Studies on the Uptake of Vitamin B12-Co60by the Hamster by olliegoblue36


       Studies on the Uptake of Vitamin B12-Co60 the Hamster
               Methylcholanthrene-induced   Sarcoma and the Rat
                            Walker Carcinosarcoma*

                        A. MILLER,t G. GAULL,H. M. LEMON,ANDJ. F. RossJ
                (Robert Dateson Erans Memorial, Massachusetts Memorial Hospitals, and Department         of Medicine,
                                      Boston University School of Medicine, Boston, Afa*«.)

    Many studies (10, 12, 15) have demonstrated                       the heavier organs 30 ml. of 2 N NaOH was used. The tissues
 the key role of vitamin Bi2 in normal growth and                     were digested in alkali at room temperature for 24 hours. Then,
                                                                      if necessary, gentle heating for 30-60 seconds brought the gel-
 development. Oleson (16) found that supplemen                        like digest into a liquid state. Measurements of radioactivity
 tary vitamin B^ caused increased growth of the                       were made on duplicate 4-rnl. aliquots of these tissue digests.
 Rous sarcoma in chicks. This observation suggest                     The total volume of the tissue digest solution was assumed to
 ed that vitamin BK might be necessary for the                        be equal to 10 ml. when the organ weight was less than 0.75
 growth of malignant neoplasms. With the use of                       gm., since the volume contributed by the dissolved organ was
                                                                      negligible. For heavier organs the total volume of the digest
 isotopically labeled vitamin Bi2, the possible rela                  was measured in a 25- or 100-ml. graduated cylinder.
 tionship of vitamin B^ to tumor metabolism has                                             In
                                                                         Urine and stools.— the rat the total urine volume was meas
 been investigated.                                                  ured in a 50-ml. graduated cylinder. Radioactivity was deter
                                                                     mined on 4-ml. aliquots of urine. For hamsters the filter paper
          MATERIALS AND METHODS                                      on which the urine had dried was cut into }-inch pieces and
                                                                     placed into If X 8¿-inchglass bottles in which radioactivity
              EXPERIMENTALLAN                                        was measured. The radioactivity in 4-day stool collections
     Adult male hamsters weighing from 75 to 100 gm., bearing        was also measured in 1} X 3i-inch glass bottles.
 a methylcholanthrene-induced sarcoma as previously de                                     rat
                                                                          Carcasses.—The carcasses were placed into i\ X 6-inch
 scribed (8, 11), and adult male Sprague-Dawley rats weighing        glass bottles. Carcass radioactivity measurements were made
 from 220 to 880 gm., bearing the Walker carcinosarcoma, were        in these bottles.
 used in these studies. These animals were housed in individual
 cages, at constant temperature, and fed stock diet and water                               DETERMINATION
 ad libitum. Under light ether anesthesia, they were given sub
cutaneous injections of vitamin Biz-Co60(secured from Merck                                                                was
                                                                         A 1:100 dilution of the injected vitamin Bi2-Co'° made,
& Co., Rahway, N.J.), receiving 0.10-0.50 pg. of vitamin Bi2,        and varying aliquots were then used as standards for the vary
equivalent to 0.09-0.40 /ic. of Co60,contained in a total volume    ing conditions employed in the counting of organ digests,
of 1.0 ml. After the injection, rats were housed in metabolic       excreta, and carcasses. By the addition of water as needed, the
cages allowing for separate urine and stool collections. The        height of the standard in the glass bottles was always the same
hamsters were housed in wire cages, under which were placed         as that of the specimen being counted, i.e., stools, urine, or
pans which were covered with two thicknesses of Whatman             carcasses. Organ and liquid urine samples were counted in a
filter paper #8. All stools fell on the paper while the urine was    scintillation well-type counter (thallium-activated, sodium
absorbed into the paper. Animals were sacrificed 4 days follow      iodide, scintillation crystal). The radioactivity count of the
ing the injection of vitamin Bi2-Co60  except in the turnover and   organ samples usually was 3-12 X background count, except
flushing experiments, in which the animals were killed at the       for the heart and spleen, for which the counts were usually
                                                                    1-3 X background. Samples were counted long enough to give
times indicated in the text.
                                                                    a counting error of less than 2 per cent, except for the low-
                                                                    count samples, for which a 5-8 per cent counting accuracy was
    PREPARATION         FORANALYSIS                                 obtained. Duplicate samples usually agreed within 1-5 per
   Organs.—Theliver, kidneys, spleen, heart, and tumor were
                                                                    cent. The radioactivity of all samples contained in the li X
removed and weighed. Organs weighing less than 5.0 gm. were         8J-inch glass bottles was determined by placing them on top of
placed into flasks containing 10 ml. of 2 N NaOH, whereas with      the above-mentioned sodium iodide well crystal. The diameter
    * This work was supported in part by grants from the Unit       of these bottles was just equal to that of the diameter of the
ed States Public Health Service and The Atomic Energy               well. Under similar geometric conditions, the radioactivity of a
Commission.                                                         standard was also determined. The 6-inch bottles containing
    t Formerly U.S. Public Health Service Fellow. Present           the rat carcasses were marked into three equal 2-inch segments.
address: Boston Veterans Administration Hospital, Boston,           With the bottle in the horizontal position and directly in con
                                                                    tact with a solid 1 X 1-inch thallium-activated, sodium iodide
                                                                    crystal, the radioactivity of each 2-inch segment of the bottle
   ÃŽresent address: University of California Medical Center,
                                                                    containing the carcass was counted. Under similar geometric
Los Angeles, Calif.
                                                                    conditions, the radioactivity of the bottle containing the stand
   Received for publication March 12, 1956.                         ard was measured.
                         MILLERet al.—Radioactive Vitamin BU in Animal Tumors                                                      843

