James G. Hamilton

Rapid separation of neutral lipids, free fatty acids and polar lipids using prepacked silica sep-Pak columns

A method is described for the separation of neutral lipid, free fatty acid and polar lipid classes using small (600 mg), prepacked silica Sep-Pak columns. Combinations of hexane and methyltertiarybutylether were used to progressively elute cholesteryl ester first then triglyceride from the column. After column acidification, fatty acids were eluted followed by cholesterol. Recoveries of these lipids were 96% or greater. Polar lipids were eluted from the column using combinations of methyltertiarybutylether, methanol and ammonium acetate. Phospholipid classes could not be separated completely from each other. Phosphatidylethanolamine and phosphatidylinositol eluted together, whereas the more polar phosphatidylcholine, sphingomyelin and lysophosphatidylcholine were eluted as a second fraction. Recoveries of each phospholipid was greater than 98%.

The effect of 5,8,11,14-eicosatetraynoic acid on lipid metabolism

The purpose of this presentation is to review the current state of knowledge regarding 5,8,11,14-eicosatetraynoic acid (ETYA, Ro 3-1428) and its effects on lipid metabolism. Accordingly, the topics discussed include hypocholesterolemic and dermatological studies involving ETYA in both animals and man, as well as the effects of ETYA on desaturate enzymes. Metabolic studies involving ETYA are also noted. Primary interest is focused on the effects of ETYA on selected processes of arachidonate metabolism, and the effect of ETYA on inflammation, platelet aggregation and tumor growth are discussed, keeping in mind the relevance of arachidonate metabolism to these processes.

Inhibition of lipogenesis in rat liver by (−)-hydroxycitrate

Abstract The purpose of these investigations was to determine the effect of the stereoisomers of hydroxycitrate on the rate of lipogenesis in rat liver. These observations were made under conditions in which there was an induced rate of lipid synthesis. Only (−)-hydroxycitrate significantly decreased the conversion of [14C]citrate into lipid in liver high speed supernatant and the conversion of [14C]alanine into lipid in vivo. The in vivo rate of lipogenesis was markedly decreased for 150 min following the administration of (−)-hydroxycitrate either iv (0.017 mmoles/kg) or ip (0.0263 mmoles/kg). Fatty acid and cholesterol synthesis were significantly inhibited by the oral administration of (−)-hydroxycitrate (5.26, 3.95 and 2.63 mmoles/kg) only when the compound was given before the feeding period.

Effect of (−)-hydroxycitrate upon the accumulation of lipid in the rat: I. Lipogenesis

The purpose of these investigations was to ascertain the effect of (−)-hydroxycitrate on the accumulation of lipid in the meal fed rat by examining the rates of lipogenesis after acute and chronic treatment. Oral administration of (−)-hydroxycitrate depressed significantly the in vivo lipogenic rates in a dose-dependent manner in the liver, adipose tissue, and small intestine. The hepatic inhibition was significant for the 8 hr period, when control animals demonstrated elevated rates of lipid synthesis. The kinetics of this reduction of in vivo hepatic lipogenesis were identical after acute or chronic administration of (−)-hydroxycitrate. However, in vitro rates of lipogenesis were elevated after chronic administration of (−)-hydroxycitrate for 30 days. Rats receiving (−)-hydroxycitrate consumed less food than the untreated controls; however, this decreased caloric intake was not responsible for the drug induced depression of hepatic lipogenesis, as shown by studies using pair fed rats.

Effect of (−)-hydroxycitrate upon the accumulation of lipid in the rat: II. Appetite

These studies were designed to determine the effect of (−)-hydroxycitrate upon the accumulation of lipid in the rat by examining appetite, wt gain, and total body lipid profiles. The chronic oral administration of a nontoxic dose of (−)-hydroxycitrate to growing rats for 11–30 days caused a significant reduction in body wt gain, food consumption, and total body lipid. The administration of equimolar amounts of citrate did not alter wt gain, appetite, or body lipid. No increase in liver size or liver lipid content occurred with either treatment. Pair feeding studies demonstrated that the reduction in food intake accounted for the decrease in wt gain and body lipid observed with (−)-hydroxycitrate treatment.

