Dual-precipitation method evaluated for determination of high-density lipoprotein (HDL), HDL2, and HDL3 cholesterol concentrations

We evaluated a dual-precipitation method for determining cholesterol in high-density lipoprotein (HDL) and its subfractions HDL2 and HDL3. After total HDL was isolated by precipitation of very-low-density (VLDL) and low-density (LDL) lipoproteins with polyethylene glycol (Mr 8000), HDL2 was isolated from total HDL by precipitation with dextran sulfate (Mr 15,000), leaving HDL3 in the supernate. Concentration of total HDL cholesterol after precipitation of VLDL and LDL with PEG showed significant proportional and constant biases of -3.8% and 0.04 mmol/L, respectively, when compared with a phosphotungstic acid-based comparison method, although results by the two methods were correlated highly (r = 0.99, P less than 0.001). HDL2 and HDL3 cholesterol concentrations measured with the present technique were not different from those obtained by density-gradient ultracentrifugation or by combined precipitation-ultracentrifugation.     

Comparison of a homogeneous assay with a precipitation method for the measurement of HDL cholesterol in diabetic patients

OBJECTIVE: To compare direct-measured HDL cholesterol with HDL cholesterol measured by a precipitation method. RESEARCH DESIGN AND METHODS: We compared a homogeneous assay for direct HDL cholesterol analysis with the phosphotungstic acid magnesium chloride precipitation method in 55 type 1 diabetic patients, 70 type 2 diabetic patients, and 82 nondiabetic normal control subjects with plasma triglyceride levels <4.6 mmol/l. The cholesterol content of HDL determined by the direct assay was overall 0.1 mmol/l higher in all three groups than HDL cholesterol measured after precipitation, but the two methods were closely correlated (r(2) = 0.98, P < 0.001). RESULTS: HbA(1c), blood glucose, serum albumin, serum bilirubin, or triglyceride did not influence the differences of the two HDL cholesterol measurements. Because we have previously shown HDL cholesterol isolated by phosphotungstic acid precipitation to be lower than that by ultracentrifugation, the positive bias found in this study was expected. It seems that the direct HDL cholesterol assay reacts with apolipoprotein (apo) B-containing lipoproteins in the fraction with a density of >1.063; these apo B-containing lipoproteins are suggested to be coprecipitated with the phosphotungstic acid method. We also measured LDL cholesterol directly by a LDL cholesterol plus method and found no significant differences between this method and LDL cholesterol calculated from Friedewald's formula. CONCLUSIONS: Direct homogeneous assay for HDL cholesterol determination in diabetic patients seems not to exhibit a negative bias, in contrast to the precipitation method, when compared with the ultracentrifugation method. In addition, the direct assay saves time and is not influenced by type of diabetes or degree of metabolic control.     

Determination of LDL cholesterol and LDL apolipoprotein B following precipitation of VLDL in blood serum with phosphotungstic acid/MgCl2

A method is described for the selective precipitation of VLDL in blood serum using phosphotungstic acid/MgCl2. The method allows for the calculation of LDL apolipoprotein B as well as for the calculation of LDL cholesterol (following the additional determination of HDL cholesterol). Dependent on the triglyceride and the cholesterol content of the serum, three different procedures were developed using phosphotungstic acid and MgCl2 in different concentrations in the precipitation assay. Within the tested range of 3-10 mmol/l total cholesterol and 1-4 mmol/l triglyceride in blood serum the VLDL were nearly completely precipitated with negligible coprecipitation of LDL and HDL, but 40-50% coprecipitation of Lp(a). Regression analysis of the cholesterol values obtained by precipitation with phosphotungstic acid/MgCl2 (= serum cholesterol - LDL cholesterol), and the cholesterol values obtained by ultracentrifugation (d greater than 1.006 kg/l) revealed a good measure of agreement (r = 0.97, y = 0.93 X + 0.35, n = 76). An equally good measure of agreement was found for the corresponding apolipoprotein B values (r = 0.96, y = 1.03 X - 0.2, n = 61). In the determination of LDL cholesterol a variation coefficient of 4.3% (n = 20) was found in relation to the precision in the series, and a variation coefficient of 4.8% (n = 25) in relation to day to day precision.     

Lipoprotein fractionation by a precipitation method. A simple quantitative procedure.

