dence Concerning the Human Requirement for Vitamin B,, Use of the Whole Body Counter for Determination of Absorption of Vitamin B,, Ricuarp C. Boziay, ap." JouN L. Fercuson, w.p.,7 Ronert M. Heysset, ¥.0.,) Geouct R. Meseuty, #0, an Waiaiast J. Dara, a. ie assessment of requirements of a nu- trient can be approached from several 1G standpeints: (1) observations of the occurrence cor induction of deficiency in man; (2) response to therapy of cli 1 sy '3) metabolic studies; and the nutrient. 2 the Die utstion and Departusents of Medicine and of Bieesenistry, Vardertilt University School «f Medicine, Nashville, Tennessee * Clinical Investigicor, U.S. Veterans Administese tion Hospital, Nashvte, Tentessce; and Department oi Bicciemintey, Vesderbilt. Caiversity School of Medicine, Nashville, Tennessee; t Assistant Resident in Medicine, Ve University Schoo! of Medivine, Nasiwite. Tenseste: } Astitant Professor of Medi ce, Vewterlile University School of Medicine, Nash tille, Tesnesce: {Formerly Associate Profesor of Medicine, Vanderbite University School of Medicine, Rashi, Tennessee, Present address, Director, Departisent wf Seientiie Assembly, American Mediesl Asoeistion, Chieage, Mois; |! Professor of Blow ‘Gremistrs and Direct, Division of Nutition, Vander- bile Caivstsity Solved -f Medicine, Nashville, Tennes: ‘These stadics were ssymorted in part by the fellow sot No. 28-5120, Army Metical Research sent Cermmiand Contract Xo. DAM. and US. Public Health Service Training Pressed at Metabuite spect ‘Semposiwia on Nutritional and Bond Cell Formation, held at the University of Arkunsas Metical Center, Lite Rock, October 29, 20i1, epesseted by The Natiowal Vita sin Fesinéation, Tne. Imericar Sours! of Clinical Nutrition This consideration of the current status of knowledge concerning vitamin Bye requirements ‘employs several of these approaches, using data oth from our studies and from others in the literature. Further, the application of the whole body counter as a safe, accurate and rela- tively simple means of appraising the absorp: tion, distribution and excretion of tagged essen- tial metabolites in man will be illustrated. BACKGROUND Tn reports!? from this laboratory it has been: argued that the development of vitamin Bue deficiency in patients with pernicious anemia, gastrectomized patients and Vegans is a com- parable event and hence that patients with pernicious anemia may be used as examples of vitamin Breepleted subjects for studies of the usual human requirements. In a group of twenty patients, the minimal dose of paren- terally administered vitamin By required to initiate maximal hemopoiesis was from 0.5 to 1.0 ug. daily.* Similar quantities of the vitamin maintained hematologic values at maximal levels. Considering the known ef- ficiency of absorption by normal persons of such amounts of vitamin By, it was estimated that the minimal dietary needs for this factor can be met by an amount of 0.6 to 28 pg, of vi- tamin By: from foods, and for most persons the range of 0.6 to 1.2 yg. should suffice? Long: term observations on these patients! that the demands for hemopoiesis equalled or exceeded those for other clinically recognizable physiologic functions and that macrocytosis Vol. 12, Pebrnary 1969 b.I6) 129us Evidence Concerning Human Requirement for Vitamin By: Tanue r Taoue Analysis of Diets Patterned After Vanderbilt Analysis of Pooled Rations, ICNND Surveys Cooperative Study of Nutrition ia Pregnancy eed Countey | Vitamin Bie |S Males 20-15 Fo of Content/"" | Years old with Hero. ] mina ofiin | Surtee) | ia 220m comic Oy | at egg) fae [se |S I= P—— Rave 8 Hon |seasy | soo | so thy. | Hens at cos bs) Huts towent fro 25 | 2g |S? xara | B5e | a : get) Baia] ites “3 Penne 2rcur8.)] aa | Erte Sica | ow Asay 1 Luma ATES, Now! Ogio, 1 kant ATCT was the most sensitive indicator of vitamin should offer some guide to a reasonable level of By deficiency, nioutherapeutic supplementation for those ‘Recently published analyses of good and poor who feel that such supplementation is de- American diets provide pertinent data.’ Table sirabl summarizes the vitamin By content of poor, A. summary of the average vitamin Bre low cost and high cost diets as analyzed. (The content in diets encountered in the course of assay procedure for vitamin By employed studies made abroad by the Interdepartmental Lactobacillus leichmannii ATCC 7830.) The Committee on Nutrition for National Defense vitamin Bus content was as low as 1.1 yg. for (ICNND) is provided by Table m. It is the poor diets and as high as 85 yg, daily for )evident that] anemia is prominent in Korea, high cost diets. The poor diets were patterned |where the vitamin By content. of the diet after the most restricted diets recorded by averages but 0.5 ng. daily. ‘The type of anemia women studied in the Vanderbilt Cooperative {was incompletely characterized although the Study of Maternal and Infant Nutrition. | mean corpuscular hemoglobin concentrations The diets with lowest content of vitamin Buz | suggest that it was a hypochromic type of (average 27 ug.) were not associated with Janemia, It is notable, however, that a level acrocytic anemia among the women. Tt is [of 1 to 2 ag, daily does not result in any signifi pertinent that in a survey of the vitamin Bio a epi- Content of foods by Lichtenstein, Beloian and demiologic studies in areas im which iron Murphy‘ of the U. S. Department of Agricul- deficiency is not limiting would be invaluable. ture, assays using L. leichmannii and Ochro- Important observations on absorption and monas mathemensis gave essentially equivalent excretion of vitamin Bye have issued irom many esults. Significantly, values oblained with laboratories, particularly pertinent are the ‘0. mathemensis were higher in fifteen of twenty- suggestions of Reizenstein* and Grasbeck*? seven foodstuffs analyzed, contrary to reported that data on vitamin Bie excretion in healthy experiences for analysis of stool and urine. subjects provide evidence that patients with ‘To quote from the summary of the Vanderbilt pernicious anemia should receive from 4 to 10 Study: “The dietary levels are equal to or yg. or more daily for the maintenance of body greater than the estimates of adult require- saturation with vitamin B; dents as based on the relatively meager evi- We wish to report some recent observa dence available for vitamin Bs, pantothenic on the absorption and biological half-life of acid, foe acid and vitamin By, ‘They provide vitamin By in normal subjects and in patients ho support for a concept of widespread dietary with vitamin By: deficiency (pernicious anemia) Tack of these factors. They afford an indica- and to interpret these in relation to vitamin tion of the expected daily intake and, thereby, requirements. tant general incidence of anemia.) Simiavai mt Subjects of the Study eases Diagnosis Diabetes mellitus orinat Normal Norinat Perniious anemia Porniiusaneria Perniious anemia Perniious anemia Perilous aneiia Pernicous anerake Penncousanernia | Pernicous snernis Perniious anemia Pericioss anes Forubious anemia Pemicious anemia Pernicions anemia Arotemia, parpura Simplex, normo: ernicios anci LO/L | Peenictows aems Mernops Data here reported are hased on sixteen subjects for whom a long series uf observations are available An for whom only’ oral absorption studies are reported, are under study at present, At the time of the stsdies, all patients With pernicious anemia were in remission induced by the administration of regular doses of witamin Bs. total quantity. of vitamin Bye given to each patient from the time of relapse on was known, Patients swere given vitamin Bie at three dosage levels and are grouped as follows (Table mm): (1) Those given weekly injections of vitamin Br just sufficient to maintain hemopciesis and normocytasis without providing a surplus for replenishment of ody stores (8.5 and 7.0-ug. weekly oF 0.3 to 10 1g, daily); (2) those given moderate doses (30 to 45 wg, monthly of 1.0 to 1.5 ag. daily); and (3) those given biteckly or monthly doses at higher levels (20 ug. per week to 60 to 100 yg. per month or 20 to.3.3 ag. daily) Isotope 8 sodium icdide, Uhalliam activated human whole howdy gamma ray scintillation spectrometer with the ditienal seven subject couintin with a was accomplished et an. 19 patient sitting beneath the crystal in a standard position’ Initial background counts of patients were made for thirty minutes. The radioactivity ‘of solutions injected or capsules ingested was counted for a one minute period, Subsequent counts on the patients were for six minutes, Reproducibility of the six minute counts was £5 per cent with most of the variation constituting that associated the randomness of the radionetive disintegration, Each subject, while in a fasting state, was given approximately 0.5 ge. of Co®-vitamin Bre (0.5 to 0.73 ye. per wg.). The residual whole hody viteri By radioactivity was determined on the next visit, ‘which was usually two to four weeks later, If ab sorption was poor, the subject was given an injec- tion of approximately 0.5 ue. (I wc. per ug.) of the vitamin and radioactivity was determined on the next visit. The count at one month following in- jection was chosen as the standard for comparison With the value obtained following oral ingestion Each patient served as his own standard for the