Sequential microultracentrifugation of lipoproteins in 100 microliters of serum.

A method is described for sequential separation of high density, very low density, and low density lipoproteins (HDL, VLDL, and LDL, respectively) from 100 microliters of serum, using an air-driven ultracentrifuge (Airfuge, Beckman). Cesium chloride was used for density adjustment. Precision-within-series (coefficient or variation) depended on the cholesterol concentration in the lipoprotein fractions, greater than 1 mmol/l, less than 2.3%. Recovery within-series was nearly 100%. The results (mmol/l) correlate well with those from an electrophoretic-enzymatic procedure (alpha-HDL: 1.49 +/- 0.34 vs. 1.48 +/- 0.33, r = 0.949; pre-beta-VLDL: 0.58 +/- 0.42 vs. 0.59 +/- 0.45, r = 0.975; beta-LDL: 3.11 +/- 0.93 vs. 3.07 +/- 0.88, r = 0.990; n = 48). Recovery of lipoprotein cholesterol from this group was 99.2 +/- 4.2%. A combination of ultracentrifugation with high-performance thin-layer chromatography for determination of lipoprotein-lipid profiles was achieved with recoveries of 98-101%, as evaluated from a group of healthy men (n = 31) and women (n = 38). The entire procedure is, therefore, suitable for compositional studies on lipoproteins from small serum samples. In particular, capillary serum from children of all ages, even from premature neonates, is quite adequate.

Early indicators for the risk of atherogenesis. A field test of a new methodology on kindergarten children in Düsseldorf.

Parameters related to the risk of atherogenesis in adults were investigated in kindergarten children living in an industrial city. Height, weight and blood pressure were measured and capillary blood obtained. This study is a field test of methods for the analysis of 11 metabolic variables in capillary serum [glucose (GLUC), uric acid (UA), total, free and esterified cholesterol (TC, C, CE), triacylglycerols (TG), phosphatidylethanolamine (PE), phosphatidylcholine (PC); beta-, pre-beta- and alpha-lipoproteins (LPs)]. For lipid and LP analysis, new micro- and ultramicroprocedures were used, which have proved to be adequate for large-scale studies outside the clinic, including young children. Among 497 German children, a reference group, an overweight group and a socially underprivileged group were distinguished. In the reference group, several sub-groups with parameters lying in the tails of the concentration distributions were considered separately, thus providing specific metabolic patterns for these groups and, also, for any individual child. The children in the overweight group exhibited significantly reduced TC and beta-LP levels. In socially underprivileged children with anthropometric data within the normal range, a reduction of several blood constituents was seen: GLUC, TG, TC, PC, pre-beta- and alpha-LP; the PC: PE ratio was diminished. The beta: alpha-LP ratio in these children was moderately increased. This effect was more pronounced in growth-retarded children, who also showed negative correlation of TG: alpha-LP.

On the composition of capillary and venous blood serum.

The concentrations of various clinical-chemical substances in the capillary and venous blood serum of apparently healthy adults (20-30 yr) were examined in the fasting state. Total protein, bilirubin, calcium, sodium and chloride concentrations were significantly lower (less than or equal to 5%) in capillary than in venous serum. In non-hemolytic capillary sera, the concentration of potassium was nearly the same as in venous samples. Inorganic phosphorus and urea concentrations were identical in both specimens. There was a tendency for glucose concentrations to be higher in capillary than in venous serum. It is concluded that capillary and venous blood serum can be used interchangeably only for certain purposes. It should be emphasized that these results are valid for apparently healthy adults in the fasting state; other population samples remain to be investigated.

Comparison of two micromethods for determination of lipoprotein cholesterol in plasma.

We compared the results obtained by a micromethod for the determination of plasma lipoprotein cholesterol, in which electrophoresis is used to separate the lipoprotein fractions (beta-, pre-beta-, and alpha-lipoproteins), with those determinations with ultracentrifugation (low-density, very-low-density, and high-density lipoproteins). Precision of determination (coefficient of variation, CV, %) was the same for beta- and low-density lipoproteins (1.6%), and for pre-beta- and very-low-density lipoproteins (3.7%); however, determination of alpha-lipoprotein cholesterol was more precise (1.4%) than that of high-density lipoprotein cholesterol (3.1%). Analytical recovery of lipoprotein cholesterol was the same for both methods (98--100%) and the results were closely correlated (r = 0.943). The procedure has been used to determine the cholesterol content of plasma lipoprotein fractions of apparently healthy adults (both sexes). Lipoprotein cholesterol concentrations in our population sample compare well with those reported for other groups of similar age, in particular Stanford long-distance runners.

Differences in lipid and lipoprotein concentrations of capillary and venous blood samples.

The concentrations of lipids and lipoproteins measured in capillary blood serum taken from young adults were significantly lower than in venous blood, although they correlated satisfactorily. These differences may reflect differences in the morphological and hemodynamic conditions existing either in large veins or in the peripheral circulatory system. It was concluded that venous and capillary blood serum cannot be used interchangeably for these estimations.

Quantitative high-performance thin-layer chromatography of lipids in plasma and liver homogenates after direct application of 0.5-microliter samples to the silica-gel layer.

Lipid profiles were determined by high-performance thin-layer chromatography (HPTLC) after direct application of 0.5 microliter plasma from capillary blood to the silica-gel layer. Coefficients of variation for the fluorescence measurements were 2.1% for phosphatidylcholine. The recovery of known amounts of lipid was 96--100%. A linear relationship between peak area and amount of lipid was found in the nmole range, corresponding to the amount of lipid in 0.125--0.75 microliter Lipid-Trol, which served as the standard reference sample. The plasma lipids of healthy subjects and of patients suffering from various illnesses were analyzed using reference methods and HPTLC. Identical values were obtained for cholesterol esters, triacylglycerols and phosphatidylcholine. Free cholesterol values determined by HPTLC were slightly lower (7%). The correlation between data obtained by reference methods and HPTLC was as follows: cholesterol, r = 0.938; cholesterol esters, r = 0.964; triacylglycerols, r = 0.985; phosphatidylcholine, r = 0.938. The separaiton and quantitation of liver lipids using HPTLC after direct application of the tissue homogenate to the silical-gel layer was carried out. Comparison with reference methods revealed that HPTLC gave higher cholesterol values (24%). The triacylglycerol concentrations, however, were identical under both methods and correalted satisfactorily (r = 0.959).