A method is described for quantitation of the three major classes of serum lipoproteins. After precipitation of very low density lipoprotein (VLDL) using sodium dodecyl sulphate, the cholesterol and triglyceride content of this lipoprotein class is directly measured. In a second aliquot serum high density lipoprotein (HDL) lipids are measured after precipitation of VLDL and low density lipoprotein (LDL). LDL cholesterol and triglyceride contents are calculated by difference. The procedure requires 2 ml serum, and sensitivity is adequate to permit lipoprotein analyses on umbilical cord serum. Close agreement is observed between this precipitation method and preparative ultracentrifugation.     

Defect in cholesterol transport in patients receiving maintenance hemodialysis

A defect in cholesterol transport was detected in patients with uremia who were receiving long-term hemodialysis when the rate of cholesterol transfer (RCT) from high-density lipoprotein (HDL) to very low-density (VLDL) and low-density lipoproteins (LDL) was compared with that in controls. The RCT (mean +/- SD) in 29 men with uremia (1.85 +/- 1.29 mg/hr/100 ml) and 11 women with uremia (1.84 +/- 1.00 mg/hr/100 ml) was significantly lower (P less than 0.001) than values in 55 healthy men (4.50 +/- 2.61 mg/hr/100 ml) and 23 healthy women (3.72 +/- 1.92 mg/hr/100 ml), respectively. Six patients, but none of the controls, totally lacked the ability for cholesterol transfer. The decreased RCT of the patients could not be completely accounted for by their decreased HDL cholesterol levels, because patients matched with controls for HDL cholesterol within 1 mg/100 ml also had lower RCT (P less than 0.0025). Recombination and crossover of serum fractions of patients and controls separated by ultracentrifugation revealed that the defect in cholesterol transfer of the patients was in the d greater than 1.063 gm/ml fraction (containing HDL and other serum proteins), which not only contained less HDL cholesterol, but was also qualitatively inferior as donor for cholesterol transfer. In one of four patients studied, the d less than 1.063 gm/ml fraction (VLDL and LDL) also had deficient ability to accept cholesteryl esters in the transfer. These in vitro data indicate a defect in cholesterol transport in the patients who are undergoing hemodialysis. Whether this defect exists in vivo and creates the risk of accelerated atherosclerosis warrants further study.     

Proton magnetic resonance spectroscopy of fractionated plasma lipoproteins and reconstituted plasma from healthy subjects and patients with cancer.

Proton nuclear magnetic resonance spectra at 500 MHz of plasma and the very low density lipoprotein (VLDL), low density lipoprotein (LDL), and high density lipoprotein (HDL) fractions isolated by KBr gradient ultracentrifugation were analysed in 16 cancer patients, six pregnant and nine non-pregnant healthy subjects. In spectra with narrow plasma composite aliphatic peaks (methylene at 1.2-1.4 p.p.m. and methyl at 0.8-0.9 p.p.m., respectively), a relative increase in either VLDL, LDL, or both, or a decrease in HDL signals was observed. The mechanism for line-width narrowing seemed different in cancer patients (less signals from HDL relative to VLDL) compared with pregnant women (more signals from LDL). By reconstitution of plasma samples from both healthy subjects and patients with malignant disease, decreased concentration of VLDL or HDL resulted in broadening or narrowing of the composite peaks, respectively. The effects of VLDL and HDL on the plasma line width were moderated by the signals from LDL. Within lipoprotein fractions, the methylene and methyl resonances were shifted to a higher field with increased observation temperature, the change in shift being greatest for HDL. The line width of composite peaks in plasma varied with the observation temperature, depending on the relative concentrations of individual lipoproteins. The correlation coefficient (r) for the relation between total plasma triglyceride level and the average of the line-width of the composite methylene and methyl peaks was -0.78 (p less than 0.001). For spectra of individual lipoproteins, statistical significant relationships were found between line-widths and triglyceride content of the LDL fraction (methyl line-width, r = -0.63) (p less than 0.001) and between methylene line-width and cholesterol of HDL (r = 0.54) (p = 0.003). In summary, the shape and width of the composite aliphatic peaks of plasma were affected by the relative concentration, chemical shift and transition temperature of both VLDL, LDL, and HDL, and by the total triglyceride level. Comparing pregnancy and malignant disease, the lipoprotein resonances contributed differently in giving narrow composite signals.     