cal- culation of percentage absorption by equation 1 uation B71 X 100 = % absorption Ry = whole body cpm. remaining following limination of unabsorbed vitamin Bix Ry = whole body cpm. per ue, in patient one ‘month following injeetion T= ne: ingested orally. ‘The number of mierocuries injected was deter ‘mined by counting the contents in the syringe before and after injection and by comparison of these ccounts with those obtained from a known amount in the svringe. Tt was assumed that the distribu: tion in the subjects after four weeks was similar whether the vitamin was received by injection or by absorption from the intestines. This procedure tends to increase the value for oral absorption in the patient with pernicious anemia by 5 to 10 per cent, for approximately this amount is Iost in the frst month following injection" which is only par- tially compensated for by the loss following oral absorption. For the normal subjects, who did not receive any injections, the LOD per cent value was taken as the activity measured immediately follow= ing ingestion of the capsule. ‘The geometry of a 4 by 8 inch crystal, 043 M. from the patient, allows this as a first approximation, Our results, compare with those of Reizenstein, Cronkite and Cobn* at Brookhaven National Laboratory For the half-life studies, the decay slope was de.termined after repeated counts and extrapolated to zero time, The average half-life was obtained from fa curve drawn theough points for maximum ft determined by the method of least squares. The ‘biological half-life was ealeulated from equation Equation 1 TBy, X TE, TR, — TEy, TB = Bij, = biological halt-ife TRig = inteinsi half-life af Co™ (1,928 ays) THY), = elective half-ife (from curve) ‘The slope of biological decay ar decay constant (K) was determined by use of equation m1 Equation 1 0.608 TB, K___ = slope of biological decay (decay constant) TB, = biological hale RESULTS Absorption Studies ‘The results of absorption studies are sum marized in Table 1v. There is a wide separa- tion between the healthy subjects and the patients with pernicious anemia. ‘The range of absorption in normal subjects is 45 to 80 per cent with a mean of 70 per cent; for the patients it ranged from 0 to 17 per cent with a ‘mean of 3.3 per cent ‘The values for per cent absorption by normal subjects and patients are similar to those of Reizenstein and colleagues’ who reported 38 to 80 per cent and 0 to 8.8 per cent, respectively. ‘Tams 1 Per Cent Oral Absorption of CoM-Vitamin Bis No. | 05 ne,/dose Bub: | 05 pe Com & § jects | Vietmin By Subjects ‘Nonpernicious Pernicious anemia. * Standard ercor. vidence Concerning Human Requirement for Vitan They are also similar to values obtained pre viously from studies utilizing measurement of urinary radioactivity, stool analysis or meas- urement of radioactivity over the liver. The present technic appears to be superior to other methods commonly used by reason of its simplicity aud accuracy and the possibility of employirig minimal dosage of radioactivity for long-term studies. Stool collections are un necessary and the added uncertainty of varia- tions in renal excretion is avoided. Further, large flushing doses of vitamin By; are not re- quired and the attendant obscuration by this flushing of subsequent study is avoided. The technic employing the whole body counter has been successfully applied at the radioisotope center at Vanderbilt University’ to the meas- urement of absorption, distribution and excre tion of numerous elements, ind sodium, potassium, cesium inter alfa Biological Half-Life Sixteen subjects have been observed frum eight months to over two years after receiving the labeled vitamin. The ealeulated biological half-life for each is listed in Table v, Repre- sentative observations are depicted in Figures 1 through 3, ‘The differences in biological hali-lize between normal subjects and patients with pernicious anemia are probably highest turnover rates were observed in two subjects, one with diabetes mellitus and one with azotemia and purpura. The explained, There is no correlation between the frequency or magnitude of the dose of vitainin Biz in patients with pernicious anemia and the rate of loss of labeled vitamin from the body. This observation is further supported by the fact that there was no statistically significant change in rate of loss of vitamin Bye alter therapy was discontinued in six patients who had been receiving vitamin Bye parenterally (Table v), not significant. The consents It is necessary to identify the assumptions in the interpretations made. It is assumed that Iabeled vitamin By: is distributed in a manner similar to nonlabeled