P.N.M. Demacker

Low-density lipoprotein receptor-deficient mice are protected against lethal endotoxemia and severe gram-negative infections.

Lipoproteins can bind lipopolysaccharide (LPS) and decrease the LPS-stimulated production of pro-inflammatory cytokines. We investigated the effect of increased plasma concentrations of low-density-lipoproteins (LDL) on survival and cytokine production after a lethal challenge with either LPS or live Gram-negative bacteria in LDL receptor deficient mice (LDLR-/-). The LDLR-/- mice challenged with LPS had an eightfold increased LD50 when compared with the wild type controls (C57Bl/6J), while tumor necrosis factor alpha (TNFalpha) and interleukin-1 alpha (IL-1 alpha) plasma concentrations were decreased twofold. LDLR-/- mice had significantly lower and delayed mortality than control mice after infection with Klebsiella pneumoniae. No differences in the outgrowth of bacteria in the organs were present between the two groups, while circulating cytokine concentrations were decreased twofold in LDLR-/- mice. In contrast, the LPS-stimulated in vitro production of cytokines by peritoneal macrophages of LDLR-/- mice was significantly increased compared with controls. This increase was associated with enhanced specific binding of LPS to the macrophages of LDLR-/- mice. In conclusion, endogenous LDL can protect against the lethal effects of endotoxin and Gram-negative infection. At least part of this protection is achieved through decreased in vivo production of pro-inflammatory cytokines, in spite of increased cytokine production capacity.

Troglitazone Decreases the Proportion of Small, Dense LDL and Increases the Resistance of LDL to Oxidation in Obese Subjects

OBJECTIVE Insulin resistance is associated with a predominance of small, atherogenic LDL particles that are more prone to oxidative modification. Treatment with the insulin-sensitizer troglitazone may improve LDL composition and resistance to oxidation. RESEARCH DESIGN AND METHODS In a randomized double-blind crossover design, 15 obese subjects were treated with either 400 mg troglitazone daily or placebo for 8 weeks. Insulin sensitivity (clamp), (apo)lipoproteins, LDL subclass pattern, plasma TBARS, and ex vivo LDL oxidation were determined. RESULTS Troglitazone treatment improved insulin sensitivity. LDL cholesterol increased from 2.58 ± 0.18 to 2.77 ± 0.20 mmol/1 (P = 0.03) because of an increase in large (buoyant) LDL1 (from 0.45 ± 0.04 to 0.62 ± 0.09 mmol/1, P = 0.008). Because small (dense) LDL3 decreased, LDL1:LDL3 ratio increased (P = 0.02). Plasma TBARS concentration declined significantly, and the lag time of ex vivo LDL oxidation showed a small but significant increase. CONCLUSIONS In obese subjects, treatment with troglitazone improves insulin sensitivity, increases the ratio of large buoyant to small dense LDL, and appears to enhance the resistance of the LDL particle to oxidation. These qualitative changes in lipoproteins may have a beneficial effect on cardiovascular risk profile and compensate for a small increase in LDL cholesterol.

The effect of concentrated n-3 fatty acids versus gemfibrozil on plasma lipoproteins, low density lipoprotein heterogeneity and oxidizability in patients with hypertrygliceridemia

