Lack of plasma lipid peroxidation during LDL-apheresis by heparin-induced extracorporeal LDL-precipitation

Abstract. The heparin-induced extracorporeal precipitation of low density lipoproteins (HELP) is a well established clinical apheresis procedure to markedly reduce cholesterol levels. The biocompatibility of this filter system was investigated by the determination of lipid peroxidation products. Both lipid hydro-peroxides and thiobarbituric acid reactive substances (TBARS) were determined before, during and after 38 aphereses in 21 patients undergoing regular HELP treatment. Although HELP patients had significantly elevated TBARS compared to 93 healthy controls (3.13 ±0.64 vs. l.66±0.50 μmol L^-1; P <0.01), no significant differences were observed compared to either 104 patients suffering from angiographically confirmed coronary heart disease (3.42 ±0.81 μmol L^-1; P > 0.05 vs. HELP patients) or 38 aged-matched hyperlipidaemic patients (3.30 ±0.75 μmol L^-1; P >0.05 vs. HELP patients), neither of which were included in the HELP programme. No lipid hydroperoxides were detected in the plasma of HELP patients either before or after the extracorporeal treatment. After the LDL-apheresis TBARS were significantly decreased (2.60 ± 0.52 μmol L^-1) compared to the values before the treatment ( P <0.01). There was no evidence for the formation of lipid hydroperoxides within the HELP circuit. It is suggested, therefore, that plasma lipids are not oxidized by the HELP procedure.     

Decreased in vitro oxidizability of low-density lipoprotein in hypercholesterolaemic patients treated with 3-hydroxy-3-methyIgIutaryl-CoA reductase inhibitors

Abstract. We studied the effects of the 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase inhibitors simvastatin and pravastatin on the in vitro susceptibility of low-density lipoprotein (LDL) to oxidation. Twenty-three hypercholesterolaemic patients (mean serum cholesterol 9.7 mmol 1^-1) were treated with increasing doses of either simvastatin or pravastatin for 18 weeks. No significant differences in effect on lipid levels between the two drugs were found. Treatment resulted in lowering of total cholesterol and LDL-cholesterol by maximally 30% and 34%, respectively. Chemical composition analysis showed that LDL particles contained relatively more protein and less free cholesterol and cholesteryl-ester after treatment. The LDL cholesterol/protein ratio decreased from 1.24 ± 0.21 to 0.97 ± 0.23 ( n = 20). By continuous monitoring of in vitro oxidation it appeared that LDL was less susceptible to oxidation after drug treatment. Maximal rate of diene production was significantly decreased from 19.7 ± 3.1 to 18.5 ± 3.3 nmol min^-1 mg^-1 LDL; total diene production decreased significantly from 420.3 ± 67.6 to 380.5 ± 49.1 nmol mg-¹ LDL; the lag time was unchanged throughout the study. These studies show that HMG-CoA reductase inhibitors reduce the oxidizability of LDL by altering its composition.     

Decreased release of glutathione into the systemic circulation of patients with HIV infection

Low glutathione (GSH) in patients with HIV infection could contribute to their immune deficiency since GSH plays an important role in the function of lymphocytes and sulphydryls decrease the expression of HIV in vitro. In order to gain more insight into the mechanisms responsible for the deranged sulphydryl homeostasis in HIV infection, the release of GSH into the circulation, an estimate of the systemic production of GSH, was determined using a pharmacokinetic approach. The basal plasma concentrations of free GSH (3.3±1.3 vs. 5.3±1.9 μmol L^-1) and cysteine (7.7±2.6 vs. 13.4±4.9 μmol L^-1) were significantly lower in eight HIV-infected patients than in eight controls. Upon infusion of GSH at a constant rate of 1 μmol min^-1 kg^-1, GSH in plasma reached a new plateau. The increment in plasma GSH was significantly larger in the HIV-infected patients than in the controls. The input of GSH into the circulation (12.9±5.7 vs. 30.1±11.7 μmol min^-1 P <0.01) and the clearance of GSH (25±7 vs. 35±7 mL min^-1 kg^-1) were significantly lower in patients with HIV-infection. During infusion of GSH the concentration of cysteine in peripheral blood mononuclear cells of the HIV-infected patients increased significantly. Nevertheless, intracellular GSH did not increase. Thus, the consumption of GSH is not increased in HIV infection. Rather, the present data suggest that GSH in patients with HIV infection is low because of a decreased systemic synthesis of GSH.     

