A DUAL MECHANISMOF VITAMIN B12 PLASMAABSORPTIONI

BY ALFREDDOSCHERHOLMENANDPAUL S. HAGENwmT THE TECHNICALASSISTANCE oF MARGARETLIU AND LOIS OLIN

(From the-Heatology Section, Medical Service, Veterans Administration Hospital, and theDepartmest of Medicine, University of Minnesota Medical School,

Minnapolis, Minn.)

(Submitted for publication May 17, 1957; accepted July 11, 1957)

Recent reports have described two types ofplasma absorption curves following the oral ad-ministration of vitamin B1.,. With microbiologicassay methods, after massive doses of vitamin. Bs,early significant plasma levels have been found in-discriminately in both pernicious anemia patientsand control subjects (1, 2). On the other hand,two independent laboratories, using radio-labeledcyanocobalamin, have successfully determinedplasma absorption curves after the oral adminis-tration of only 0.46 to 1.0 microgram doses (3, 4).With these small amounts of vitamin B12 a dis-tinctly different type of absorption curve was foundin normal subjects and in pernicious anemia pa-tients when intrinsic factor was added. Thiscurve was characterized by little or no plasmaradioactivity during the first four hours of thetest and a peak concentration in the 8- to 12-hour interval. Furthermore, there was clear dif-ferentiation between control subjects and patientswith pernicious anemia, because at these dosagelevels the latter without added intrinsic factorshowed insignificant plasma radioactivity.

In this investigation plasma absorption curveswere obtained in pernicious anemia patients withand without intrinsic factor, as well as in controlsubjects after the oral administration of test dosesof radio-labeled cyanocobalamin which rangedfrom 0.56 to 500 micrograms. This included adosage range not previously examined.

MATERIAL AND METHODS

Cobalt'-labeled vitamin B. 2 used in test doses had aninitial specific activity of 1,137 microcuries per mg.,while that of the Cotm-labeled vitamin Bn- 2 used was 893microcuries per mg. Crystalline non-radioactive vitaminB,2 was added as necessary to make up the desired test

1 This investigation was supported by a grant from EliLilly and Company to the University of Minnesota.

2 Kindly supplied by Dr. N. S. Ritter, Merck and Co.,Rahway, N. J.

dosages. In each case-the test. dose was administered inapproximately 10 ml.. of water- after an- overnight fast;breakfast was withheld for two; hours. When used, in-trinsic factor concentrate (1FC)' was added in 100- or200-mg; amounts to the solution of- cyanocobalamin. Thismaterial had been found- active in pernicious anemia pa-tients when tested by the methods of. Hinle, Welch,Scharf, Meacham, and Prusoff (5) and that of Schilhing(6). Blood was withdrawn at different time intervalsafter the test. Radioactivity measurements ' were madeas detailed elsewhere (3). Briefly, a 20-ml. well-typescintillation counter was used with an automatic vialsample-changer and a recording timer. The overall, effi-ciency of this counter was 18.4 per cent for Co' and 24.0per cent for Co. A pre-set count of 32,000 was used foreach determination in this study. At this count, threetimes the standard deviation of the background was fivecounts per minute (cpm). Generally, in each study a pre-test plasma sample was used for the background count.Altogether, eight tests were carried out in eight controlsubjects and 12 tests in eight patients with perniciousanemia in remission. The diagnosis in each of the latterhad been substantiated by absorption tests with and with-out the addition of IFC.

External monitoring over the liver area was carriedout one to two weeks after the test dosages according tothe method of Glass, Boyd, Gellit, and Stephanson (7)with the use of a NaI (thallium-activated) probe scintil-lation counter. The crystal, 1% inches in diameter and 1inch in thickness, was mounted on an RCA 5819 photo-multiplier tube and shielded by % inch of lead. Ten-minute counts were registered on a Tracerlab Superscaler,model SC 18A.

