MP480SIMPLE MEASUREMENT OF VITAMIN B12 BY OPTICAL SPECTROSCOPY

Abstract

Introduction and Aims: Vitamin B12 is well known to be a useful marker as a middle molecular weight substance like inulin for evaluation of dialyzer clearance. Usual methods for measuring vitamin B12 are microbiological assay or chemiluminescence enzyme immunoassay, but spectroscopy offers an alternative approach for clinical analysis. Spectroscopy does not require reagents, thereby permitting real-time analysis. In this study, we investigated direct measurement of vitamin B12 by optical spectroscopy. Methods: Vitamin B12 (Wako Pure Chemical Industries, Ltd.) was dissolved in pure water at a concentration of 0.78-25.00 mg/dl. Measurement was performed with an ultraviolet spectrophotometer and spectra were recorded over the 200-600 nm wavelength range. The concentration of vitamin B12 was determined from the absorption band of the spectrum. A compact diffusion apparatus with two chambers and one membrane partition was made with a polyacrylonitrile dialysis membrane (AN69 H12 Hospal), and the permeability coefficients of vitamin B12 (1355 daltons), glucose (180 daltons), creatinine (113 daltons) and urea (60 daltons) were measured to evaluate this optical spectroscopy method. Concentrations of glucose, creatinine, and urea were measured by an auto-chemical analyzer. Results: The absorbance spectrum of vitamin B12 in pure water showed a large peak at 363 nm and a small peak at 550 nm. Therefore, the vitamin B12 was concentration determined from the peak at 363 nm. There was a significant positive correlation between the peak value and the predetermined concentration of vitamin B12 (r=0.952, P<0.001). The permeability coefficient of vitamin B12 was 18.2×10-5 cm/s while the coefficients of glucose, creatinine, and urea were 25.6×10-5 cm/s, 44.6×10-5 cm/s, and 65.1×10-5 cm/s, respectively. Conclusions: The permeability coefficients of these four different substances corresponded to their molecular weights. Therefore, it is possible to determine the vitamin B12 concentration by measuring peak values using a spectrophotometer. This optical spectroscopy method could be useful for evaluating dialyzer clearance of vitamin B12.