Lipoprotein cholesterol concentrations in patients with Beh?et's disease

Serum cholesterol concentrations among very low, low, and high density lipoproteins (VLDL, LDL and HDL) in 12 male patients with Beh?et's disease were compared with those of 12 normal male subjects. Serum lipoproteins were separated by a combination of ultracentrifugation and gel filtration chromatography. The patients had significantly (p less than 0.001) lower concentrations of HDL-cholesterol than the control subjects (356 +/- 62 mg/l vs. 573 +/- 108 mg/l, means +/- SD). The cholesterol concentrations in apolipoprotein B-containing lipoproteins (VLDL and LDL) from the patients tended to be reciprocally higher than those of the controls, though not statistically significant. There was no difference in serum total cholesterol concentrations. The chemical composition of HDL from the patient group was characterized by higher protein and lower cholesterol (both esterified and free) contents compared with the control HDL.     

Precipitation of LDL with sulphopolyanions: a comparison of two methods for LDL cholesterol determination

Two commercially available tests for the determination of LDL cholesterol were compared. The determination principle lies in the precipitation of LDL using heparin (Merck, Darmstadt test) or using polycyclic surface activated anions (bioMérieux test), as well as ascertaining LDL cholesterol by subtracting cholesterol in the supernatant from total cholesterol (Merck, Darmstadt test) or the direct determination of cholesterol in the redissolved precipitate (bioMérieux test). Regression analysis of the LDL cholesterol values obtained by precipitation and the LDL cholesterol values obtained by the combination of ultracentrifugation and HDL cholesterol determination (cholesterol in the d greater than 1.006 kg/l ultracentrifugation fraction- HDL cholesterol) resulted in a good correlation (precipitation using polycyclic surface activated anions: r = 0.95, y = 1.01x - 0.14, n = 34; heparin precipitation: r = 0.94, y = 0.86x + 0.10, n = 28). In sera containing triglycerides ranging from 2.28 mmol/l to 10.9 mmol/l there was an adequate agreement between the values obtained by precipitation with polycyclic surface activated anions and those of the reference method (r = 0.94, y = 0.9x + 0.47, n = 51), whereas the data obtained by heparin precipitation clearly deviated from the data of the reference method (r = 0.69, y = 0.75x + 0.003, n = 32).     

Accelerated cholesteryl ester transfer in patients with insulin-dependent diabetes mellitus

Abnormalities in cholesteryl ester transfer (CET) may play a role in the development of diabetic arterial vascular complications. To assess this important step systematically in reverse cholesterol transport, we have studied 20 treated, clinically stable, normolipidaemic patients. Contrary to the impairment in CET described previously in NIDDM, the mass of CE transferred from HDL to VLDL + LDL was significantly greater in IDDM patients than in controls at 1,2, and 4 h (P less than 0.001). When the d less than 1.063 plasma fractions from IDDM subjects were combined with controls d less than 1.063 fractions, an accelerated CET response was observed which was identical to that found in intact IDDM plasma. This finding, which indicates that this disturbance in CET was associated with the acceptor lipoproteins, was confirmed when we found that it was reproduced by the addition of IDDM VLDL and not LDL to control d greater than 1.063 fractions. Changes observed in lipoprotein core lipid composition were consistent with accelerated CET occurring in IDDM in vivo: the TG/CE core lipid ratio was decreased in VLDL from six subjects (diabetic 9.5 +/- 0.8 vs control 12.9 +/- 3.4; P less than 0.1) and increased in their HDL (diabetic 0.55 +/- 0.11 vs control 0.42 +/- 0.04; P less than 0.025). No correlation was demonstrable between estimates of diabetic control (glycoalbumin, fasting glucose) and CET. These data indicate that CET may be abnormally increased in normolipidaemic IDDM patients. A defect of this type may be atherogenic because it increases the number of lipoprotein particles in plasma which resemble cholesteryl ester-enriched chylomicron and VLDL remnants but whose normal receptor-mediated catabolism may be altered.     

