Control of ACAT2 liver expression by HNF4{alpha}: lesson from MODY1 patients.

OBJECTIVE: ACAT2 is thought to be responsible for cholesteryl ester production in chylomicron and VLDL assembly. Recently, we identified HNF1alpha as an important regulator of the human ACAT2 promoter. Thus, we hypothesized that MODY3 (HNF1alpha gene mutations) and possibly MODY1 (HNF4alpha, upstream regulator of HNF1alpha, gene mutations) subjects may have lower VLDL esterified cholesterol. METHODS AND RESULTS: Serum analysis and lipoprotein separation using size-exclusion chromatography were performed in controls and MODY1 and MODY3 subjects. In vitro analyses included mutagenesis and cotransfections in HuH7 cells. Finally, the relevance in vivo of these findings was tested by ChIP assays in human liver. Whereas patients with MODY3 had normal lipoprotein composition, those with MODY1 had lower levels of VLDL and LDL esterified cholesterol, as well as of VLDL triglyceride. Mutagenesis revealed one important HNF4 binding site in the human ACAT2 promoter. ChIP assays and protein-to-protein interaction studies showed that HNF4alpha, directly or indirectly (via HNF1alpha), can bind to the ACAT2 promoter. CONCLUSIONS: We identified HNF4alpha as an important regulator of the hepatocyte-specific expression of the human ACAT2 promoter. Our results suggest that the lower levels of esterified cholesterol in VLDL- and LDL-particles in patients with MODY1 may-at least in part-be attributable to lower ACAT2 activity in these patients.

Plasma lipid transfer protein as a determinant of the atherogenicity of monkey plasma lipoproteins.

This study was undertaken to determine potential tissue sources of plasma cholesteryl ester transfer protein (CETP), and to assess the influence of CETP on lipoprotein concentrations and atherosclerosis. In a group of 28 cynomolgus monkeys fed high fat, high cholesterol diets, plasma CETP concentration was strongly correlated with the abundance of CETP mRNA in liver and in adipose tissue, and with the output of CETP in liver perfusates. Plasma CETP concentration showed a strong inverse correlation with HDL cholesterol concentrations (r = -0.62, P less than 0.001) and a positive correlation with LDL cholesterol concentration (r = 0.54, P less than 0.005) and molecular weight (r = 0.57, P less than 0.001). The extent of coronary artery atherosclerosis was positively correlated with LDL cholesterol concentration and molecular weight, and with plasma CETP concentration. Thus, in monkeys fed an atherogenic diet, individual variation in CETP mRNA abundance in liver and adipose tissue probably plays a major role in the determination of plasma CETP levels. In plasma, CETP influences the distribution of cholesteryl esters between LDL and HDL, and CETP concentration appears to be a key determinant of the relative atherogenicity of the plasma lipoproteins.

The transport of exogenous cholesterol in the rabbit. I. Role of cholesterol ester of lymph chylomicra and lymph very low density lipoproteins in absorption.

Thoracic lymph duct cannulations were performed shortly after a meal in rabbits trained to ingest a moderate fat, low cholesterol diet. A tracer dose of cholesterol-(3)H was administered to label exogenous (dietary) cholesterol during absorption. Sequential lymph samples were collected up to 24 hr postprandially, after which ultracentrifugal fractionation of lymph lipoproteins was carried out. The d < 1.006 lipoproteins were separated into two classes, chylomicra and very low density lipoproteins (VLDL).A comparison was made between chylomicra and VLDL of lymph in the transport of exogenous cholesterol after ingestion of a single meal. The per cent of exogenous cholesterol present in VLDL of sequential lymph collections progressively increased with time after a meal and by 18 hr had reached a value of 80% or greater. In chylomicra the per cent of exogenous cholesterol of sequential lymph collections progressively decreased. Therefore, exogenous cholesterol was preferentially transported in VLDL compared with chylomicra. Cholesterol ester specific activity (CESA) of lymph chylomicra and VLDL increased at a more rapid rate than free cholesterol specific activity (FCSA). CESA of VLDL was three times higher than FCSA at the maximum. Exogenous cholesterol which appeared in both chylomicra and VLDL was consistently 80% esterified. while the per cent of total cholesterol esterified decreased with time and was significantly lower than that for exogenous cholesterol from 6 to 24 hr postprandially. These results demonstrate preferential esterification of exogenous cholesterol during absorption and indicate that a mechanism exists within the intestinal mucosal cell to maintain both free and esterified exogenous cholesterol in a chemically distinct pool from endogenous cholesterol during incorporation into both chylomicra and VLDL.