Microdialysis assessment of adipose tissue metabolism in post-absorptive obese NIDDM subjects

Abstract. Lactate and glycerol turnover is enhanced in obesity and NIDDM. To evaluate the influence of NIDDM on subcutaneous adipose tissue metabolism microdialysis combined with ¹³³Xe clearance and measurements in arterialized plasma were carried out using samples of subcutaneous abdominal fat from nine obese NIDDM subjects (glucose, 7.9 ± 0.7 mmol L^-1) (mean±SEM) and nine obese non-diabetic subjects (glucose, 4.9 ±0.1) matched for age, BMI and body fat. After an overnight fast arterialized plasma levels were 1145 ±110 vs. 876 ±59 μmol L^-1 ( P <0.05) for lactate and 75±10 vs. 66 ±8 μmol L^-1 for glycerol in the diabetic and control group, respectively. The corresponding abdominal subcutaneous interstitial lactate and glycerol concentrations were 1278 ± 63 vs 1107 ±64 μmolL^-1 and 314 ±28 vs. 311 ± 17μmol L^-1, respectively. However, adipose tissue blood flow in the same region was lower in NIDDM subjects (1.5±0.2 vs 2.4±0.3 mL 100g^-1 min^-1) ( P <0.05). Consequently, apparent subcutaneous lactate and glycerol release, estimated according to Fick, were not statistically different in the two groups (1.8 ±0.4 vs 2.4 ±0.8 and 2.1 ±0.4 vs 3.1 ±0.5 μmol kg^-1 min^-1 in NIDDM and control subjects, respectively). Thus, in the post-absorptive state apparent lactate and glycerol release by the abdominal subcutaneous tissue in obese NIDDM subjects was similar to that in a matched group of obese non-diabetic controls. The data suggest that no primary defect is evident in adipose tissue metabolism in well controlled NIDDM subjects.     

Effect of insulin treatment on serum lipoprotein(a) in non-insulin-dependent diabetes

Abstract. In order to evaluate whether Lp(a), a lipoprotein that is potentially thrombogenic and atherogenic, is a potential risk factor for CAD in non-insulin-dependent diabetes (NIDDM), we compared the Lp(a) and its distribution in 145 NIDDM patients with that in 94 healthy control subjects. Furthermore, we studied the effect of insulin treatment on serum Lp(a) in 108 patients with NIDDM. Male and female NIDDM patients had similar Lp(a) concentrations to healthy controls (median value 167 mg L^-1, range 15–1550 mg L^-1 vs. 157 mg L^-1, range 15–919 mg L^-1, NS and 92, range 15–1190 mg L^-1 vs. 103 mg L^-1, range 15–842 mg L^-1, NS). Also, the cumulative distribution of Lp(a) did not differ between the NIDDM patients and healthy subjects. Insulin treatment increased Lp(a) in diabetics with a Lp(a) concentration of less than 300 mg ^-1L, but this effect was not related to the concomitant improvement in metabolic control (mean change (±SEM) of HbA_1c from 9.80±0.15 to 8.00±0.12; P < 0.001). In subjects with elevated Lp(a) concentrations (>300 mg L^-1) the Lp(a) concentration was unaffected by insulin, despite a similar improvement in glycaemic control. These results suggest that insulin may modulate the concentration of Lp(a).     