Simplified methods for measuring cholesterol concentrations of high-density lipoprotein subclasses in serum compared

We used a combination of heparin micro-affinity column chromatography and heparin-Mn2+/dextran sulfate (DS) precipitation procedures to measure directly the total high-density lipoprotein (HDL)-cholesterol and HDL3-cholesterol in serum. The value for HDL2-cholesterol was obtained by subtracting the value for HDL3-cholesterol from that for total HDL-cholesterol. Results of this methodology and of the original heparin-Mn2+/DS double-precipitation method were compared with those of preparative ultracentrifugation. Concentrations of HDL- and HDL2-cholesterol by the original double-precipitation method were respectively 26 and 33 mg/L lower than by the ultracentrifugation method (p less than 0.001). Corresponding values by the micro-affinity/precipitation method were 36 and 29 mg/L higher than by the ultracentrifugation method (p less than 0.001). The values for HDL3-cholesterol by ultracentrifugation and by precipitation differed only by 7 mg/L (p greater than 0.05). Micro-affinity/precipitation and double-precipitation results both correlated well with those by ultracentrifugation (r = 0.84 to 0.94); the y-intercept of the comparison with the micro-affinity/precipitation method was close to zero. Use of the micro-affinity/precipitation method with samples from black or white adolescents showed that the blacks had higher concentrations of HDL2-(p less than 0.01) and HDL3-cholesterol (p less than 0.02) than did the whites.     

High protein diet complements resin therapy of familial hypercholesterolemia.

The intermediate-term effects on plasma lipoprotein lipids of substituting meat and dairy protein for carbohydrate in the diets of five subjects (three women, two men) with familial hypercholesterolemia receiving cholestyramine (mean dose, 18 g/d) were studied. Subjects were randomly allocated to either the high or low protein diets (mean 27 versus 10% of energy as protein, 25% as fat, and 48 versus 65% as carbohydrate) for 4 to 5 weeks and then switched to the other diet for another 4 to 5 weeks. Mean fasting plasma HDL cholesterol rose significantly by 17 +/- 3% (1.11 +/- 0.12 vs 0.95 +/- 0.11 mmol/L, p less than 0.005, n = 5), whereas total triglycerides fell by 23 +/- 2% (1.7 +/- 0.3 vs 2.2 +/- 0.3 mmol/L, p less than 0.005, n = 5), VLDL triglycerides fell by 28 +/- 5% (0.88 +/- 0.15 vs 1.18 +/- 0.19 mmol/L, p less than 0.02, n = 5), VLDL cholesterol fell by 32 +/- 7% (0.39 +/- 0.08 vs 0.56 +/- 0.09 mmol/L, p less than 0.01, n = 5), the ratio of LDL cholesterol: HDL cholesterol by 19 +/- 5% (4.7 +/- 0.7 vs 5.7 +/- 0.7, p less than 0.05) and that of total cholesterol: HDL cholesterol by 16 +/- 5% (6.6 +/- 0.5 vs 8.0 +/- 0.7, p less than 0.05) on the high versus low protein diet. Increasing dietary protein intake at the expense of carbohydrate may be useful in treating hypoalphalipoproteinemia and/or hypertriglyceridemia in patients with familial hypercholesterolemia.     

Effect of ileal exclusion on kinetics of very low density lipoprotein triglycerides in familial hypercholesterolemia

Plasma lipoproteins, VLDL triglyceride kinetics, and bile acid and cholesterol synthesis were measured in 21 patients heterozygous for familial hypercholesterolemia with (n = 11) or without (n = 10) ileal bypass. LDL cholesterol and apoprotein B concentrations were lower, and cholesterol and bile acid synthesis, the VLDL triglyceride/cholesterol ratio, and the HDL cholesterol concentration were higher in the operated than the control patients. The VLDL triglyceride production rate was increased in the operated normotriglyceridemic patients by about 65%, whereas the fractional catabolism of VLDL triglycerides and the calculated VLDL cholesterol transport were similar in the operated and control groups. VLDL triglyceride production was not correlated with cholesterol or bile acid synthesis. The VLDL triglyceride concentration was positively correlated with the production and negatively with the fractional catabolism of VLDL triglycerides. In unoperated normotriglyceridemic patients the VLDL triglyceride production was positively correlated with LDL cholesterol (r = 0.69, p less than 0.05), LDL triglyceride (r = 0.84, p less than 0.01) and LDL apoprotein B (r = 0.80, p less than 0.01) concentrations, and with the LDL triglyceride/apoprotein B (r = 0.72, p less than 0.05) and LDL triglyceride/cholesterol (r = 0.68, p less than 0.05) ratios. None of these correlations was significant in the operated patients. We conclude that in heterozygous familial hypercholesterolemia VLDL triglyceride level depends on both VLDL triglyceride synthesis and catabolism, LDL level is proportionate to VLDL triglyceride production in the unoperated patients but not in the patients with ileal bypass, ileal exclusion results in an increase in the production rate of VLDL triglycerides in normotriglyceridemic patients but otherwise VLDL triglyceride production is poorly associated with cholesterol and bile acid synthesis, ileal exclusion may induce hepatic secretion of triglyceride-rich VLDL.     

