L-carnitine administration and withdrawal affect plasma and hepatic carnitine concentrations, plasma lipid and lipoprotein composition, and in vitro hepatic lipogenesis from labeled mevalonate and oleate in normal rabbits.

Carnitine was administered to normal rabbits to investigate the possible effects of pharmacologic doses on various aspects of normal lipid and lipoprotein metabolism. Carnitine concentrations were measured in the plasma and liver of normal rabbits that received L-carnitine orally [40 mg/(kg.d)] for 21 d and after withdrawal from the carnitine supplement for 21 d. Plasma lipids, plasma lipoprotein composition and in vitro hepatic lipid biosynthesis from [2-14C]mevalonate and [1-14C]oleate were also measured. Threefold elevations in plasma carnitine with carnitine treatment were essentially reversed after 48 h of carnitine withdrawal, but elevated hepatic carnitine accumulation (twofold) persisted for 21 d, suggesting that the accumulated carnitine constituted a pool that is only slowly miscible with plasma. The rabbits withdrawn from L-carnitine for 21 d experienced a 35% decrease in plasma cholesterol, a 50% decrease in VLDL cholesterol, and an increase in the protein content of HDL and of intermediate density lipoprotein + LDL. Additionally, the proportion of [14C]oleate incorporated into hepatic phospholipids increased 35% at the expense of triglyceride and the ratio of hepatic [14C]cholesterol to [14C]squalene derived from [14C]mevalonate increased over twofold following carnitine withdrawal. These studies provide evidence that normal lipid homeostasis can be altered by supplemental carnitine and that the perturbations are reflected by changes in plasma lipids and lipoproteins and in the proportions of the hepatic lipids synthesized.

In vitro incubation of low-density lipoproteins with inflammatory cells causes enhanced degradation by macrophages in culture.

Utilizing the polyvinyl sponge-implant model, we have reported in vivo modification of low-density lipoproteins (LDL) isolated from interstitial inflammatory fluid (IF) of the rabbit. Further studies on the metabolism of IF-LDL by resident mouse peritoneal macrophages (MPM), demonstrated enhanced uptake and degradation of these modified lipoproteins by scavenger mechanisms. Based upon these studies, we attempted to examine the mechanisms of these observed in vivo modifications in IF-LDL by in vitro incubation of plasma LDL with inflammatory fluid subfractions. Incubation of LDL with inflammatory cells at 37 degrees C resulted in an increased anodal electrophoretic mobility and alteration in apolipoprotein (APO) composition. Subsequent incubation of this modified plasma LDL with MPM resulted in a significant increase in cell surface binding and an increase in the appearance of degradation products in the medium. The formation of lipid peroxides, measured as thiobarbituric acid-reacting substances (T Bars), increased with the time of LDL incubation with inflammatory cells. Conversely, incubation of LDL with cell-free, lipoprotein-deficient IF (LPDIF, d greater than 1.210 g/ml) significantly inhibited LDL degradation by MPM. LPDIF did not alter the electrophoretic mobility of LDL or result in the appearance of T Bars in the medium. These results implicate peroxidative reactions associated with an inflammatory response as mediators of the in vivo modifications in IF-LDL which facilitates enhanced uptake via the scavenger receptor in MPM.

The influence of diet and carnitine supplementation on plasma carnitine, cholesterol and triglyceride in WHHL (Watanabe-heritable hyperlipidemic), Netherland dwarf and New Zealand rabbits (Oryctolagus cuniculus).

1. Plasma carnitine levels in the spontaneously (endogenously) hyperlipidemic Watanabe (WHHL) rabbit are approximately 2-fold higher (P less than 0.001) than in normal rabbits of the New Zealand (NZ) or Netherland Dwarf (NDw) breeds. 2. Plasma carnitine levels in WHHL (44 +/- 3 nmol/ml) can be approximated in NZ and NDw which are rendered exogenously hyperlipidemic by supplementation of the stock chow diet with cholesterol and peanut oil. 3. The induction of endogenous hyperlipidemia in NZ by feeding a sucrose casein rich diet results in a biphasic response of plasma carnitine (elevation followed by normalization). 4. Plasma carnitine in WHHL is readily elevated by supplemental L-carnitine and the elevation is associated with a reduction in plasma triglyceride which shows differences in individual response time; plasma cholesterol is unaffected by supplemental L-carnitine.

The effect of oral l-carnitine on lipoprotein composition in the Watanabe Heritable Hyperlipidemic Rabbit (Oryctolagus cuniculus).