Studies on the structure of low density lipoproteins isolated from Macaca fascicularis fed an atherogenic diet.

Cynomolgus monkeys, Macaca fascicularis, fed cholesterol-containing saturated-fat diets develop increased levels of high molecular weight plasma low density lipoproteins (LDL), associated with accelerated atherosclerosis. To study the composition and structure of these abnormal particles, LDL from monkeys, fed atherogenic and control diets, were characterized chemically and examined by differential scanning calorimetry and low-angle X-ray scattering. LDL from animals on the experimental diet showed an increase in molecular weight (4.0 to 7.0 x 10(6), experimental diet compared with 3.0 to 3.7 x 10(6), control diet) associated with a large increase in cholesterol ester content and concomitant smaller increases in protein, phospholipid, and free cholesterol. There was a strong positive correlation between molecular weight and the number of saturated and monounsaturated cholesterol esters in the particle. In contrast, particle content of polyunsaturated cholesterol esters remained constant despite large changes in total particle cholesterol esters.When examined by calorimetry and X-ray scattering, LDL from monkeys on both diets diplayed a reversible transition of cholesterol esters from an ordered smeticlike (layered) structure to a more disordered state. For all animals on the experimental diet, the peak temperature of the cholesterol-ester transition (42-48 degrees C) was above body temperature (39 degrees C), but below body temperature on the control diet (34-38.5 degrees C). In the experimental group, the transition temperature was correlated with the LDL molecular weight. However, after thermal disruption of LDL, liquid-crystalline transitions of LDL cholesterol esters were observed in the same temperature range as in the intact lipoprotein, which shows that changes in particle size had little effect on the cholesterol-ester transition temperature. Rather, the transition temperature was determined by the degree of saturation of the LDL cholesterol ester fatty acids and the LDL cholesterol ester: triglyceride ratio, both of which correlated with increased LDL molecular weight.The existence of smectic-like cholesterol ester in LDL at body temperature was clearly a discriminating feature between monkeys on control and experimental diets. Diet-induced changes in the lipid composition of precursor lipoproteins of LDL appeared to lead to the existence of smectic-like cholesterol ester in LDL above body temperature. The altered composition and structure of the core lipids of high molecular weight LDL probably account, in part, for the previously documented correlation between increased LDL molecular weight and atherosclerosis in this species.

Dietary cholesterol and downregulation of cholesterol 7 alpha-hydroxylase and cholesterol absorption in African green monkeys.

In this study, hepatic production of bile acid was considered together with intestinal cholesterol absorption as potential regulatory sites responsive to dietary cholesterol. Sequential liver biopsies were taken from 45 feral African green monkeys studied during three different diet periods. Low-fat Monkey Chow was fed during the baseline period, a cholesterol and fat-enriched diet was then fed for 12 wk during period 2, and finally, after a washout period of 10 wk, three subgroups were fed low-, moderate-, and high-cholesterol diets for 12 mo during period 3. The percentage of cholesterol absorbed in the intestine was significantly lower when higher levels of cholesterol were fed; however, this percentage was significantly and positively correlated to plasma cholesterol concentration at each dietary cholesterol level. Hepatic free and esterified cholesterol content were significantly elevated by dietary cholesterol challenge and remained elevated even after 20 wk of low-cholesterol diets. Hepatic mRNA abundance for cholesterol 7 alpha-hydroxylase (C7H) was significantly lower (approximately 60%) when the high-cholesterol diet was fed, with the decrease being greater than that seen for low density lipoprotein (LDL) receptor mRNA. At the same time, hepatic mRNA abundance for apolipoprotein B and hepatic lipase were not diet sensitive. C7H activity was decreased to a similar extent by diet as was C7H mRNA, although the correlation between enzyme activity and mRNA abundance was only r = 0.5, suggesting that dietary regulation includes factors in addition to transcriptional regulation. Activity and mRNA abundance of C7H remained decreased when liver esterified cholesterol content was reduced to only a two- to three-fold elevation over baseline, at a time when plasma cholesterol and hepatic LDL receptor mRNA abundance had returned to baseline levels. These data on liver C7H, obtained in one of the few primate species predisposed to cholesterol gallstone formation, support the hypothesis that the liver may attempt to downregulate intestinal cholesterol absorption by decreasing bile acid production when increased amounts of absorbed dietary cholesterol reach the liver. Presumably this represents attempted downregulation of intestinal cholesterol absorption by limiting bile acid availability as a means to maintain hepatic cholesterol balance.

