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Specific activity of radiovitamin B12 in organs and subcellular liver fractions after injection of 58Co-labelled vitamin B12
CLINICA CHIMICA ACTA
56
SPECIFIC
ACTIVITY
AND SUBCELLULAR
OF RADIOVITAMIN LIVER
OF 58Co-LABELLED R. GRP;SBECK,
R. IGNATIUS,
Department
B,, IN ORGANS
FRACTIONS VITAMIN
J. JARNEFELT,
ofMedical Chemistry,
AFTER
INJECTION
Blz*
H. LINDfiN
AND
A. MALI
University of Helsinki, Helsinki
AND
W. NYBERG Laboratory
Division,
Vasa Central Hospital,
(Received
Vasa (Finland)
July znd, 1960)
In clinical laboratories radioactive cyanocobalamin is widely used in the measurement of intestinal vitamin B,, absorption. However, during recent years there has been a growing tendency to employ it as a tracer for vitamin B,, contained in tissues and body fluids in states such as leukemia, liver disease, etc. The question then arises to what extent does the behavior of the radioactivity reflect that of the nonradioactive vitamin. In long-term experiments in which radiovitamin B,, is injected into man’, 2 or animalsap 4 it has usually been assumed that the labelled compound ultimately mixes uniformly with the nonradioactive tissue vitamin, and there is some circumstantial evidence that this is truest B. Moreover, it has been demonstrated that some, at least, of the radioactivity stored in the livers of such animals persits in the form of vitamin B,,‘. However, final proof of total mixing can only be obtained by demonstrating that the specific activity of the labelled vitamin becomes the same everywhere in the body. For this purpose, rats were injected with radiovitamin B,, and subsequently the ratio of radioactive to microbiological vitamin B,, activity was determined in various organs and subcellular liver fractions at different time intervals. With similar aims in mind, COOPERMAN et aZ.* determined the distribution of radioactive
and non-radioactive
vitamin
B,, in two dogs.
MATERIAL Twelve male rats of the Wistar strain, weighing 160-310 g, were given a subcutaneous injection of 40 mpg of %o-labelled cyanocobalamin in I ml of physiological saline. The radiovitamin was purchased from Philips-Duphar, the Netherlands, and had a specific activity of 18 mC/mg at the start of the experiment. The animals were kept on a normal stock diet throughout the experiment and housed in metabolic cages. Three rats at a time were killed at 3, IO, 17, and 31 days. For three days before killing, their excreta were collected three times daily. After killing, the organs were removed immediately, cleaned, weighed, and homogenized as described below. * Aided by grants from the Sigrid JusClius Foundation. Reported briefly in a discussion 7th European Congress of Haematology, London, September 7-12, 1959.
at the
SPECIFIC ACTIVITYOF RADIOVITAMIN B,,
57
METHODS Tissue preparation
The livers were homogenized in 0.25 M sucrose containing 0.1 M K,HPO,, in a Potter-Elvehjem type homogenizer with a Teflon pestle. The homogenates were fractionated essentially according to the technique described by SCHNEIDERANDHOGEBOOMS.The nuclei were separated by centrifuging at 700 x g, the mitochondria at 6,500 x g, and the microsomes at 105,000 x g, the two last-mentioned centrifugations being performed in the Spinco model L centrifuge. All the particulate fractions were washed once with 0.25 M sucrose containing phosphate buffer of pH 7.4 in a final concentration of 0.1 M, and finally suspended in this medium and diluted to a suitable volume. All operations were performed at o to 4’. The other organs were homogenized in distilled water. 