Carbamylation is a rapid, nonenzymatic reaction occurring between isocyanic acid, the spontaneous dissociation product of urea, and proteins. This process is increased in vivo in chronic renal failure patients, who have high urea levels.
Human albumin (40 mg/mL) was incubated at room temperature with 0.1M sodium cyanate in 0.1M sodium phosphate buffer, pH 7.4, for four days. During the incubation, aliquots were removed and the albumin separated from excess cyanate by means of a P-6DG exclusion column. The desalted albumin samples were applied to an ultrathin polyacrylamide gel (Bio-Lyte 4/6) and focused using a Bio-Rad ยฐ Mini IEF Cell. The degree of carbamylation was measured colorimetrically using diacetyl monoxime and expressed as absorbance units/mg albumin. The pI of the carbamylated albumin decreased from 4.9 (native albumin) to 4,2 after 40 h of incubation. Similarily, the number of carbamyl groups increased in proportion to the change in pI.
These results indicate that the carbamylation process can be easily detected in vitro and that a saturating level of carbamylation occurs. The techniques employed in this study may be useful in evaluating the extent of carbamylation in patients with chronic renal failure.
other. The reaction mixture is transferred to streptavidincoated microtiter wells where the PCR product is captured. Afterwards, alkaline phosphatase-labelled antidigoxigenin antibody is added. Fluorosalicylphosphate is used as substrate. The released fluorosalicylate, forms highly fluorescent complexes with Tb3+-EDTA. The fluorescence is measured with a time-resolved fluorometer. Amplified product corresponding to a single leukemic cell can be detected. Only Ph-positive cells give a double-labelled product. The whole procedure takes about 6 h. The proposed method avoids time-consuming steps such as electrophoresis and Southern blot and the use of radioisotopes.
Fq Quantitative polymerase chain reaction analysis of pathogenic DNA sequence using an internal DNA sequence standard constructed by recombinant DNA methodology