Christine C. Lee

Separation of three compositionally distinct subclasses of rat high density lipoproteins by heparin-affinity chromatography

The composition of 3 subclasses of plasma high density lipoproteins (HDL) separated by heparin-affinity chromatography was characterized. Plasma was obtained from Fischer-344 adult male rats fed a semi-purified diet containing 1% cholesterol. HDL particles were isolated by ultracentrifugation and agarose column chromatography. The purified HDL fraction was applied to a column (1.0 X 28 cm) packed with heparin-Sepharose CL-6B and eluted at 4 degrees C with 5 mM Tris buffer (pH 7.4) with varying concentrations of NaCl. The first peak (P1) eluted with 50 mM NaCl and 25 mM MnCl2 was albumin; the second peak (P2) eluted at 70 mM NaCl accounted for 78% of total plasma HDL-cholesterol (HDL-C) and 82% of total HDL protein. The particles of this HDL subclass measured 113 A in diameter and were devoid of apolipoprotein (apo) E, but high in apo A-I. The third peak (P3) eluted with 290 mM NaCl represented 4.3% of total HDL-C and 6.0% of total HDL protein, and contained apo E (25% of its protein). The average size of the particles was 126 A. The last peak (P4) eluted at 0.6 M NaCl accounted for 18% of total HDL-C and 12% of HDL protein. The particles of P4 were considerably larger in size (156 A) relative to those of P2 and P3, and rich in apo E (73% of its protein) with relatively low concentrations of apo A-I and C. Based on the compositional characteristics and sizes of the particles, the HDL subclasses of P2, P3 and P4 were designated as HDL2 with no apo E, HDL2 with moderate apo E, and HDL1 (or HDLc), respectively. The above results provide evidence for the existence of 3 compositionally distinct subclasses of plasma HDL in the rat, which may differ with regard to their roles in the transport and metabolism of cholesterol.

Effect of copper deficiency on the lymphatic absorption of cholesterol, plasma chylomicron clearance, and postheparin lipase activities

The lymphatic absorption of cholesterol and plasma clearance of chylomicrons were investigated in Cu-deficient rats (CuD) fed 0.5 mg Cu/kg diet, as compared with Cu-adequate control rats (CuA) fed 7.5 mg/kg diet. Cholesterol absorption was measured by the 14C-radioactivity appearing in the mesenteric lymph at hourly intervals for 8 hr after an intraduodenal dose of [14C]cholesterol. The plasma clearance of chylomicrons was measured at 3, 6, and 10 min after an intravenous dose of chylomicrons labeled in vivo with [3H]retinyl ester. Cumulative [14C]cholesterol absorption and total lymphatic output of cholesterol were significantly decreased in CuD at 4 hr and thereafter, with no change in percentage distribution of free and esterified cholesterol. Over an 8-hr period, 7.3% of the dose was absorbed by CuD and 9.2% by CuA. When [3H]chylomicrons, obtained from a CuD or CuA donor rat, were injected into CuD and CuA recipient rats, the label was cleared faster in CuD during the first 3 min. At 6 and 10 min, however, no significant difference in percentage clearance of the dose was observed between the groups. The half-life (t1/2) of [3H]chylomicrons and the total 3H-radioactivity taken up by the liver during the entire 10-min period did not differ between the groups, regardless of the source of chylomicrons. The activities of both endothelial lipoprotein lipase (LPL) and hepatic lipase (HL) in postheparin plasma were markedly lower in CuD. As expressed in micromoles fatty acid released/hr/ml plasma, the activities of LPL in CuD and CuA were 32.6 +/- 1.9 and 45.6 +/- 1.3, respectively. A similar magnitude of difference was also observed in HL activity. The data provide evidence that copper deficiency impairs the intestinal transport of cholesterol and the peripheral lipolysis of chylomicrons. The data, however, strongly suggest that the hepatic uptake of chylomicron remnants via the apo-E-dependent mechanism may not be impaired in Cu deficiency.

