Seasonal variation in plasma lipids, lipoproteins, apolipoprotein A-I and vitellogenin in the freshwater turtle, Chrysemys picta.

An analysis of plasma lipids and lipoprotein fractions was performed over the course of the annual ovarian cycle of the female turtle, Chrysemys picta. Determinations of total plasma triglycerides, cholesterol, vitellogenin and apolipoprotein A-I (apoA-I) were made. The lipid and protein composition of the lipoprotein fractions [very low density lipoprotein (VLDL), low density lipoprotein (LDL), high density lipoprotein (HDL) and very high density lipoprotein (VHDL)] were also observed over the same period. Plasma triglyceride and vitellogenin levels were significantly increased in the spring preovulatory period and fall recrudescent phase. Total plasma cholesterol levels were significantly elevated only at the onset of the fall recrudescent phase and apoA-I levels were highest during the postoviposition/ovarian arrest phase. The triglyceride content of VLDL was highest in preovulatory animals and there were apparent seasonal changes in the expression of apoA-I and apoE of HDL/VHDL. We conclude that the coordinate regulation of lipids and protein contributes to seasonal ovarian growth and clearance of lipids from plasma, both of which are most likely under hormonal control.

Determination of human coronary artery composition by Raman spectroscopy.

BACKGROUND: We present a method for in situ chemical analysis of human coronary artery using near-infrared Raman spectroscopy. It is rapid and accurate and does not require tissue removal; small volumes, approximately 1 mm3, can be sampled. This methodology is likely to be useful as a tool for intravascular diagnosis of artery disease. METHODS AND RESULTS: Human coronary artery segments were obtained from nine explanted recipient hearts within 1 hour of heart transplantation. Minces from one or more segments were obtained through grinding in a mortar and pestle containing liquid nitrogen. Artery segments and minces were excited with 830 nm near-infrared light, and Raman spectra were collected with a specially designed spectrometer. A model was developed to analyze the spectra and quantify the amounts of cholesterol, cholesterol esters, triglycerides and phospholipids, and calcium salts present. The model provided excellent fits to spectra from the artery segments, indicating its applicability to intact tissue. In addition, the minces were assayed chemically for lipid and calcium salt content, and the results were compared. The relative weights obtained using the Raman technique agreed with those of the standard assays within a few percentage points. CONCLUSIONS: The chemical composition of coronary artery can be quantified accurately with Raman spectroscopy. This opens the possibility of using histochemical analysis to predict acute events such as plaque rupture, to follow the progression of disease, and to select appropriate therapeutic interventions.

Renal mouse proximal tubular cells are more susceptible than MDCK cells to chemical anoxia.

To elucidate the mechanisms responsible for the resistance of continuous cell lines to anoxic injury, we have compared the effects of ATP depletion induced by chemical anoxia on primary cultures of mouse proximal tubular (MPT) cells and on Madin-Darby canine kidney (MDCK) cells. Inhibition of ATP production by cyanide and 2-deoxyglucose (CN+DOG) in the absence of dextrose reduced cell ATP content to < 5% of control values in MPT cells and caused progressive deterioration in mitochondrial function as well as loss of cell viability in these cells. Cell free fatty acid (FFA) content rose from 4.3 +/- 0.9 to 23.7 +/- 2.0 micrograms/mg of total lipid weight after 4 h of CN + DOG (P < 0.05). The mitochondrial injury and cell death induced by CN + DOG in MPT cells was ameliorated by the addition of fatty acid-free bovine albumin to the cell medium, which reduced cell FFA content during chemical anoxia from 25.0 +/- 3.0 to 10.4 +/- 2.0 micrograms/mg (P < 0.05). The phospholipase A2 (PLA2) inhibitor, mepacrine, also resulted in functional protection and reduction of cell FFA content from 20.2 +/- 2.3 to 15.9 +/- 1.7 micrograms/mg (P < 0.05). These data suggest a role for phospholipase activation and accumulation of toxic lipid metabolites in the pathophysiology of MPT cell injury. We then compared cell injury induced by CN + DOG in MPT and MDCK cells. Despite comparable reduction in cell ATP content in the two cell types, injury was far more severe in MPT than MDCK cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of human apolipoprotein E but not that of apolipoprotein A-I by mouse C127 cells is associated with increased secretion of lipids in the form of vesicles and discs.