1. We have recently reported the ability of orally administered l-carnitine to lower plasma triglyceride in the Watanabe Heritable Hyperlipidemic Rabbit (WHHL), an animal model of familial hyperlipoproteinemia. 2. In the present studies we examined the effect of l-carnitine administration upon individual lipoprotein subfractions in this animal model. 3. Carnitine feeding resulted in a reduction in very low density lipoproteins (VLDL) and high density lipoprotein (HDL). 4. Compositional analysis revealed a reduction in core triglyceride content with a concomitant increase in protein and phospholipid in VLDL and low density lipoproteins (LDL). 5. Conversely, electrophoretic mobility and apolipoprotein composition were unchanged with l-carnitine. 6. These results further demonstrate the ability of l-carnitine to modulate lipoprotein lipid composition in this animal model of familial hyperlipoproteinemia.

Turnover of low-density lipoprotein isolated from interstitial inflammatory fluid of the rabbit.

Utilizing the polyvinyl sponge-implant model in the rabbit, we have described in vivo modification of low-density lipoproteins (LDL) in interstitial inflammatory fluid (IF). In the present studies, plasma clearance rates of IF-LDL were determined and compared with normal whole plasma LDL (WP-LDL) as well as plasma LDL modified by chemical reaction with acetic anhydride (Ac-LDL). Lipoproteins were labeled with 125I and injected into the lateral ear vein of recipient rabbits. At 10 min after injection, only 51.5% of IF-LDL could be accounted for in recipient plasma, as compared to 91.9% for WP-LDL, and 2.4% of Ac-LDL. Subsequent log-linear decay rates were similar for IF-LDL and WP-LDL (t 1/2 = 9.5 vs. 11.0 h). Autoradiography of plasma obtained from recipient animals at 15 min and 1 h after injection revealed a return to normal electrophoretic mobility of [125I]IF-LDL. These results indicate that IF-LDL is a mixture of both modified and essentially unmodified particles. We propose that the modified particles may be removed from the circulation by mechanisms described for Ac-LDL.

Lipoproteins of the extravascular space: enhanced macrophage degradation of low density lipoproteins from interstitial inflammatory fluid.

Current evidence has demonstrated that cholesteryl ester-loaded macrophages are important components of the atherosclerotic lesion. Additional studies have implicated low density lipoproteins (LDL) and circulating monocytes as central to the origin of lipid-laden foam cells found in the arterial wall. This is a result of the finding of accelerated macrophage uptake of LDL chemically modified by reaction with malondialdehyde (MDA-LDL), acetic anhydride (Ac-LDL), or incubation with arterial cells in vitro. In concert with these chemical modifications, we have previously demonstrated selective in vivo modification of LDL isolated from interstitial inflammatory fluid (IF) of the rabbit. Utilizing the polyvinyl sponge implant model, we reported that IF-LDL had an altered chemical composition, electrophoretic mobility, and particle size distribution when compared to LDL isolated from homologous plasma (WP-LDL). In this study reported herein, we examined the metabolism of IF-LDL by resident mouse peritoneal macrophages (MPM) in culture. IF-LDL was degraded substantially faster by MPM, and resulted in a substantial increase in cellular cholesteryl ester when compared to cells incubated with WP-LDL. IF-LDL binding to MPM was inhibited by Ac-LDL derived from WP-LDL, but only minimally by unmodified WP-LDL. Transmission electron microscopy of MPM revealed extensive lipid deposition in cells incubated with Ac-LDL and IF-LDL. These results implicate LDL from interstitial inflammatory fluid as an in vivo modified lipoprotein that can enhance uptake via the acetyl LDL receptor pathway in resident macrophages.

Direct modification of plasma low-density lipoproteins in interstitial inflammatory fluid of the rabbit.

Utilizing the polyvinyl sponge-implant model in the rabbit, we have previously demonstrated modification in low-density lipoproteins (LDL) in the extravascular space in association with a cellular inflammatory response. In an attempt to isolate the source of these modifications, plasma LDL was labeled with 125I, and introduced directly into the extravascular space at the time of sponge implantation. [125I] plasma LDL recovered from interstitial inflammatory fluid (IF) at 24 h after implantation demonstrated increased electrophoretic mobility as well as heterogeneity in particle size and hydrated density. These results are in agreement with our previous observations and indicate that modification in IF-LDL probably occurs after it has entered the extravascular space across the vascular wall.

Effect of cholesterol feeding on lipoprotein distribution in interstitial inflammatory fluid of the rabbit.