vitamin By: in the body| we | ibjeces| Vitamin Poe Sevan) tig | Uangth | Xo ot | ge Lone Given Obser- | “per | Period | vations | Day Daring Vitamin Bh: Therapy Auer Vitanin Bye Therapy ogth | Xo. of of | Ohser Perind | i Biological Half-Life ays) 1 Day Gays) vations Normal Subjects Dietary Dietary Patients without Pernicions Anemia Dietary + we [05/8 wk, Lt | Diewry | 487 7 | 02 01 ea | Patients with Pericions Anemia | | } ors | os son ae a 3 | on (Base pod ous fO/4 we eecee a0 5a. | corms > | on FL | Ghawe 4 {en ci. [Snowe i 3 [ow Be laiewe | 8 [oe ash i | O44 | 586 ii | oto | as 387 7 | Ol | 630 HS 6 }ou} oso | | | 3 | 18 | ote | tt * Azotemta plus purpura 4 Dishetes mellitus after 2 period of approximately sixty to ninety. days and that the labeled cobalt in the tissue is in the form of vitamin By. It is further assumed that the “pools” or “compartments” of vitamin By: attain equilibrium and that losses irom any compartment are reflected through- ‘out alter this period. There is evidence to support each of these assumptions. During the first several weeks following ab- sorption or injection of CoM.vitamin Bi, there are wide fluctuations of counts which probably correspond to redistribution of the vitamin through various compartments, Usually after a month, and invariably after two months in the case of injected vitamin By, the decay rate of radioactivity in the body becomes relatively constant. Normal subjects who did not re ceive an injection maintained a steady rate of decline of radioactivity following the stabil tion period which corresponds to a biological half-life of 462 to 770 days and a decay constant 0f 0.09 to 0.15 per cent of residual counts daily (Fig. 1 and 2, Table v). Similarly, patients With pernicious anemia displayed decay con- stants which ranged from 0.1] to 0.17 per cent per day (Fig. 3, 4 and 5, Table v) ‘Asan approximation, this would imply that the daily loss of vitamin Bye in both normal subjects and patients with pernicious anemia approaches @ first order function with the absolute amount lost being a function of the sizeof the pool. ‘Table vr gives data from RossEvidence Concerning Human Requirement for Vitamin e ResONR/Oay S Fic. 1, Loss of Cot-vitamin Bi: in forty-one year old norma! man (R. B.) (Oral tracer dose—0.53 ye. CoMvitamin Bre nee 0.75 us.) » st Fie.2. Lossof Co®-vitamin Buin twenty-seven year old norial man (T. B.) (Oral tracer dose 0.5 ye. Co" vitamin Bus per O75 ns.) ‘and Mollin obtained on autopsy material" with pernicious anentia in relapse. Table vit and from Wolff," Swendseid" and their as-is a compilation of data on the vitamin Bre sociates on the vitamin By content of the content of the liver in control subjects and in human liver in healthy subjects and in patients patients with pernicious anemia in remissionasae/ ween Fre. anemia (S.N.). Bozian et al. $f rrrerers Loss of Cofcitainin Bis in seventy-five year old woman with pernicious (Subcutaneous tracer dase of 0.58 we. Cofvitamin Br per 0.58 wg.) ——orr enary —— =) & Fic. 4. Loss of Co-vitamin Buin fifty: (RR) It is evident that the stores of vitamin Bie in both treated patients and in patients in relapse are well below those of normal subjects, Table vuit shows the comparison of tumover rates in three normal subjects and in two patients treated with minimal doses of vitamin By. One patient (R. R.) was in hematologic seven year old man with pernicious anemia (Subcstaneons tracer dose of 0.58 ne. CoM vitamin By per 0.53 ua) and neurologic relapse, and remission occurred on a regimen of 7 ng. of vitamin By per week. His serum vitamin Br levels have been con- sistently below 100 wag. per ml. In the other patient (S. N.) relapse occurred while on a regimen of 3.5 ug. per week. Values for decay constants in such close agreement for personsEvidence Concerning Human Requirement for Vitamin By — z 5 nena — : wl LL L Fic, 5. Loss of CoMvitamin Bus in sixty-two year old man with pernicious anemia Ge. (Gubeutancous tracer dose of 0.51 we. CoM-vicamin Bis per 0.51 ws.) Tance vt Vitamin By Content of Human Liver | Subjects wi Pernicious Ameria Relapse Investigator Normal Subjects Swendseid, Hvotboll, Schick and Halsted Ross and Mottin Wolf, Drovet, Karlin and Rauber! Pitney and Ouest 0.75 to 1 wg em 1,50 yg, Roa iver 0°8 ./em. 0.78 ag. /a | I | 0.1 ua./am, to as tow as [1 e./toal liver ‘Tanue ver Vitamin Bus Concentration in Liver of Noriaal Control Subgeets and Patients with Pernicious Anemia in Remission ‘Material Studied Liver biopsy proaches a first order function. An of the pool of the patient with Concentration a | Concentration Liver biopsy" 19 ve/em tier | 3 3 oxen iver Astopsy"™ 1.5 mug /eotal liver 5 0:7 