Effects of dietary cholesterol and fasting on hamster plasma lipoprotein lipids

Hamsters are commonly utilized for comparative study of cholesterol metabolism. The present study was conducted to assess the effects of fasting on the plasma lipoprotein cholesterol concentrations of hamsters. Over a period of 3 weeks, adult male Golden Syrian hamsters (n = 32) were fed chow with or without the addition of 2 g/kg cholesterol. Half of the animals consuming each diet were fasted for 18 hours prior to blood sampling. Comparison of diets showed the following increases in those animals receiving cholesterol: total plasma cholesterol (180%) and triacylglycerols (75%), high density (75%), low density (250%), and very low density (560%) lipoprotein cholesterol. Compared with fasted animals, total plasma triacylglycerols were higher in both non-fasted diet groups. Compared with fasted hamsters that had received cholesterol, total plasma cholesterol (mean +/- SE mmol/l) was greater (6.36 +/- 0.18 vs 5.43 +/- 0.21; p less than or equal to 0.05) in the non-fasted group, due primarily to higher VLDL cholesterol (2.07 +/- 0.18 vs 1.58 +/- 0.18; p less than or equal to 0.05). There were no differences in HDL cholesterol (2.07 +/- 0.05 vs 2.17 +/- 0.08) or LDL cholesterol (1.29 +/- 0.08 vs 1.37 +/- 0.05) between fasted and non-fasted hamsters fed cholesterol. Fasting is not necessary for the study of the plasma HDL cholesterol and LDL cholesterol of hamsters fed cholesterol.     

Lipoprotein changes during continuous subcutaneous insulin infusion in insulin-dependent diabetic patients

We have studied the long-term effects (9 months) in plasma lipoprotein concentrations during continuous subcutaneous insulin infusion (CSII) (n = 11, six females, five males) and compared these changes to conventional insulin therapy (CIT) (n = 12, six females, six males). The two groups were allocated to CSII or CIT randomly, and were comparable as regards lipoprotein values at the start of the study. There were initially normal total plasma cholesterol values in both groups (CSII group: mean plasma cholesterol 3.77 +/- 0.57 mmol/l, CIT group: mean plasma cholesterol 4.37 +/- 0.55 mmol/l, means +/- SD). Further, there were normal total plasma triglyceride values at the start of the study (CSII group: mean plasma triglyceride 0.86 +/- 0.23 mmol/l, CIT group: mean plasma triglyceride 0.84 +/- 0.26 mmol/l, means +/- SD). There were no alterations seen in total plasma cholesterol and total plasma triglyceride in either groups during a 9 months observation period. In the same period no changes in LDL and HDL levels were registered. The very low density lipoprotein (VLDL) was separated into VLDL-1 and VLDL-2 by its binding to heparin-sepharose columns. It was found that CSII treatment for 9 months resulted in a decline in VLDL-2-triglyceride values (0.18 +/- 0.07 mmol/l before versus 0.10 +/- 0.07 mmol/l after, p less than 0.05, means +/- SD) which was not seen in the CIT group. Decline in VLDL-2-triglyceride might delay the development of late diabetic manifestations.     