Studies on the production of low density lipoproteins by perfused livers from nonhuman primates. Effect of dietary cholesterol.

Nonhuman primates consuming diets containing cholesterol develop coronary artery atherosclerosis that we have found to be highly correlated with an increase in the size and cholesteryl ester content of plasma low density lipoproteins (LDL). The present studies were designed to determine whether the enlarged plasma LDL are produced directly by the liver of cholesterol-fed monkeys. African green monkeys were fed a diet containing 40% of calories as butter fat and either 0.16 mg cholesterol/kcal (control diet) or 0.78 mg cholesterol/kcal (test diet). The livers of these monkeys were perfused by recirculation with a lipoprotein-free medium for 4 h. The rate of accumulation of perfusate cholesterol was linear and greater in liver perfusates from test diet-fed vs. control diet-fed monkeys and was positively correlated with both the plasma cholesterol concentration and LDL size in the donor animal. All perfusate d less than 1.063 g/ml lipoprotein subfractions from livers of test diet-fed monkeys were enriched in cholesteryl ester severalfold over the corresponding subfractions from control diet-fed monkeys and contained only the larger form of apolipoprotein B typical of plasma LDL. However, the perfusate lipoproteins in the LDL density range did not have an average size or composition typical of LDL from plasma. Rather, they were relatively enriched in phospholipid and unesterified cholesterol and were deficient in cholesteryl esters. In addition, perfusate high density lipoproteins were discoidal particles. These data show that the enzyme lecithin:cholesterol acyltransferase (LCAT) was essentially inactive in these perfusates and, as a result, the dietary cholesterol-induced enrichment of perfusate d less than 1.063 g/ml lipoproteins with cholesteryl esters probably resulted from increased hepatic secretion of cholesteryl esters and not from modification of lipoproteins by LCAT during recirculating perfusion. In spite of this increase, enlarged cholesteryl ester-rich LDL were not found in the perfusate, suggesting that large molecular weight plasma LDL are not directly secreted by the liver but instead probably result from further intravascular metabolism of cholesteryl ester-enriched hepatic precursor lipoproteins.

Hepatic origin of cholesteryl oleate in coronary artery atherosclerosis in African green monkeys. Enrichment by dietary monounsaturated fat.

Relationships among plasma lipoprotein cholesterol, cholesterol secretion by the isolated, perfused liver, and coronary artery atherosclerosis were examined in African green monkeys fed diets containing cholesterol and 35% of calories as fat enriched in polyunsaturated, monounsaturated, or saturated fatty acids. The livers of animals fed monounsaturated fat had significantly higher cholesteryl ester concentrations (8.5 mg/g wet wt) than the livers of the other diet groups (3.65 and 3.37 mg/g wet wt for saturated and polyunsaturated fat groups, respectively) and this concentration was highly correlated with plasma cholesterol and apoB concentrations in each diet group. Cholesteryl oleate was 58 and 74. 5% of the liver cholesteryl ester in the saturated and monounsaturated fat groups. In each diet group, perfusate cholesteryl ester accumulation rate was highly correlated to liver and plasma cholesterol concentrations, and to plasma LDL cholesteryl ester content. Cholesteryl oleate was 48 and 67% of the cholesteryl esters that accumulated in perfusate in the saturated and monounsaturated fat animals, and this percentage was very highly correlated (r = -0.9) with plasma apoB concentration. Finally, in these two diet groups, liver perfusate cholesteryl ester accumulation rate was well correlated (r >/= 0.8) to coronary artery cholesteryl ester concentration, a measure of the extent of coronary artery atherosclerosis that occurred over the five years of diet induction in these animals. These data define an important role for the liver in the cholesteryl oleate enrichment of the plasma lipoproteins in the saturated and monounsaturated fat groups, and demonstrate strong relationships among hepatic cholesteryl ester concentration, cholesteryl ester secretion, and LDL particle cholesteryl ester content. The high correlation between liver cholesteryl ester secretion and coronary artery atherosclerosis provides the first direct demonstration of the high degree of importance of hepatic cholesteryl ester secretion in the development of this disease process. The remarkable degree of enrichment of cholesteryl oleate in plasma cholesteryl esters of the monounsaturated fat group may account for the relatively high amount of coronary artery atherosclerosis in this group.

