Carbamylation of erythrocyte membrane proteins: an in vitro and in vivo study

Trepanier, Daniel J.; Thibert, R.J.; Draisey, T.F.; Caines, P.S.M.

doi:10.1016/0009-9120(96)00038-0

Cited by 24 publications

(19 citation statements)

References 31 publications

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“…The classic methods are mainly based on gradient ultracentrifugation followed by LDL isolation and measurement of protein modification (32,33 ). Protein carbamylation in serum can be assayed by a colorimetric method using diacetyl monoxime as a detector and homocitrulline as a calibrator (14 ). Alternatively, carbamylated proteins can be measured by HPLC ( 34 -37 ).…”

Section: Discussionmentioning

confidence: 99%

“…nLDL was dialyzed separately the same way. A colorimetric method using diacetyl monoxime was used to measure the degree of carbamylation in LDL preparations (14 ). The electrophoretic mobilities of nLDL and cLDL were determined by electrophoresis in 0.5% agarose gel containing 2 g/L BSA as suggested by Noble (16 ).…”

Section: Materials and Methods Chemicals And Buffersmentioning

confidence: 99%

“…At present, cLDL can be quantified by colorimetric measurement of protein carbamylation in the LDL fraction after isolation by ultracentrifugation, but this is an expensive, complicated, and time-consuming procedure that requires considerable amounts of serum (14 ). On the other hand, measurements of other LDL isoforms by immunoassays using specific antibodies are more precise, simpler, and usually can be performed in a clinical laboratory (15 ).…”

confidence: 99%

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

Apostolov

Shah

et al. 2005

Clinical Chemistry

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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.

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Section: Discussionmentioning

confidence: 99%

Section: Materials and Methods Chemicals And Buffersmentioning

confidence: 99%

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

Apostolov

Shah

et al. 2005

Clinical Chemistry

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“…A colorimetric method using diacetyl monoxime was used to measure the degree of carbamylation in LDL preparations (50). Oxidation of LDL was quantified by thiobarbituric acid reactive substance assay (15).…”

Section: Methodsmentioning

confidence: 99%

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

Apostolov¹,

Basnakian²,

Yin³

et al. 2007

American Journal of Physiology-Heart and Circulatory Physiology

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Apostolov EO, Basnakian AG, Yin X, Ok E, Shah SV. Modified LDLs induce proliferation-mediated death of human vascular endothelial cells through MAPK pathway. Am J Physiol Heart Circ Physiol 292: H1836 -H1846, 2007. First published December 22, 2006; doi:10.1152/ajpheart.01079.2006.-The ability of modified low-density lipoptoteins (LDLs) to induce both proliferation and death of endothelial cells is considered to be a mechanism of early atherosclerosis development. We previously showed that carbamylated LDL (cLDL) induces human coronary artery endothelial cell (HCAEC) death in vitro. This effect is similar to the atherogenic action of oxidized LDL (oxLDL) that induces the proliferation and death of endothelial cells. The present study was designed to analyze a potential proliferative effect of cLDL and whether proliferation caused by modified LDLs is related to cell death. Cultured HCAECs were exposed to different concentrations of modified LDL or native LDL for varying periods of time. Cell proliferation measured by bromodeoxyuridine incorporation and S-phase analysis was dosedependently increased in the presence of cLDL (6.25-200 g/ml). The proliferation induced by cLDL or oxLDL was associated with cell death and increased phosphorylation of extracellular signal-regulated kinase (ERK) and c-Jun NH 2-terminal kinase (JNK). Inhibition of cLDL-or oxLDL-induced proliferation by aphidicolin (1 g/ml) was protective against both short-term cell death measured by lactate dehydrogenase release into the medium and long-term cell viability visualized by cell multiplication. Inhibition of ERK phosphorylation led to a significant decrease of DNA synthesis and cell rescue from injury by modified LDLs, while inhibition of JNK phosphorylation had an only partial rescue effect without involvement in cell proliferation. These data are the first evidence that endothelial cell death induced by cLDL or oxLDL is mediated by cell proliferation through the mitogen-activated protein kinase pathway. carbamylation; carbamylated low-density lipoprotein; oxidized lowdensity lipoprotein; mitogen-activated protein kinase; atherosclerosis CARBAMYLATION is a spontaneous, nonenzymatic reaction of protein modification by cyanate derived from urea, which is normally present in human plasma and is elevated in uremic patients (27). Chronic kidney disease in humans is associated with a several times increased risk of developing cardiovascular diseases because of accelerated atherosclerosis (12,32,47). 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.It is commonly accepted that modified LDLs are important for early atherosclerosis initiation and progression (39, 43). Endothelial dysfunction i...

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“…Assays were validated for mouse plasma before use. Total plasma protein carbamylation was assayed using diacetyl monoxime, 24 and total plasma protein oxidation was measured by the thiobarbituric acid assay. 25 …”

Section: Blood Collection and Measurementsmentioning

confidence: 99%