                       CALCULATIONS                       ranged from 11.4 to 29.6 per cent at both dosage
  The per cent radioactivity in organ =                   levels. Furthermore, the tumor was the major
                                                          site of vitamin B12-Co60localization with these
 counts/min/ml organ digest X total volume digest
                counts/min injected
                                                   X 100; implants in the back, which grow to much larger
                                                          sizes than the cheek pouch implants.
                            total counts/min in organ         The radioactivity uptake of the rat Walker carcino-
    counts/min/gm organ
                               weight of organ (gm)       sarcoma.—Eight rats with Walker carcinosarco-
                                                          mas weighing from 5.1 to 82.7 gm. took up from
                                                           1.5 to 16.2 per cent of the injected radiovitamin
   The radioactivity uptake of the hamster methyl-
                                                           (Table 3). In four of the eight rats, the tumor was
cholanthrene-induced sarcoma.—Hamster methyl-
                                                          the organ with the greatest content of radioac
cholanthrene-induced sarcomas, implanted in the
                                                          tivity. Except for a decreased kidney uptake, the
cheek pouch, weighing from 0.25 to 1.00 gm., took distribution of radioactivity in the other organs
up 1.2-3.1 per cent of the injected radioactivity
                                                          was similar to that found for normal rats. When
                         TABLE 1
                                                          the total uptake was related to organ weight, the
                                                          tumor uptake was approximately one-sixteenth
THE TISSUE    DISTRIBUTION   OFRADIOACTIVITY       FOLLOW that of the kidneys and approximately one-half
   Bi2-CoM TOHAMSTERS                                      that
                          BEARINGHEMETHYLCHOLAN- of the liver, spleen, and heart (Table 3).
                                                                                                  TABLE 2
                                                                     COMPARISON                     T
                                                                                      BETWEENwo GROUPS FHAMSTERS      O             IN
                                                                       JECTED    WITH    DOTERENT OSES  D        OFVITAMIN   Bn-Co60
                                                                             ASTOSARCOMA                     O
                                                                                                  UPTAKE FRADIOACTIVITY
                                                                                                              Per cent of
                                                                                                  Weight of      injected
                                                                                                  larcoma     radioactivity   Counts/rain/
                                                                                        Animal      (gm.)      in sarcoma          gm
                                                                                          1        16.35          26.5            1301
                                                                         Group I*         2         7.20          12.6            1178
                                                                                          3        31.70          29.5             771
                                                                           Mean                    18.42          22.8            1083
                                                                                                   33.96          25.2            2513
                                                                         Group Hf                  21.00          22.8            6487
                                                                                                    5.97          11.4            6145
                                                                            Mean                    20.30          19.8           5048
                                                                             * Dose = 0.09 /ic. = 0.10 /ig. of vitamin BH.
                                                                             t Dose = 0.36 ite. = 0.40 ¿ig- vitamin B1S.

