Hyperinsulinemia and abdominal obesity affect the expression of hypertriglyceridemia in heterozygous familial lipoprotein lipase deficiency.

We have reported three missense mutations (G188E, P207L, and D250N) in the lipoprotein lipase (LPL) gene among French-Canadians, resulting in the absence of measurable postheparin plasma LPL activity in homozygotes. Presence of triglyceride- and cholesterol-rich VLDL, as well as cholesterol-poor HDL particles, has been shown in heterozygotes affected by partial reduction in postheparin LPL activity. However, significant heterogeneity in their plasma triglyceride levels has been found, even among individuals carrying the same LPL gene mutation, indicating that factors other than LPL deficiency could affect the phenotypic expression of hypertriglyceridemia in the heterozygous state. The aim of the present study was to examine the combined effects of abdominal fat accumulation and hyperinsulinemia on plasma triglyceride levels among heterozygous patients for familial LPL deficiency. Based on sex and BMI, 43 heterozygotes (25 women and 18 men) were matched with noncarrier control subjects. Our data indicate that heterozygotes with higher abdominal fat deposition, as defined as waist girth values above the 50th percentile, had higher plasma triglyceride levels than nonobese heterozygotes. However, an important proportion of male heterozygote subjects were hypertriglyceridemic, even in absence of abdominal obesity, suggesting that another factor(s) was involved in the modulation of hypertriglyceridemia in these subjects. Indeed, multivariate analyses revealed that fasting hyperinsulinemia was a significant correlate of hypertriglyceridemia among these heterozygotes. Results of the present study indicate that abdominal obesity and hyperinsulinemia both have deleterious effects on plasma triglyceride levels in familial LPL deficiency. It is suggested that heterozygotes with moderate obesity and/or insulin resistance may be at higher risk of coronary artery disease because of the expression of an atherogenic lipoprotein phenotype among these patients.

A monoclonal antibody against human lipoprotein lipase inhibiting heparin binding without affecting catalytic activity.

A fragment of the human lipoprotein lipase (LPL) cDNA (405 bp, 5' terminal end) was cloned in an expression vector to produce a approximately 17 kDa fusion peptide and was used as antigen to produce a high titre anti-LPL monoclonal antibody (10C3 MAb). This antibody reacts with both native and denatured forms of LPL from different tissue and animal sources. Competition studies with heparin indicate that 10C3 MAb is specific for an epitope at a heparin binding site. The antibody does not inhibit LPL enzyme activity, indicating that the antigenic epitope is not situated within or in the proximity of the LPL catalytic region. With these characteristics, 10C3 MAb should prove to be a useful immunochemical tool in clinical as well as in fundamental investigations on the metabolism of triglyceride-rich lipoproteins and in studies on the functional anatomy of LPL.

Difference spectroscopic and kinetic studies on the interaction of lactate dehydrogenase with structurally related triazine dyes.

Difference spectroscopy and enzyme kinetics were employed to study the interaction of lactate dehydrogenase (LDH) from rabbit muscle with the azo-dye Procion Red HE-3B and two of its structural variants in order to follow the significance of the sulphonated terminal rings for the strength and specificity of binding. Procion Red HE-3B possesses a significantly higher affinity to LDH compared to the dye Cibacron Blue F3G-A, a well characterized pseudo-biospecific ligand of dehydrogenases. Moreover, Procion Red HE-3B showed competition towards the cofactor NAD+/NADH. The enzyme-dye complex is mainly stabilized by hydrophobic interactions, but other binding forces cannot be excluded. LDH possesses one dye-binding site per subunit. As a binding region the active center of LDH, preferentially the hydrophobic nicotinamide pocket is involved. Removal of the negatively charged sulphonic acid group from the terminal rings of Procion Red HE-3B decreases the affinity to LDH significantly but does not change the type of binding. Addition of an anilino group to the terminal rings of Procion Red HE-3B does not affect the affinity to the active site significantly but enables the binding on other sites with lower affinity in dependence on the dye concentration.

Interaction of lactate dehydrogenase with structurally related triazine dyes using affinity partitioning and affinity chromatography.

Affinity partitioning in aqueous two-phase systems consisting of dextran and dye-liganded polyethylene glycol was employed to study the interaction of lactate dehydrogenase (LDH) from rabbit muscle (E.C. 1.1.1.27) with Procion Red HE-3B and four structurally related derivatives of this dye in order to follow the significance of the terminal rings of Procion Red HE-3B for the strength of interaction. The study revealed that the arrangement of the two 1-amino-8-naphthol-3,6-disulphonic acid rings seems to be a prerequisite for the interaction of azonaphthol dyes with LDH. The negatively charged sulfonic acid group at the terminal rings of Procion Red HE-3B enhances the affinity of the ligand for LDH significantly. The removal of this sulphonic acid group or splitting off the complete terminal rings decreases the affinity to LDH and improves the competitive effect of NAD+. The results of affinity partitioning are compared with those of affinity chromatography and kinetic data. The usefulness and the choice of parameters of affinity partitioning as an analytical tool to predict the chromatographic behaviour of dye ligands are discussed.