Hypertriglyceridemic hyperapoB in type 2 diabetes

OBJECTIVES: Much less attention has been paid to LDL in type 2 diabetes than to VLDL or HDL. In particular, there are few data on apoB levels in these patients. Moreover, most reports have focused on mean lipoprotein levels and consequently there is little information on the frequencies of the various dyslipidemic phenotypes. RESEARCH DESIGN AND METHODS: Plasma and lipoprotein lipids, apoB and apoA1 were measured by standardized methods. LDL particle size was determined by PAGE. The total cohort was divided into phenotypes by two different methods. The first was based on triglycerides (> or = or <1.5 mmol/l) and LDL cholesterol (> or = or <4 mmol/l), whereas the second was based on triglycerides (> or = or <1.5 mmol/l) and apoB (> or = or <120 mg/dl). RESULTS: For the overall cohort, plasma triglycerides were elevated (2.13 +/- 1.6 mmol/l), total and LDL cholesterol were normal (5.34 +/- 1.1 and 3.28 +/- 0.88 mmol/l, respectively), and peak LDL size was reduced (252.9 +/- 5.8 A). HDL cholesterol was between the 25th and 50th percentiles of the general population (1.12 +/- 0.36 mmol/l). The average level of apoB was 114 +/- 29 mg/dl, a value that is between the 50th and 75th percentiles of the general population and is higher than that for LDL cholesterol, which was between the 25th and the 50th percentiles of the population. The results of the phenotyping analysis were as follows. Using the conventional approach, only 23% has abnormal LDL, i.e., an elevated LDL cholesterol level. Using the new approach, almost 40% has an elevated apoB and therefore an elevated LDL particle number. Only 12.8% has combined hyperlipidemia based on the conventional approach, whereas almost one-third had the equivalent, hypertriglyceridemic hyperapoB-based on the new algorithm. The severity of the dyslipoproteinemia in this group was noteworthy. Although the average LDL cholesterol was 3.91 mmol/l, a value just below the 75th percentile of the general population, the average apoB was 145 mg/dl, a value that approximates the 95th percentile of the population. CONCLUSIONS: The dyslipidemic profile of patients with type 2 diabetes is not uniform. A substantial group have normal lipids and normal LDL particle number and size whereas others have markedly abnormal profiles. Diagnosis based on triglycerides and apoB rather than triglycerides and LDL cholesterol revealed that more than one in five had hypertriglyceridemic hyperapoB, which is characterized by hypertriglyceridemia, marked elevation of LDL particle number, small dense LDL, and low HDL, a constellation of abnormalities that is associated with markedly accelerated atherogenesis and therefore justifies intensive medical therapy.     

HDL phosphatidyl choline and risk-factors of coronary heart disease

As part of our epidemiological study of employees in Westphalia, the concentration of HDL phosphatidyl choline was measured in 1546 men and 778 women. The results were analysed in relation to the corresponding HDL cholesterol values, as well as the various risk factors for coronary heart disease. HDL phosphatidyl choline values were found to be age independent, higher in women than in men (p les than 0.001), and lognormally distributed in both sexes (men: mean 1.162 mmol/l, median 1.13 mmol/l, minimum 0.60 mmol/l, maximum 2.46 mmol/l; women: mean 1.370 mmol/l, median 1.34 mmol/l, minimum 0.55 mmol/l, maximum 2.46 mmol/l). A positive correlation (p less than 0.001) was found in both sexes between HDL phosphatidyl choline and HDL cholesterol (men: r = 0.588; women r = 0.605). A negative correlation was found in both sexes between HDL phosphatidyl choline values and body weight (men: r = -0.102 (p less than 0.001) women: r = -0.129 (p less than 0.001); and in men, but not in women, there was a negative correlation between HDL phosphatidyl choline values and triglycerides (men: r = -0.190 (p less than 0.001) women: r = -0.042). A negative correlation between HDL phosphatidyl choline and cigarette smoking was found only in female smokers (r = -0.121 (p less than 0.05). The correlation coefficients between HDL cholesterol and triglycerides as well as HDL cholesterol and relative body weight in both sexes were clearly higher than the corresponding correlation coefficient of HDL phosphatidyl choline. In men as well as in women the HDL phosphatidyl choline/HDL cholesterol ratio decreased with increasing HDL cholesterol values or decreasing triglyceride values in blood serum.     

Change in very low-, low-, and high-density lipoproteins during lipid lowering (bezafibrate) therapy: studies in type IIA and type IIb hyperlipoproteinaemia

