The Plasma Disappearance of Intravenously Administered

The combination of low sernmii content and low specific activity tracer material has presented a difficult technical barrier to the study of the plasma disappearance of tracer amounts of vitamin BI,2 Three groups (1-3) have studied the rate of plasma disappearance of intravenous (loses ranging fromi 0.5 to 4.0 jg. These (loses were not and could not be considere(l to be of tracer mlagnitu(le since even 0.5 K.g mnay e(qual the entire plasmna content. Estren, Brody an(l W;assermiian (3) in their review gave 2 x 10-4 to 9 x 10-4 jig per ml as the normal range of serumiii vitamiiin B1 2. Thtus, persons with plasnma volumiies of 3,000 mil would have a total plasm-ia content of between 0.6 andl 3 ,ug. All three grotups referred to above found a rapid plasma loss of the labeled B,., in normal subjects. Patients with chronic mvelogenous leukemia (1-3) retained more of the vitamin in the plasma, and it was assumied that the greater plasma-binding capacity of these patients prevented the escape of the vitamini. If plasmiia binding determiiines the rate of loss from the circulation, the rapidl loss described in normal subjects (1-3) may have been a consequence of the relatively large amiiounts of labeled material used. The mleasturemiient of the disappearance of truly tracer amiiounts of vitamiin B1 . appeared necessary for a proper tinderstanding of the process. The term "binding" is tised here according to cturrent concepts of vitamin Bl.2)laslmia binding. A detailed disctussion is beyond the scope of this paper, but the many approaches which give similar but not identical values can be sumnmarized unnder three categories: 1) the use of dialysis or a related process for the separation of free and bound vitanmin; 2) the use of charcoal extraction for the samiie purpose; 3) miiaking use of the unavailability of bound vitamin to certain microorganisms * Supported iln part by Grant A-2808 from the Nationial Ii,stituites of Health, Bethesda, Md. tintil the serum is heated. Estren andl associates (3) stated the tunsaturated sertumii linding capacity to be on the order of 7 x 10--4 Mg per ml. This amotunt can be bound in addition to that already present and in a bound fornm. A person with a plasmiia volumiie of 3,000 nml wouild then have a total tinsatturate(l binding capacity on the ordler of 2 Mg. An imlportant contribution to the lprobllelml of binding was recently miiade bv Miller and Sullivan (4) who fotund a seromuiticoid with specific moI,ilitv on electrophoresis to be the binding site of native vitamin Bt . However, a(lde(l B2 cou(ld be bound to otlher sites firmly eniough to resist removal l)y dialysis, and it appears that binding as imieasured by current in v,itro techniqtues may measure more than that vitamin attached to the native hinding site. Too little is known al)out the relationships between the plasmla binding of vitamiiin B,2 and vitamiiin B12 metabolism to permit thl assumpltion that binding at a site different fromii that of the native B (l does not have biological significance. This recently described imore restricte(l concept of binding is presented here mlainly to emiiphasize that while in zvitro binding of B,., canI be measured easily and precisely by charcoal extraction or dialysis techniquies, one miiust b)e cautious in relating it to in zizvo events until it is better understood. For the present study, bound vitamlin was considered to be that vitamin which could not be removed by charcoal extraction. Fortunately, a high specific activity cobalt s vitamin B1. (Co15B,2) has recently become available.' This development and the adaptation of miiore sensitive counting techniques nmade it possible to uise verv smiiall amotunts of labeled material in the present study. The smlaller doses use(l were on the order of 1/50 to 1/250 of the entire