The radioassays of vitamin B12 and folic acid can be carried out in a single tube to give the simultaneous assay of both vitamins in 100 microliter of serum. Release of bound vitamins from their endogenous binders and the destruction of these binders are effected by a heating step at pH 9.3. The subsequent binding reactions with hog intrinsic factor and milk binder protein proceed advantageously and simultaneously in the same tube at pH 9.3. A single set of dual reagents replaces two sets of reagents that would normally be used for separate radioassays. Complete separation of bound radioactivities, [57Co]cyanocobalamin and 125I-labeled folate derivative, is obtained in a dual-channel gamma counter with no requirement for any correction for spill-over of counting data. Analytical results are comparable to those found for previously developed individual radioassays. The simultaneous assay has decreased technical time of analysis for these interrelated vitamins by about 50%.

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SummaryConditions for the efficient conversion of commercial RNA to nucleoside 5'-monophosphate by means of a phosphodiesterase in malt sprouts have been determined. A comparison of the enzyme content of the rootlets, stems, and kernels of various plant seedlings, including barley, rye, oat, wheat, rice, and beans shows maximum amounts in the rootlets, and minimum quantities in the ungerminated kernels. Of all the seedlings tested, (mung bean, soy bean, oat, wheat, rice, barley) barley gave the highest conversion of RNA to 5'-nucleotides. Commercial malt sprouts prepared from 6 different malted barleys including 2-rowed and 6-rowed amples all showed about the same amount of phosphodiesterase content. Be des phosphodiesterase, other enzymes capable of hydrolyzing KNA and 5'-nucleotides were found in sprouts. These included 3'-phosphodiesterases, 5'-nucleotidases, and nucleosidases. By carefully pretreating both extracts and the solid sprouts at elevated temperatures for a limited time and by the addition of minimum amounts of k + 2 , the action of these undesirable enzymes was either effectively destroyed or minimized so that the production of 5'-nucleotides was maximized. I t was found that suspensions of appropriately washed and treated barley malt rootlets are substantially more effective than aqueous extracts for converting RNA to Z'-nucleotides.

We compare the binding of folic acid and N-5-methyltetrahydrofolic acid to milk binder in the pH range 7.4 to 10.1. At pH 7.4 the relative affinities are quite disparate, with folic acid showing the greater affinity for milk binder. As the pH is increased from 7.4 to 9.3, the difference in affinities becomes smaller, and at pH 9.3 the affinities are nearly the same. As the pH is Increased from 9.3 to 10.1, the relative affinities again begin to differ, with N-5-methyltetrahydrofolic acid displaying the greater affinity. These results indicate that at a pH of 9.3 the more stable folic acid may be used as the standard in radioassay of endogenous folates.