A sandwich enzyme-linked immunosorbent assay for human plasma apolipoprotein A-V concentration

Abstract

Apolipoprotein A-V (apoA-V) is a recently discovered apolipoprotein that appears to have a role in plasma triglyceride (TG) transport. We have developed an ELISA for apoA-V using monoclonal antibodies that has a lower limit of detection of 0.3 ng/ml and linearity up to 20 ng/ml. The ELISA was then used to quantify plasma apoA-V in 196 healthy subjects and 106 patients with insulin-resistant diabetes mellitus. In the healthy subjects, total apoA-V concentration was 179.2 ؎ 74.8 ng/ml, and it was greater in females than in males ( P Ͻ 0.005). It was correlated positively with the plasma HDL cholesterol ( r ‫؍‬ 0.32, P Ͻ 0.0001), apoA-I ( r ‫؍‬ 0.27, P ‫؍‬ 0.0001), and apoE ( r ‫؍‬ 0.18, P ‫؍‬ 0.011) concentrations and negatively with plasma TG concentration ( r ‫؍‬ ؊ 0.22, P ‫؍‬ 0.021). In relation to single nucleotide polymorphism 3 ( ؊ 1131C/T) of the apoA-V gene, apoA-V concentration was higher in the T/T type than in the C/C type ( P Ͻ 0.01). Plasma TG concentration was lower in the T/T type than in the C/C or C/T type ( P Ͻ 0.05). ApoA-V concentration was lower in the diabetic patients (69.4 ؎ 44.3 ng/ml; P Ͻ 0.01) than in the healthy controls. Plasma triglyceride (TG) levels are influenced by both genetic and environmental factors and are a major independent risk factor for coronary heart disease (1, 2). Plasma TG concentration is influenced by many factors. These include apolipoproteins A-I, A-IV, C-II, and C-III, LPL, LCAT, cholesteryl ester transfer protein, and phospholipid transfer protein (3-11). These factors and their associated gene-environment interactions are of importance in the pathogenesis of coronary heart disease.Apolipoprotein A-V (apoA-V) has recently been identified by comparative sequencing of human and mouse DNA and is located ‫ف‬ 27 kb distal to the apoA-IV gene in the APOA1/C3/A4 gene cluster on chromosome 11q23 (12). ApoA-V, shown to be expressed mostly in liver and independently named regeneration-associated protein 3, is upregulated after the early phase of liver regeneration after hepatectomy in rat (13). In mice overexpressing the human apoA-V gene, TG concentrations decreased by 50-70%, and in apoA-V gene knockout mice, plasma TG concentrations increased ‫ف‬ 4-fold (12-14). These results suggest that apoA-V expression may strongly influence, and be negatively associated with, plasma TG concentrations. ApoA-V both enhances lipoprotein lipase-mediated hydrolysis of plasma TG and inhibits hepatic VLDL-TG production (15). ApoA-V also stimulates the efflux of cholesterol from cells by a mechanism independent on the ABCA1 protein, as do other exchangeable apolipoproteins, such as apoA-I and apoA-IV (16). It was recently described that apoA-V mRNA is regulated by peroxisome proliferator-activated receptor ␣ agonists (17,18) and that the liver X receptor ligand T0901317 decreases apoA-V mRNA through the activation of sterol-regulatory element binding protein 1c (SREBP-1c) (19). These results raise the possibility that some TG-lowering agents, such as fenofibrate, may act by alte...

Plasma apolipoprotein A5 and triglycerides in type 2 diabetes

“…In contrast, all VLDL particles are carrying one or more APOC3 molecules. Average plasma APOA5 levels have been reported in healthy normolipidaemic volunteers of Caucasian origin as 16 μg/100 ml [19] and 1 μg/100 ml [22], and in healthy Japanese subjects as 18 μg/100 ml [17]. In our patients with type 2 diabetes, who were of Caucasian origin and had moderately elevated plasma triglyceride levels, plasma APOA5 levels were 7 to 139 μg/100 ml.…”

“…270-0003, Bio-Rad, Richmond, CA, USA). Plasma APOA5 concentration was measured using a recently developed sandwich ELISA as described [17]. The monoclonal antibody B10E was used for capture and biotinylated monoclonal antibody E8E was used for detection.…”

A single nucleotide polymorphism in APOA5 determines triglyceride levels in Hong Kong and Guangzhou Chinese

“…3 Single nucleotide polymorphisms (SNPs) in this gene, such as rs662799 (À1131T4C), have been reported to be associated with hypertriglyceridaemia in Caucasians and Asians. [3][4][5][6] It is not clear if other SNPs in APOA5 might also be related to triglyceride level. Therefore, we investigated the relationship between tagging SNPs in the APOA5 gene with triglyceride level in the Hong Kong Cardiovascular Risk Prevalence Study and confirmed the results in a larger group of subjects from the Guangzhou Biobank Cohort Study.…”

“…3 The plasma level of apo A5 correlates positively with high-density lipoprotein (HDL) cholesterol, but negatively with triglycerides. 4 It is lower in men and patients with diabetes. 4 In man, the APOA5 gene is located on chromosome 11q23.…”

“…4 It is lower in men and patients with diabetes. 4 In man, the APOA5 gene is located on chromosome 11q23. 3 Single nucleotide polymorphisms (SNPs) in this gene, such as rs662799 (À1131T4C), have been reported to be associated with hypertriglyceridaemia in Caucasians and Asians.…”