The amounts of vitamin B.. absorbed into the plasmawere calculated from the radioactivity measurements withthe following formula: micromicrograms of vitamin B,,per ml. of plasma equal pr/20 mik; where p is the oral doseof. vitamin B. in micromicrograms; r is the experimentalnet cpm in 20 ml. of plasma; mis the amount of radio-activity in the test dose in microcuries; k is the overallefficiency of the counter times disintegrations per minuteper microcurie (2.22 X 10'). This calculation was basedupon the assumption that the radioactivity represented

3 Kindly supplied by Dr. R. W. Heinle, The UpjohnCompany, Kalamazoo, Michigan.

4 Facilities of the Radioisotope Unit of. the MinneapolisVeterans Administration Hospital. were. used for radio-activity measurements.

1551

ALFRED DOSCHERHOLMENAND PAUL S. HAGEN

X50 ILK s I FC --v R.K. c IFC

CL~~~~~~A40 -L I FC~40- °A.L.E IFC

0 30C6

120

2 4 6 8 10 12 24Hours

FIG. 1. RADIoAcTIVmY OF PLASMA IN Two PATIENTSWITH PERNICIOUS ANEMIA IN REMISSION Arm ToORAL ADMINISTRATION- OF 0.56 MICROGRAMVITAMIN B.,CONTAINING 0.5 MICROCURIE CO" WITH AND WrTHOUTINTRINSIC FACTORCONCENTRATE(IFC)

cyanocobalamin and not a split-off cobalt salt. This wasa reasonable presumption because the radioactivity isstored in the same way after oral and parenteral ad-ministration of Co'-labeled vitamin B.2, but in a com-pletely different manner from that observed after pa-renterally injected CoCl, (8).

In a few instances the large oral doses of vitamin B2,contained radioactivity in amounts above those calcu-lated to be safe (Co', 8 microcuries; Co", 3 microcuries)if entirely absorbed. This was justifiable because only asmall fraction of these test doses was expected to be ab-sorbed (9). Moreover, the highest liver radioactivityfound in any of the patients in this study was within thegeneral range reported by others after Co' (7, 10).

RESULTS

Two male patients with pernicious anemia, aged61 and 64, were given oral test doses of 0.56 mi-crogram (0.5 microcurie Co60), first without andlater with the addition of 100 mg. IFC. Fourweeks elapsed between the repeated tests. Figure1 shows that there was but little radioactivity inthe plasma unless intrinsic factor was given withthe test dose. When IFC was added, absorptioncurves were obtained similar to those found incontrol subjects given test doses of like amount(3) (Figure 2); negligible radioactivity appearedin the plasma during the first four hours of the test,and peak concentrations in the plasma were notattained until eight hours following dosage.

Differing absorption curves were observed infive of six pernicious anemia patients given from50- to 300-microgram doses of radio-labeled vita-min B12 without IFC. The results are summarized

60

Q. 50

~40

20

2 .4 6 8 10 12 24 72 120 168 21GHouirs

FIG. 2. RIAroAcTwvTY OF PLASMA IN Six CONTROLSUBJECTS (SoLm LINES, TEST DOSE 0.46 MICROGRAMVrTAMIN B.2, 0.5 MICROCURIECo) ANDIN ONEPATIENTWITH LAENNEC'S CnIuOSIS AFM A PORTACAVALSHUNT OPERATION (BROKEN LINE, TEST DOSE 0.56MICROGRAMVITAMIN B.2, 0.5 MicRocuiRi CO)

in Table I and are shown graphically in Figure 3.One patient with the 50-microgram test dose hadlittle plasma radioactivity, but each of the otherfive had relatively high values by three hours withpeak concentrations usually in four to six hours.

Two of these patients were retested with thesame doses of vitamin, but with the addition of 200mg. IFC. The early rise in plasma radioactivitywas abolished in each case (Figure 4). Insteadthere was a gradual rise in radioactivity to peakvalues at 12 hours. While there was inhibition ofthat phase of absorption not mediated by intrinsicfactor, radioactivity measurements over the livers

Hour

FIG. 3. RADIoAcTIVrIY OF PLASMA IN SIX PATIENTSWITH PmERIcIoUS ANEMIA AFTER THE ORAL ADMINIS-TRATION OF 50 To 300 MICRoGRAMSOF RADIOACTIVE Vi-TAMIN B22,

