Jerome L. Hojnacki

Oral Nicotine Induces an Atherogenic Lipoprotein Profile

Abstract Male squirrel monkeys were used to evaluate the effect of chronic oral nicotine intake on lipoprotein composition and metabolism. Eighteen yearling monkeys were divided into two groups: 1) Controls fed isocaloric liquid diet; and 2) Nicotine primates given liquid diet supplemented with nicotine at 6 mg/kg body wt/day. Animals were weighed biweekly, plasma lipid, glucose, and lipoprotein parameters were measured monthly, and detailed lipoprotein composition, along with postheparin plasma lipoprotein lipase (LPL) and hepatic triglyceride lipase (HTGL) activity, was assessed after 24 months of treatment. Although nicotine had no effect on plasma triglyceride or high density lipoproteins (HDL), the alkaloid caused a significant increase in plasma glucose, cholesterol, and low density lipoprotein (LDL) cholesterol plus protein while simultaneously reducing the HDL cholesterol/plasma cholesterol ratio and animal body weight. Levels of LDL precursors, very low density (VLDL) and intermediate density (IDL) lipoproteins, were also lower in nicotine-treated primates while total postheparin lipase (LPL + HTGL) activity was significantly elevated. Our data indicate that long-term consumption of oral nicotine induces an atherogenic lipoprotein profile (+LDL, +HDL/total cholesterol ratio) by enhancing lipolytic conversion of VLDL to LDL. These results have important health implications for humans who use smokeless tobacco products or chew nicotine gum for prolonged periods.

Separation of six lipid classes on one thin-layer chromatogram

Since the concentrations of squalene’ and cholesteryl esters’ in the aorta have been related to the severity of atherosclerosis, it seems highly desirable to be able to separate these two lipid classes, along with triglycerides, fatty acids, sterols, and complex lipids, on one thin-layer chromatogram. Such a separation would allow the six lipid classes to be analyzed fluorometrically or densitometrically insitu on one chromatogram3 and prevent losses of microgram quantities of these two lipids when scraped, eluted, and rechromatographed for separation by conventional procedures. Previously, Blank et a/.4 separated five lipid classes on a single chromatogram. However, an overlap of the sterol ester and hydrocarbon fractions necessitates the scraping of this region followed by elution and development in a different solvent system (e.g. diethyl ether-petroleum ether (b.p. 38.5-40.0”), 7: 93) if these two classes are to be studied. This communication describes a technique for the separation of six lipid classes on one chromatogram through the sequential use of two solvent systems.

Effect of drinking pattern on plasma lipoproteins and body weight

The effect of drinking pattern on plasma lipoproteins and body weight was examined in three groups of squirrel monkeys: (1) controls fed isocaloric liquid diet; (2) regular drinkers given liquid diet containing ethanol (EtOH) substituted isocalorically for carbohydrate at 12% of calories daily; and (3) binge drinkers fed 6% EtOH calories daily for a four-day period followed by three days of 20% EtOH to mimic a weekend bout drinking cycle. The number of calories offered per day was the same for all groups, and the average weekly EtOH consumption (12% calories) was identical for the two alcohol treatments. The entire study lasted six months. There were no significant differences in plasma cholesterol, triglyceride or liver function tests. Regular drinkers had the highest high density lipoprotein2/high density lipoprotein3 (HDL2/HDL3) protein and apolipoprotein A-I/B ratios of any group and exhibited a significant elevation in the molar plasma lecithin:cholesterol acyltransferase (LCAT) rate (nmol/min/ml). Binge drinking produced a selective increase in low density lipoprotein (LDL) cholesterol and apolipoprotein B, and a depression in the fractional LCAT rate (% esterified/min). During the course of the study, controls ate 92% of their diet while the alcohol groups each consumed 95% of the liquid diet. Despite this difference, body weight and Quetelet index (weight/height2) decreased progressively in the order controls greater than regular drinkers greater than binge drinkers. Results from our study indicate that moderate, regular daily consumption of EtOH at 12% of calories causes a modest reduction in body weight and produces a coronary protective lipoprotein profile (increases HDL2/HDL3, increases apolipoprotein A-I/B, low LDL cholesterol). By contrast, when this same average weekly dose is concentrated in a binge cycle, unfavorable alterations in lipoprotein composition (increases LDL cholesterol, increases apolipoprotein B) and metabolism (decreases LCAT activity) occur along with weight loss and depletion of body fat. These studies point to the value of the squirrel monkey model in evaluating both favorable and pathophysiological effects of chronic EtOH intake.

Densitometric quantitation of neutral lipids on ammonium sulfate impregnated thin-layer chromatograms.