Decreased clearance of uraemic and mildly carbamylated low-density lipoprotein

Abstract. Low-density lipoprotein (LDL) was in vitro carbamylated with potassium cyanate and the clearance was studied in man. A minor carbamylation of LDL decreased the clearance of LDL by 41% (94% of amino groups free) and by 18% (90% of amino groups free). When LDL was extensively carbamylated its clearance was substantially accelerated. Moreover, the clearance of LDL isolated from 14 haemodialysis patients (uremic-LDL) was studied in rabbits. Uraemic-LDL, injected into rabbits simultaneously with the LDL of a healthy control subject, was cleared more slowly than the control-LDL (difference in fractional catabolic rate –6·5%, P = 0·02). We also examined the lipid peroxidation of the carbamylated LDL by measuring the amount of thiobarbituric-acid reactive substances (TBARS) and formation of conjugated dienes during exposure of carbamylated LDL to 5 μ M Cu²⁺. The carbamylated and native LDL had similar lipid peroxidation and propensity for oxidation. In summary, both the uraemic-LDL and minimally carbamylated LDL had a decreased clearance in vivo , which may contribute to the accelerated atherosclerosis in uraemic patients.     

Plasma aluminium in a reference population: the effects of antacid consumption and its influence on biochemical indices of bone formation

Abstract. Aluminium is involved in the etiology of several complications of chronic renal failure and has been firmly established as having toxic effects on bone tissue. We have measured plasma aluminium together with serum osteocalcin, procollagen I C-terminal peptide and total alkaline phosphatase activity in healthy subjects and in a group of subjects who consumed aluminium-containing and non-aluminium containing antacid preparations, with normal renal function. Age-related healthy reference ranges for plasma aluminium are presented and the effects of chronic antacid consumption on plasma aluminium and biochemical markers of bone formation investi gated.
In 172 healthy subjects the mean plasma aluminium concentration was 4.4 ± 2.9 μ g L^-1, men having a significantly greater circulating aluminium load than women (5–4 ± 2.8 μg L^-1 vs. 4.0 ± 2.8 μg L^-1 respect ively ( P = 0.0039)). Older men were found to have significantly higher plasma aluminium levels than younger men. Increased plasma aluminium was seen in subjects taking antacids although this was not asso ciated with significant changes in most indices of bone formation.     

δ-Aminolaevulinic acid dehydratase as an index of lead toxicity. Time for a reappraisal?

Abstract. δ-Aminolaevulinic acid dehydratase activity is traditionally accepted as the most sensitive measurable biological index of lead toxicity. We have measured δ-aminolaevulinic acid dehydratase activity and blood lead concentration in 47 healthy controls (A), 42 iron deficient patients (B) and 38 occupationally exposed to lead subjects (C). Blood lead levels [^x (SD)] did not differ between groups A and B [0.51 (0.21) and 0.43 (019) μmol L^-1, respectively] while those of group C [2.28 (0.56) μmol L^-1 were significantly higher ( P < 0.001) as compared to the controls. δ-Aminolae-vulinic acid dehydratase activity [^x (SD)] was significantly increased [3599 (1909) μmol L^-1 h^-1] in group B and decreased in group C [1052 (532) μmol L^-1 h^-1] as compared to the controls [2034 (446) μmol L^-1 h^-1] ( P < 0.001). There was a significantly negative correlation of logarithm of δ-aminolaevulinic acid dehydratase with lead in both groups B ( P < 0.05) and C ( P < 0.001) but not in group A ( P = 0.1). δ-Amino-laevulinic acid dehydratase activity had a high specificity (100%) but a low sensitivity (37%) as an index of toxic lead exposure. According to our data the value of δ-aminolaevulinic acid dehydratase measurement in the diagnosis of lead intoxication is doubtful in cases with low blood lead levels, while in the presence of iron deficiency its reliability is further reduced, since low blood lead levels may be falsely predicted. δ-Amino-laevulinic acid dehydratase activity should be restricted only to monitoring cases with moderate or severe lead poisoning.     

