Tracer Studies. Measurement of the endogenous production rate of 25-OH- vitamin D3 (25OHD) has been accomplished using 6,19,19-trideutero-25-OH- vitamin D3 as a tracer. The 25OHD measurements were done in conjunction with a study investigating the effects of dietary fiber on calcium absorption and 25OHD production rates. We have determined that half like of 25OHD to be about 9.5 days in 5 healthy young men. In contrast to earlier studies using radiolabelled tracers, the present results show: a) half life that is reduced by about 50% from earlier results (mean 20 days); b) no perturbation of the half life by high levels of dietary fiber, and c) reduction of the fractional absorption of dietary calcium by the high fiber diet. Vitamin D metabolites. We have demonstrated the HPLC separation and thermospray LC/MS analysis of the two major metabolic end products of 24,25-dihydroxy vitamin D, which is the principal unregulated pathway used for the disposition of 25OHD. The two water soluble oxidations products, 23-carboxy-24,25,27-tetranor vitamin D (C23 acid) and 24-carboxy-25,26,27- trinor vitamin D (C24 acid), are separated with a typical reverse phase column using 70% methanol solvent at pH 2.5 from acetic acid. We have observed a 4:1 ratio of C23 acid to C24 acid, produced by a perfused rat kidney model. This is the inverse of what had been observed previously by GC/MS. Instrumentation Development. An electrospray mass spectrometer interface for an existing quadrupole mass filter is being developed. The electrospray instrument will permit characterization of molecules in the 20,000 to 50,000 dalton range of molecular weights. Results to date include demonstrating that the vacuum system can accommodate flows of a 1:1 water methanol mixture flowing at 0.002ml/min through a 3 kV potential between a syringe pump needle and the sampling capillary orifice. This yields an ion beam of 60 picoamps at the center of the quadrupole entrance lens, 30 cm from the capillary entrance. Such high currents suggest that fmole sensitivity for certain large molecules may be anticipated.