A procedure is described which extends the densitometric quantitation of phospholipids on ammonium sulfate impregnated thin-layer chromatograms by Gluck et al. to include total lipid, free and esterified cholesterol, free fatty acid and triglyceride. Lipids separated on thin-layer plates containing silica gel G impregnated with ammonium sulfate were charred upon heating and absorbance was measured densitometrically. Thus, the necessity of spraying or submersing in a charring agent was eliminated, uniform charring became possible, and quantitation over a wider range of sample sizes than most densitometric procedures was obtained. One linear relationship existed for concentrations of standards over the range of 0.0-3.0 mug and another line from 4.0-50.0 mug. Both accuracy and precision of the method were highly reliable.

Ethanol Induced Alterations in Low and High Density Lipoproteins

Abstract Male squirrel monkeys fed ethanol (ETOH) at variable doses were used to determine whether alcohol modifies levels of plasma low density lipoproteins (LDL) in addition to increasing high density lipoproteins (HDL). Because we earlier showed that high alcohol consumption enhances lipoprotein cholesterol synthesis, experiments were also performed to further assess whether ETOH alters lipoprotein clearance and plasma transfer processes in vivo- Monkeys were divided into three groups: Controls fed isocaloric liquid diet; and Low and High ETOH animals fed liquid diet with vodka substituted isocalorically for carbohydrate at 12 and 24% of calories, respectively. High ETOH primates had significantly more LDL lipid and protein while serum glutamate oxaloacetate transaminase was similar for the three groups. Although removal of 3H LDL cholesteryl ester (CE) from the plasma compartment was not affected by dietary ETOH, transfer of LDL CE to HDL was impaired in the High ETOH group suggesting a mechanism for the enlarged circulating pool of LDL. Transfer of 14C HDL CE to lower density lipoproteins was similar for the three groups. However, ETOH at both doses delayed clearance of radiolabeled HDL CE from circulation. Thus besides enhancing synthesis of lipoproteins, ETOH at a moderately high dose (24% of calories) influences lipoprotein levels in primates by modifying lipid transfer processes (LDL) as well as by altering clearance (HDL) without adversely affecting liver function.

Alcohol delays clearance of lipoproteins from the circulation

Long-term (18-month) consumption of high-dose ethanol ([EtOH] 24% of total calories) by squirrel monkeys results in marked elevations in plasma antiatherogenic high-density lipoprotein (HDL) cholesterol and apolipoprotein (apo) A-1, and atherogenic low-density lipoprotein (LDL) cholesterol and apo B. In an effort to determine whether alterations in lipoprotein turnover could explain the above findings, 131I-HDL apo A-1 and 125I-LDL apo B were injected into EtOH and control animals, following which in-vivo catabolic and production rates were determined. For both lipoproteins, synthetic rates were unaltered, while fractional catabolic rates (FCR) were significantly reduced in EtOH monkeys. Results from this study implicate EtOH-induced changes in hepatic metabolism as the basis for delayed lipoprotein clearance and hence elevated plasma apolipoprotein levels.

Oral nicotine impairs clearance of plasma low density lipoproteins.

Abstract The effect of chronic oral nicotine intake on plasma low density lipoprotein (LDL) clearance, lipid transfer protein, and lecithimcholesterol acyltransferase (LCAT) was examined in male atherosclerosis susceptible squirrel monkeys. Eighteen yearling primates were divided into two groups: 1) Controls fed isocaloric liquid diet; and 2) Nicotine monkeys given liquid diet supplemented with nicotine at 6 mg/kg body wt/day for a two-year period. Averaged over 24 months of treatment, animals in the Nicotine group had significantly higher levels of plasma and LDL cholesterol compared to Controls while plasma LCAT activity was similar for both groups. Following simultaneous injection of 3H LDL and 14C high density lipoprotein (HDL) cholesteryl ester (CE), removal of the latter was not altered by oral nicotine while plasma clearance of 3H LDL was dramatically delayed in Nicotine monkeys. Transfer of 14C HDL CE to very low density lipoprotein (VLDL)-LDL particles was greatly accelerated in the Nicotine group vs Controls while the reciprocal movement of 3H LDL CE to HDL was only higher in experimental animals at two time points following injection of the isotopes. Results from this study provide evidence that one major detrimental effect of long-term oral nicotine use is an increase in the circulating pool of atherogenic LDL which is due to: 1) accelerated transfer of lipid from HDL; and 2) impaired clearance of LDL from the plasma compartment. Diminished removal of LDL is of particular importance because an extended residence time of these particles in circulation would increase the likelihood of their deposition in the arterial wall.