Effect of marginal zinc deficiency on the apolipoprotein-B content and size of mesenteric lymph chylomicrons in adult rats

To investigate the mechanisms underlining the impaired intestinal absorption of lipids in zinc deficiency, the apo-B content and chemical composition of chylomicrons from marginally zinc-deficient rats fed 2.8 ppm of dietary zinc (ZD) were compared with those from pair-fed (PF) and ad libitum control (CT) groups fed an adequate level (30.8 ppm) of zinc. Chylomicrons, obtained by cannulating the mesenteric lymph, were isolated by ultracentrifugation at 1.3×106 g/min at 12 C and purified by 2% agarose column chromatography. Apolipoprotein- (apo) B was separated by the method of isopropanol precipitation. The apo-B concentration of chylomicrons was lowered significantly in ZD group. The apo-B contents of chylomicrons in ZD, PF and CT rats, as expressed as % chylomicron protein, were 8.7±0.1, 11.5±0.5 and 10.7±0.7%, respectively. No significant differences were noted between ZD and PF groups in total protein (TP), phospholipid (PL), triglyceride (TG) and cholesterol (CH), although there was a slight decrease in TG and an increase in CH in CT rats compared with ZD and PF groups. The ratio of the core to surface constituents, as determined by TG/(TP+PL), was significantly higher in ZD group relative to the controls, suggesting that chylomicrons from ZD rats were larger. This finding was consistent with the appearance of larger chylomicron particles in the lacteal of the intestinal mucosa following lipid ingestion. These findings suggest that the intestinal synthesis of apo-B may be defective in zinc-deficient rats and may explain in part the impaired absorption of dietary lipids observed in zinc deficiency.

Effect of copper deficiency on the composition of three high-density lipoprotein subclasses as separated by heparin-affinity chromatography.

Copper deficiency in rats produces a hypercholesterolemia with a marked increase in HDL fraction. This study investigated changes in the plasma distribution and composition of HDL subclasses as affected by copper deficiency. Plasma HDL were separated into the following three subclasses by heparin-affinity chromatography: HDL containing no apo E but high in apo A-I (HDL-E0); HDL with an intermediate level of apo E (HDL-E1); and HDL highly enriched in apo E but low in apo A-I (HDL-E2). The compositional analysis showed that the hypercholesterolemia observed in copper-deficient rats was due specifically to an increase in plasma cholesterol carried by HDL-E0. Copper deficiency did not alter the percent distribution of apo A-I in HDL-E0, but lowered the apo A-I content in HDL-E1 and HDL-E2, with an increase in apo E in these subclasses. The total plasma concentration of apo A-I was, however, significantly elevated in Cu-deficient rats, which was attributable to an increase in the total number of circulating HDL particles. No difference was noted between Cu-deficient and control groups in the distribution of free cholesterol or the ratio of free cholesterol to esterified cholesterol in any of the HDL subclasses. The present results and earlier observations suggest that copper deficiency may produce a defect in the plasma clearance or tissue uptake of the HDL subclass high in apo A-I but devoid of apo E (HDL-E0), which may be mediated by the specific apo A-I receptor or non-endocytotic transfer of HDL-E0 cholesterol to the liver. Such metabolic defects may partly explain the simultaneous increases in both plasma HDL cholesterol and apo A-I and altered cholesterol homeostasis observed in copper deficiency.

Effect of marginal zinc deficiency on lipoprotein lipase activities in postheparin plasma, skeletal muscle and adipose tissues in the rat

The activities of lipoprotein lipase in postheparin plasma, retroperitoneal adipose and gastrocnemius muscle tissues were determined in the rats fed 2.8 ppm of dietary zinc for eight weeks, as compared with pair-fed and ad libitumfed rats given 30.8 ppm of zinc. The postheparin lipoprotein lipase activity, as determined by using a lipid emulsion labeled with [3H]triolein as the substrate, was significantly lower in the first group of rats, relative to that in the second and third groups. Tissue lipoprotein lipase activities were compared using the lipid emulsion and activator serum obtained from the zinc-deficient rats and the ad libitum-fed rats. The activator sera were devoid of very low density and low density lipoproteins, but enriched in high density lipoproteins. Muscle lipoprotein lipase activities were significantly lower when assayed with the activator serum from the zinc-deficient compared with the activities determined with the activator serum from the ad libitum-fed. Similarly, muscle lipoprotein lipase activities were lower in all groups when [3H]-triolein-labeled chylomicrons from the zinc-deficient were used as the substrate, compared with the activities determined using the chylomicrons from the ad libitum-fed. Lipoprotein lipase activities in the adipose tissues were not affected by the different sources of the activator sera and chylomicrons. The results strongly suggest that the decrease in postheparin lipoprotein lipase activity in zinc deficiency is not due to changes in tissue lipoprotein lipase enzyme per se, but to compositional alterations in with regard to C apolipoproteins, modulators of lipoprotein lipase activity.