Failure of complete suppression of endogenous glucose production by euglycaemic hyperinsulinaemia in normal humans

Abstract. In normal subjects, endogenous glucose production (EGP) is usually assumed to be completely suppressed during euglycaemic clamp studies performed at high insulin levels (>100 mU L^-1). However, this assumption is based on non-steady-state tracer measurements of EGP which are prone to negative errors. We have used purified [6-³ H]glucose in an optimal tracer infusion protocol to assess the suppression of EGP during 4 h euglycaemic clamps in eight normal men. An insulin infusion rate of 5 mU kg^-1 min^-1 was chosen to achieve supraphysiological (> 500 mU L^-1) plasma insulin concentrations. Using a labelled exogenous glucose infusion, plasma glucose (mean ± SEM 5.3 ± 0.1 mmol L^-1) and glucose specific activities (mean 100 ± 3% of basal) were maintained constant from 80 to 240 min. During hyperinsulinaemia, isotopically determined glucose appearance rates (Ra) were greater than glucose infusion rates (GIR) throughout the euglycaemic clamp period ( P <0.001) and EGP (Ra-GIR) was always greater than zero. In seven of the eight subjects studied EGP was partly suppressed but showed a wide variation (EGP 5 to 91% of basal at 80–120 min and 12 to 87% of basal at 200–240 min) while in one subject EGP rose above basal (by 72% at 80–120 min and 49% at 200–240 min). We conclude that EGP is not completely suppressed during euglycaemic clamps at high insulin levels.     

Susceptibility of LDL to oxidation in vitro and antioxidant capacity in familial combined hyperlipidemia: comparison of patients with different lipid phenotypes

BACKGROUND: There is increasing evidence that oxidation of low-density lipoprotein (LDL) plays an important role in atherogenesis. AIM: To explore the LDL oxidizability and its determinants in familial combined hyperlipidemia (FCHL) patients with different phenotypes. METHOD: The study included 59 FCHL family members with different lipid phenotypes, 39 non-affected relatives, and 30 spouses as healthy controls. RESULTS: The lag time for LDL oxidation was significantly shorter in FCHL patients with different lipid phenotypes as compared to healthy controls. There were no significant differences in the propagation rate and conjugated diene formation and alpha-tocopherol content in LDL between the FCHL groups and healthy controls. Plasma concentrations of alpha-tocopherol in all FCHL patients and uric acid in FCHL patients with IIB and IV phenotypes were significantly higher than in healthy controls. Plasma total peroxyl radical trapping capacity measured (TRAPmea) and TRAPcalc tended to be higher in affected FCHL groups, but the difference was significant only for IIB phenotype. The peak LDL particle size in the combined group of FCHL patients was significantly smaller than in healthy controls. The lag time for LDL oxidation correlated significantly with LDL size both in the group of FCHL family members (r = 0.477, P<0.001) and in the healthy controls (r = 0.482, P<0.01). CONCLUSIONS: LDL from FCHL patients irrespectively of lipid phenotypes is more susceptible to oxidation in vitro than LDL from healthy controls. This increased susceptibility of LDL to oxidation in vitro seems to be attributed to the abundance of small dense LDL particles and not to the defect of antioxidant capacity in FCHL     

Identification of defective binding of low density lipoprotein by the U937 proliferation assay in German patients with familial defective apolipoprotein B-100