mg ftotal liver Liver biopss™ 082 gm fem. liver 5 0.2 nga. iver th body stores at such widely differing levels support the view that daily loss ap- 0.78 ug. /ae liver | anemia by vigorous therapy theoretically would then result in the loss of increased amounts vitamin By: via stool, urine and po: thelial desquamation. expansion bly epi pernicious‘Tanue vor som of Patients with Pevuiciogs \neinia aad ‘cally Depleted Bod Stores of Vitanin By: to Noninal Subjects Length of No.of | Biological | % Study Period | Otner- | HalLite | Lossy vations | lays) | Day days) re | ss } 495 fou SN" jaz fos RB us. a7 fa TR 130 42 | 0.15 Wot 480 | | | 668 * Patients with percious aneinis This concept of excretion as a first order function has been expressed by Grasheck*? and hinted at by Reizenstein’ and Adams.” Our data, which are comprised of a longer series bservations, extend this concept and we interpret it and its ultimate significance dif- ferently. slier it was stated that certain assump- tions are inherent to the interpretation of data on biological half-life; namely, the identity with vitanin By of the residual radioactivity ssues, the similarity of distribution of injected and body vitamin content and reflee- tion by all body compartments of losses from any compartment. The following evidence supports these considerations. Schiloesser, Desphande and Schilling,” Glass and Mer sheimer"® and Griisbeck et al.*! have estab: Jished in man and animals that the radioactiv stored component corresponds to vitamin B by cliromatographic and microbiologic correla- tion of vitamin Bie activity and radioactivity. ‘The latter group has shown that the specific a tivity within various organs in the rat is es- sentially the same after au equilibration period ‘of one month, Data presented in Tables vr and vitend by Blum and Heinrich** indicate that the relative vitamin Bye content in various organs of normal subjects and of patients with pernicious anemia, with or without treatment, remains proportional. Baker and associates have published data indicating that the vitamin By: level of erythrocytes and plasma of patients with pernicious anemia is reduced in value to an extent paralleling the other organs. These ndings support the thesis that the various Bozian et al. pools are in equilibrium and that the stored radioactivity corresponds to vitamin Bu. In an abstract, Reizenstein** reports on studies of the half-life of Co™vitamin Bis utilizing the whole body counter. He found that there was a progressively decreasing rate of. excretion of radioactivity with time and that even at 250 days the rate continued to de- Details of this study are not yet avail- able, but we have followed our normal subjects for over a year and have not observed any evidence of a decrease in the slope of decay after 8 period, at most, of several months. Loss of radioactivity in each of the three normal sub- jects has maintained a constancy whieh has extended to as long as 479 days to date. This constancy warrants the assumption that general equilibration is complete in a quantitative sense even though final equilibration may be incom- plete for certain tissue components which have exceedingly low turnover rates. Tt is recog nized that each tissue has @ spectrum with respect to constituent turnover time and it is not unreasonable that certain constituents involved with vitamin Byg do have ‘an even slower turnover rate than the period here involved. ‘The half-life figures probably represent an average figure covering a wide spectrum of half-fife values for various tissue components. The implications of the concept presented are as follows: The body is eapable of storing significant amounts of vitamin Br: (790 to 11.900 1g, The size of the body store is the major determinant of the quantity of vitamin Bylost daily. Other contributing factors, such as rate of degrada tion, turnover and inactivation of the active fora of vitamin are of a maguitude which prob- ably is minor when compared with the contri bution of the pool size. ‘The larger the body stores, the larger the excretion. This rela~ to consider in the normal subject) tionship of body stores to excretion is even more important in patients with pernicious anemia who may well have a limited reabsorption capacity for vitamin By which enters the intestine in the bile." ‘The body maintains fake and storage. Law levels of intake and small pool size are associated with low levels of excretion. It126 does not follow that there is attached to any particular level of storage or excretion an especial advantage with respect to optimal health. Grasbeck and Reizenstein have argued that the conventional treatment for pernicious anemia is inadequate; that maintenance ther- apy should be such as to saturate the body stores to the levels of healthy normal subjects. ‘The amounts they suggest approximate 4 to 10 ug. per day!