We evaluated in a double-blind randomized trial with a double-dummy design in 28 patients with primary hypertriglyceridemia, the effect of gemfibrozil (1200 mg/day) versus Omacor (4 g/day), a drug containing the n-3 fatty acids eicosapentaenoic (EPA) and docosahexaenoic acid (DHA), on lipid and lipoprotein levels, low density lipoprotein (LDL) subfraction profile and LDL oxidizability. Both Omacor and gemfibrozil therapy resulted in a similar significant decrease in serum triglyceride (TG), very low density lipoprotein (VLDL) triglyceride and VLDL cholesterol concentrations and an increase in high density lipoprotein (HDL) and LDL cholesterol concentrations. The increase in LDL cholesterol was due to a significant increase in cholesterol content of the relatively buoyant LDL subfractions LDL1, LDL2 and LDL3, whereas the relative contribution of the dense LDL subfractions LDL4 and LDL5 to total LDL tended to decrease. So, both therapies resulted in a more buoyant LDL subfraction profile, reflected by a significant increase of the value of parameter K (+10.3% on Omacor vs. +26.5% on gemfibrozil therapy, gemfibrozil vs Omacor P>0.05). Cu(2+)-induced oxidation of LDL was measured by continuous monitoring of conjugated dienes. After 12 weeks of Omacor treatment LDL appeared more prone to oxidative modification in vitro than LDL after gemfibrozil treatment, as measured by the significantly decreased lag time, preceding the onset of the lipid peroxidation. In both groups the rate of oxidation did not change with therapy. The amount of dienes formed during oxidation increased significantly on Omacor treatment, but not on gemfibrozil treatment. Plasma thiobarbituric acid reactive substances were higher after Omacor and lower after gemfibrozil treatment, although not significantly. We conclude that both Omacor and gemfibrozil have favorable effects on lipid and lipoprotein concentrations and the LDL subfraction profile. However, Omacor increased the susceptibility of LDL to oxidation, whereas gemfibrozil did not affect the resistance of LDL to oxidative modification in vitro. The clinical relevance of these changes remains to be established in the light of other postulated favorable effects of n-3 fatty acids on the course of cardiovascular disease.

Plasma levels of lipid and cholesterol oxidation products and cytokines in diabetes mellitus and cigarette smoking: effects of vitamin E treatment

To evaluate the role of both oxidation and inflammation in atherosclerosis, we compared LDL oxidizability, in vivo lipid and cholesterol oxidation, and basal and lipopolysaccharide (LPS)-stimulated production of various cytokines in normolipidemic patients with diabetes mellitus (DM: n = 11), cigarettes smokers (n = 14). In addition, the effects of vitamin E (600 I.U./day for 4 weeks) on these parameters were evaluated. Initial LDL oxidation characteristics before and after vitamin E were identical in the 3 groups. Plasma thiobarbituric acid reactive substances were higher in DM and smokers versus controls (0.77 +/- 0.22, 0.74 +/- 0.14 versus 0.62 +/- 0.10 mumol malondialdehyde equivalents/l, respectively; P versus controls < 0.05) and normalized after vitamin E supplementation. Total plasma oxysterols were higher in smokers versus controls (354 +/- 104 versus 265 +/- 66 nmol/l, P < 0.05) and unaffected by vitamin E. The basal and LPS-stimulated levels of interleukin-1 beta and tumour necrosis factor alpha (TNF alpha) and the basal level of interleukin-1-receptor antagonist (IL-1RA) were identical for the 3 groups. LPS-stimulated IL-1RA was higher in DM versus controls (10.7 +/- 2.0 versus 8.1 +/- 1.7 pmol/l, P < 0.05). After vitamin E, TNF alpha dropped in controls and smokers, and IL-1RA in smokers only. Results suggest increased in vivo oxidative stress and inflammation in DM and smoking, which is partly overcome by vitamin E.

Pro- and anti-inflammatory cytokines in healthy volunteers fed various doses of fish oil for 1 year

Dietary supplementation with n‐3 fatty acids from fish oil alleviates inflammation in various chronic inflammatory disease states. Reductions in the production of pro‐inflammatory cytokines interleukin 1β (IL‐1β), tumour necrosis factor alpha (TNF‐α), and interleukin 6 (IL‐6) have been seen in humans after short‐term n‐3 fatty acid supplementation. We investigated long‐term effects of dietary n‐3 fatty acids on circulating cytokine concentrations and on ex vivo stimulated whole‐blood production of IL‐1β, TNF‐α and interleukin 1 receptor antagonist (IL‐1Ra), the naturally occurring antagonist of IL‐1. A total of 58 monks with a mean age of 56 years were randomized into four groups and their diets were supplemented with 0, 3, 6, or 9 g of fish oil, providing 0, 1.06, 2.13 or 3.19 g of n‐3 fatty acids per day. Subjects received equal amounts of saturated fatty acids, vitamin E and cholesterol. Compliance was excellent and erythrocyte fatty acid profiles closely reflected the amounts of n‐3 fatty acids ingested. In the group receiving 9 g of fish oil per day, no influence of n‐3 fatty acids on circulating cytokine concentrations was observed relative to placebo. Endotoxin‐stimulated whole‐blood cytokine production was measured at 26 and 52 weeks after the start and at 4, 8 and 26 weeks after cessation of supplementation. In all groups, the production of IL‐1β and IL‐1Ra was higher during supplementation than afterwards. However, no differences in cytokine production were noted between the placebo group and the various treatment groups at any point in time. Our results suggest that long‐term supplementation of fish oil does not affect ex vivo cytokine production in man.