Transfected mouse mammary-derived cells (C127) expressing human apolipoprotein (apo) E (C127E) were used a) to determine whether the lipid-binding character of apoE is sufficient to promote its assembly with lipid to form lipoprotein-like particles when expressed by cells not normally expressing apolipoproteins; b) to characterize the secreted complexes in terms of morphology, size, and composition; and finally c) to determine whether apoE or apoA-I gene expression by these transfected cells has any effect on the levels and the profiles of the synthesized and secreted lipids. The findings of the present study demonstrate that: a) as determined by density gradient ultracentrifugation and gel filtration chromatography, about 20% of the secreted [35S]methionine-labeled apoE expressed by C127E cells is lipid-associated. b) Negative-stain electron microscopic analysis of the lipid-protein complexes recovered in the lipoprotein fractions (d less than 1.21 g/ml) revealed that approximately 13% of the total population of particles were discs (16 +/- 5 nm mean diameter and 4-6 nm thick), resembling nascent high density lipoproteins (HDL). The majority of the particles however (greater than 82%) appeared vesicular with varying diameters (48 +/- 40 nm mean diameter). The discoidal and the vesicular appearance of the particles secreted by C127E cells is consistent with the composition of lipids. These consisted mostly of surface lipids, phospholipids (45 +/- 18%), diacylglycerols (36 +/- 17%), and free cholesterol (17 +/- 7%) (by weight). c) Expression of apoE by C127E cells was associated with an increased release of [35S]methionine-labeled protein and [3H]glycerol-labeled lipid (3- to 5- and 4- to 8-fold, respectively) compared to nontransfected C127 cells. Expression of mutant apoE or normal apoA-I, however, was not associated with increased release of the major lipid classes compared to the parent C127 cells, strongly suggesting that this character of C127E cells is specific to apoE expression. The release of lipids by C127E cells could be reduced considerably by the addition of the metabolic inhibitors, colchicine or cycloheximide (10 and 1 microM, respectively), suggesting that lipid release by C127E cells is an active process requiring both protein synthesis and functional secretory mechanisms. Taken together the findings suggest that apoE expression by C127 cells promotes the formation of nascent discoidal lipoprotein-like particles and enhances the release of vesicular lipids, possibly by promoting shedding of cell plasma membrane fragments.

A comparison of lipoprotein secretion, bile production and hepatic morphology in isolated rat livers perfused with a perfluorocarbon emulsion or rat erythrocytes.