-? ‘They advance the suggestion that neurologic complications, decreased fer~ tility, degenerative states and possibly the development of gastric neoplasm, gallbladder disease and other usual complications could be alleviated or possibly averted by the use of saturating doses of vitamin By. There is no acceptable evidence for these suggestions. ‘There is no evidence at present that the ad- ministration of large doses of vitamin Bu prevents any of the corollaries of pernicious anemia such as gastric atrophy, gastric polyps or achlorhydria, The toll of subacute com- bined degeneration appears to be a function of the completeness of depletion aud length of time the deficiency existed, rather than being related. to the treatment dose. Indeed, less vitamin Bis seems to be required to prevent combined system disease than macrocytosis in pernicious As previously stated, one of the patients in this series (R. R.), a fifty-five-year old Negro man, has been treated with a weekly dose of 7 ug. of vitamin By without preliminary re~ plenishment of body stores; he experienced complete remission of the hematologic picture in a matter of weeks and of the neurotogi defects over a period of six months. This occurred in the face of body stores which remained “depleted” as measured by serum Ievels* of 50 and 90 ung. per ml., two and six months after the start of treatment. ‘The ‘experience at the Vanderbilt Hospital and other clinies over the years does not provide evidence to suggest that failure to saturate body stores, with vitamin Bie (by present standards) is detrimental to patients. The protracted well- * Courtesy of Dr. G. Goldsmith, Departient of Medicine, Tulane University Schoo! of Medicine, New Orleans, Louisiana, Assay orgauism, L,leichmanl Saree Evidence Concerning Human Requirement for Vitamin Bye being of the patient, the maintenance of func- tional good health without evidence of glositis, neurologic deficit of macrocytosis cannot be disregarded. .On the basis of this and previous data, one might predict that it would be difficult to attain and sustain full body stores at saturation of vitamin Br: by injection. The estimates at which we have arrived with regard to turnover of vitamin By: are compatible with clinical observations concerning the length of time required for the signs and symp- toms of a lack of vitamin By to develop in patients who either have undergone total gastrectomy or from whom vitamin By: therapy has been withdrawn.’ Ordinarily relapses occur in six months to six years, an observation made repeatedly. This period of time for exhaustion of body stores is predicted from our data Relapse would be expected to occur shen the body stores of vitamin By: are approximately 500 wg. oF ess.* If initial body stores are 4,000 1g., then this level would be expected in about five years aud if they are 10,000 wg. at about eight to ten years. A slower turnover rate, as suggested by Reizenstein,** does not coincide with the clinical observations and, in fact would have abnormalities occurring at fourteen to twenty years under similar cixeumstances, An additional factor for consideration sug- gested by the rate of loss of isotope from the body (a body store of 700 to 11,900 ug. would imply a loss of 0.79 to 11.9 ng. daily at the decay rate of 0.1 per cent) is the limits of absorption of vitamin Bis by the normal subject. Pre vious studies in normal adults, oF in patients with pernicious anemia given supplements of intrinsic factor, indicated that absorption is approximately proportional to the log dose of vitamin Be with an upper limit of approxi mately 2 yg. for a 100 yg. dose." Glass, Bord and Stephanson® have postulated a mucosal block, analogous to that with iron, which prevents the absorption of more than 13 ws. from a 50 ug. dose. Swendseid ct al.* set an upper limit of Lug. ‘These studies, in com: mon, used erystalline vitamin Bye as the test material, Vitamin By. oceurs in food in other than the cyano or crystalline form."* Cy- anide treatment of bound vitamin By: displaces intestinal symptoms,(il it from its binding, yet it is the bound form in liver which is absorbed at a level of 80 per cent. of a 22 ug. dose.” The effect of food and di- gestion upon the secretion of intrinsic factor or any other absorption-stimulating factor which may be present in gastric or intestinal seere- tions, should be considered. This isillustrated by the increased absorption of erystalline vita- niin Byfollowing stimulation of gastric secretion by carbamyleholine.