No abstract

A liquid chromatography/thermospray mass spectrometry method has been developed and used to measure the plasma levels and half-life of 25-hydroxyvitamin D3 in adults. The mean plasma levels of 25-hydroxyvitamin D3 averaged 40 ng ml-1 (n = 4) in summer and 30 ng ml-1 (n = 6) in winter. The method was also used to determine the half-life of serum 25-hydroxyvitamin D3 in subjects maintained on either high or low-fiber diets who had been given an intravenous infusion of (6,19,19-2H3)25-hydroxyvitamin D3 sufficient to label 5% of their estimated body pools. The half-life was determined to be 10.4 days (n = 4), which is approximately 50% of the currently accepted value of 19 days, determined using radiolabeled methods. This difference may be due to kinetic isotope effects arising as a result of the tritiated compounds being labeled at sites that undergo Cyt-P450-catalyzed oxidations.

In the determination of the free mobility, related to the surface net charge, by quantitative gel electrophoresis, the previous arbitrary extrapolation of Ferguson plots from the lowest gel concentrations that give a mechanically stable gel to 0% T has recently been replaced by measurement of mobilities across that concentration range, using the addition of 0.5% agarose to polyacrylamide at the various low concentrations in application to a DNA fragment 155 bp in size (Orbán, L. et al., in preparation). The present study applies that approach to several proteins and DNA fragments smaller than 1300 bp, using 0.4% agarose in polyacrylamide gels of varying concentration. The intercepts of the plots with the mobility axis provide experimental data by which the free mobility in polyacrylamide gel electrophoresis can be estimated for molecules not significantly retarded in their migration at the agarose concentration admixed to polyacrylamide. Across the gel concentration range below 3% T, in the presence of agarose, the Ferguson plots of proteins and DNA fragments are convex. It was shown by mass spectrometry that this convex curvature of the plots in the mixed polymer is not significantly due to low polymerization efficiency in the concentration range of liquid polyacrylamide (below 3%T).

No abstract

Five proteins with molecular mass in excess of 200 kDa were found to adsorb onto gels during isoelectric focusing on immobilized pH gradients (IPGEF). To probe for the mechanism of that adsorption, the homogeneity of the six Immobiline preparations used to make IPGEF gels was tested. Five of these Immobiline preparations appear homogeneous in gel filtration of Sephadex G-10. The sixth Immobiline (pK 9.3) exhibits a minor component eluting ahead of the major peak and comprising less than 4% of the total Immobiline absorbing at 226 nm. The proportion of the minor component increases with column load. Major and minor components when isolated appear to equilibrate with one another. Judging by the results of mass spectrometry, all 6 preparations are free of small aggregates of less than 500-600 Da molecular mass. Ultrafiltration of the Immobiline preparations through a membrane with 500 Da nominal cutoff leads to partial desorption of only 3 of the 5 adsorbed proteins. CHAPS is ineffectual in desorbing the 5 proteins from the IPG gel made with ultrafiltered Immobilines. None of the 6 Immobiline preparations used precipitates ferritin. All large proteins that adsorb onto IPGEF gels in the pH range 4-9.5 also adsorb onto commercial IPGEF gels in the pH range 4-7.