Isolated rat livers were perfused with an oxygenated perfluorocarbon emulsion, FC-43 emulsion for 1 to 4 hr. FC-43 emulsion contained 20% FC-43 (wt/vol) perfluorotributylamine (the fluorocarbon component for the transport of oxygen and carbon dioxide) emulsified with 2.56% Pluronic F-68 (a nonionic surfactant) in Krebs-Ringer bicarbonate buffer. FC-43 emulsion also contained 3% hydroxyethyl starch as an oncotic agent and 1.8 mg/ml glucose. The viability (oxygen consumption), bile secretion, structural integrity and secretion of nascent lipoproteins by FC-43-perfused rat livers was compared with livers perfused with Krebs-Henseleit bicarbonate buffer that contained rat erythrocytes (25% hematocrit) and 1.5 mg/ml glucose (red blood cell medium). Oxygen consumption was somewhat higher in livers perfused with FC-43 emulsion. Bile secretion of livers perfused with FC-43 emulsion for 4 hr was reduced significantly to 40% of that by red blood cell medium. The structural integrity of livers perfused with FC-43 emulsion varied from normal to marked cellular damage. Light-microscopical examination of rat livers perfused with FC-43 emulsion showed ballooning of sinusoids, presence of vacuoles in sinusoidal lining cells in some hepatocytes and detachment of endothelium in sinusoids. The number of vacuoles progressively increased in longer perfusions. Electron-microscopical studies showed the presence of small (60 to 100 nm) vesicles of varying electron density, presumably fluorocarbon particles inside the vacuoles in sinusoidal lining cells (Kupffer and endothelial) and hepatocytes. After 4 hr of perfusion with FC-43 emulsion, most of the sinusoidal endothelia were denuded, and the microvilli of the hepatocytes all but disappeared. In contrast, the ultrastructure of rat livers perfused with red blood cell medium for 4 hr was unaltered. The accumulation of nascent lipoproteins in perfusates of FC-43-perfused livers was markedly reduced, and no normal very-low-density lipoprotein, low-density lipoprotein or high-density lipoprotein were isolated. Chemical analysis showed the presence of Pluronic F-68 in all lipoprotein fractions. Our data strongly suggest that, during recirculating liver perfusions with FC-43 emulsion (between 1 and 4 hr), the nonionic surfactant detergent Pluronic F-68 dissociated from the emulsion and markedly affected hepatic structure, lipoprotein secretion and the composition of lipoproteins isolated from perfusate. Therefore FC-43 emulsion is not a suitable liver-perfusion medium for studies of lipoprotein metabolism.

Chemistry in the kitchen. Making ground meat more healthful.

BACKGROUND: The National Cholesterol Education Program recommends a diet containing less than 30 percent of calories in the form of fat, less than 10 percent in the form of saturated fat, and less than 300 mg of cholesterol per day. Since Americans' diets generally exceed these recommendations, we wished to find an easy kitchen method to reduce substantially saturated fat and cholesterol in ground meat. METHODS: Raw ground meat was heated in vegetable oil and rinsed with boiling water to extract fat and cholesterol. The fat-free broth was recombined with the meat to restore flavor. The amounts of total fat, saturated fat, and cholesterol in the meat after extraction were compared with the amounts in meat cooked as patties and in stir-fried, rinsed meat. RESULTS: When raw ground beef containing 9.6 to 20.8 percent fat was cooked as patties and the fat poured off, 6 to 17 percent of the fat and 1.3 to 4.3 percent of the cholesterol were lost. In stir-fried, rinsed ground beef, 23 to 59 percent of the fat and 9.0 to 18.8 percent of the cholesterol were lost. When vegetable oil was used to extract fat and cholesterol from beef containing 20.7 percent fat, a mean (+/- SD) of 67.7 +/- 1.6 percent of the fat and 39.2 +/- 5.1 percent of the cholesterol were lost. The differences between conventionally cooked meat and meat prepared by the extraction of fat were significant (P less than 0.001). An average of 43 percent (range, 38 to 49) of cholesterol was extracted from a wide variety of ground meats. Although conventional cooking produced no change in fatty-acid composition as compared with raw meat, our extraction process greatly increased the ratio of unsaturated to saturated fat, from 1.32 in conventionally cooked meat to 2.92 to 4.56 in meat after extraction. Extraction resulted in the loss of 72 to 87 percent of saturated fat. CONCLUSIONS: This method produces a tasty meat product that is much lower in saturated fat and cholesterol than conventionally cooked meat, and that can be used in sauces, soups, and solid meat products.

Colorimetric assay for pluronic F-68 as measured in isolated rat liver perfusion systems.

A colorimetric assay has been developed for the quantitation of Pluronic F-68, a nonionic detergent (surfactant) which is a polyoxypropylene-polyoxyethylene (POP-POE) block copolymer. We measured this substance in organic extracts of rat liver perfusates from livers which had been perfused with an oxygenated perfluorocarbon, FC-43 Emulsion (Oxypherol).