7 ml aliquots of these and the unfractionated liver homogenates and the subcellular liver fractions were pipetted into test-tubes of equal size, fitting into a well-type scintillation crystal having the internal dimensions I g/16” x 11/16”. The remainder of each preparation was frozen and stored at -15’ until subjected to microbiological assay. The feces samples and the combined carcasses and skins were ashed in porcelain cups in a muffle furnace kept at 600’. The urines were first dried by evaporation on a steam bath and then treated in the same way. The ashes were packed into counting tubes and made up to 7-ml volume with finely ground rat food. Radioactivity
counting
The radioactivity of the samples was measured in an Ekco model N550 A scintillation counter having a model N597 2” x 2 5116” thallium-activated sodium iodide crystal. The counter was equipped with extra lead shielding which reduced the background considerably. When counted in this manner the samples having the lowest radioactivity gave a count about 1.5 x that of the background. For these, zo-min counting times were used. Each sample was counted twice, and the mean count used in calculating the result. Standards containing radioactive vitamin in carrier solution were counted after every 10th sample. The radioactivity of the samples was converted into micrograms of radiovitamin B,,. Microbiological
assay
The tissue suspensions were diluted with distilled water to contain between 0.1 and 0.02 g of tissue per ml. To one volume of the dilution was added one volume of papain (Merck), suspended in 0.1 M sodium acetate buffer containing a trace of sodium cyanide and with a pH of 4.6. The amount of papain used was 30 mg per g of tissue. The mixture was incubated for three hours at 57”, half of the enzyme suspension being added at the beginning and the remainder half-way through the incubation period. The digest was then adjusted to the original volume with distilled water, and finally appropriately diluted and assayed for vitamin B,, after heating in steam for 30 min lo. The microbiological vitamin B,, assay was performed by the method of HUTNER et al.ll without modification, Euglena gracilis of the z strain being employed. Papain suspensions in the dilutions used in the digestions had no effect on the growth of the assay organism. Cl&. Chim.
Acta,
6 (1961)
56-62
R. GRiiSBECK et al.
58
RESULTS
The data for the distribution of microbiological activity are given in Table I. In this table all the rats are treated as one group, though the animals killed soon after the TARLE MEAN
MICROBIOLOGICAL
(ranges
Kidneys
ACTIVITY
tissue
1137 (483-1690) 202.3
Liver
(115.0-391.0)
Intestine Spleen
42.7 (21.3-63.5)
Testes
36.7 (14.4-54.0) 28.8 (‘0.1-44.0)
Lungs
.
Psoas muscle (piece) _~ _____~
2064 (900-3100) 1438 100.9 (52.5-138.5) ‘I54 (466-2642) 34.4 (13.2-63.0) 74.3 (38.9-112.5) 35.2 (17.2-52.2)
17.3 (6.1-27.0)
__~
Kidneys
mpg zn whole organ
(575-2972)
150.6 (87.5-277.0) 111.2 (35.6254.0)
Heart
OF RAT ORGANS
in parentheses)
mpg lg
OYpZ
I
B,,
VITAMIN
_
1
\
100
\ \ \ Lungs
Liver Psocs Heart
Days
after
injection
of
rodiovitomin
8,~
Fig. I. The specific activity in organs at different times following the injection B,,. Each point represents the average of three observations.