Effects of dietary cholesterol and fatty acids on plasma lipoproteins.

The effects of dietary cholesterol and fatty acids on low density and high density lipoproteins (LDL and HDL) were studied in 20 young men. After 2-3 wk of evaluations on ad lib. diets, basal diets, which consisted of 15% protein, 45% carbohydrates, 40% fat, and 300 mg/day of cholesterol, were given for 4-5 wk (Basal). The ratio of dietary polyunsaturated to saturated fatty acids (P/S) for different groups of subjects were 0.25, 0.4, 0.8, or 2.5. 750 and 1,500 mg/d of cholesterol were added to the basal diets as 3 and 6 eggs, respectively. Total cholesterol and LDL cholesterol were lower in all subjects on the basal diets than on the ad lib. diets. Addition of 750 mg cholesterol to the diet with P/S = 0.25-0.4 raised LDL cholesterol by 16 +/- 14 mg/dl to 115% of basal diet values (n = 11, P less than 0.01); 1,500 mg increased LDL cholesterol by 25 +/- 19 mg/dl to 125% (n = 9, P less than 0.01). On the diet with P/S = 0.8, 750 mg produced insignificant increases in LDL cholesterol, but 1,500 mg produced increases of 17 +/- 22 mg/dl to 115% of basal (n = 6, P less than 0.02). On the P/S = 2.5 diet, neither 750 nor 1,500 mg produced significant changes. Thus, both the cholesterol contents and P/S ratios of diets were important in determining LDL levels. The lipid and apoprotein compositions, flotation rates, molecular weights, and binding by cellular receptors of LDL were virtually unchanged by the addition of cholesterol to the diets high in saturated fat. These diets, therefore, caused an increase in the number of LDL particles of virtually unchanged physical and biological properties. On the diet with low P/S ratio, HDL2 rose, whereas this effect was absent on diets with high P/S ratios. The response of LDL to dietary manipulations is consonant with epidemiologic data relating diets high in cholesterol and saturated fat to atherogenesis. The response of HDL2, however, is opposite to that of its putative role as a negative risk factor. Further work is needed to clarify this interesting paradox.

Defining the atherogenicity of large and small lipoproteins containing apolipoprotein B100.

Apo-E-deficient apo-B100-only mice (APOE:(-/-)APOB:(100/100)) and LDL receptor-deficient apo-B100-only mice (LDLR:(-/-)APOB:(100/100)) have similar total plasma cholesterol levels, but nearly all of the plasma cholesterol in the former animals is packaged in VLDL particles, whereas, in the latter, plasma cholesterol is found in smaller LDL particles. We compared the apo-B100-containing lipoprotein populations in these mice to determine their relation to susceptibility to atherosclerosis. The median size of the apo-B100-containing lipoprotein particles in APOE:(-/-)APOB:(100/100) plasma was 53.4 nm versus only 22.1 nm in LDLR:(-/-)APOB:(100/100) plasma. The plasma levels of apo-B100 were three- to fourfold higher in LDLR:(-/-)APOB:(100/100) mice than in APOE:(-/-)APOB:(100/100) mice. After 40 weeks on a chow diet, the LDLR:(-/-)APOB:(100/100) mice had more extensive atherosclerotic lesions than APOE:(-/-)APOB:(100/100) mice. The aortic DNA synthesis rate and the aortic free and esterified cholesterol contents were also higher in the LDLR:(-/-)APOB:(100/100) mice. These findings challenge the notion that all non-HDL lipoproteins are equally atherogenic and suggest that at a given cholesterol level, large numbers of small apo-B100-containing lipoproteins are more atherogenic than lower numbers of large apo-B100-containing lipoproteins.