Abstract. Familial defective apolipoprotein B-100 (FDB) is a dominantly inherited disorder characterized by decreased binding of low density lipoprotein (LDL) to the LDL receptor due to a substitution of glutamine for arginine in residue 3500 of apolipoprotein B-100. We present the results of the U937 cell proliferation assay for the detection of familial defective apo B100 in 13 German FDB patients. Due to a defect in the pathway of cholesterol synthesis the human myelomonocytic tumour cell line U937 lacks the ability to synthesize cholesterol which makes proliferation of these cells dependent on the presence of exogenous LDL-cholesterol. U937 cells were incubated with LDL from 13 FDB-patients, 10 healthy normocholesterolaemic individuals (NC) and 26 patients with familial hypercholesterolaemia due to a defective LDL-receptor (FH). At LDL-cholesterol concentrations below 1 μg ml^-1 no proliferation occurred. In the presence of LDL from FDB patients at concentrations between 2·5 μg ml^-1 and 15·0 μg ml^-1, the proliferation was significantly reduced compared to LDL from FH-patients and normocholesterolaemic controls. At 5 μg ml^-1 the reduction was 31–80% regardless of age, sex, apo E genotype, Lp (a)- and lipid levels. At concentrations above 25·0 μg ml^-1 no further differences were observed. The present results indicate that the U937 proliferation assay is a reliable test for the detection of defective LDL-binding due to the 3500 mutation in FDB patients. It may be useful for the detection of defective binding of LDL due to other mutations in the apo B-100 gene.     

Hyperlipidaemia in renal transplantation—risk factor for long-term graft outcome

Abstract. The role of hyperlipidaemia for the outcome of renal transplantation was evaluated in a prospective study involving 151 patients. Graft losses were associated with more pronounced pre-transplant lipid abnormalities. An increased risk of graft loss during the first two post-transplant years was found in patients with marked pre-transplant hypercholestero-laemia (≥6.9 mmol L^-1, P = 0.014; relative risk 2.2). Hypercholesterolaemia ≥6.9mmol L^-1 at 6 months after transplantation, present in 41/115 patients, was associated with a lower GFR ( P = 0.007) and more pronounced albuminuria ( P = 0.009) at 2 years. In patients with graft dysfunction (serum creatinine >160μmol L^-1) at 2 years, more pronounced lipid abnormalities before and at 6 months after transplantation were found. Between 6 months and 2 years, total and LDL cholesterol did not change significantly, but HDL cholesterol decreased ( P = 0.03). In conclusion, hyperlipidaemia is also a risk factor for the long-term outcome in renal transplantation. Further investigations are needed to determine whether graft losses and late graft failure can be prevented or ameliorated by treating hyperlipidaemia in renal transplantation.     

Susceptibility of LDL to oxidation in vitro and antioxidant capacity in familial combined hyperlipidemia: comparison of patients with different lipid phenotypes

BACKGROUND. There is increasing evidence that oxidation of low-density lipoprotein (LDL) plays an important role in atherogenesis. AIM. To explore the LDL oxidizability and its determinants in familial combined hyperlipidemia (FCHL) patients with different phenotypes. METHOD. The study included 59 FCHL family members with different lipid phenotypes, 39 non-affected relatives, and 30 spouses as healthy controls. RESULTS. The lag time for LDL oxidation was significantly shorter in FCHL patients with different lipid phenotypes as compared to healthy controls. There were no significant differences in the propagation rate and conjugated diene formation and &#102 -tocopherol content in LDL between the FCHL groups and healthy controls. Plasma concentrations of &#102 -tocopherol in all FCHL patients and uric acid in FCHL patients with IIB and IV phenotypes were significantly higher than in healthy controls. Plasma total peroxyl radical trapping capacity measured (TRAP mea ) and TRAP calc tended to be higher in affected FCHL groups, but the difference was significant only for IIB phenotype. The peak LDL particle size in the combined group of FCHL patients was significantly smaller than in healthy controls. The lag time for LDL oxidation correlated significantly with LDL size both in the group of FCHL family members (r = 0.477, P < 0.001) and in the healthy controls (r = 0.482, P < 0.01). CONCLUSIONS. LDL from FCHL patients irrespectively of lipid phenotypes is more susceptible to oxidation in vitro than LDL from healthy controls. This increased susceptibility of LDL to oxidation in vitro seems to be attributed to the abundance of small dense LDL particles and not to the defect of antioxidant capacity in FCHL.     

