Branum, Earl L.

Abstract Background: Recent evidence suggests that the presence of small, dense LDL is independently associated with increased risk of developing coronary artery disease. Current methods to subfractionate LDL are time-consuming and/or technically demanding. Therefore, we have sought the development of a less complex LDL subfractionation procedure. Methods: LDL subfractions were separated using the Quantimetrix LipoprintTM LDL System. High-resolution 3% polyacrylamide gel tubes were scanned densitometrically (610 nm) with a Helena EDC system. A computerized method to identify and quantitatively score the resolved LDL subfractions was developed. Results from the Quantimetrix method were compared using 51 plasma samples with values obtained by nondenaturing gradient gel electrophoresis (NDGGE) and nuclear magnetic resonance (NMR) spectroscopy. Results: LDL subfractionation scores correlated significantly (P <0.05) with triglyceride, HDL-cholesterol, apolipoprotein B100, and LDL-cholesterol/apolipoprotein B100 (r = 0.591, −0.392, 0.454, and −0.411, respectively). For 51 samples, the Quantimetrix method classified 21 with small, 14 with intermediate, and 16 with large LDL. Of the 21 samples classified as small by Quantimetrix, 20 (95%) were classified as small (n = 18) or intermediate (n = 2) by NDGGE. All of the 16 specimens classified as large by Quantimetrix were either large (n = 14) or intermediate (n = 2) by NDGGE. LDL score was inversely correlated (r = −0.674; P <0.0001) with LDL particle size determined by NMR spectroscopy. Conclusions: A quantitative method for the assessment of LDL particle size phenotype was developed using the Quantimetrix Lipoprint LDL System. The method can be performed in less than 3 h in batch mode and is suitable for routine use in clinical laboratories.

Abstract We used wheat-germ-lectin affinity chromatography as a tool to investigate the structure of alkaline phosphatase (ALP, EC 3.1.3.1) and to obtain fractions enriched in either bone or liver ALP activity. Liver and bone isoenzymes in serum samples were incompletely resolved except that the activity in the nonretained fraction (fraction 1) always represented pure liver isoenzyme and constituted a larger percentage of total activity in pooled sera with increased liver ALP activity than in pooled sera with increased bone activity. In contrast, a more avidly retained ALP activity, presumably with high glycosylation, was found in human serum with high activity of bone ALP. Using a solid-phase immunoassay, we examined the fractions obtained from the wheat-germ-lectin-Sepharose 4B column to determine whether the isoenzyme preference of the monoclonal antibody was markedly influenced by the degree of glycosylation. Whether samples contained high proportions of liver or of bone isoenzyme activity, the nonretained fraction contained a higher percentage of liver ALP, whereas the more strongly bound fraction contained a higher percentage of bone ALP. Except for eluted fractions that either contained no detectable N-acetylglucosamine or the highest percentage of it, the avidity of the liver-isoenzyme-specific monoclonal antibody for ALP seemed to be independent of the degree of glycosylation, suggesting that the epitope for monoclonal antibody may be expressed in some structure other than the carbohydrate moieties.

Abstract We compared acid citrate-dextrose (ACD-B) and heparin to determine which anticoagulant better preserves leukocytes for lysosomal enzyme assays if processing was done immediately or delayed for 24 h or more. Twenty normal subjects had blood drawn into tubes containing either ACD-B or heparin. The leukocytes were isolated by sedimentation in dextran (50 g/L) less than 2, 24, 48, and 72 h later. The most apparent difference was that cell counts indicated a 30% reduction in the number of leukocytes for ACD-B and a 95% reduction for heparin-treated cells at 48 h. The neutrophil function assay indicated that leukocyte processing must be done in less than 24 h regardless of the anticoagulant used, and that heparin is to be preferred. A comparison of heparin and ACD-B for maintenance of the activity of arylsulfatase A (EC 3.2.6.1) and hexosaminidase (EC 3.2.1.50) indicates that there is no effect of anticoagulant. However, at 48 h after venipuncture, there is an 80% reduction in the number of heparin-treated samples that are suitable for use in the assay. Those laboratories doing lysosomal enzyme tests on mailed specimens, which are most often greater than 24 h old, should use ACD-B as anticoagulant.

Abstract To separate liver and bone alkaline phosphatase (ALP) isoenzymes in human serum, we used high-performance affinity chromatography (HPAC) on a column of wheat-germ lectin conjugated to 7-microns-diameter silica particles and an eluent containing N-acetyl-D-glucosamine (NAG). On-line spectrophotometric detection of ALP involved pumping diethanolamine-buffered p-nitrophenyl phosphate solution post-column. Bone and liver isoenzymes could be separated into two peaks with only 10% overlap when an exponential gradient was used. A linear-step gradient separated 80.9% of liver ALP and 91.6% of bone ALP in two distinct peaks. True bone and liver ALP peak areas for the linear-step gradient were determined by using correction factors, because each peak contained a co-eluted portion of the other ALP isoenzyme. The detection limit improved 10-fold over those of other techniques for ALP isoenzymes, owing to the relatively large sample that could be applied to the column. Correlation with a urea-inactivation procedure was reasonable for patients' serum samples (r = 0.98 and 0.79 for liver ALP and bone ALP, respectively).