Effect of copper deficiency on the plasma clearance of native and acetylated human low density lipoproteins

Abstract The rates of plasma clearance of human native low density lipoproteins (LDL) and acetylated human low density lipoproteins (acetyl-LDL) were compared between copper-deficient (CuD) and copper-adequate (CuA) rats. Purified human LDL (d 1.02–1.063) were labeled with 125I and injected to fasted recipient rats intravenously. At different time intervals plasma clearance of 125I radioactivity was measured. The percent of clearance was calculated based on the total plasma volume, as determined by a radioisotopic dilution method. Native human 125I-LDL were cleared at a faster rate in CuD, compared with CuA rats. The half-times (t 1 2 ) of 125I-LDL clearance are 4.90 ± 0.20 and 5.80 ± 0.30 hours in CuD and CuA rats, respectively. The plasma trichloroacetic acid-soluble 125I-radioactivity was significantly and steadily increased in CuD rats at each interval, reflecting the faster clearance and degradation of LDL in those rats. The plasma removal of 125I-acetyl-LDL was faster compared with that of 125I-LDL. The half-times (t 1 2 ) of acetyl-LDL in CuD and CuA rats are 5.20 ± 0.06 and 5.16 ± 0.08 minutes, respectively, with no significant difference between the groups. The data indicates that the uptake of LDL via the “scavenger” receptor remains unaffected in copper-deficient rats. The faster removal of the unmodified (native) LDL in CuD group suggests that the apoB,E receptor is up-regulated in copper-deficient rats and that the hypercholesterolemia observed in copper deficiency is not associated with the defective uptake of LDL by the apoB,E-receptor dependent mechanism.

Effect of copper deficiency on the hepatic synthesis and rate of plasma release of cholesterol

Abstract The relative rates of hepatic 3 H incorporation into digitonin-precipitable sterols (DPS) and release of labeled DPS into the plasma were compared between copper-deficient (CuD) and copper-adequate (CuA) rats. Following intravenous injection of 3 H 2 O and Triton WR 1339, the liver and plasma 3 H-DPS radioactivities were measured at 2, 4, 6, and 8 hr, and the net total accumulation of plasma cholesterol (CH) and triglyceride (TG) was determined during the 8-hr period. In both groups, the liver synthesis of 3 H-DPS displayed a biphasic curve, with an initial slow rate up to 4 hr, followed by a rapid increase starting at 6 hr. During the latter phase, a notable increase in liver 3 H-DPS radioactivity was observed in CuD rats, but the plasma release of 3 H-DPS did not significantly change, despite the increased synthesis of 3 H-DPS in the liver. In both groups, the net total accumulation of CH in the plasma, as expressed in mmol CH/100 g body weight, increased steadily from 1.2 mmol at 2 hr to 4.7 mmol at 8 hr, while plasma TG accumulated at a much greater rate ranging from 3.3 mmol at 2 hr to 13.6 mmol at 8 hr. There was no significant difference in net total accumulation of CH or Tg in the plasma between the groups at any given interval. The results provide evidence that copper deficiency stimulates the liver synthesis of cholesterol, but that it does not result in an accelerated release of newly synthesized cholesterol from the liver involving very low density lipoproteins (VLDL). Thus, the present findings suggest that the hypercholesterolemia induced in copper deficiency is not attributable to a stimulation of the hepatic release of VLDL cholesterol into the plasma.

HPLC Separation of Geometric Isomers of Carbamyl Peptides

Abstract Carbamylated peptides that were studied showed much improved resolution on C-18 reversed phase HPLC columns compared to the parent peptides. A number of dipeptides were carbamylated with ethyl-, n-propyl- or isopropyl isocyanates. The three carbamyl derivatives of each dipeptide could easily be resolved. Carbamylated dipeptides with reversed amino acid sequences were also easily separated. Methionine-enkephalin, leucine-enkephalin, Angiotensins I, II and III and substance P were carbamylated with isocyanates derived from certain carcinostatic 2-chloroethyl nitrosoureas. 1-(2-Chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU), 1-(2-chloroethyl)-3-(cis-4-hydroxycyclohexyl)-1-nitrosourea (cis-4-hydroxy-CCNU) and 1-(2-chloroethyl)-3-(trans-4-hydroxycyclohexy1)-1-nitroso urea (trans-4-hydroxy-CCNU) gave carbamylated derivatives of each peptide and each mixture of derivatives from a single parent peptide could be resolved. Conditions were found in each case whereby baseline resolution of the corresponding ...