Quantification of Carbamylated LDL in Human Sera by a New Sandwich ELISA

Abstract

Background:We previously suggested that increased carbamylated LDL (cLDL), a product of nonenzymatic modification of LDL in human serum by urea-derived cyanate, may cause cardiovascular complications in patients with chronic renal insufficiency. An assay for precise measurement of cLDL in serum was not previously available. Methods: Polyclonal antibodies against human cLDL and nonmodified, native LDL (nLDL) were raised in rabbits and extensively purified by affinity chromatography. New sandwich ELISAs to measure cLDL and nLDL with use of these antibodies were developed. Serum concentrations of cLDL and nLDL were measured by the sandwich ELISAs in 41 patients with end-stage renal disease (ESRD) and 40 healthy controls. Results: Both assays showed satisfactory reproducibility, linearity, and recovery. The assays could detect 2.7 mg/L cLDL with a linear detection range of 5-1000 mg/L and 5 mg/L nLDL with a linear detection range of 50 -1000 mg/L. These measurements showed that patients with ESRD have significantly increased serum cLDL [281.5 (46.9) mg/L compared with 86.1 (29.7) mg/L in a control group; P <0.001]. There was no significant difference in nLDL concentrations between the groups. Conclusions: These assays are a potentially valuable tool for cardiovascular research in renal patients and healthy individuals. The cLDL concentration appears to be the highest among all previously described modified LDL isoforms in both controls and ESRD patients.

Modified LDLs induce proliferation-mediated death of human vascular endothelial cells through MAPK pathway

“…Human native LDL (nLDL) and all chemicals were purchased from Sigma (St. Louis, MO) unless otherwise stated. cLDL was prepared as previously described (2,38). oxLDL was prepared as described by Kume et al (28).…”

“…The mechanism of uremia-induced atherosclerosis is not quite understood. We previously showed (2,36,37) that carbamylated low-density lipoprotein (cLDL) induces injury or dysfunction of endothelial cells and is present in human plasma in high quantities. Our recent observation (38) that cLDL induces cell death of human coronary artery endothelial cells (HCAECs) in vitro provided support for this hypothesis.…”

Chronic Uremia Stimulates LDL Carbamylation and Atherosclerosis

“…11 Wang et al 12 reported carbamylated proteins in atherosclerotic plaques that mainly colocalized with macrophages. Our findings suggest that cLDL may compose one of these carbamylated proteins, and the majority of it is likely to derive from urea, not thiocyanate, as suggested by Wang et al We also observed colocalization of cLDL and macrophages and detected cLDL accumulation in macrophage-free zones of intima and plaques.…”

“…9,10 Using a new ELISA assay, we recently confirmed that cLDL is significantly elevated in the plasma of uremic patients. 11 The carbamylation of LDL occurs by spontaneous, nonenzymatic chemical modification of apolipoprotein B, the protein component of LDL, by isocyanic acid derived from urea. An alternative myeloperoxidase-mediated mechanism of LDL carbamylation has been suggested by Wang et al, 12 who also observed a correlation between total plasma protein carbamylation and the incidence of cardiovascular disease in nonuremic patients.…”

Are oxidised low-density lipoproteins the true inducers of inflammation in atherosclerosis?

“…It is proposed that the generation of carbamyl-LDL is a common injurious event that links inflammation, smoking, and uremia as risk factors in atherogenesis [95]. Consequently levels of carbamyl-LDL in plasma are higher than levels of ox-LDL and in both uremic patients and in smokers levels are considerably higher [96,97]. The scavenger receptor LOX-1 receptor mainly recognise oxidised and carbamylated proteins as well as carbohydrate moieties.…”