Maureen K. Anderson

Rapid separation and quantitation of combined neutral and polar lipid classes by high-performance liquid chromatography and evaporative light-scattering mass detection

Modifications are described for an innovative and widely used high-performance liquid chromatography technique that resolves a very broad spectrum of lipids for quantitation by evaporative light-scattering detection. Substitution of acetone for 2-propanol in a portion of the solvent gradient program yields consistent resolution of diacylglycerol and cholesterol without sacrificing baseline resolution of the remaining major lipid classes. Moreover, previously noted instabilities in triacylglycerol retention time are eliminated. The introduction of acetone also enables a 20% reduction in flow-rate without an increase in total run time. As a further modification of the mobile phase composition, acetic acid and ethanolamine are substituted for the serine-ethylamine combination that was originally shown to improve column performance. The combination of acetic acid and ethanolamine yields the same result but the increased volatility of these solutes over serine results in decreased baseline noise. Finally, 1,2-hexadecanediol is introduced as an internal standard that is well suited for this method. The chromatographic performance obtained with these modifications is demonstrated in compositional analyses of lipid extracts from rat liver, heart, kidney and brain.

Hypercholesterolemia Causes Mechanical Weakening of Rabbit Atheroma: Local Collagen Loss as a Prerequisite of Plaque Rupture

Hypercholesterolemia may render atherosclerotic plaques prone to rupture. To test this hypothesis, catheters with matrix-covered balloons were implanted into the aorta of rabbits fed standard or 0. 5% cholesterol chow (n=70). In 1 month, fibrous plaques developed around the balloon. Time-dependent accumulation of cholesteryl esters and free cholesterol was detected in the plaques of the cholesterol-fed group only. The pressure needed to rupture the plaque by balloon inflation was used as an index of plaque strength. Three months after the catheter implantation, the breaking pressure was 2.1 times lower (P<0.05) in cholesterol-fed rabbits. It was accompanied by collagen loss, as measured by plaque hydroxyproline content, but not with deficiency of collagen cross-linking. Sirius red staining showed preservation of collagen originally covering the balloon and accumulation of nascent collagen in the lesions of standard chow-fed rabbits. In the cholesterol-fed group, both mature and new collagen underwent degradation predominantly in the plaque shoulders. Collagen breakdown was associated with local accumulation of foamy macrophages. Gel zymography demonstrated relative enhancement of gelatinolytic activity at 92 and 72 kDa, as well as caseinolytic activity at 57, 45, and 19 kDa in the lipid-laden plaques. Lipid accumulation in the plaque was also associated with a loss of smooth muscle cells, the cellular source of the collagen fibers. The remaining smooth muscle cells showed increased collagen synthesis, although it was insufficient to counterbalance collagen degradation and cell loss. Thus, we have obtained direct evidence that hypercholesterolemia is accompanied by enhanced local collagen degradation, which is potentially responsible for plaque weakening.

Lipid-lowering effects of WAY-121,898, an inhibitor of pancreatic cholesteryl ester hydrolase

WAY-121,898 is an inhibitor of pancreatic cholesteryl ester hydrolase (pCEH). After confirming its in vitro potency and relative lack of a major effect on acyl-CoA:cholesterol acyltransferase (ACAT), it was found that this compound lowers plasma cholesterol in cholesterol-fed, but not chow-fed, rats. Measures of liver cholesteryl ester content and the direct determination of cholesterol absorption (lymph-fistula model) show that inhibition of cholesterol absorption is at least one mechanism for the observed cholesterol lowering. However, WAY-121,898 was also active when administered parenterally to cholesterol-fed rats, and in cholesterol-fed hamsters cholesterol-lowering occurred with oral dosing despite no change in cholesterol absorption, suggesting other modes of action possibly relating to inhibition of liver CEH. Combination treatment in cholesterol-fed rats with the ACAT inhibitor CI-976 resulted in a greater-than-additive reduction in plasma cholesterol, imlying that both pCEH and ACAT may play a role in cholesterol absorption in this species. In rabbits, WAY-121,898 prevented the rise in plasma cholesterol due to the feeding of cholesteryl ester but not in rabbits fed (free) cholesterol. In guinea pigs, the compound induced an increase in adrenal cholesteryl ester mass. Taken together, the overall profile in these animal models suggests that WAY-121,898 inhibits more than just the intestinal (lumenal) pCEH, and that the role of this enzyme in cholesterol metabolism may be different within and across species, the former depending upon the dietary cholesterol load.

Inhibitors of acyl-CoA: Cholesterol O-acyl transferase (ACAT) as hypocholesterolemic agents. 13. Design, synthesis and biological evaluation of tetrazole anilides as potent inhibitors of ACAT in vitro and hypocholesterolemic agents in vivo

Abstract The syntheses and biological activities for anilides derived from 2-phenyl-2-(dodecyl-2H-tetrazol-5-yl)acetic acid are described. Evidence is provided that one of these compounds, (+)- 8b , stereoselectively inhibits ACAT in vitro and possesses superior efficacy in vivo compared to (−)- 8b or the recemic mixture (±)- 8b .

Inhibitors of acyl-coa:cholesterol acyltransferase (ACAT). 15. sulfonylurea inhibitors with excellent hypocholesterolemic activity in vivo.

Abstract A series of sulfonylureas were prepared and tested for the ability to inhibit the enzyme acyl-CoA: cholesterol acyltransferase (ACAT) in vitro and lower plasma cholesterol in cholesterol-fed rats in vivo. Although compounds from this series were generally weak inhibitors of ACAT in vitro, several displayed excellent hypocholesterolemic activity in vivo.

Inhibitors of acyl-CoA: Cholesterol O-acyl transferase (ACAT) as hypocholesterolemic agents. 12. Syntheses and biological activity of structurally novel tetrazole amides

Abstract The identification of tetrazole benzamide and nicotinamide derivatives as new structural classes of potent ACAT inhibitors is described. The ensuing structure-activity relationship (SAR) studies revealed that retroamide 8c and 7q possesses comparable in vitro potency and efficacy to that of the fatty acid anilide, CI-976 ( 1 ).