Chemical and physiochemical comparison of advanced atherosclerotic lesions of similar size and cholesterol content in cholesterol-fed New Zealand White and Watanabe Heritable Hyperlipidemic rabbits.

Watanabe Heritable Hyperlipidemic (WHHL) and cholesterol-fed New Zealand White (CH-FED NZW) rabbits were sacrificed at 15 months of age or after 16 weeks of cholesterol feeding, respectively. During the experimental period, the arterial walls of both the CH-FED NZW and WHHL rabbits were exposed to similar amounts of cholesterol and the lesions which developed at the aortic arch had similar intimal thicknesses, total lipid and cholesterol content. However, the lesions of the WHHL rabbits morphologically resembled human plaques, and contained lipid in the form of smectic liquid crystalline droplets and cholesterol monohydrate crystals. The CH-FED NZW rabbits had lesions which were fatty streak-like, containing liquid crystalline cholesteryl ester droplets but few crystals. The aortic arch intimas of the CH-FED NZW rabbits contained significantly more cholesteryl ester, and less unesterified cholesterol and triglyceride, than those of the WHHL rabbits. The intimal compositions of the two rabbit models did not overlap. Analysis of the compositions predicted precipitation of cholesterol monohydrate crystals in the WHHL but not the CH-FED NZW. The physical state of the deposited cholesterol esters was similar in both with about half being in smectic liquid crystalline form at body temperature. Since the size and total lipid content of the lesions of the CH-FED NZW and WHHL rabbits were similar, we suggest that the greater time of exposure to hypercholesterolemia was important in the formation of cholesterol monohydrate crystal-containing plaques in the aortic arch of the WHHL rabbits.

Acyl migration in 1,2-dipalmitoyl-sn-glycerol.

The acyl migration of 1,2-dipalmitoyl-sn-glycerol (1,2-DPG) to 1,3-dipalmitoylglycerol (1,3-DPG) in different states, neat, in the presence of egg yolk lecithin (sonicated and unsonicated) and on silica gel was studied. The isomerization was quantitated by scanning densitometry of charred TLC plates, at different temperatures and for varying periods of time. At equilibrium the amount of 1,3-DPG was found to be 56%. The rates of initial isomerization, and the time required to isomerize to half the equilibrium quantity (i.e., t1/2 eq. = 1,3-DPG 28%) under the above conditions was estimated. In the case of neat melt at 74 degrees C and in an organic solvent the time required to t1/2 eq. is 18 h and a few days, respectively. However, at 62 degrees C in the presence of a polar solvent (sodium phosphate buffer at pH 7.0) the t1/2 eq. is 1-2 h. On dry silica gel (TLC plate) at 24 degrees C the t1/2 eq. is reached in less than 1 h.

Physico-chemical properties of cholesterol-fed rabbit beta-VLDL are not affected by different dietary oils.

Various oils have been used as vehicles for cholesterol in diets used to produce atherosclerosis in rabbits. Because such oils may affect the physico-chemical properties of the beta-VLDL produced in response to cholesterol feeding, we have studied the physico-chemical characteristics of beta-VLDL isolated from cholesterol-fed rabbits using several different oils as vehicles (corn, safflower, cod liver, and peanut oils). All animals developed severe hypercholesterolemia by 2 weeks. During the second and third weeks on diet the apo beta-containing lipoproteins began to develop thermal transitions due to order-disorder cholesterol ester transitions in the lipoproteins. By 4 weeks the apo beta-containing lipoproteins (overwhelmingly beta-VLDL) had transitions which were quite similar (transition temperatures 41.0-42.5 degrees C). No statistical differences were noted between the transition temperatures or enthalpies of any of the dietary groups. Thus, the physico-chemical properties of the beta-VLDL of rabbits fed cholesterol appear to be quite similar, despite the vehicles used to carry the cholesterol in the diet. Thus other mechanisms must be looked for to explain the different atherogenicity of different oils used as cholesterol carrying vehicles.