Lipoprotein lipase gene mutations D9N and N291S in four pedigrees with familial combined hyperlipidaemia

Abstract. The role of the lipoprotein lipase (LPL) gene in familial combined hyperlipidaemia (FCH) is unclear at present. We screened a group of 28 probands with familial combined hyperlipidaemia and a group of 91 population controls for two LPL gene mutations. D9N and N291S. LPL-D9N was found in two probands and one normolipidaemic population control. LPL-N291S was found in four probands and four population controls. Subsequently, two pedigrees from probands with the D9N mutation and two pedigrees from probands with the N291S mutation were studied, representing a total of 24 subjects. Both LPL gene mutations were associated with a significant effect on plasma lipids and apolipoproteins. Presence of the D9N mutation (n = 7) was associated with hypertriglyceridaemia [2.69± 1.43 (SD) mmol L^-1] and reduced plasma high-density lipoprotein cholesterol (HDL-C) concentrations (0.92± 0.21 mmol L^-1) compared with 11 non-carriers (triglyceride 1.75± 0.64 mmol L^-1; HDL-C 1.23± 0.30 mmol L^-1, P = 0.03 and P = 0.025 respectively). LPL-D9N carriers had higher diastolic blood pressures than non-carriers. LPL-N291S carriers ( n = 6) showed significantly higher (26%) apo B plasma concentrations (174± 26 mg dL^-1) than non-carriers (138± 26 mg dL^-1; P = 0.023), with normal post-heparin plasma LPL activities. Linkage analysis revealed no significant relationship between the D9N or N291S LPL gene mutations and the FCH phenotype (hypertriglyceridaemia, hypercholesterolaemia or increased apo B concentrations). It is concluded that the LPL gene did not represent the major single gene causing familial combined hyperlipidaemia in the four pedigrees studied, but that the LPL-D9N and LPL-N291S mutations had significant additional effects on lipid and apolipoprotein phenotype.     

Insulin resistance shows selective metabolic and hormonal targets in the elderly

Abstract. There has been no simultaneous evaluation of different aspects of insulin action in ageing. We studied 12 elderly (77 ± 2 years) and 12 young (26 ±1 years) subjects with normal glucose tolerance and matched for sex, body mass index, lean body mass (LBM), blood pressure and physical activity, using a euglycaemic-hyperinsulinaemic clamp at about 350 pmol L^-1 in combination with [³H]-glucose infusion. In the elderly group, hepatic glucose production was normal, fasting serum insulin and C-peptide were significantly increased ( P = 0.001) and glucose utilization (34.4 ±2.4 vs. 44.4± 3.2 μ mol kg^-1 LBM min^-1, P = 0.02) and the percentage maximal suppression of C-peptide (58 ± 6% vs. 79 ±5%, P = 0.02) during the clamp were reduced. Fasting plasma free fatty acid (FFA) and glycerol levels were similar in the two groups, but their percentage maximal suppression during the clamp was reduced in the elderly group (FFA 45± 5% vs. 77 ± 6%, P = 0.001; glycerol 43 ± 5% vs. 76± 3%, P = 0.001). Branched-chain amino acids (valine, leucine, isoleucine) and glucagon levels were similar in the two groups, both while fasting and during the clamp. Thus, insulin resistance in ageing appears selective on glucose utilization, inhibition of lipolysis and feedback inhibition of the B-cell secretion.     