%! Abels and associates** have reported absorption of vitamin By as high as 35 per cent of a 10 ug. dose in a normal person by the concomitant administration of human gastric juice. ‘This is to be compared with much poorer absorption with the use of heterologous fonns of intrinsic factor.** Studies need to be made in man to determine (under varying conditions) the limits of absorption of labeled vitamin By: present in foodstufis in its natural state. CconcLustons ‘The considerations presented provide indica tions as to a “floor’ on vitamin By needs. Minimal requirements will maintain health, nd normal hemopoiesis in most subjects who have a small body pool and who are operating at an eficient physiologic economy. These are at the level of 0.6 to 1.2 yg. daily. Storage bins filled to the “ceiling,” if achieved, would be associated with a considerable loss of physiologic economy. A larger body pool results in a larger loss; to maintain maximum, body stores woutld require high levels of intake, especially when injected as crystalline co: balamine. One may argue the philosophical question whether the patient benefits from a full body store of the nutrient, but it is evident that there is a wide range of healthy adapt- ability for vitamin By (as with most nutrients). ‘These limits, concerning which we believe one miay be reasonably definite, represent varia tions in rate of use, loss and excretion (prin- cipally the latter) of the injected, crystalline vitamin, when added to body pools of various ‘Knowledge concerning the nornial absorption from the gastrointestinal tract of different forms of the vitamin in the natural state, the influence cof various foods upon absorption, the mecha 127 nisms for and limits of normal gastrointestinal absorption and the distribution in foodstuffs and diets must be clarified before the dietary requirements of vitamin By: ean be stated pre~ cisely and with assurance. SUMMARY An example of the use of the whole body couniter as a safe, simple and accurate means of assessment of absorption, distribution and excretion of a metabolite, vitamin By, is pre- sented. Results of absorption studies in five subjects without pernicious anemia and eighteen pa- tients with pernicious anemia are presented. ‘The range of absorption in the subjects without pernicious anemia is 45 to $0 per cent with ‘a mean of 70 per cent and in those with per- nicious anemia 0 to 17 per cent with a mean per cent, Studies of biological half-tife of vitamin Bra are presented in sixteen subjects (cleven with pernicious anemia and five without), including. three normal subjects, who have been observed for as long as two years. There was no sig- nificant difference in decay constants between those with pernicious anemia and the normal subjects, The values ranged from 0.09 to 0.15 per cent Toss per day of labeled vitamin in normal subjects, and from 0.11 to 0.17 per cent Joss per day in eleven patients with pernicious anemia. ‘These figures correspond to a bio- logical half-life of 407 to 770 days. ‘The proposal is made and support cited for the concept that the daily need for injectable vitamin By: in patients with pernicious anemia reflects almost entirely the loss of the vitamin from the body. Despite the marked difference in the size of the body stores of vitamin Bry in normal subjects and in those with pernicious anemia, daily loss rate constants are essentially identical, Parameters are identified which require study hefore dietary requirements levels can be set more precisely. ACKNOWLEDGMENT We wisi to acknowledge the invaluable advice and assistance at various stages of this study of Dr. Charles F. Federspiel and Mr. Edvsia Bridgforth of the Division of Biostatistics, Vanderbilt University.128 We wish to express our appreciation for the faithful and competent technical assistance of Mrs. George R, Meneely and Mr. Meredith Cowsert -ROFERENCES 1. Daswy, W. J., Jones B, Cuare, $. L., Je Gastry, W. j,, Durea pe Ouwvers, J, Plies, C, Kevany, J. and ts Brocavy, J. The development of vitamin By, deficiency’ by un treated patients with pernicious semis, Amv. J. Clin, Nutrsion, 6: 313, 1958, 2. Dagny, W. J. Beiboroxme, EB, ue Beovovy, Ja, Cuark, SL. JR, DUTRA DE Ouivera, J Kevany, J., McGastry, W. J. and Pérez, © Vitamin By requirement of adult man, ston J Med., 25:72, 1958, 3, Masoav CHUNG, AS, Pransos, W. N., Daxsy W. J. Muutgr, O. N. and Goosen, G. A. Folic acid, vitamin By, pantothenic acid. and vitamin Bre in ume dictaries, am J. Clix, Nutrition, 9: 873, 1961 4, Licurexsrins, H., Butotas, A. and Mearuy, E. W. 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