Uptake of artificial model remnant lipoprotein emulsions by the perfused rat liver.

In comparison with their precursor lipoproteins, the remanants of the triacylglycerol-rich lipoproteins are reduced in contents of triacylglycerols and apolipoproteins AI and AIV, whereas the contents of cholesterol (free and esterified) and apolipoprotien E are increased. In this study, lipid emulsion models of remnant lipoproteins were used to explore which of these factors are necessary for physiological rates of remnant uptake by the perfused rat liver. Uptake rates of lipid emulsion models of remnant lipoproteins in the presence of apolipoprotein E were similar to in vivo uptake rates.

Plasma lipoproteins of free-ranging howling monkeys (Alouatta palliata).

1. Plasma lipids and lipoproteins of free-ranging howling monkeys from Costa Rica (Alouatta palliata), aged 5 months to 23 years, were characterized. 2. High density lipoproteins were lipid-rich, similar to HDL2 of human plasma. 3. Fatty acid compositions of major lipid classes of very low, low and high density lipoproteins differed among social groups, possibly due to both dietary and genetic factors. 4. Low and high density lipoprotein phospholipids were enriched in phosphatidylethanolamine. 5. Howler plasma cross reacted with antihuman apoA-I antibodies but not with antihuman LDL antibodies. 6. No dimeric form of apoA-II was present, unlike human apoA-II.

Effects of cholesterol content on the metabolism of protein-free emulsion models of lipoproteins.

After intravenous injection, emulsions with compositions similar to chylomicrons behaved metabolically as described for chylomicrons, with faster removals of triacylglycerols than cholesteryl esters from the blood after injection into rats, and with greater uptakes of cholesteryl esters than triacylglycerols by the liver. In contrast, emulsions with a high content of free cholesterol showed equal removal rates from the blood of triacylglycerols and cholesteryl esters; and similar uptakes by the liver. This pattern of metabolism was that expected for a chylomicron core remnant particle. Emulsions poor in cholesteryl ester but rich in free cholesterol showed remnant-like behavior, whereas emulsions rich in cholesteryl ester but poor in free cholesterol were metabolized like nascent chylomicron particles. The amount of free cholesterol appeared to regulate metabolism by affecting the binding of apolipoproteins to the particle surface. Emulsions with a high content of free cholesterol bound less A-I, A-IV and C apolipoproteins, and the relative amount of apolipoprotein E was increased. All of these effects are consistent with the metabolic differences between chylomicrons and remnant particles, suggesting that the amount of free cholesterol plays a regulatory role in chylomicron metabolism.

Reduced cholesterol transmucosal transport in rats with inhibited mucosal acyl CoA:cholesterol acyltransferase and normal pancreatic function.

Absorption of cholesterol during inhibition of mucosal acyl CoA:cholesterol acyltransferase was studied in mesenteric lymph fistula rats with normal pancreatic function. The specific inhibitor used (Sandoz Compound 58-035; 3-(decyldimethylsilyl)-N-[2-(4-methylphenyl)-1-phenylethyl]prop anamide) greatly reduced cholesterol esterification in vitro and decreased lymphatic secretion of esterified cholesterol in vivo, but did not affect triglyceride metabolism by the gut in vitro or in vivo. During steady state lymphatic transport of cholesterol, unesterified cholesterol was increased and cholesteryl esters were decreased in whole lymph, lymph chylomicrons, and very low density lipoproteins. Incorporation of labeled cholesterol infused into the lumen into cholesteryl esters of lymph very low density lipoproteins was particularly suppressed. Labeled cholesterol incorporation into individual cholesteryl esters differed and was differentially affected when total cholesteryl ester synthesis was inhibited. The results support a major regulatory role for mucosal acyl CoA:cholesterol acyltransferase in cholesterol absorption and imply differences in the metabolism of endogenous and exogenous cholesterol by the intestinal mucosa.