Tubular site of renal sodium retention in ascitic liver cirrhosis evaluated by lithium clearance

Abstract. Renal tubular sodium handling was evaluated in 27 non-azotemic cirrhotic patients with ascites and positive sodium balance and in 17 controls after at least 5 days of a constant sodium intake using the lithium clearance as an index of fluid delivery to the distal tubule. Plasma renin activity and plasma aldos-terone were also evaluated. Sodium fractional excretion, filtered sodium load, absolute sodium distal delivery, lithium fractional excretion and absolute distal sodium reabsorption were significantly lower in cirrhotics than in controls (0.58 ± 0.11 vs. 1.29 ± 0.12%, < 0.001; 12529± 677 vs. 15707±796 μEq min^-1 1.73 m^-2 BSA, <0.005; 2384±135.2 vs. 3685±219.3 μEq min^-1 1.73 m^-2 BSA, < 0.001; 19.5±1.0 vs. 24.2±l.3%, < 0.01; 2299±127 vs. 3485±214 μEq min^-1 1.73 m^-2 BSA, <0.001, respectively). A correlation was found between lithium clearance and sodium clearance only in cirrhotic patients ( r = 0.62; <0.01). Distal sodium reabsorption evaluated as a per cent of filtered sodium load was lower in cirrhotics than in controls (19.1 ±1.0 vs. 22.4±1.2%, <0.05) while distal sodium reabsorption evaluated as a per cent of sodium distal delivery was higher in cirrhotics than in controls (96.7 ± 0.4 vs. 94.4± 0.5%,< 0.005). In both groups a correlation was found between log plasma aldosterone and distal sodium reabsorption evaluated as a per cent of absolute sodium distal delivery ( r = 0.61, <0.01 and r =0.52,<0.05 respectively).
Our study indicates that a decrease in filtered sodium load and an increase in proximal sodium reabsorption play a critical role in the impairment of renal sodium handling in non-azotemic cirrhotic patients with ascites.     

No evidence for feedback inhibition of hepatic apolipoprotein B (apo B) production after extracorporeal low density lipoprotein precipitation as determined by [I-13C]leucine infusion in normal volunteers

Abstract. To determine the impact of an acute reduction of the circulating mass of apolipoprotein B (apo B) on apo B metabolism we studied six healthy male volunteers before (day 0), 1 day after (day 2), and 7 days after (day 8) an LDL apheresis treatment which reduced apo B mass by 59%. Appearance of newly synthesized apo B in plasma VLDL and LDL was studied using a primed-constant infusion of [I-¹³C]-leucine. VLDL apo B pool size and fractional VLDL apo B production rate calculated using a one-compartment model were similar on all 3 study days. Absolute VLDL apo B production was not statistically different throughout the study (19.7±12.3, 19.5 ± 7.5, 29.1 ± 17.7 mg kg^-1 day^-1). LDL apo B fractional production rate was increased on day 2 (0.38 ± 0.17, 0.68±0.08, 0.37±0.06 pools day^-1on days 0, 2, and 8; P <0.01). Absolute LDL apo B production, however, remained constant throughout the study (10.8 ± 3.3, 11.0±1.9, 10.8 ± 3.1 mg kg^-1 day^-1). We conclude that in healthy male volunteers acute reduction of the circulating apo B mass by LDL apheresis does not affect apo B metabolism significantly.     

Low-density lipoprotein-induced formation of nitric oxide by cultured vascular smooth muscle cells of the rat

There is evidence that low-density lipoprotein (LDL) plays a crucial role in atherogenesis. On the cellular level, LDL has been shown to activate a number of mechanisms involved in atherogenesis and vasoconstriction. Local immoderate vasoconstriction is physiologically antagonized by nitric oxide, which is released from the endothelium. To find out whether LDL also influences the synthesis of nitric oxide in vascular smooth muscle cells, both the conversion of arginine to citrulline and the production of nitrite were determined as a measure of nitric oxide formation. After incubation of rat vascular smooth muscle cells with native LDL (25 μg mL⁻¹) for 24 h, the production of both l -[¹⁴C]-citrulline [39 600 (3600) cpm mg⁻¹ cell protein] and nitric oxide [2.95 (0.56) μmol L⁻¹] were about twice and 1.5-fold the amount of the corresonding values in untreated cells (mean ± SD, P  < 0.05, n  = 4). Oxidized LDL was less effective than the native form. The presence of the arginine analogue N ^G-methyl- l -arginine reduced citrulline production dose-dependently but augmented DNA synthesis, both induced by LDL. In addition, the lipoprotein caused a 1.6-fold increase in cyclic GMP production following a 24-h incubation [control = 10.9 (3.8) pmol mg⁻¹ cell protein, P  = 0.016]. The results suggest that native LDL might partly impair its atherogenic potential on the vasculature by stimulating the production by smooth muscle cells of both nitric oxide and cyclic GMP.     