of radiovitamin
injection may not be strictly comparable to those killed later. The lowest concentration of microbiological activity was found in the muscle. If the mean concentration in muscle is denoted with the figure I, the corresponding figures for the other organs are: lungs 1.7, testes 2.1, spleen 2.5, stomach and intestine 6.4, heart 8.7, liver 11.7, kidneys 65.7. Of the microbiological activity contained in the unfractionated liver C/in.Chim. Acta,
G (1961)
jh-61
SPECIFIC ACTIVITY OF RADIOVITAMIN
B,,
59
homogenate an average of 71% was recovered in the washed subcellular particles and the supernatant liquid remaining after the sedimentation of the particulate elements. In the subcellular fractions the microbiological activity had the following average distribution: microsomes 2.3%, mitochondria 14.6%, supernatant liquid 15.6%, nuclei and debris 67.5%. The relatively high activity found in the nuclear fraction is best explained on the basis of incomplete rupture of cells. The ratios of radioactivity to microbiological activity “B,,*/B,, ratio”, are given in Table II. Figs. I and 2 describe this ratio as a function of time. For the sake of simplicity the B,,*/B,, ratio is used to express the specific activity; the conventional units, mC/mg, extrapolated to the time of the injection, may be obtained by multiplying by the specific activity of the injected vitamin. TABLE II THE
B,,* IB,,
RATIO
( X
Id)
AT DIFFERENT
TIMES
AFTER
THE
INJECTION
OF RADIOVITAMIN
B,,
Mean values for three rats (ranges in parentheses) Days after injection of radiovitamin B,, _____ Kidneys
Organs
3
10
(55-180) Lungs
17
31
26 (22-28)
108
(67%2)
27 (20-35)
23 (11-33)
(16?zh)
(52%
(245-594)
(24%
(rG3)
Spleen
(,7,,
18 (‘4-22)
(334-047)
(27%
(2z531
(&o)
25 (20-29)
25 (11-43)
Intestine (&O)
(&:6)
Testes (3Z54) Liver (193-‘2, Psoas muscle (17%
(4%7)
Heart (1527) Liver fractions Supernatant
(13:5,
(r4::8) 18 (15-22)
37 (11-82)
(*&;I,
Microsomes (&o) Nuclei and debris
68 (28-106)
Mitochondria (2830,
In the rats killed three days after the injection, the specific activities of the tissues and the subcellular fractions varied over a considerable range, being highest in the kidneys, lungs, and spleen, and among the liver fractions, in the supernatant liquid remaining when the particulate elements had been removed by centrifugation. Later the variation in the B,,*/B,, ratio tended to decrease. At 17 and 31 days there was no clearcut difference in the specific activities of different organs, or in those of the liver fractions if compared with each other. Clin. Chim. Acta, 6 (1961) 56-62
R. GRiiSBECK et al.
60
Since the B,,*/B,, ratio tended to become equal in different parts of the body, it was suspected that the slight differences observed in the specific activities could be explained on the basis of normal errors of observation. Therefore, the data for the rats killed at 17 and 31 days were subjected to statistical analysis. The statistical method was two-dimensional variance analysis, one argument being the variation from one organ to another. The possible disturbance caused by the specific activity attaining a different value in different rats was eliminated by taking the variation from one rat to another as the second argument. At 17 days the specific activities of
Supernatant
Microsomes Nuclei L debris Mitochondria
Days oftcr
injection
of radiovitamin
Bt2
Fig. z. The specific activity in subcellular liver fractions following the injection of radiovitamin B,,. Each point represents the average of three observations.
TABLE MEAN
RADIOACTIVE
III
AND MICROBIOLOGICAL VITAMIN B,, ACTIVITIESIN TISSUES AND EXCRETA (ranges in parentheses) Days after injection 3
Radioactivity
IO
of radiovitamin B,, =7
31
11.4
11.1 (9.6-12.4)
fm,ug)
A. Organs
14.8
19.2
(15.5-22.3) IO.1 (9.2-10.9)
B. Carcasses C. Total body (A + B)
29.3 (25.7-31.6)
(12.6-16.1)
(9.5-12.5)
8.9 (7.4-10.4) 23.6 (21.5-26.5)
7.3 (6.3-7.9) 18.8
0.25 (0.20-0.30)
3.5% (2.9-4.0)
0.5 (0.5-0.5) 2.5% (2.3-2.7)
4.9 (3.2-6.7) -
5.9 (5.7-6.2) -
5.4 (4.0-6.3) 8.2
D. Daily excretion*
1.9 (1.7-2.0)
0.8 (OS0.9)
E. D as % of C
6.5% (5.5-7.5) 3.6 (2.2-4.9)
Microbiological
activzty (yg)
F. Organs G. Total body
(17.4-20.3)
1.5% (1.0-1.7)
(5.8-10.1)
(A + B) x F A I [ H. Estimated daily excretion (E x G) * The radioactivity
5.5 (5.1-5.8) 16.6 (15.4-18.2)
-
in the three-day
-
-
0.11 (0.10-0.14)
colleckion of excreta divided by 3. Cl&. Chim. Acta, 6 (1~61) 56-62
SPECIFIC ACTIVITY
the different
B,,
OF RADIOVITAMIN
organs still differed from each other significantly
at 31 days no significant
difference
could be detected
61 at the 97.5%
[F(7,13)
= 0.761.