1552

A DUAL MECHANISMOF VITAMIN B12 PLASMAABSORPTION

TABLE IRadioactivity (cfm) ian 20 mtiilirs pf plasta at dijffe .t tie interalsOft the oraladmi~nistration of.

various test doses of Co' vitamin B,

calculatedamdoutt Radio.labeled activity

Radio- Cpmat different hours after oral test ose vitamin Bs overtheVit. Bns activity -wsg./ml. liver,

Name* Age Diagnosis . pc. 12 3 4 5 6 8 10 12 24 36. : sa ebcpm

H. M. 23 No disease 10 5.0 .8 8 8 13 38 66 56 31 16G. B. 21 No disease 50 8.0 7 9 11 11 15 25 34 32 29 23 26 1,125R. R. 40 Psychoneurosis 50 8.0 3 7 6 9 15 23 24 16 18E. J. 25 Appendicitis 100 8.0 1 2 4 9 12 14 16 14 11 24E. E. 59 Ess. hypertension 100 8.0 13 9 17 20 20 34 23 21 S1A. H. 58 Emphysema 200 16.0 11 15 19 26 27 36 33 26 54 1,123M.F. 44 Duodenal ulcer 300 16.0 14 21 20 17 20 22 25 27 21 61G. G. 40 Br. asthma, diabetes 500 16.0 7 15 19 18 19 22 21 21 19 83A. O.t 60 Pernicious anemia 50 8.0 6 16 19 24 21 21 21 15 18 670L. B.t 63 Pernicious anemia so 8.0 2 3 5 2 0 1 -2 1 4 118C. L.t 64 Pernicious anemia 50 8.0 3 10 15 15 16 18 12 10 14 254C. L.tt 64 Pernicious anemia 50 8.0 2 3 4 6 8 14 21 14 16 721A. B. 68 Pernicious anemia 100 9.8 16 45 55 58 62 51 41 30 76 760A. B.4 68 Pernicious anemia 100 8.9 5 4 6 10 15 20 22 11 30 946J. P. 45 Pernicious anemia 200 16.0 12 13 21 22 23 17 12 9 35 170P. E. 66 Pernicious anemia 300 16.0 17 21 28 32 28 19 20 15 72 370

* All were male patients.t Approximately 4.0 microcuries COOOB1, included.$ Two hundred mg. intrinsic factor concentrate added.

after IFC showed higher counts in each instance(Table I).

Figure 5 depicts the absorption curves in eightcontrol subjects given 10- to 500-microgram test

Id I

.

E 3

Oral dos 50, A+2o_ o PA IFC (C.L.)

PA IFC (C.L)to

10

0:

00rt -ratJm6 100 1

At -a - --PA a I F (A.B)i- * -- PAlIFC(A.

M0-lo

10 -,,

0f *li2f 4 6 --8 IO 12 24Hours

FIG. 4. COMPARISONOF THE PLASMA RADIoAcTIvITyIN Two PATIENTS WITH PERNICIOUS ANEMIA AFirRTHE ORAL ADMINISTRATION OF 50 AND 100 MICROGRAMSOF RADIOACTIVE VITAMIN B1, WITH AND WITHOUT IN-TRINSIC FACTORCONCENTRATE(IFC)

doses of radio-labeled cyanocobalamin. Threekinds of curves were obtained: the familiar delayedtype; a new, somewhat biphasic, plateau-shapedconfiguration; and curves intermediate betweenthese two types. None of the curves were the same

as that found with comparable dosages of vitaminwithout addition of IFC in patients with perniciousanemia. The early, proportionately large amountof radioactivity in the plasma found in perniciousanemia was not obvious until the 200- to 300-microgram dosage. Then, in contrast to the con-

;060

40

a0u.2% 0

-c

_ SO

a 4030

t0

10

2 4 * 10 a Z4 L 46 8105 Z U4 t 4 i> 10 eHours

FIG. 5. RADIoAcTIVIrY OF PLASMAIN EIGHT CONTROLSUBJECTS AFTER THE ORAL ADMINISTRATION OF 10 TO

500 MICROGRAMSOF VITAMIN B1. CONTAINING 5.0 TO 16.0MICROCURIESOF CO'

Chwl do". Op1.B5m Oral de". 50~mi.aI.B Oral dAm 0vil.111

,: iS1 Wosos "o~~~ft LE.