Alcohol Produces Dose-Dependent Antiatherogenic and Atherogenic Plasma Lipoprotein Responses

Abstract A comprehensive assessment of lipoprotein compositional/metabolic response to incremental caloric ethanol (EtOH) doses ranging from low to moderate to high was undertaken using male squirrel monkeys. Control monkeys were maintained on a chemically defined, isocaloric liquid diet, while experimental primates were fed increasing doses of alcohol (6, 12, 18, 24, 30, and 36% of energy) substituted isocalorically for carbohydrate at 3-month intervals. Liver function tests and plasma triglyceride were normal for all animals. Plasma cholesterol showed a transient increase at the 12% caloric dose that was attributed solely to an increase in high density lipoprotein (HDL). A more pronounced increase in plasma sterol, beginning at 24% and continuing to 36% EtOH, was the result of increments in both HDL and low density lipoprotein (LDL) cholesterol, although the contribution by the latter was substantial primarily at the 36% dose. Plasma apolipoprotein elevations (HDL apolipoprotein A-1, LDL apolipoprotein B) generally accompanied the lipoprotein lipid increases, although the first atherogenic response for LDL became manifest as a significant increase in apolipoprotein B at 18% EtOH calories. Postheparin plasma lipoprotein lipase was not affected by dietary alcohol, whereas hepatic triglyceride lipase activity showed significant increases at higher (24 and 36%) EtOH doses. Plasma lecithin-cholesterol acyltransferase activity was normal at the 6 and 12% EtOH doses, but exhibited a significant reduction beginning at 18% and continuing to 36% EtOH. Alterations in these key lipoprotein regulatory enzymes may represent the underlying metabolic basis for the observed changes in lipoprotein levels and our earlier findings of HDL2/HDL3 subtraction modifications. Results from our study indicate that in squirrel monkeys, moderate (12%) EtOH caloric intake favors an antiatherogenic lipoprotein profile (↑HDL, normal LDL levels, and lecithin-cholesterol acyltransferase activity), whereas higher doses (24–36%) produce both coronary-protective (↑HDL) and atherogenic (↑LDL) responses. Moreover, the 18% EtOH level represents an important transition dose which signals early adverse alterations in lipoprotein composition (↑apolipoprotein B) and metabolism (↓lecithin-cholesterol acyltransferase).

Ethanol enhances de novo synthesis of high density lipoprotein cholesterol.

Abstract Male squirrel monkeys fed ethanol at variable doses were used to assess whether alcohol enhances de novo synthesis of high density lipoprotein (HDL) cholesterol in vivo. Monkeys were divided into three groups: 1) Controls fed isocaloric liquid diet; 2) Low Ethanol monkeys fed liquid diet with vodka substituted isocalorical-ly for carbohydrate at 12% of calories; and 3) High Ethanol animals fed diet plus vodka at 24% of calories. High Ethanol primates had significantly higher levels of HDL nonesterified cholesterol than Control and Low Ethanol animals while serum glutamate oxaloacetate transaminase was similar for the three treatments. There were no significant differences between the groups in HDL cholesteryl ester mass or specific activity following intravenous injection of labeled mevalonolactone. By contrast, High Ethanol monkeys had significantly greater HDL nonesterified cholesterol specific activity with approximately 60% of the radioactivity distributed in the HDL3 sub-fraction. This report provides the first experimental evidence that ethanol at 24% of calories induces elevations in HDL cholesterol in primates through enhanced de novo synthesis without adverse effects on liver function.

Effect of ethanol on lipoprotein synthesis and fecal sterol excretion

Abstract The effect of variable doses of ethanol on plasma lipoprotein composition, lipoprotein synthesis and fecal sterol excretion was examined in male, atherosclerosis susceptible squirrel monkeys. Primates were divided into three groups: 1) Controls fed isocaloric liquid diet; 2) Low Ethanol monkeys given liquid diet with vodka substituted isocalorically for carbohydrate at 12% of calories; and 3) High Ethanol animals fed diet plus vodka at 24% of calories. Circulating high density lipoprotein (HDL) free cholesterol and phospholipid, very low density-low density lipoprotein (VLDL-LDL) total cholesterol, and total plasma cholesterol and triglyceride were significantly elevated in High Ethanol primates compared to the other treatments. However, the percent distribution of cholesterol among the lipoprotein fractions was identical for the three groups. There were no significant differences in serum glutamate oxalo-acetate transaminase. High Ethanol primates also had significantly greater HDL free cholesterol specific activity following intravenous injection of 3 H mevalonolactone compared to the other groups while radioactive VLDL-LDL free cholesterol was elevated in both High and Low Ethanol animals. Although, total fecal bile acid mass was significantly greater in both alcohol treatment groups compared to Controls, fecal neutral sterol specific activity was only higher in monkeys fed the high ethanol diet. This study provides evidence that ethanol at 24% of calories: 1) raises HDL cholesterol levels by enhancing lipoprotein synthesis; 2) increases the fecal output of newly synthesized cholesterol without causing liver dysfunction; and 3) maintains a constant relative distribution of cholesterol among lipoprotein classes.