level, but When
the
liver fractions were compared with each other no significant differences in the specific activities could be detected in rats killed at 17 and 31 days [F(3,6) = 1.9 at 17 days, and = 2.5 at 31 days]. The radioactivity found in the organs, carcasses, and three-day collections of excreta is reported in Table III. In calculating the percentage of the total body radioactivity excreted per day during the period when excreta were collected (line E in the table) the assumption was made that at the time when the excretion occurred the total body radioactivity was the same as that measured after the killing of the rat. The mathematical error caused by this simplification is negligible, at least in the rats killed at 31 days. The table demonstrates that the percentage of the total body radioactivity excreted per day decreases with time. This is apparently a phenomenon related to the mixing of the isotope with the tissue vitamin. In the rats killed at 31 days this value figure correvaried between 1.0 and 1.7% with a mean of 1.50/b. The last-mentioned sponds to a biological half-life of about 46 days. Since there is evidence of total mixing of the isotope with the body vitamin B,, pool, the value 1.5%/day should represent a fairly good estimate of the turnover rate of this pool. An estimate of the corresponding loss of microbiological activity is therefore included in the table. The microbiological vitamin B, 2 activity contained in the part of the body which was not assayed, was estimated by assuming that the distribution was the same as that of the radioactive vitamin. The calculations indicate that the rats lost an average of 0.11 pug of microbiological vitamin B,, activity per day. DISCUSSION
The distribution of microbiological activity observed in the present series resembles that described by othersr21 13, and the same applies to the subcellular distribution in the liver14. However, it is evident that the vitamin B,, level in the tissues is subject to considerable individual variation. The fact that after a sufficient lapse of time injected radiovitamin B,, appears to have mixed uniformly with the microbiological vitamin B,, activity of the tissues, indicates that radiovitamin B,, behaves as a true tracer. It should perhaps be mentioned that the results of a preliminary series consisting of IO rats are in complete agreement with the present data, but owing to the low specific activity (I mC/mg) of the injected vitamin, the methodological variations were too great to permit determination of the time required for total mixing with any accuracy. The fact that total mixing appears to occur should be a welcome observation to those who have tried to study the behavior of vitamin B,, in the tissues with the radioisotope technique. However, the time required for equilibration is relatively long. Also, the reservation must be made that in the present study the number of organs and tissues examined was limited, and that if the experimental error could have been diminished, slight differences in the specific activities might have been detected. Our present results agree with those obtained by COOPERMANet al.8 in studies on dogs, though in that species the time required for equilibration appears to be 14 days or less. This slight discrepancy may naturally be due to a species difference, but also Clin. Chim.
Ada,
6 (1961)
gh-1.2
62
R. GR;iSBECK Etd.
to our more rigid, statistical criteria of complete mixing. In view of the finding of complete mixing in two species, the dog and the rat, there is no reason to doubt that mixing occurs in man, though the time required for equilibration still remains to be determined. The calculated mean daily loss of vitamin B,, in the rats, 0.11 ,ug/day, is of the same order of magnitude as the daily requirement, 0.125 fug/day, observed by EMERSONI5 in thyroid-fed growing rats weighing about ISO g. I’c is interesting to note that vitamin B,, app ears to be a very stable compound in the body. At least the cobalt atom seems to be continuously associated with microbiological vita.min B,, activity, and in the event of its dissociation, it must be rapidly excreted. Since EzGglena is relatively selective in its vitamin B,, requirement 16, the radiocobalt introduced in the form of vitamin B,, appears to be still associated with a corphinamide (Factor B)-like structure carrying a nucleotide.
