George V. Vahouny

Purification and properties of subunits of sterol ester hydrolase from rat pancreas.

Abstract A procedure has been developed for solubilizing subunits of sterol ester hydrolase (EC 3.1.1.13) from rat pancreas by treatment of a tissue homogenate with 0.5% ( w v ) digitonin. The crude, solubilized enzyme subunit was shown to have a molecular weight of approximately 70,000 by Sephadex G-200 gel filtration. This enzyme subunit has been purified 500-fold by a combination of ammonium sulfate fractionation, hydroxylapatite, and gel-filtration chromatography. The 70,000 molecular-weight subunits have been shown to aggregate in the presence of cholic acid or sodium taurocholate to a 400,000 molecular-weight form which is the active enzyme. Studies on binding of cholic acid to the subunit protein suggest that after the binding of one molecule of the bile acid, the subunit undergoes a conformational change(s) which makes additional binding sites for cholic acid available. Three types of differential inactivation studies (thermal, guanidine hydrochloride, and pH) indicated significantly greater stability of the active enzyme when compared to the subunits. These data are consistent with the tentative conclusion that a conformational change(s) accompanies the binding of the bile salt to the enzyme subunits, which results in their aggregation and enzyme activity. The theoretical and physiological significance of this interaction between the subunit protein and bile salt is discussed.

Activation of adrenal Sterol Ester Hydrolase by dibutyryl cAMP and protein kinase

Direct activation of adrenal Sterol Ester Hydrolase (EC 1.1.1.13) by dibutyryl cAMP, ATP and Mg+2 has been demonstrated in adrenal homogenates from three species. Variability in the degree of activation was minimized by preincubation of the tissue homogenate for two hours prior to addition of the cofactors and subsequent enzyme assay. Although baseline sterol ester hydrolytic activity, independent of cofactors, was present in all subcellular fractions, the cAMP-dependent enzyme was primarily associated with the 105,000 × g soluble fraction of the cell. A requirement for protein kinase in the system was demonstrated with a partially-purified enzyme from bovine adrenal.

Cholesterol esterase — A polymeric enzyme

Abstract A direct interaction of cholic acid and pancreatic cholesterol esterase has been shown using labeled bile salt of high specific activity and Sephadex G-50 chromatography. This interaction results in the formation of a protein of M.W. 400,000 which appears to be a hexamer of the protein in pancreatic juice (M.W. 65,000 – 69,000). Rechromatography of the polymer on Sephadex G-200 results in dissociation to the monomeric form. Evidence is provided, suggesting that the polymeric form of cholesterol esterase represents the active enzyme responsible for sterol ester synthesis and hydrolysis.

Dietary fiber supplements: Effects on serum and liver lipids and on liver phospholipid composition in rats

Rats (6 per group) were fed semipurified diets containing either particulate fibers (alfalfa, 10%; cellulose, 10%; bran, 10%), a soluble ionic fiber (pectin 5%), soluble, nonionic fibers (guar gum, 5%; Metamucil, 10%), a mixed fiber preparation (Fibyrax, 10%, or an insoluble, ionic bile acid-binding resin (cholestyramine, 2%). The control group was fed the unsupplemented diet. The feeding period, during which diet and water were provided ad libitum, was 28 days.Compared with the control group, serum total cholesterol levels were increased by more than 10% in rats fed alfalfa and decreased by more than 10% in rats fed cellulose, guar gum, Fibyrax and cholestyramine. There were no significant differences in percentage of plasma HDL cholesterol. Serum triglycerides were elevated in the groups fed alfalfa, pectin, guar gum or Fibyrax and reduced in the group fed Metamucil. Plasma phospholipids were elevated in rats fed alfalfa or bran, unaffected in rats fed pectin or Metamucil and reduced in the other groups. Liver total cholesterol was elevated in all groups but those fed wheat bran and cholestyramine. The percentage of liver cholesterol present as ester was elevated in every group except that fed cholestyramine. Liver triglycerides were reduced in rats fed guar gum or Metamucil and elevated in those fed alfalfa. Liver phospho-lipids were lowered in the group fed cellulose.Liver phospholipids were fractionated by thin layer chromatography to give phosphatidylcholine (PC), phosphatidylethanolamine (PE), sphingomyelin (Sph), lysophosphatidylcholine (LPC) and phosphatidylinositol plus phosphatidylserine (PI+PS). PC was elevated in all test groups (7–25%); PE levels ranged from 14% below to 0.3% above controls; Sph levels were sharply lower (20–53%) in all groups. LPC and PI+PS levels were close to the control value in all test groups.The results demonstrate that different dietary fibers can affect liver phospholipid composition. In view of the critical roles of phospholipids in many biological reactions, it will be interesting to survey the influence of dietary fiber on phospholipid spectra of other tissues.

Alterations in calcium, magnesium, iron, and zinc metabolism by dietary cholestyramine

Cholestyramine is an effective drug for the reduction of plasma cholesterol because of its ability to sequester intestinal bile acids. Since metabolic alterations, including diminished intestinal absorption of vitamin D and osteomalacia have been reported with long-term use of this resin, the influence of cholestyramine on dietary balance of four mineral elements has been investigated. Wistar-strain rats were fed either a 2% cholestyramine or control diet for one month. Dietary intakes and fecal and urinary excretions of calcium, magnesium, iron, and zinc were determined using atomic absorption spectrophotometry during three, 3-day balance periods. Cholestyramine-fed rats had a net negative balance for calcium and a lower net positive balance for magnesium, iron, and zinc than the controls. Other effects of cholestyramine were an increased urinary excretion of calcium and magnesium, a decreased urinary zinc, and an alkalinization of urine. Blood and tissue cation content was unchanged except for a rèduction in serum magnesium with resin feeding. Alterations in calcium, magnesium, and zinc metabolism might be explained by inadequate vitamin D absorption from the intestine followed by an increased secretion of parathyroid hormone. A diminished iron absorption due to resin binding could account for the reported disturbance in iron balance.

