepared. When emulsion particles were incubated with rat high-density lipoproteins (HDL) in the presence of lipid transfer activity (d greater than 1.21 g/ml fractions) from rabbit or human plasma there was a rapid bi-directional exchange of cholesteryl oleate (CO) and phospholipid (PL) labels between lighter and heavier fractions of HDL and emulsion particles. The transfers of CO and PL labels between both light and heavy fractions of HDL and the emulsion particles were increased with increasing amounts of emulsion added to the incubations. Incubation with the d greater than 1.21 g/ml fraction from rat plasma resulted in only a small exchange of CO whereas PL exchange was similar to rabbit and human plasma. Retinyl palmitate label was not transferred from emulsion particles to the HDL fractions even in the presence of lipid transfer activity from rabbit or human plasma. The present study shows that the transfer protein-mediated exchanges of surface and core lipids between HDL and the triacylglycerol-rich lipoproteins are affected by the quantity of triacylglycerol-rich particles in the system. This mechanism may contribute to the inverse relationships between plasma triacylglycerol concentrations and HDL concentrations in normal and hypertriglyceridemic individuals.     

Agarose--starch gel electrophoresis of rat serum lipoproteins

Rat serum lipoproteins were separated into at least four fractions by agarose-starch gel electrophoresis. The system used was discontinuous in that glycine and sodium barbitone buffer was used in the reservoirs and Tris buffer was used for the gels. The four major bands could be related to the pattern obtained by ultracentrifugation. The high density lipoproteins consisted of at least two poorly resolved bands and were not separated from albumin. The vertical gel apparatus was further modified to accept 0.4 ml of rat plasma, which was prestained with Sudan black. After electrophoresis the different lipoprotein bands could conveniently be cut out and the lipid phosphorus determined. The addition of Sudan black B decreased the recovery of the low and high density lipoproteins by 5-9%. However, the recovery of phospholipids was reproducible (80 +/- 2%) and the high density lipoproteins contained over two-thirds of the plasma lipid phosphorus.