2 4 6 a 10 12 36 2 4 60 10 It 4 Z 4 b 10 It

Oral dos" Z0Wa,. . Oral de". VOOe,.D Orsl Jose 500Fsq5tz

A.AM.F. -~~~~~66

1553

3

ALFRED DOSCHERHOLMENAND PAUL S. HAGEN

tinued fall-away after the early peak in the pernici-ous anemia patient a second phase was observedin the control subjects, in which the plasma radio-activity increased and persisted for many hours.At dosage levels of 50 to 100 micrograms inter-mediate types of curves were found.

Table I includes the maximum amounts of vita-min B12 absorbed into the plasma. Sixteen micro-micrograms per ml. of plasma were found when the10-microgram test dose was given to a controlsubject. In general, with increasing size of testdoses larger amounts of cyanocobalamin werefound, 83 micromicrograms per ml. being observedin the subject who received the 500-microgramtest dose. This same tendency was seen in thesix pernicious anemia patients to whom IFC wasnot given. However, the individual results showedwide variation.

The results of measurements of radioactivityover the liver one to two weeks after dosage arerecorded in Table I. Consistently higher valueswere obtained in normal subjects and in patientswith pernicious anemia when IFC accompaniedthe test dose, than in patients with pernicious ane-mia without IFC. This was true even when higherpeak plasma radioactivity had been found in thelatter. The effect of intrinsic factor can be il-lustrated by comparison of ratios of liver radio-activity to the peak radioactivity of 20 ml. ofplasma. The average ratio was 16 in six patientswith pernicious anemia without the addition ofIFC to the test dose (range, 7 to 28). On theother hand, the ratio was raised to 34 and 43 intwo pernicious anemia patients retested with addedIFC, and was 31 and 33, respectively, in two nor-mal subjects.

DISCUSSION

These studies show that patients with perniciousanemia absorb significant amounts of radio-labeledcyanocobalamin in the dosage range of 50 to 300micrograms of the vitamin. The resulting plasmaabsorption curves were unique with rapid rises inplasma radioactivity to peaks usually in four tosix hours. The peaks were followed by relativelymore rapid declines of radioactivity than in con-trol subjects given comparable dosages. For ex-ample, in the pernicious anemia patients the av-erage plasma concentration after 24 hours was45.4 per cent of the peak plasma level, compared

to 71.6 per cent in control subjects. The differ-ence is statistically significant, t = 3.9, p < 0.01.

Of particular interest was the finding that IFCabolished this early type of absorption curve andgave rise instead to gradual increases of radioac-tivity in the plasma with delayed peaks (Figure 4)such as found with smaller test doses with in--trinsic factor present. Although excessive amountsof intrinsic factor can inhibit the absorption ofsmall test doses (e.g., 0.5 microgram) of vitaminB12 (11, 12), inhibition of the absorption of largertest doses has rarely been found (13). The re-tarded absorption presently observed might be dueto interference with absorption by some non-specific material in the IFC (11). However, theintrinsic factor may have been responsible, sincethe resulting absorption curves were similar tothose observed in control subjects given compara-ble amounts of cyanocobalamin. Moreover, theaddition of IFC resulted in each instance in higherhepatic radioactivity. This suggests that more,rather than less, cyanocobalamin was absorbed inspite of the change in the contour of the absorptioncurves.

Another possibility must be considered to ex-plain the delayed rise of radioactivity in peripheralblood samples in the presence of intrinsic factor.With intrinsic factor there could be early removalof the absorbed vitamin B1, by peripheral tissuesor organs such as the liver. Simultaneous radio-activity measurements of blood samples from theportal vein would be most helpful to place thecause of the slower rise in -plasma radioactivity.Such studies are not available. However, Boothand Mollin (4) found maximum radioactivity inthe plasma 8 to 12 hours after dosage while thehighest counts over the liver were not obtaineduntil two to six days later. On the other hand,we have obtained a plasma absorption curve in onepatient with an effective portacaval shunt. Thesubject, a 46-year-old man with Laennec's cir-rhosis, was studied 17 months after operation. Asseen in Figure 2, there was a delay in the peak ofradioactivity in the plasma identical to that foundin normal subjects. These observations make itunlikely that the liver immediately removes cyano-cobalamin from the blood under the influence ofintrinsic factor and thereby causes the delay in therise of radioactivity in the plasma. Moreover,we are not aware of any evidence which indicates