Nomogram to Diagnose Familial Combined Hyperlipidemia on the Basis of Results of a 5-Year Follow-Up Study

Background—Familial combined hyperlipidemia (FCH) is traditionally diagnosed by total plasma cholesterol and/or triglyceride levels above the 90th percentile adjusted for age and gender. In a recent study, we showed that the diagnosis of FCH on the basis of these diagnostic criteria was inconsistent in 26% of the subjects over a 5-year period. This result emphasizes the need for reevaluation of the diagnostic criteria for FCH. Methods and Results—A total of 32 families (299 subjects) were studied in 1994 and 1999. A subject was defined “truly” FCH when diagnosed FCH in 1994 and/or 1999 on the basis of traditional plasma lipid criteria. Additional lipid and lipoprotein parameters, including apolipoprotein B (apoB) and small, dense LDL, were measured at both time points. In total, 121 subjects (40%) were defined as truly FCH. Multivariate analysis revealed that absolute apoB values combined with triglyceride and total cholesterol levels adjusted for age and gender best predicted truly FCH. A nomogram including these parameters is provided to simply and accurately calculate the probability to be affected by FCH. Furthermore, it is shown that when percentiles of triglyceride and total cholesterol adjusted for age and gender are not available in a population, the definition of FCH can be established on the basis of hypertriglyceridemia (>1.5 mmol/L) and hyper-apoB (>1200 mg/L). Conclusions—The diagnosis of FCH is best predicted by absolute apoB levels combined with triglyceride and total cholesterol levels adjusted for age and gender and can accurately be calculated by a nomogram. This definition is also a good predictor of cardiovascular risk in FCH.

Intra-individual variation of serum cholesterol, triglycerides and high density lipoprotein cholesterol in normal humans

The intra-individual variation in the concentrations of serum cholesterol, triglycerides and high density lipoprotein cholesterol (HDL-chol) was determined in 53 healthy subjects, without extreme standardization of test subjects and sampling conditions. Within 1 year, the intra-individual variation of the subjects ranged from 3.9 to 10.9% for cholesterol; from 12.9 to 40.8% for triglycerides, and from 3.6 to 12.4% for HDL-chol. More than 60% of the average total intra-individual variation was caused by biological fluctuations and the remainder was the result of analytical variation. Thus, a single measurement of these serum constituents in an individual can be misleading or meaningless, unless the value is considerably outside the normal range. No significant diurnal variation was found in the concentrations of serum cholesterol and HDL-chol. The maximal post-prandial increase of the serum triglycerides was twice as great in the men than in the women. Finally, significant trends in the fluctuations of serum lipids and HDL-chol during 1 year were not found.

Circulating soluble tumor necrosis factor receptors, interleukin‐2 receptors, tumor necrosis factor α, and interleukin‐6 levels in rheumatoid arthritis.