                                                            Relationship of the size of the tumor to vitamin
   * Dose—0.45MC.Co« 0.50 /ig. Vitamin B12.
                       =                                 Bi2-Cow uptake.—Chart 1 demonstrates the corre
                                                         lation between tumor size and the per cent uptake
(Table 1). The distribution of radioactivity in the of vitamin Bi2-Co80by the hamster sarcoma both
liver, kidney, and other viscera was similar to that in the cheek pouch and on the back. There was a
found in normal hamsters. When the total uptake          good correlation between the uptake of radioac
was related to organ weight, the tumor uptake was tivity and the tumor size for tumors weighing less
approximately ^ that of the kidney, | that of the than 17.5 gm. With the heavier tumors the in
liver, similar to that of the spleen, and 3 times that crease in vitamin Bu-Co60 uptake was not com
of the heart (Table 1).                                  mensurate with increases in their weight. The rat
   Effect of size of dose on the uptake of radioactivity Walker carcinosarcoma showed a similar relation
in hamster methylcholanthrene-induced sarcoma.— ship between tumor radioactivity uptake and size.
Two groups of hamsters with large methylcholan           Thus, with Walker carcinosarcomas weighing less
threne-induced sarcomas implanted in the back than 40.3 gm., a fair correlation was found be
were given injections of 0.1 and 0.4 ¿tg. vitamin
                                            of           tween vitamin Bw-Co60uptake and size. However,
Biz-Co60.No major difference in the sarcoma con          with the heavier tumors, this relationship no long
tent of radioactivity (expressed as per cent of ad       er obtained (Table 3).
ministered dose) was found at these two dosage               The turnover of radioactivity in the hamster sarco
levels (Table 2). The tumor uptake of radioactivity      ma.—The turnover of radioactivity in the tumors
844                                                Cancer Research

and organs of hamsters sacrificed at varying inter            spleen, and heart also remained relatively con
vals up to 45 days was studied. The per cent radio            stant. After the first 4 days urinary excretion of
activity in the sarcoma over a 45-day period was              radioactivity  was no longer present, whereas the
relatively constant, i.e., there was a negligible rate        daily excretion of radioactivity  in the stools, al
of turnover (Chart 2). The 9.7-19.5 per cent range            though very small, continued over the whole 45-
in the sarcoma content of radioactivity found dur             day period.
                                                                 "Flushing" experiment in rats bearing Walker
 ing the 45 days was probably secondary to the dif
 ference in the weights of the tumors (3.37-99.20             carcinosarcomas.—Two groups of rats bearing
gm.) and to the varying degrees of necrosis found.            Walker carcinosarcomas     were given injections of
The per cent radioactivity       in the liver, kidney,        vitamin Biz-Co60. Beginning \\ days after injec-

                                                       TABLE 3
                     THETISSUE                       FOLLOWING
                                       OFRADIOACTIVITY      THEPARENTERAL
                           ADMINISTRATION*      B     TORATSBEARING
                                                                                               Per cent of injected
                     Organ               Rat no.                                                     in organ
            Kidney                         1                                                            8.6
                                           2                                                            8.4
                                           3                                                           21.9
                                           4                                                            6.1
                                           6                                                            8.7
                                           6                                                              .0
                                           7                                                            4.9
                                           8                                                            5.0
                             Mean and S.E. of mean          +
           Liver                            1                                                     10.3
                                           2                                                       8.4
                                           8                                                            5.4
                                           4                                                            7.9
                                           5                                                            8.9
                                           6                                                           10.9
                                           7                                                           18.7
                                           8                                                           12.8
                             Mean and S.E. of mean                                              10.4 + 1 4
            Walker carcinosarcoma          1                                                            7.3
                                           2                                                            2.8
                                           9             1143                                           1.5
                                           4             5163.4037                                      9.2
                                           5                                                           16 2
                                           6             5382.7169.20Specific                          15 6
                                            7                                                          15.6
                                            8                                                          15.2

                             Mean and S.E. of mean    43.20 + 9.78              365 ±34            10.4±1.7
            Spleen                          1                78                     861                 10
                                           2                 32                     758                 06
                                           3                 70                     538                 0.6
                                           4                 08                     608                 18
                                           5                 86                   1,219                 1.2
                                           6               1.46                     771                 0.8
                                           7               1.52                     788                 09
                                           8               1.48                   1,214                 1.4