1554

A DUAL MECHANISMOF VITAMIN B12 PLASMAABSORPTION

that peripheral tissues selectively remove and tem-porarily store newly absorbed vitamin B1, afteroral administration. Therefore, -it seems morelikely that the intestine is in some way responsiblefor the slower rise in radioactivity in the plasma inthe presence of intrinsic factor. This view is inagreement with that suggested by Booth and Mol-lin (4).

Tests in control subjects showed that the in-hibition of the early rise of radioactivity in theplasma could be overcome by larger doses ofvitamin B12. Examination of Figure 5 shows thatin one of two control subjects given 100 micro-grams and in the subjects tested with 200 micro-grams there was considerable plasma radioactivityby four hours, as compared with the results at the10- and 50-microgram levels. With 300- and 500-microgram doses relatively high plasma radioac-tivity was found at two and three hours. Then, incontrast to the results obtained in patients withpernicious anemia, the peak was followed by aplateau-like curve. This prolongation was prob-ably due to the superimposition of absorption medi-ated by intrinsic factor.

The results of these studies lend support to thehypothesis that there are two modes of intestinalabsorption of cyanocobalamin: one, mediated byintrinsic factor, is characterized -by more gradualand prolonged plasma absorption curves; theother, found with much larger doses of cyanoco-balamin, is characterized by early rises in plasmalevels of vitamin B12, as indicated by measurementsof radioactivity. It has been suggested that thislatter mechanism is due to a passive diffusionacross the intestinal barrier (9). However, inpernicious anemia the four- to six-hour peakamounts of plasma radioactivity (Figure 3) ap-peared later than would be expected from a processof simple diffusion. The particular curves can becompared to the plasma absorption curves of ironwhich are believed to be related to an active processof absorption. Oral administration of iron re-sults in peak plasma concentrations two-and-one-half to five hours after test doses (14). Untilmore details are known it is perhaps best to referto the two mechanisms of absorption -of vitaminB12 as the one mediated by intrinsic'factor, -and-theother as absorption independent of this factor.

Recently, it has been estimated that .100 gramsof undried beef liver may contain from 60 to 118

micrograms of vitamin B12 (15, 16). Figure 3includes plasma absorption curves with dosages inthis general range. Such absorption suggests thepossibility that the success of the dietary livertherapy of Minot and Murphy (17) was due to thecyanocobalamin content itself, and not necessarilydue -to other factors present in the liver whichmay have promoted hematopoiesis or vitaminB12 absorption.

Clinical experience has indicated a wide varia-tion in results in the treatment of pernicious anemiapatients with oral doses of cyanocobalamin.Whereas some may show an almost optimal re-sponse to daily doses of 15 micrograms (18) andsome can be successfully treated with daily oraldoses of 50 micrograms (19), failures have beenreported with daily doses of as much as 250 mi-crograms (20-22). This variation is well illus-trated in Figure 3 at the 50-microgram dose. Twopatients had significant plasma absorption curvesand hepatic radioactivity (Table I), while a thirdpatient showed little absorption and only one-halfto one-sixth the liver radioactivity of the othertwo. These findings indicate great individual vari-ation in the absorption gradient in patients withpernicious anemia for doses of vitamin B12 abovethe physiologic range. Such a variation in the ab-sorption gradient is well known also for test dosesof a more physiologic magnitude, when a sourceof intrinsic factor is added (23).

The plasma absorption curves determined bybioassay of vitamin B12 after the administration ofmassive doses of the vitamin have shown peakplasma concentrations 1 to 24 hours after theadministration of the tests, and no definite separa-tion between patients with pernicious anemia andcontrol subjects has been claimed (1, 2). This isin contrast to the definitive results obtained whentest doses of 50 to 300 micrograms were given.However, with the larger test doses there was lessdifference between the absorption curves of per-nicious anemia-patients and normal subjects, This-was probably .due to the fact that as the test doseswere increas, te absorption mechanism inde-pendent of intrinsic factor gradually 'became themore dominant also in the control subjects.