Objective. To assess whether circulating concentrations of soluble tumor necrosis factor receptors (sTNFR; p55 and p75), soluble interleukin-2 receptors (sIL-2R), tumor necrosis factor α (TNFα), and interleukin-6 (IL-6) reflect clinical response and whether changes are dependent on the drug used in rheumatoid arthritis (RA) patients taking methotrexate (MTX) or azathioprine (AZA). Methods. These cytokines and soluble receptors were assessed in 20 control subjects and serially for up to 48 weeks in 61 RA patients, by bioassay (IL-6) and immunoassays (sTNFR, sIL-2R, TNFα, and IL-6). Results. Concentrations of p55 and p75, sIL-2R, and TNFα (but not IL-6) were significantly higher in RA patients than in controls. Significant decreases in sIL-2R and p55 concentrations were associated with clinical improvement and were observed in patients treated with MTX, but not AZA. Both treatments induced decreases in IL-6 concentrations, but circulating AZA (or its metabolites) appears to interfere with the measurement of IL-6 bioactivity. TNFα and p75 levels did not show significant changes. Conclusion. Measurement of circulating sIL-2R, p55, and IL-6 may be useful in the evaluation of RA disease activity and response to therapy. Interference by circulating levels of drugs must be ruled out when bioassays are used to evaluate cytokine levels.

Improved Cardiovascular Risk Profile and Renal Function in Renal Transplant Patients after Randomized Conversion from Cyclosporine to Tacrolimus

Cyclosporine is considered to contribute to the high cardiovascular morbidity and mortality in patients after renal transplantation. Tacrolimus may be more favorable in this respect, but controlled data are scarce. In this prospective randomized study in 124 stable renal transplant patients, the effects of conversion from cyclosporine to tacrolimus on cardiovascular risk factors and renal function were investigated. Follow-up was 6 mo. Statistical analysis was performed by ANOVA for repeated measurements. The serum creatinine level decreased from 137 +/- 30 micromol/L to 131 +/- 29 micromol/L (P < 0.01). Three months after conversion from cyclosporine to tacrolimus, mean BP significantly decreased from 104 +/- 13 to 99 +/- 12 mmHg (P < 0.001). Serum LDL cholesterol decreased from 3.48 +/- 0.80 to 3.11 +/- 0.74 mmol/L (P < 0.001,) and serum apolipoprotein B decreased from 1018 +/- 189 to 935 +/- 174 mg/L (P < 0.001). Serum triglycerides decreased from 2.11 +/- 1.12 to 1.72 +/- 0.94 mmol/L (P < 0.001). In addition, both rate and extent of LDL oxidation were reduced. The fibrinogen level decreased from 3638 +/- 857 to 3417 +/- 751 mg/L (P < 0.05). Plasma homocysteine concentration did not change. Three months after conversion, plasma fasting glucose level temporarily increased from 5.4 +/- 1.3 mmol/L to 5.8 +/- 1.9 mmol/L (P < 0.05). Conversion to tacrolimus resulted in a significant reduction of the Framingham risk score. In conclusion, conversion from cyclosporine to tacrolimus in stable renal transplant patients has a beneficial effect on renal function, BP, serum concentration and atherogenic properties of serum lipids, and fibrinogen.

Oxidation resistance, oxidation rate, and extent of oxidation of human low-density lipoprotein depend on the ration of oleic acid content to linoleic acid content studies in vitamin E deficient subjects

The purpose of this study was to understand better the factors providing oxidation resistance to human low-density lipoprotein (LDL). Therefore, the susceptibility to copper-induced in vitro oxidation of LDL from vitamin E deficient patients and normal healthy subjects was studied. Surprisingly, the LDL of vitamin E deficient patients appeared less susceptible to oxidation than control LDL. Both oxidation rate and extent of oxidation, measured as diene production, were reduced when compared to control LDL. The lag time, a measure of resistance to oxidation, was not different from the lag time of LDL from healthy subjects. No relation was found between vitamin E content and resistance against oxidation. LDL from vitamin E deficient patients contained lower amounts of vitamin E, less cholesteryl esters, and increased amounts of triglycerides. Furthermore, its oleic acid content was increased and its linoleic acid content decreased. Linear regression analyses revealed that the ratio of oleic acid content to linoleic acid content was strongly correlated with the lag time, and inversely correlated with oxidation rate and extent of oxidation. Thus, LDL rich in oleic acid and poor in linoleic acid was less easily oxidized. It is concluded that the susceptibility of LDL to oxidation is determined not only by its antioxidant content, but also by other compositional factors, and more specifically by the ratio of oleic acid content to linoleic acid content.