Separation of neutral lipid free fatty acid and phospholipid classes by normal phase hplc

Normal phase high performance liquid chromatography methods are described for the separation of neutral lipid, fatty acid and five phospholipid classes using spectrophotometric detection at 206 nm. Separations were accomplished in less than 10 min for each lipid class. A mobile phase consisting of hexane/methyltertiarybutylether/acetic acid (100∶5∶0.02) proved effective in separating cholesteryl ester and triglyceride with recoveries of 100% for radiolabeled cholesteryl oleate and 98% for radiolabeled triolein. Free fatty acid and cholesterol were separated by two different mobile phases. The first, hexane/methyltertiarybutylether/acetic acid (70∶30∶0.02) effectively separated free fatty acids and cholesterol, but did not separate cholesterol from 1,2-diglyceride. A mobile phase consisting of hexane/isopropanol/acetic acid (100∶2∶0.02) effectively separated free fatty acid, cholesterol, 1,2-diglyceride and 1,3-diglyceride. Recoveries of oleic acid and cholesterol were 100% and 97%, respectively. Five phospholipid classes were separated using methylteriarybutylether/methanol/aqueous ammonium acetate (pH 8.6) (5∶8∶2) as the mobile phase. The recoveries of phosphatidylinositol, phosphatidylethanolamine, phosphatidylcholine, sphingomyelin and lysophosphatidylcholine were each greater than 96%.

An improved competitive protein binding assay for 1,25-dihydroxyvitamin D

Abstract A practical procedure for the routine analysis of blood levels of 1,25-dihydroxyvitamin D has been developed. 1,25-Dihydroxyvitamin D was extracted from serum with methylene chloride:methanol. The concentration of acidic lipids in the extract was reduced with an alkaline wash. The serum extract was chromatographed on Sephadex LH-20, which was extracted with methanol prior to use in the columns. The chromatography was aided by a device which simultaneously collected and evaporated the eluates. High pressure liquid chromatography to further purify 1,25-dihydroxyvitamin D was found to be unnecessary. These modifications resulted in negligible 1,25-dihydroxyvitamin D values ( 3 standard curve capable of measuring 1.5 pg of 1,25-dihydroxyvitamin D 3 was achieved with the use of high specific activity 3 H-labeled 1,25-dihydroxyvitamin D 3 (92 Ci/mmol). The sensitivity of the standard curve, combined with changes in the resuspension of the assay sample, reduced the required amount of serum for an assay from 5 to 2 ml. A simplified centrifugation procedure for the separation of bound from free 1,25-dihydroxyvitamin D was attained by the inclusion of bovine γ-globulin in the polyethylene glycol precipitation.

Hypolipidemic activity of (−)-hydroxycitrate

The influence of (−)-hydroxycitrate, a potent competitive inhibitor of adenosine triphosphate (ATP) citrate lyase, on serum triglyceride and cholesterol levels, and in vitro and in vivo rates of hepatic fatty acid and cholesterol synthesis was investigated in normal and hyperlipidemic rat model systems. (−)-Hydroxycitrate reduced equivalently the biosynthesis of triglycerides, phospholipids, cholesterol, diglycerides, cholesteryl esters, and free fatty acids in isolated liver cells. In vivo hepatic rates of fatty acid and cholesterol synthesis determined in meal-fed normolipidemic rats were suppressed significantly by the oral administration of (−)-hydroxycitrate for 6 hr, when control animals exhibited maximal rates of lipid synthesis; serum triglyceride and cholesterol levels were significantly reduced by (−)-hydroxycitrate. In two hypertryglyceridemic models—the genetically obese Zucker rat and the fructose-treated rat—elevated triglyceride levels were due, in part, to enhanced hepatic rates of fatty acid synthesis. (−)-Hydroxycitrate significantly reduced the hypertriglyceridemia and hyperlipogenesis in both models. The marked hypertriglyceridemia exhibited by the triton-treated rat was only minimally due to increased hepatic lipogenesis; (−)-hydroxycitrate significantly inhibited both serum triglyceride levels and lipogenesis in this model.