The calculated amount of vitamin B12 absorbedwas found to range from about 18 micromicro-grams per anl. of -plasma in a control subject giventhe 50-microgram test dose to 83 micromicrograms

1555

ALFRED DOSCHERHOLMENAND PAUL S. HAGEN

per ml. of plasma when 500 micrograms were ad-ministered. The figures were not too different inthe patients with pernicious anemia, except in thepatient who absorbed only a minor amount whenthe 50-microgram test dose was given. Our dataare too few to make a more detailed comparisonbetween the amounts calculated to have been ab-sorbed in each group.

Because such small amounts of cyanocobalaminwere absorbed it is understandable that bioassayhas failed to reveal definite changes in the concen-tration of vitamin B12 in the serum after single oraltest doses of less than 500 micrograms (9, 18).Tracer techniques, being more sensitive, have thefurther advantage that they can determine absorp-tion without measurable changes in the totalamount of vitamin B12 in the serum.

SUMMARYAND CONCLUSIONS

1. Studies of the rate of entrance of vitamin B12into the plasma have been carried out in eight per-nicious anemia patients and eight control subjectsafter the oral administration of test doses varyingfrom 0.56 to 500 micrograms of Co58- or Co6°-labeled vitamin B12.

2. Negligible or no plasma absorption was ob-served in two patients .with pernicious anemiawhen oral test doses of 0.56 microgram of vitaminB12 were administered without intrinsic factorconcentrate. When the latter was administered,absorption curves were obtained which were simi-lar to those previously found in control subjectsgiven test doses in the same range. They werecharacterized by delayed plasma radioactivity ap-pearing after four hours, with a peak concentra-tion at eight hours following dosage.

3. Oral test doses of 50 to 300 micrograms ofvitamin B12, given without IFC to patients withpernicious anemia, were followed by differing ab-sorption curves characterized by an early rise inplasma radioactivity and peak concentrations fourto six hours after the test. The addition of IFCabolished the early rise in plasma concentrationand resulted in a delayed peak concentration at 12hours. Although IFC delayed the appearance ofvitamin B1, in the plasma, it enhanced hepaticuptake of radioactivity.

-4. Control subjects given test doses of 200 to 500micrograms of vitamin B12 displayed a slightly di-phasic absorption into the plasma, with an early

rise in the radioactivity. A test dose of 10 micro-grams caused a delayed absorption similar to theresults observed with the much smaller test doses,while doses of 50 to 100 micrograms resulted inabsorptions of an intermediate nature.

5. The present findings lend support to theconcept of a dual mechanism of absorption of vi-tamin B12 from the gastrointestinal tract.

ACKNOWLEDGMENT

The authors wish to thank Dr. C. J. Watson for hishelpful criticisms in the preparation of the manuscript

REFERENCES

1. Ross, G. I. M., Mollin, D. L, Cox, E. V., and Ungley,C. C., Hematologic responses and concentrationof vitamin Be, in serum and urine following oraladministration of vitamin B. without intrinsic fac-tor. Blood, 1954, 9, 473.

2. Unglaub, W. G., Rosenthal, H. L., and Goldsmith,G. A., Studies of vitamin B, in serum and urinefollowing oral and parenteral administration. J.Lab. & Clin. Med., 1954, 43, 143.

3. Doscherholmen, A., and Hagen, P. S., Radioactivevitamin B1, absorption studies: Results of directmeasurement of radioactivity in the blood. Blood,1957, 12, 336.

4. Booth, C. C., and Mollin, D. L., Plasma, tissue andurinary radioactivity after oral administration of'Co labelled vitamin B1.. Brit. J. Haemat., 1956,2, 223.

5. Heinle, R. W., Welch, A. D., Scharf, V., Meacham,G. C., and Prusoff, W. H., Studies of excretion(and absorption) of Cow labeled vitamin B., inpernicious anemia Tr. A. Am. Physicians, 1952,65, 214.

