Detection of PCR-amplified chronic myelogenous leukemia-specific mRNA sequences by immunofluorometry

✍ Scribed by S. Bortolin; T.K. Christopoulos

• Book ID: 103039923
• Publisher: Elsevier Science
• Year: 1993
• Tongue: English
• Weight: 116 KB
• Volume: 26
• Category: Article
• ISSN: 0009-9120
• DOI: 10.1016/0009-9120(93)90072-e

✦ Synopsis

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 ° 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.