                             Mean and S.E. of mean     1.7 ±0.21               845 ±87             1.0±0.1
            Heart                          1              1.04                      404                 0.3
                                           2              1.28                      703                 0.7
                                           3              1.02                      896                 06
                                           4              1.02                      637                 0.5
                            Mean and S.E. of Mean       1.09 ±0.07             660 ±102            0.5±0.1
               ' Dose = 0.25 ite. Coâ„¢ 0.30 pg. vitamin BU.
                            MILLERet al.—Radioactive Vitamin Bn in Animal Tumors                                                                                        845
tion, one group of rats was given five daily "flush                                           nary excretion of radioactivity rose to a mean of
ing" doses of nonradioactive vitamin BU. A                                                    11.0 per cent in the flushed group as compared
marked decrease in radioactivity was found in the                                             with a mean of 1.8 per cent in the nonflushed
liver, carcass, and Walker carcinosarcoma of the                                              group (Chart 3).
flushed animals as compared with the control
group (Chart 3). Slight differences in tumor size                                                                DISCUSSION
between the two groups were present, but these                                                   The hamster sarcoma and the Walker carcino
                                                                                              sarcoma concentrated appreciable amounts of the
                                                                                              injected vitamin Bi2-Co60.The uptake correlated
                                                                                              fairly well with the weight of the tumor. This rela
                                                                                              tionship was true for all but the heaviest tumors,
                                                                                              in which increases in tumor weight were associated
                                                                                              with only minimal increases in the uptake of vita
                                                                                              min Bi2-Co60. The necrosis found in the larger
                                                                                              tumors probably explains this finding.
        10 - . •¿

               5      IO     15       20        25         30        35        40
                       WEIGHT OF SARCOMA (CM.)

   CHART1.—Correlation between the size of the inethyl-
cholanthrerie-induccd sarcoma and the tumor uptake of vita
min Bi2-CoM.

                                                                                                    *AOlO*Crmrr      >nKIDNEY
                                                                                                                  —¿          mURINE   .nCARCASS   itiLIVER   m TUMOR

                                                                                                     cttlmnvat— 12912.9       ;02t44    16.UZ.5 11.4:1.2 160112

                                                                                                  CHART3.—  Changes in the tissue distribution and excretion
                                                                                              of a parenteral dose of vitamin Bij-CoMfollowing large flushing
                                                                                              doses of nonradioactive vitamin BU in rats bearing the Walker
                                                                                                  * Refers to number of animals in each group.
                                                                                                  ** Flushed group received five daily SO-ftg. injections of
                                                                                              vitamin Hi- beginning I Õ ays after the injection of 0.3 p%.of
                                                                                              vitamin B|2-Co60.

                5      IO        15        20        25         30        35        40   45       In this study it would have been desirable to
                    DAYS AFTER    S.C. INJECTION          OF Co"     Vitamin 3g               administer a dose of vitamin Bj2 that did not ap
    CHART2.—Theturnover of tumor and organ radioactivity                                    preciably alter the blood concentration of vitamin
after the parenteral administration of vitamin Bu-Co80 to
                                                                                              BIZ, i.e., a "tracer" dose. The smallest doses of
hamsters bearing the methylcholanthrene-induced sarcoma.                                      vitamin Bu-Co60 compatible with accurate count
    At each indicated time, the tumor and organ radioactivity                                 ing were thus employed. However, these doses
(per cent of injected dose) of two hamsters are given, except at
45 days, when only one hamster was used. The tumor and
                                                                                              still exceeded the total blood vitamin Bw of the rat
organ turnover curves shown above were drawn through the
                                                                                              (3) by about 10-11 times. The organ and tumor
mean of each of the two given values. The weights of the ham                                  uptake of the radiovitamin at such pharmacologi
ster sarcomas at each indicated time are also shown.                                          cal dosage levels may not represent their uptake
                                                                                              of endogenous vitamin B^. Because of the size of
differences were probably not large enough to ac                                              dose and the necrosis in the larger tumors, com
count for the results noted. In contrast, the kid                                             parison of the organ and tumor uptake of vitamin
neys of the flushed animals contained only about                                              Bi2-Co«° is difficult.
one-half the radioactivity of the kidneys of the                                                  In some of the larger hamster and rat neo
control animals.                                                                              plasms, the tumor uptake of B^-Co60 was greater
   Urinary excretion during the first 1| days (be                                             than that of any other organ. It is possible that
fore flushing) was identical for both groups (an                                              depletion of vitamin Bi2 stores, with or without
average of 5.7 per cent). Following flushing, uri-                                            evidence of tissue depletion of this vitamin, may
846                                         Cancer Research