Quantitative and Qualitative Adaptations in Gastrointestinal Mucin with Dietary Fiber Feeding

The viscous multicomponent mucin gel that coats the epithelial lining of the gastrointestinal tract is a heretofore neglected component of this organ system. The unique structural and functional characteristics of this biological material enable it to act as a protective physiological barrier to potentially deleterious agents. In addition, it may function to limit access to the intestinal surface and consequently limit absorption of nutrients.

Lipid Accumulation in Jejunal and Colonic Mucosa Following Chronic Cholestyramine (Questran) Feeding

The hypolipidemic agent, cholestyramine (Questran), when fed to rats inhibits intestinal absorption of cholesterol and triglycerides and causes significant epithelial cell damage in both small and large intestine. In this study, we report significant accumulation of lipids in the mucosal layer of both jejunum and colon in rats administered 2% cholestyramine for a four-week period, when compared to a control group maintained on regular chow. The total lipid increment with cholestyramine was 4.7-fold in the jejunum and 3.7-fold in the colon. The triglyceride fraction increased substantially in the small but not the large intestine. Relative phospholipid levels decreased in the treated jejunum but not in the colon. The biochemical data were reflected in morphological evidence of lipid-laden enterocytes obtained by light and transmission electron microscopy. Since cholestyramine has been shown to sequester 99.8% of micellar phospholipidin vitro, it is concluded that the presence of cholestyramine in the intestinal lumen may interefere with phospholipid availability for chylomicron synthesis and serosal lipid exit from the epithelium. This unusual deposition of lipid within the mucosal layer may also be correlated with the known cocarcinogenic effect of this resin in experimentally induced intestinal cancer.

Quantitative extraction of methylmalonic and succinic acids and their determination by gas-liquid chromatography

Abstract A method for routine small-volume solvent extraction, derivitization and gas-liquid chromatographic analysis of methylmalonic and succinic acids is described. The procedure allows for quantitative determination of mass and radioactivity of these acids and can be applied to metabolic studies of the genetic disorder methylmalonylacidurea.

Acetyl carnitine formation in rat heart

The comparative incorporation of acetate into long chain fatty acids and acetyl carnitine by cell-free preparations of rat heart has been investigated. Whereas the addition of 1 mM carnitine stimulated (45%) fatty acid synthesis by liver preparations in citrate-containing media, fatty acid synthesis from acetate in rat heart homogenates under the same incubation conditions was markedly depressed. This depression by carnitine of acetate incorporation into long chain fatty acids in 105,000 × g soluble fractions of heart was associated with increased acetyl carnitine formation. Thus in heart tissue acetyl CoA is effectively shuttled into acetyl carnitine and is unavailable for synthesis of fatty acids. These data are in agreement with results obtained earlier in studies with perfused rat heart. A similar conversion of added acetyl CoA to the carntine derivative occurred when labeled malonyl CoA was used as fatty acid precursor, again resulting in reduced fatty acid synthesis. It was shown by direct measurement that acetyl carnitine formation in the absence of carnitine was greatest in heart mitochondria and least in microsomes. In the presence of carnitine, acetyl carnitine formation was increased in all subcellular fractions, with the greatest change again occurring with mitochondria.

Dietary Fiber and Atherosclerosis

The relationship between the intake of dietary fiber materials and a lowered risk of cardiovascular disease is principally derived from the lipid hypothesis as an etiologic factor in atherosclerosis. High values of high-density lipoprotein (HDL), HDL-cholesterol, or the ratio of HDL-to LDL-cholesterol are believed to be negative correlation factors in coronary heart disease. It has been estimated that a 12% decrement in plasma cholesterol reduces the risk of myocardial infarction by 24%. The data supporting the ameliorative effect on atherosclerosis strongly suggest that only certain types of fiber are important in this regard, particularly in man. Wheat and corn bran have little impact on these plasma parameters, whereas oat bran is moderately effective. Soluble fibers such as oat gum, guar gum, and pectin demonstrate a significant hypocholesteremic effect and also lower LDL levels. Although coronary heart disease is a multifactorial development, the nature of the dietary intake is recognized as a contributory factor. Animal models offer the advantage of short-and long-term studies, direct lymphatic and vascular cannulation or reinfusion to test putative mechanisms, and both pre-and postmortem structural and functional data accrewal. We have focused on the effects of dietary fiber derivatives on specific aspects of structural and functional adaptation of the rat gastrointestinal tract. The basic postulate of this approach is that physiological, health-positive sequelae of fiber intake must result from signals perceived and/or relayed systemically by the mucosal lining, since it is generally agreed that these materials are exposed to this organ system only. In the rat model, viscous fiber derivatives delay but do not impede fatty acid absorption, although cholesterol absorption into lymph is significantly diminished. There is no difference in chylomicron sizing as assayed morphometrically. Alfalfa and guar gum feeding causes a greater recovery of oleic acid and cholesterol in the chylomicron fraction. Pectin decreased this expression of absorbed lipid with a corresponding increment in VLDL. The effects of dietary fiber supplementation are not solely explained by alterations in gastric emptying or interference with bulk-phase diffusion of lipids.