Antioxidant action of Vaccinium myrtillus extract on human low density lipoproteins in vitro: initial observations

Summary— Oxidative modifications of low density lipoproteins (LDL) are now recognised as one of the major processes in atherogenesis. Various drugs, as well as a number of natural products, have been proposed to inhibit such processes. Among the naturally-occurring constituents of plants which appear to possess antioxidant activity are polyphenolic compounds such as flavonoids. The aqueous extract of Vaccinium myrtillus is rich in such molecules. In this report, we describe the in vitro antioxidative potential of this extract on human LDL. The copper-induced oxidative modification of these lipoproteins was assessed using 1) measurement of oxidative resistance as determined by the lag-phase preceding conjugated diene formation; 2) quantification of the amount of lipoperoxides and thiobarbituric acid-reactive substances generated, and measurement of the modification in the net negative electrical charge of the lipoproteins, over a 7-hour time course experiment. Trace amounts of V myrtillus extract (15 to 20 μg/mL) induce statistically significant changes in the oxidation behaviour of LDL, which include 1) prolongation of the lag-phase of conjugated diene production ( P < 0.01); 2) reduction in the formation of lipoperoxides and of thiobarbituric acid-reactive substances up to 7 hours and especially between 1 and 5 hours ( P < 0.01); and 3) inhibition of modification in the net negative charge of LDL. These results demonstrate that V myrtillus extract exerts potent protective action on LDL particles during in vitro copper-mediated oxidation. Calculation of IC₅₀ values indicates that, on a molar basis, this extract may indeed be more potent than either ascorbic acid or butylated hydroxytoluene in the protection of LDL particles from oxidative stress.     

Paraoxonase-1 (PON-1) genotype and activity and in vivo oxidized plasma low-density lipoprotein in Type II diabetes

The HDL (high-density lipoprotein)-associated enzyme PON (paraoxonase)-1 protects LDL (low-density lipoprotein) from oxidative modification in vitro, although it is unknown if this anti-atherogenic action occurs in vivo. In a cross-sectional study of 58 Type II diabetic subjects and 50 controls, we examined the fasting plasma LDL basal conjugated diene concentration [a direct measurement of circulating oxLDL (oxidatively modified LDL)], lipoprotein particle size by NMR spectroscopy, PON-1 polymorphisms (coding region polymorphisms Q192R and L55M, and gene promoter polymorphisms -108C/T and -162G/A), PON activity (with paraoxon or phenyl acetate as the substrates) and dietary antioxidant intake. Plasma oxLDL concentrations were higher in Type II diabetic patients (males, P = 0.048; females, P = 0.009) and unrelated to NMR lipoprotein size, PON-1 polymorphisms or PON activity (with paraoxon as the substrate) in any group. In men with Type II diabetes, however, there was a direct relationship between oxLDL concentrations and PON activity (with phenyl acetate as the substrate; r = 0.611, P = 0.0001) and an atherogenic NMR lipid profile in those who were PON-1 55LL homozygotes. Circulating oxLDL concentrations in vivo were unrelated to PON-1 genotypes or activity, except in male Type II diabetics where there was a direct association between PON activity (with phenyl acetate as the substrate) and oxLDL levels. These in vivo data contrast with in vitro data, and may be due to confounding by dietary fat intake. Male Type II diabetic subjects with PON-1 55LL homozygosity have an atherogenic NMR lipid profile independent of LDL oxidation. These data do not support an in vivo action of PON on LDL oxidation.