Relationship of an abnormal plasma lipoprotein to protection from atherosclerosis in the cholesterol-fed diabetic rabbit.

Alloxan-diabetic rabbits develop a pronounced hypercholesterolemia and hypertriglyceridemia in response to cholesterol feeding. Despite higher levels of plasma cholesterol, these animals have much less atherosclerosis than cholesterol-fed nondiabetics. To determine whether this effect is due to properties of the lipoproteins, we compared chemical, physical, and metabolic characteristics of a very low density lipoprotein (VLDL) fraction (d less than 1.019 g/ml) from the diabetic and nondiabetic cholesterol-fed rabbits. The molar ratio of triglyceride to cholesteryl ester in the particles from diabetic animals ranged from 2:1 to 6:1, and this ratio remained constant in subfractions from individual rabbits. Triglyceride from nondiabetic control animals was a minor component. Differential scanning calorimetry showed a distinct order-disorder phase transition for cholesteryl ester at approximately 42 degrees C in the fractions from control animals, whereas in fractions from most of the diabetics no such transition was observed, indicating that both triglyceride and cholesteryl ester are present in the core of the same particle. The relative amount of apoprotein E in particles from diabetic animals was much less than that of cholesterol-fed controls. The ability of the lipoproteins from both groups to stimulate cholesteryl ester formation in mouse peritoneal macrophages also was tested. Lipoproteins from cholesterol-fed controls stimulated cholesteryl ester formation in a dose-dependent manner, but particles from the diabetic group had little or no effect. The results suggest that the presence of unusual VLDL particles in diabetic cholesterol-fed rabbits is responsible, at least in part, for the reduced incidence of atherosclerosis in this animal model.

Properties of angiotensin-converting enzyme in intact cerebral microvessels.

Angiotensin-converting enzyme (ACE) was studied in preparations of microvessels isolated from rabbit cerebral cortex. Activity was determined by measuring the degradation of hippuryl-histidyl-leucine (Hip-His-Leu) by the intact microvessels in a physiological salt solution at pH 7.4. ACE activity was dependent on both substrate and chloride ion concentration and was inhibited by captopril in a manner similar to that observed previously with tissue homogenates. Angiotensin I was rapidly degraded by the intact microvessels, even in the presence of 10(-6)M captopril. An advantage of the methodology employed was the ability to pretreat the microvessels and then assess the effect of pretreatment by transfer to a postincubation assay system. Pretreatment with a hyperosmolar urea solution did not change ACE activity or cause release of ACE from the microvessels, although lactic dehydrogenase and lysosomal enzymes were released. Pretreatment with captopril caused a lag in the subsequent degradation of Hip-His-Leu, presumably reflecting dissociation of inhibitor from the cell-associated enzyme. ACE activity was unaffected by hypoxic or anoxic incubation conditions. The ability to measure ACE activity of the microvessels in vitro provides a unique opportunity to study the properties of the enzyme in intact cerebrovascular endothelial cells.

Peptidyl dipeptidase in rabbit brain microvessels.

The activity of peptidyl dipeptidase (peptidyldipeptide hydrolase, EC 3.4.15.1), also known as angiotensin-converting enzyme, was studied in small blood vessel preparations isolated from rabbit brain. The vascular preparation contained arterioles and capillaries and was essentially free of extravascular material. Enzymatic activity was demonstrated in microvessel homogenates using both hippuryl-histidyl-leucine and tritium-labeled angiotensin I as substrates. Activity in the microvessels was dependent on the presence of chloride ion and was sensitive to inhibition by converting enzyme inhibitors previously shown to be effective in both vivo and in vitro. Specific activity in the micro-vessels was approximately 20 times that in homogenates of brain, and was almost 60% of that found in rat lung homogenates. The data were consistent with an endothelial localication for peptidyl dipeptidase in the cerebral vasculature and supports the proposal that this enzyme has a physiological role in extrapulmonary vascular beds.