Utilizing the polyvinyl sponge-implant model in the rabbit we have previously demonstrated modification in low density lipoproteins (LDL) of interstitial tissue fluid obtained in association with a cellular inflammatory response. In order to examine the interaction between the inflammatory response and lipoproteins from hypercholesterolemic rabbits, 30 male, New Zealand White rabbits were fed standard chow supplemented with 0.5% cholesterol for 4 weeks prior to sponge implantation. Lipoproteins were prepared from interstitial inflammatory fluid (IF) as well as homologous whole plasma (WP). Total IF cholesterol was positively correlated with plasma cholesterol (459 +/- 43 vs. 1485 +/- 130 mg/dl, means +/- SEM, r = 0.81, P less than 0.01). Distribution of lipoproteins in IF was similar to WP in both particle size and density. Beta-migrating VLDL were the predominant particles in both WP and IF, containing 43.7 +/- 3.4 and 42.2 +/- 5.1% of WP and IF cholesterol, respectively. IF-VLDL were similar to WP-VLDL in lipid and apoprotein composition, morphology and particle size distribution. We conclude from these data that the observed dramatic alterations in lipoprotein distribution in response to a dietary cholesterol challenge in rabbit plasma is essentially unaltered in interstitial inflammatory fluid obtained from these animals.

Lipoprotein metabolism in the extravascular space. Cyclophosphamide inhibits modification of low-density lipoproteins in interstitial inflammatory fluid of the rabbit.

We have recently reported preferential modification and reduction in low-density lipoproteins (LDL) of inflammatory exudates in the rabbit. In an attempt to establish the role of inflammatory cells in these specific alterations, using the sponge-implanted rabbit model we characterized extravascular lipoproteins in animals with severely induced leukopenia. Under these conditions we were unable to demonstrate alterations in the distribution of lipoproteins in inflammatory fluid as compared to homologous plasma. Characterization of LDL from both plasma and inflammatory fluids revealed close similarity in molecular composition and electrophoretic mobility. These studies further implicate the role of scavenger cell systems as a significant component of daily lipoprotein homeostasis.

Lipoproteins of the extravascular space: alterations in low density lipoproteins of interstitial inflammatory fluid.

Although extrahepatic degradation of low density lipoproteins (LDL) by peripheral cells is considered to be a significant component of daily cholesterol homeostasis, the nature of lipoproteins in the extravascular space has not been well described. Using a sponge implantation model in the rabbit, we examined lipoproteins prepared from interstitial inflammatory fluid. Inflammatory fluid cholesterol is correlated with plasma values, (r = 0.735, P less than 0.01), but triglyceride values are not. Examination of inflammatory fluid lipoproteins by agarose gel electrophoresis, column chromatography, and density gradient centrifugation revealed a marked reduction in LDL concentration as compared to plasma LDL. Inflammatory fluid low density lipoproteins prepared by sequential density flotation had a larger mean diameter, they were erratic in shape, and contained more triglyceride and less cholesterol and cholesteryl ester than plasma LDL. Total cholesterol to protein ratio was significantly reduced in inflammatory fluid LDL (0.73 vs. 1.10, P less than 0.05). Inflammatory fluid LDL migrated further than plasma LDL on agarose electrophoresis, despite similar apoprotein patterns. These data concur with findings of altered composition and electrophoretic mobility of plasma LDL modified in vitro by exposure to acetylating agents, malondialdehyde, or aortic cells in culture, and they may represent the actual form of LDL in the extravascular space.

Circulating phospholipids in interstitial inflammatory fluid of the rabbit.

Using a sponge implantation model in the rabbit, we have examined the distribution and fatty acid composition of extravascular phospholipids in interstitial inflammatory fluid. Inflammatory fluid contained less phosphatidylserine and -inositol and a complete absence of phosphatidylethanolamine as compared to autologous plasma. Inflammatory fluid phospholipids contained an increased amount of oleic acid, decreased linoleic acid, and undetectable levels of arachidonic acid, despite unaltered levels of these fatty acids in other extravascular lipid classes as compared to plasma. Potential metabolic consequences of these findings are discussed.

In vitro injury of porcine aortic endothelial cells by very-low-density lipoproteins from diabetic rat serum.