6. Schilling, R. F., Intrinsic factor studies. II. The ef-fect of gastric juice on the urinary excretion ofradioactivity after the oral administration of radio-active vitamin B.. J. Lab. & Clin. Med., 1953, 42,860.

7. Glass, G. B. J., Boyd, L. J., Gellin, G. A., and Steph-anson, L., Uptake of radioactive vitamin B. by theliver in humans: Test for measurement of intesti-nal absorption of vitamin B., and intrinsic factoractivity. Arch. Biochem. & Biophys., 1954, 51,251.

8. Meyer, L. M., Berlin, N. I., Jiminez-Casado, M., andArkun, S. N., Vitamin B. distribution, determinedby surface body counting following parenteral ad-ministration of Co vitamin B1,. Proc. Soc. Exper.Biol. & Med., 1956, 91, 129.

9. Ungley, C. C, The chemotherapeutic action of vitaminaction of vitamin B1,. Vitamins and Hormones,1955, 13, 137.

10. Glass, G. B. J., Pack, G. T., and Mersheimer, W. L.,Uptake of radioactive vitamin B., by the liver in

1556

A DUAL MECHANISMOF VITAMIN B12 PLASMAABSORPTION

patients with total and subtotal gastrectoomy. Gas-troenterology, 1955, 29, 666.

11. Glass, G. B. J., Boyd, L. J., Stephanson, L., andJones, E. L, Metabolic interrelations between in-trinsic factor and vitamin B,,. III. B,, absorptionat varied intrinsic factor doses. Proc. Soc. Exper.Biol. & Med., 1955, 88, 1.

12. Callender, S. T., and Evans, J. R., Observations onthe relationship of intrinsic factor to the absorptionof labeled vitamin B., from the intestine. Clii.Sc., 1955, 14, 387.

13. Chow, B. F., Wiiams, W. LI, Okuda, K, and Gras-beck, R., The urinary excretion test for absorptionof vitamin B,,. IL Effect of crude and purifiedintrinsic factor -preparation. Am J. Clin. Nutr.,1956, 4, 147.

14. Moore, C. V., Arrowsmith, W. R., Wek:h, J., andMinnich, V., Studies in iron transportation -andmetabolism. IV. Observations on the absorptionof iron -from the gastrointestinal tract. J. Clin.Invest, 1939, 18, 553.

15. Scheid, H. E., and Schweigert, B. S., Vitamin B.,contents of organ meats. :J. Nutrition, 1954, 53,419.

16. Shenoy, K. G., and Ramasarma, G. B., Extractionprocedure and determination of the vitamin B.,

content of some animal livers. Arch. Biochem. &Biophys., 1954, 51, 371.

17. Minot, G. R., and Murphy, W. P., Treatment of per-nicious anemia by a special diet, J. A. M. A., 1926,87, 470.

1& Mollin, D. L., and Baker, S. J., The absorption andexcretion of vitamin B., in man in Biochemical So-ciety Symposia, Number 13, The Biochemistry ofVitamin B,,, R. T. Williams, Ed. Cambridge,University Press, 1955, p. 52.

19. Chalmers, J. N. M-, and Hall, Z. M., Treatment ofpernicious anemia with oral vitamin B. withoutknown source of intrinsic factor. Brit. M. J., 1954,1, 1179.

20. Spies, T. D., Stone, R. E., Lopez, G. G., Milanes, F.,Tocs, R. L, ,and rambur T., Vitamin B, bymt ipeiniious and mt4ritional macrocytic

anenija and spre. Lancet, 1949, 2, 454.21. Hall, B. E., Studies on the nature of the intrinsic fac-

tor of Castle. Brit. M. J., 1950, 2, 585.22. Mey, -L M., Sawltey, A., Cohen, B. S., Krini, M.,

and Fadeni, R, Oral treg: of p us anemiawi vitamin B=.. Am. J. M. Sc., 1950, 220, 604.

23. Baker, S. J., and Mollin, D. L., The relationship be-tween intrinsic factor and the intestinal absorptionof vitamin B.2. Brit. 3. Haemat., 1955, 1, 46.

1557