ACAT1 and ACAT2 membrane topology segregates a serine residue essential for activity to opposite sides of the endoplasmic reticulum membrane.

A second form of the enzyme acyl-CoA:cholesterol acyltransferase, ACAT2, has been identified. To explore the hypothesis that the two ACAT enzymes have separate functions, the membrane topologies of ACAT1 and ACAT2 were examined. A glycosylation reporter and FLAG epitope tag sequence was appended to a series of ACAT cDNAs truncated after each predicted transmembrane domain. Fusion constructs were assembled into microsomal membranes, in vitro, and topologies were determined based on glycosylation site use and accessibility to exogenous protease. The accessibility of the C-terminal FLAG epitope in constructs was determined by immunofluorescence microscopy of permeabilized transfected cells. Both ACAT1 and ACAT2 span the membrane five times with their N termini in the cytosol and C termini in the ER lumen. The fourth transmembrane domain is located in a different region for each protein, placing the putative active site ACAT1 serine (Ser(269)) in the cytosol and the analogous residue in ACAT2 (Ser(249)) in the ER lumen. Mutation of these serines inactivated the ACAT enzymes. The outcome is consistent with the hypothesis that cholesterol ester formation by ACAT2 may be coupled to lipoprotein particle assembly and secretion, whereas ACAT1 may function primarily to maintain the balance of free and esterified cholesterol intracellularly.

Heterogeneity in the low density lipoproteins of cholesterol fed African green monkeys (Cercopithecus aethiops).

The low density lipoproteins of cholesterol-fed African green monkeys were isolated by either preparative ultracentrifugation or agarose column chromatography, then radioiodinated and fractionated by agarose column chromatography. A marked variation was noted in the specific radioactivity of the column fractions regardless of the method used to obtain low density lipoproteins. The radioactivity in each fraction migrated with the beta-globluins, but when the fractions were mixed with carrier plasma, alpha-migrating radioactivity was also detected which was associated with the apo-A-I and apo-C of the high density lipoproteins. The uptake of these apoproteins by the high density lipoproteins reduced the variation in specific radioactivity among the low density lipoprotein fractions. By successive injections of the various 125I-labeled low density lipoprotein fractions into the same recipient, it was also possible to compare the kinetic behavior of the fractions. Large and small low density lipoprotein particles were metabolized differently, indicating that the low density lipoproteins from cholesterol-fed African green monkeys are heterogeneous.

Detection of immunological heterogeneity of an isolated, purified protein (vervet apolipoprotein A-I).

Using a single goat antiserum, we have identified immunological heterogeneity of purified apolipoprotein A-I from high density lipoprotein of vervet monkeys. We examined whether the apparent heterogeneity was due to separate antigenic sites within the polypeptide sequence or rather on the different isoproteins, which result in charge heterogeneity of this protein. The apolipoprotein A-I was cleaved with cyanogen bromide and the resulting three fragments were purified and characterized. By using immunodiffusion, each of the fragments was found to show a characteristic and different reaction to the antiserum. By contrast, apparent identity was found by immunodiffusion among the separate isoprotein forms of apolipoprotein A-I. We have concluded that the immunological heterogeneity of apolipoprotein A-I was due to different antigenic sites within the primary sequence of apolipoprotein A-I.

Metabolism of lipoprotein acylglycerols by liver parenchymal cells.

We investigated the metabolism by hepatocyte suspensions of the acylglycerols in lipoprotein remnants as well as those associated with albumin and low or high density lipoproteins. Remnants, albumin and plasma lipoproteins, rich in monoacylglycerol were prepared by short-term incubations of radio-labeled chylomicra or very low density lipoproteins with extrahepatic lipoprotein lipase in the presence of albumin and low and high density lipoproteins. We demonstrated that liver parenchymal cells contain an active monoacylglycerol acyltransferase that is located on the extracellular surface of the cell plasma membrane. Further, the enzyme is capable of degrading the monoacylglycerol in all the above forms. Triacylglycerol in intact chylomicra and very low density lipoproteins were not metabolized by the cells to any appreciable degree. The degradation of the remnant triacylglycerol appeared to depend solely on the activity of the lipoprotein lipase bound to the lipoprotein remnants. Little uptake of intact lipoprotein acylglycerols by the hepatocytes was observed; instead, hydrolysis of the substrates in the medium always preceded the uptake of the products. The products were then utilized for the synthesis of triacylglycerol and phospholipid within the cells.