Primary cultures of porcine aortic endothelial cells were used to detect injurious agents in diabetic rat serum. Media containing normal or diabetic serum (streptozotocin-induced) were incubated with subconfluent primary cultures of endothelial cells. Cells exposed to 17% diabetic serum were severely contracted and permeable to trypan blue after 18 h. Cultures in normal serum contained significantly more protein (P less than 0.005) than cultures in diabetic serum after 1 day. When normal and diabetic serum were mixed in different proportions and added to cells at 17% total serum concentration, a 50% reduction from growth in normal serum occurred at a ratio of 1 part diabetic serum to 9 parts normal serum. The toxicity of diabetic serum was not altered by heat inactivation. Administration of insulin to diabetic rats reversed both cytotoxicity and elevated triglyceride levels. Serum triglyceride levels were inversely correlated with endothelial cell growth (r = -0.878, P less than 0.001). In vitro addition of up to 1 U/ml insulin to cultures in diabetic serum did not alter toxicity. Ultracentrifugal flotation of diabetic serum demonstrated the toxic substance to be entirely localized in the very-low-density lipoprotein fraction (d less than 1.006 g/ml). A pool of d greater than 1.006 fractions from diabetic serum supported growth equivalent to normal serum. Diabetic serum contains a substance localized in the very-low-density lipoprotein fraction that is severely toxic to aortic endothelial cells in vitro. Very-low-density lipoprotein injury of endothelial cells may play an important role in the development of arterial vascular disease in insulin-dependent diabetes.

Failure of chronic cigarette smoke exposure to alter plasma lipoproteins of stumptailed macaques (Macaca arctoides).

Twenty-one 8-14 kg adult male stumptailed macaques, Macaca arctoides, were fed a standard laboratory diet and divided into 3 groups. The high-dose group and low-dose group were exposed to cigarette smoke at the human equivalent of 3 packs and 1 pack per day, respectively, 7 days per week, for 3-5 years. Eight animals served as cage an sham controls. Peak blood carboxyhemoglobin (COHb) levels measured immediately after smoking showed levels of 0.5+/- 0.1%, 3.6+/-1.0%, and 5.7+/-2.8% for sham controls, low, and high dose smokers, respectively. Hemoglobin and hematocrit values were 2-7% higher (N.S. to P less than 0.05) for smoking groups, presumably as a consequence of chronically elevated COHb levels. No significant differences were seen in total plasma cholesterol and lipoprotein cholesterol concentration measured at four intervals over period of one year. We conclude from these data that, while fed a low fat diet, chronic cigarette smoke inhalation fails to alter plasma lipoprotein levels in this animal model.

The interaction of dietary fibers and cholesterol upon the plasma lipids and lipoproteins, sterol balance, and bowel function in human subjects.

To identify any metabolic effects of dietary fiber upon cholesterol metabolism in man, six adult volunteer subjects were fed eucaloric cholesterol-free formula diets, with and without added dietary fiber for two 4-wk periods. A large quantity of dietary fiber was fed, some 60 g of plant cell wall material (or 16 g of crude fiber) derived from corn, beans, bran, pectin, and purified cellulose. This provided about five times the fiber intake of the typical American diet. The addition of fiber to the cholesterol-free diet did not change either the plasma cholesterol level (171+/-21 mg/dl, SEM, to 167+/-18) or the triglyceride (103+/-39 to 93+/-27 mg/dl). The excretion of both endogenous neutral steroids and bile acids were unchanged with fiber (505+/-41 to 636+/-75 mg/day and 194+/-23 to 266+/-47 mg/day, respectively.) However, total fecal steroid excretion was increased 699+/-29 to 902+/-64 mg/day, P < 0.025). With fiber, intestinal transit time was decreased (59+/-9 to 35+/-8 h, P < 0.005), and both the wet and dry stool weights were greatly increased.A second group of six subjects was fed similar diets containing 1,000 mg cholesterol derived from egg yolk. The addition of fiber to the 1,000-mg cholesterol diet did not alter either plasma cholesterol level (233+/-26 to 223+/-36 mg/dl) or triglyceride (102+/-19 to 83+/-11 mg/dl). The excretion of endogenous neutral steroids (618+/-84 to 571+/-59 mg/day), of bile acids (423+/-122 to 401+/-89 mg/day), and of total fecal steroids (1,041+/-175 to 972+/-111 mg/day) were unchanged by fiber. The absorption of dietary cholesterol was not altered when fiber was added to the 1,000-mg cholesterol diet (44.0+/-3.3 to 42.9+/-2.5%). A two-way analysis of variance utilizing both groups of subjects indicated a significant (P < 0.001) effect of dietary cholesterol upon the plasma cholesterol concentration. We concluded that a large quantity of dietary fiber from diverse sources had little or no effect upon the plasma lipids and sterol balance in man in spite of the fact that intestinal transit time and stool bulk changed greatly.