Determination of carbamylated hemoglobin by high-performance liquid chromatography

Abstract

We have developed an HPLC method for measuring carbamylated hemoglobin (CarHb), based on the quantification of valine hydantoin formed from the released NH2-terminal carbamyl valine residue after acid hydrolysis of hemoglobin. In uremia, CarHb is produced by nonenzymatic post-translational modification of the terminal amino group of hemoglobin monomers by isocyanic acid, derived from the spontaneous dissociation of urea. We measured CarHb in 25 nonuremic control subjects, 24 nonuremic diabetic subjects, and 30 patients with stable chronic renal failure. There was no significant difference between the controls and diabetic patients, their mean (SD) CarHb values being 41 (11.5) and 38 (10.8) micrograms of carbamyl valine per gram of hemoglobin (microgram CV/gHb), respectively. Mean (SD) CarHb values in the uremic patients were much greater, 164 (87.7) microgram CV/gHb. There was significant correlation between the concentrations of CarHb and plasma urea in the uremic subjects. Thus CarHb provides a urea-derived index of chronic uremia.

Evaluation of retinol binding protein 4 and carbamoylated haemoglobin as potential renal toxicity biomarkers in adult mice treated with 177Lu-octreotate

“…VH is an indicator of average urea levels over the lifetime of Hb, while serum urea gives the renal status at the moment the sample is taken. In the present work, the HPLC-MS/MS technique was introduced for quantification of VH [ 36 , 37 ].…”

Reactions of the melatonin metabolite N1‐acetyl‐5‐methoxykynuramine with carbamoyl phosphate and related compounds

“…Both urea and carbamoyl phosphate are known to cause non‐enzymatic N ‐carbamoylation of free amino groups under physiological conditions, phenomena which have been particularly studied in sickle cell anemia. In this disease, carbamoylation of the N‐terminal valine of the β‐chain, followed by release of the modified amino acid as valine hydantoine, has received some attention, because it attenuates the symptoms and favors the lifetime of erythrocytes [15–19]. The effect of urea is caused by spontaneous decomposition to isocyanate, which represents the carbamoylating agent [18, 19].…”

“…In this disease, carbamoylation of the N‐terminal valine of the β‐chain, followed by release of the modified amino acid as valine hydantoine, has received some attention, because it attenuates the symptoms and favors the lifetime of erythrocytes [15–19]. The effect of urea is caused by spontaneous decomposition to isocyanate, which represents the carbamoylating agent [18, 19]. Carbamoyl phosphate can likewise generate this compound [20] and has been used to carbamoylate proteins, not only hemoglobin, but also carbonic anhydratase [21], other blood and brain proteins [20].…”

Correction formula for carbamylated haemoglobin in diabetic uraemic patients

“…Results were expressed as a percentage ratio to total haemoglobin. Carbamylated haemoglobin was measured as valine hydantoin, from carbamyl valine, in the acid hydrolysate of haemoglobin according to the method of Kwan et al 1 Red blood cells from 0´5 ml of heparinized blood were washed in isotonic sodium chloride in a screw-capped glass vial and 1 mL of concentrated hydrochloric acid and 1 mL of glacial acetic acid were added. The capped vial was then placed in a heating block at 1108C for 2 h. After cooling the hydrolysate in tap water, 2 mL of 10 N sodium hydroxide were added, followed by 100 mL of the internal standard solution (carbamyl norvaline, 96 mg/ L) and 5 mL of ethyl acetate.…”

“…Cyanate in its reactive form, isocyanic acid, reacts with free amino groups of proteins leading to the formation of carbamylated proteins. 1 Carbamylation of proteins may contribute to the pathophysiology of uraemia. An association of carbamylation with pathological changes or loss of function, such as cataract, neuropathy, nausea, drowsiness, memory loss, altered low-density lipoprotein metabolism and drug-binding defects has been reported.…”