Characterization of modified low density lipoprotein subfractions by capillary isotachophoresis.

Oxidative modification of low density lipoproteins (LDLs) is an important pathogenetic factor in atherosclerosis. The various steps in oxidative modifications of LDL can be monitored using different methodologies with varying degrees of complexity. In this study, we propose capillary isotachophoresis (CITP) as a suitable tool to detect and measure the degree of oxidation of LDL. LDL was isolated from pooled plasma of healthy volunteers by sequential ultracentrifugation, and oxidation was performed in vitro as well as in cell culture experiments. Native LDL and oxidatively modified LDL were characterized by apo B-100 fluorescence and conjugated diene formation. Samples were separated by CITP combined with sudan black B staining. To underline the inherent advantages of this approach, CITP was compared with classical lipoprotein electrophoresis using agarose gel. We demonstrate the CITP method to be highly sensitive, as changes in peak area of the separated LDL subfractions were detected after only 2 h of oxidation. The leading LDL peaks increased, while the terminating LDL peaks decreased in parallel throughout the duration of oxidation. The LDL samples, oxidized for 4-24 h, also exhibited an increased migration velocity of the fractions. In summary, we present the first study investigating LDL-subfractions separated by CITP and the alterations of these LDL-subfractions after gradual in vitro oxidation and after oxidative modification by monocyte-derived macrophages and vascular smooth muscle cells.

Separation of lipoproteins by capillary isotachophoresis combined with enzymatic derivatization of cholesterol and triglycerides.

Combining specific enzymatic derivatization of cholesterol or triglycerides with capillary isotachophoresis (CITP), human serum lipoproteins are separated into 14 lipoprotein subfractions, monitored and quantitated by direct capillary UV detection. By comparing the separation patterns of human serum with the patterns of lipoprotein particles isolated by sequential ultracentrifugation it became evident that peaks 1-5 represent lipoproteins of the high density lipoprotein (HDL) fraction, peaks 6-8 embody the very low density lipoprotein (VLDL) fraction and chylomicrons, and peaks 7-14 represent the low density lipoprotein (LDL) fraction. Peaks 7 and 8 were found in the VLDL as well as in the LDL fraction. Using triglyceride-specific staining peaks 6-8 occurred prominently; and with cholesterol-specific staining, peaks 1-5 and 7-14 were prominent. The coefficient of variation, for the sum of the peak heights of a pooled serum, was 3.94 for triglyceride-specific staining and 2.32 for cholesterol-specific staining. A linearity range between 0.23 and 2.29 mM/L was found for triglyceride-specific staining and between 0.043 and 4.33 mM/L for cholesterol-specific staining. The practicability of the method was evaluated (i) using blood of humans before and 45 min after an oral fat load. Triglyceride-specific staining revealed a prominent increase in the VLDL fraction and chylomicrones containing peaks 6 and 7, and a minor increase in the HDL fraction containing peaks 3 and 4, and (ii) in patients with manifest hypothyroidism before and after thyroxine therapy. Cholesterol-specific staining demonstrated a massive decrease in the first peak of the HDL fraction and in peaks 9 and 11 of the LDL fraction regarding the hypo versus hyperthyroid state.

Aetiology and clinical significance of thrombocytosis: analysis of 732 patients with an elevated platelet count.

OBJECTIVE: To determine the aetiology and clinical significance of an elevated platelet count (thrombocytosis) in a large cohort of patients. DESIGN: A retrospective review of the medical records was performed on all patients, who had at least one platelet count > or = 500 x 10(9) L-1. SETTING: Departments of Medicine and Surgery, University of Ulm, Germany. SUBJECTS: A total of 732 patients with thrombocytosis. MAIN OUTCOME MEASURES: Classification of thrombocytosis and thromboembolic complications, and evaluation of laboratory parameters distinguishing between primary and secondary thrombocytosis. RESULTS: Of the total of 732 patients, 89 (12.3%) had primary and 643 (87.7%) had secondary thrombocytosis. Essential thrombocythaemia was observed in 40 of 89 patients (45%) with primary thrombocytosis. The most frequent causes of secondary thrombocytosis were tissue damage (42%), infection (24%), malignancy (13%) and chronic inflammation (10%). Primary thrombocytosis was significantly associated with a higher platelet count and an increased incidence of both arterial and venous thromboembolic complications. In secondary thrombocytosis, thromboembolic events were restricted to the venous system and occurred only in the presence of other risk factors. Mean values of leucocyte count, haematocrit, erythrocyte sedimentation rate, fibrinogen, serum potassium and lactate dehydrogenase were significantly different in primary and secondary thrombocytosis. CONCLUSIONS: The finding of an elevated platelet count on routine blood examination has diagnostic, prognostic and therapeutic implications. It is of clinical importance to distinguish between primary and secondary thrombocytosis, as thrombotic complications occur more frequently in primary thrombocytosis. Unless additional risk factors are present, secondary thrombocytosis is not associated with a significant risk for thromboembolic events.