occur in such animals. In the human, widespread        radioactivity in normal rat liver, kidney, and
carcinomatosis has not been associated with the        spleen has also been described by Harte (5). As
usual evidence of vitamin BIZ deficiency. The          tissue radioactivity measurements do not neces
peripheral neuropathy reported with some cases         sarily measure the intact radiovitamin molecule,
of bronchogenic carcinoma (6, 9, 18) does not ap       these findings do not necessarily reflect the true
pear to be due to a deficiency of vitamin Bi2.         metabolic turnover of vitamin Bi2-Co60in these
   The flushing experiment was conducted to de         tissues.
termine to what extent the radiovitamin was                                      by
                                                           The uptake of Bi2-Co60 tumor tissue suggests
bound or altered 36 hours after injection. Since       that this vitamin may be important for the growth
large injections of vitamin Bi2 are largely excreted   of malignant tumors as well as for normal tissues.
in the urine (2, 14, 19), the appearance of radio      Further studies should help in clarifying the role
activity in the urine following such an injection      of vitamin Bw in the metabolism of malignant
indicates exchange between the previously admin        tumors.
istered radiovitamin and the nonradioactive
parenteral dose. The increase in kidney concentra              SUMMARY AND CONCLUSIONS
tion of radioactivity found in the "flushed" ani
mals is undoubtedly a reflection of this excretion,        1. The uptake of parenterally administered
                                                       vitamin B^-Co60 by the hamster methylcholan-
further evidenced by the increase in urinary radio
                                                        threne-induced sarcoma and the rat Walker car
activity. This increase in urinary radioactivity was
accompanied by a reduction in the radioactivity        cinosarcoma was studied.
                                                           2. Vitamin Bu-Co60 was taken up by both the
in the liver, Walker carcinosarcoma, and carcass
of the "flushed" animals to about hah' that of the     hamster sarcoma and the Walker carcinosarcoma.
                                                       The uptake of vitamin Bi2-Co60correlated well
control animals. This would indicate that at least
hah" of the administered radiovitamin was not          with tumor weight except for the heavier tumors.
firmly bound or materially altered 36 hours after          3. Large tumors were the major sites of vita
                                                       min Bis-Co60localization in some animals.
injection, since exchange between the parenterally
                                                           4. The turnover of radioactivity in the methyl-
administered vitamin and some radioactive inter
mediate seems unlikely. Approximately one-half         cholanthrene-induced sarcoma of the hamster, as
of the radioactivity in the carcass, liver, and Walk   well as in the kidneys, liver, and spleen, was neg
er carcinosarcoma was not exchangeable, indicat        ligible.
ing either firm "binding" or chemical alteration of        5. Large doses of nonradioactive vitamin BU
this portion of the injected radiovitamin. It is of    administered If days after the injection of vitamin
                                                       Biz-Co80were associated with: (a) a decrease in
interest that the radiovitamin taken up by the
Walker carcinosarcoma was exchangeable, which          liver, carcass, and Walker carcinosarcoma radio
observation suggested that this tumor is not an        activity; (6) an increase in kidney radioactivity;
absolute metabolic "trap" for vitamin Bi2 analo        and (c) an increase in urinary excretion of radio
gous to that suggested for amino acids and pro         activity.
teins (4, 13). The results of this "flushing" experi
ment differ from those of Harte et al. (5) in a simi                     ACKNOWLEDGMENTS
lar study on normal rats.                                 The authors wish to acknowledge the valuable technical
                                                       assistance of Mrs. Mary Lou Turner.
   Radioactivity as measured in this study merely
determines the presence of the Co60moiety of the
vitamin B^ molecule. Thus, the radioactivity may                            REFERENCES
be due to vitamin B^ and/or to an intermediate                      K                   B.
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