Neutral lipid transfer and lipolysis convert high molecular weight LDL from cholesterol-fed nonhuman primates towards normal: a molecular analysis.

In cynomolgus monkeys (Macaca fascicularis) fed an atherogenic diet, large, cholesterol ester-rich LDL (Mr greater than 3.5.10(6] are found at the same time that the plasma triacylglycerol levels are low. We studied whether the presence of higher concentrations of triacylglycerol-rich lipoproteins (VLDL) during in vitro incubations would allows depletion from LDL of cholesterol ester and a decreased LDL molecular weight. Three high Mr LDL (Mr = (3.7-4.8).10(6)), rich in cholesterol ester (50 +/- 1.4% by weight), were isolated from three animals by zonal ultracentrifugation, and were then incubated with human VLDL at 37 degrees C for 18 h in lipoprotein-deficient human plasma containing neutral lipid transfer activity. After incubation, modified LDL (M-LDL) was isolated by zonal ultracentrifugation. M-LDL was triacylglycerol-rich (36 +/- 5% by weight) and cholesterol ester-poor (20 +/- 3%), and cholesterol ester had transferred into VLDL. Purified lipoprotein lipase was added to the M-LDL, and triacylglycerol was hydrolyzed. The size of the post-lipolysis M-LDL (Mp-LDL) particles became smaller (mean diameters of 253 A and 228 A for two native LDLs and 215 A and 193 A for Mp-LDL, respectively). Both analytical and zonal ultracentrifugation showed Mp-LDL to be more dense than native LDL. Estimated molecular weights for Mp-LDL were 40%-50% less than that of the original LDL, and fell within the molecular weight range for normal human and monkey LDL. Lipid exchanges, but not apoprotein transfers, were responsible for LDL remodelling, as supported by three separate methods of analysis. Cholesterol ester losses accounted for about two-thirds of the molecular weight decrease. These in vitro results suggest that cholesterol ester enrichment of apoprotein B lipoprotein particles can be reversed by providing adequate levels of VLDL in the presence of neutral lipid transfer processes and lipolytic activity.

Determination of cholesterol using o-phthalaldehyde.

A simple, rapid method for the determination of cholesterol in plasma and tissue using o-phthalaldehyde is presented. Comparison of this method with the FeCl(3) method gave identical results. However, the o-phthalaldehyde determination is three times more sensitive than the FeCl(3) determination (molar extinction coefficients of 11,610 and 33,440 for FeCl(3) and o-phthalaldehyde, respectively), it takes less time to complete, and the color developed is more stable. The o-phthalaldehyde method can be used to assay free and esterified cholesterol directly after thin-layer chromatographic separation.

Increased circulating levels of an endogenous digoxin-like factor in hypertensive monkeys.

An endogenous, immunoreactive digoxin-like factor (endoxin) was measured in the plasma of nonhuman primates with hypertension. Both normotensive and hypertensive rhesus monkeys had levels of endoxin that significantly correlated with their systolic or diastolic blood pressure. Vervet monkeys with experimentally produced chronic Goldblatt hypertension had significantly elevated endoxin, but not plasma renin. These data suggest that increased plasma endoxin may be a contributing factor in the development of hypertension.

A comparison of the kinetics of low-density lipoprotein oxidation initiated by copper or by azobis (2-amidinopropane).