Organic solvent extraction of liver microsomal lipid: I. The requirement of lipid for 3,4-benzpyrene hydroxylase

Summary Extraction of lyophilized microsomes from 3-methylcholanthrene treated rats with n-butanol and acetone removed all of the neutral lipid, 80% of the phosphatidylcholine and phosphatidylethanolamine, and 75% of the total phospholipid phosphorus. Recoveries of cytochrome P-448 and NADPH-cytochrome c reductase were 70–85%. The 3,4-benzpyrene hydroxylase activity was reduced to 40–60% of control and could be restored to full activity by the addition of liposomes of total microsomal lipid, or synthetic phosphatidylcholine. These results indicate that lipid is an essential component of the microsomal hydroxylation system.

Distribution and metabolism of two orally administered esters of tocopherol

A comparison of the distribution of total radioactivity in rat tissue lipids after the oral administration of d,1-3,4-3H2-α-tocopheryl nicotinate and d,1-α-tocopheryl-1’,2’-3H2-acetate in equimolar concentrations has demonstrated that there is considerable variation in the concentration of vitamin E in organs at different times after dosing. A higher total radioactivity was found in the tissues of animals receiving α-tocopheryl nicotinate than after α-tocopheryl acetate 12 hr after feeding with an emulsion, but not at most other time intervals studied. These findings indicate that the tissue uptake of vitamin E after oral dosage with nicotinate ester is, perhaps, poorer than that occurring after feeding with tocopheryl acetate, or that α-tocopheryl nicotinate has a faster turnover than the acetate ester. Although total radioactivity in the blood and liver of those animals dosed with α-tocopheryl acetate varied slightly with time, there was a high peak of radioactivity at 12 hr after dosage with nicotinate ester. In both groups of rats, the adrenals, ovaries, adipose tissue and heart appeared to extract vitamin E from the blood for a period of up to 48 hr postabsorptively. Metabolic products of tocopherol detected by glass-fiber paper chromatography were found in both instances. This analysis revealed that, when orally administered, both α-tocopheryl nicotinate and α-tocopheryl acetate are extensively metabolized by the tissues of the rat. The metabolite most abundantly occurring under these conditions was α-tocopheryl quinone. In the adrenal glands, however, the most highly labeled compound was unesterified tocopherol, which increased with time and comprised up to 90% of the chromatographed radioactivity. From the data obtained, it can be assumed that the adrenal tissue plays a definite role in the metabolism of vitamin E.A comparison of the distribution of total radioactivity in rat tissue lipids after the oral administration of d,1-3,4-3H2-α-tocopheryl nicotinate and d,1-α-tocopheryl-1’,2’-3H2-acetate in equimolar concentrations has demonstrated that there is considerable variation in the concentration of vitamin E in organs at different times after dosing. A higher total radioactivity was found in the tissues of animals receiving α-tocopheryl nicotinate than after α-tocopheryl acetate 12 hr after feeding with an emulsion, but not at most other time intervals studied. These findings indicate that the tissue uptake of vitamin E after oral dosage with nicotinate ester is, perhaps, poorer than that occurring after feeding with tocopheryl acetate, or that α-tocopheryl nicotinate has a faster turnover than the acetate ester. Although total radioactivity in the blood and liver of those animals dosed with α-tocopheryl acetate varied slightly with time, there was a high peak of radioactivity at 12 hr after dosage with nicotinate ester. In both groups of rats, the adrenals, ovaries, adipose tissue and heart appeared to extract vitamin E from the blood for a period of up to 48 hr postabsorptively. Metabolic products of tocopherol detected by glass-fiber paper chromatography were found in both instances. This analysis revealed that, when orally administered, both α-tocopheryl nicotinate and α-tocopheryl acetate are extensively metabolized by the tissues of the rat. The metabolite most abundantly occurring under these conditions was α-tocopheryl quinone. In the adrenal glands, however, the most highly labeled compound was unesterified tocopherol, which increased with time and comprised up to 90% of the chromatographed radioactivity. From the data obtained, it can be assumed that the adrenal tissue plays a definite role in the metabolism of vitamin E.