The effects of lipid lowering therapy (bezafibrate) on plasma lipoproteins was investigated in twelve patients with familial hypercholesterolaemia (type IIA) and eight with familial combined hyperlipidaemia (type IIB). Bezafibrate caused a decrease of plasma cholesterol, plasma triglycerides, plasma apolipoprotein B, VLDL cholesterol and LDL cholesterol and an increase of HDL cholesterol. Post-heparin plasma lipoprotein and hepatic lipase activities increased in both groups (significant only in type IIB). Lipoprotein composition showed the following changes: Increased protein and phospholipids and decreased triglycerides and cholesteryl esters in VLDL. Decreased protein and triglycerides and increased free and esterified cholesterol in LDL. Decreased triglycerides and increased phospholipids in HDL. Cholesteryl ester to protein ratios decreased in VLDL and increased in LDL. The hydrated density of LDL (both groups) and of HDL3 (type IIB) decreased following bezafibrate therapy. These changes were in general similar to those observed in hypertriglyceridaemic patients and could be ascribed, at least in part, to the increase of plasma lipase activities and the decrease of lipid transfer reactions. Comparing the present data with that previously reported, it was found that bezafibrate caused decreased LDL cholesterol in types IIA and IIB but increased levels in type IV. This change was correlated with the initial plasma triglycerides (r = 0.74, P less than 0.0001) and initial plasma LDL cholesterol (r = 0.66, P less than 0.001). It is concluded that varied response of LDL to therapy reflects a complex interaction of metabolic events, including changing rates of VLDL conversion to LDL, lipoprotein compositional changes and effects of therapy on LDL degradation rates.     

A long-term study of the effects of norethisterone on lipoprotein metabolism in menopausal women

Long-term effects of the androgenic progestogen norethisterone on lipoprotein metabolism were studied by measuring lipoprotein concentrations in 21 women during one year on treatment for the relief of climacteric symptoms. There were significant reductions in total serum triglyceride (p less than 0.01), very low density lipoprotein (VLDL) cholesterol (p less than 0.01) and high density lipoprotein (HDL) cholesterol (p less than 0.001) after two months on treatment, all of which were still in evidence after one year. Low density lipoprotein (LDL) cholesterol levels climbed gradually becoming significantly higher than baseline after one year on treatment (p less than 0.01). Comparison of cholesterol concentrations in HDL subfractions in the one year treated subjects with those in a control group of untreated subjects suggests that the fall in HDL cholesterol is due to reductions in both the HDL2 and HDL3 fractions. We conclude that norethisterone adversely affects the important lipoprotein risk factors for coronary heart disease.     

Cholesterol distribution between high-density-lipoprotein subfractions HDL2 and HDL3 determined in serum by discontinuous gradient gel electrophoresis

We used discontinuous gradients of polyacrylamide gel to determine the high-density-lipoprotein (HDL) subfractions HDL2 and HDL3 of serum lipoproteins. Serum (40 microL) prestained with Sudan Black was electrophoresed in cylindrical tubes over successive layers of 3.5%, 6%, 13%, and 17.5% acrylamide gels in a Tris.glycine buffer (3-4 h, 300 V). Very-low- (VLDL) and low-density lipoprotein (LDL) were retained by the 3.5% and 6% gels. HDL2 was concentrated at the interface between the 13% and 17.5% gels, and HDL3 migrated into the 17.5% gel. The distribution between HDL2 and HDL3 was obtained by densitometric scanning. Application of the respective percentages to HDL cholesterol assayed after phosphotung-state-Mg2+ precipitation of VLDL and LDL gave calculated concentrations of HDL2 and HDL3 cholesterol. The calculated values for HDL2 cholesterol were in excellent agreement with those for HDL2 isolated by ultracentrifugation (r = 0.920 for n = 120 sera; differences nonsignificant by Student's paired t-test). Besides being highly discriminating, the method is rapid, easily performed, and economical.     

Falsely high high-density lipoprotein triglyceride values by the heparin-manganese precipitation method

Recent evidence indicates that high-density lipoprotein triglyceride (HDL-Tg) may be a predictor of coronary artery disease. We examined three methods for HDL-Tg measurement, comparing results obtained by measurement of Tg in the supernate after heparin-manganese chloride (heparin-Mn) precipitation of EDTA-treated plasma (I) with results obtained after preparative ultracentrifugation (II and III). In II, we used heparin-Mn precipitation of low-density lipoprotein (LDL) from the infranate after ultracentrifugation at d 1.006 to remove very-low-density lipoprotein (VLDL). In III, we performed sequential flotation ultracentrifugation at d 1.006 and 1.063, then measured Tg in the d greater than 1.063 fraction. Method I gave significantly higher HDL-Tg results than II and III, which gave essentially identical results. The difference in results between I and II was not caused by the presence of heparin or manganese chloride, because these were used in both methods. Prior removal of VLDL in II and III resulted in lower HDL-Tg values, and subsequent removal of LDL by precipitation or ultracentrifugation did not alter final HDL-Tg values. The higher values obtained in I were the result of the presence of VLDL-rich unsedimented precipitate in the supernate.