This article describes the kinetics of low density lipoprotein (LDL) oxidation catalyzed by azobis (2-amidinopropane) dihydrochloride, ABAP, or by copper. The LDLs were isolated from nonhuman primates fed diets enriched in one of three types of fatty acids: saturated fatty acids, monounsaturated fatty acids, predominantly, oleic acid, or polyunsaturated fatty acids, predominantly linoleic acid. Oxidation was followed by monitoring the formation of conjugated diene hydroperoxides from polyunsaturated fatty acids (PUFA). For both copper and ABAP-initiated oxidation, the rate of LDL oxidation depended on the concentrations of initiator, PUFA, and LDL. Except for the dependence on PUFA concentration the rate of LDL oxidation was not directly influenced by the fatty acid composition of the LDL particle. The two initiators had very different dependence on initiator concentration. Because LDL particles are essentially small, lipid-rich droplets, the kinetic descriptions of LDL oxidation assumed: (1), that there was only one chain per particle, and (2) that the radical chain was terminated when a second radical either entered or was formed in the particle. When two LDL samples having very different lag times were mixed, the oxidation profile was bimodal. This finding demonstrated that the oxidation of native LDL particles was independent of the oxidation state of the other native LDL particles in solution, i.e., LDL particles do not rapidly exchange radicals, for example, hydroperoxyl radicals. Oxidation initiated by ABAP was proportional to [ABAP]0.5, suggesting that hydroperoxyl radical recombination between the lipid hydroperoxyl radical and the ABAP-hydroperoxyl radical was the chain-terminating step. The reciprocal of the rate of copper oxidation was linearly related to the reciprocal copper concentration, demonstrating that the binding of copper to LDL was necessary to initiate oxidation. This binding constant showed considerable variability among LDL samples. The kinetic descriptions of LDL oxidation reflect the differences in the mechanisms of initiation and termination.

Isoprostane levels in lipids extracted from atherosclerotic arteries of nonhuman primates.

Nonhuman primates used in these studies had been fed for 5 years diets enriched with cholesterol and one of three classes of fatty acids: saturated, monounsaturated, or polyunsaturated fatty acids. Atherosclerotic iliac artery lipid extracts were quantitatively analyzed for cholesterol, cholesteryl esters, fatty acid composition, and a marker of lipid oxidation, the F(2)-isoprostanes. There was no significant difference in the mean accumulation of F(2)-isoprostanes among the different diet groups. To account for the small, individual variation in the arachidonate concentration the F(2)-isoprostane mass from each sample was normalized by dividing by arachidonate mass: F(2)-isoprostane mass/(mass arachidonate). At lower levels of cholesterol accumulation, the F(2)-isoprostane mass/(mass arachidonate) ratio was greater in lipids from POLY arteries compared to SAT arteries, but the reverse was true at high levels of cholesterol. F(2)-isoprostane/(mass arachidonate) increased with mole fraction linoleate for the SAT group, but decreased for the POLY group. In summary, these studies demonstrated that there is no simple explanation of how F(2)-isoprostane accumulation did not depend on the concentration of oxidizable lipids that promote free-radical lipid oxidation.

Dietary polyunsaturated fat modifies low-density lipoproteins and reduces atherosclerosis of nonhuman primates with high and low diet responsiveness.

We tested the hypothesis that an increased content of n-6 polyunsaturated fatty acids (principally linoleic acid) in an atherogenic diet of nonhuman primates would decrease atherosclerosis by modifying the composition and decreasing the concentration of plasma low-density lipoprotein (LDL). A species readily susceptible to diet-induced atherosclerosis (cynomolgus monkey) was compared with a less-susceptible species (African green monkey) with dietary cholesterol concentration and saturated or polyunsaturated fat (40% of energy) as variables. In both species, cholesterol concentrations in whole plasma, LDL, and high-density lipoprotein (HDL) were 20-30% lower when polyunsaturated fat was fed, whereas dietary cholesterol increased LDL cholesterol three- to fourfold. LDL was enriched in cholesteryl oleate when saturated fat and cholesterol were fed. Dietary linoleic acid prevented cholesteryl oleate enrichment and promoted cholesteryl linoleate accumulation in LDL. At the same plasma cholesterol concentration, cynomolgus monkeys had higher LDL cholesterol and lower HDL-cholesterol concentrations than did African green monkeys. LDL particle size was significantly (P < 0.001) larger in the group of cynomolgus monkeys fed polyunsaturated fat but tended to be smaller in African green monkeys fed polyunsaturated fat. Dietary polyunsaturated fat protected against coronary artery atherosclerosis in both species. Thus, LDL particle size, per se, was not atherogenic; instead, coronary artery atherosclerosis and cholesteryl oleate enrichment of LDL were more highly correlated. This outcome suggests that information about LDL composition may be more important for understanding the pathogenesis of atherosclerosis than previously suspected.