The ratio of radioactive to microbiological vitamin B,, activity was determined in organs and subcellular liver fractions of rats which had been given a subcutaneous injection of 5sC,o-labeIled vitamin B,,. In the rats killed 31 days after the injection there was no statistical difference between these ratios in different parts of the body; but in the animals killed earlier such a difference could be detected. This study establishes that radiocobalt-labelled vitamin B,, may be used as a physiological tracer for tissue vitamin B,,, but that a sufficient equilibration time is needed before the labelled compound mixes uniformly with the body stores of vitamin B,,. Data are given for the distribution of microbiological vitamin B,, activity in rat organs and subcellular liver fractions. The turnover of vitamin B,, in the rat appears to be of the order of 0.11 pglday. REFERENCES 1 R, GR~SBZCI-Z, W. NYBERG AND P. REIZENSTEIN, PYOC. Sac. Exptl. BioE. Med., 97 (1955) 780. 2 L. M. MEYER, N. I. BERLIN, M. JIMINEZ-CASADO AND S. N. ARKUN, Proc. SW. ExptE. Sol. Med., 9’ (1956) 129. 2 C. A. LANG, D. M. GLEYSTEEN AND B. F. CHOW, J. Nutrilion, 50 (1953) 213. 4 W. D. WOODS, W. B. HAWKINS AND G. H. WHIPPLE, J. Exptl. Med., 108 (1958) I. 6 R. GRKSBECK, &and. J. Clin. & Lab. Invest., II (1959) 250. fl P. G. REIZENSTEIN, Acta Med. Scasd., 165 (1959) 467. 7 G. 3. J. GLASS, Haematol. katinn,2 (1959) 279. * J. M. COOPERMAN, A. L. LUHBY, D. ?iT.TELLER AND J. F. MARLEY, J. Riot. Chmz., 235 (rg60)
sg1. Q W. C. SCHNEIDER AND G. H. HOGEBOOBI, J. Biol. Cketiz., 183 (1950) 123. 10 M. E. GREGORY AND E. S. HOLDSWORTH, Biochcm. J., 59 (1955) 335. 11 S. H. HUTNER, M. K. BACH AND G. I. M. Ross, I. Prolozool., 3 (1956) IOT. 12 U. J. LEWIS, W. D. REGISTER AND C. -4. ELVEHJEM, Proc. Sot. Exptl. Viol. Med., 71 (1949) 509. 13 R. WOLFF in H. C. HEINRICH (Ed.), Vitamin B,, und Intrinsic Factor, Ferdinand Enke Verlag, Stuttgart, 1957. p. 519. 14M. RACHMILEWITZ, Y. STEIN, 0. STEIN, J. ARONOVITCH AND N. GROSSOWIC~, H~e~~a~o~. Latin% 2 (‘959) 297. 15 G. A. EMERSON, Proc. Sac. Exptl. Biol. Med., 70 (19.49) 392. 18M. E. COATES AND S. K. KON in 1-i. C. HEINRICH (Ed.), Vitamin B,, und Intrinsic nand Enke Verlag, Stuttgart, 1957, p. 72
Factor, Ferdi-
C&n. China. Acta, 6 (1961) 56-62
56
SPECIFIC
ACTIVITY
AND SUBCELLULAR
OF RADIOVITAMIN LIVER
OF 58Co-LABELLED R. GRP;SBECK,
R. IGNATIUS,
Department
B,, IN ORGANS
FRACTIONS VITAMIN
J. JARNEFELT,
ofMedical Chemistry,
AFTER
INJECTION
Blz*
H. LINDfiN
AND
A. MALI
University of Helsinki, Helsinki
AND
W. NYBERG Laboratory
Division,
Vasa Central Hospital,
(Received
Vasa (Finland)
July znd, 1960)
In clinical laboratories radioactive cyanocobalamin is widely used in the measurement of intestinal vitamin B,, absorption. However, during recent years there has been a growing tendency to employ it as a tracer for vitamin B,, contained in tissues and body fluids in states such as leukemia, liver disease, etc. The question then arises to what extent does the behavior of the radioactivity reflect that of the nonradioactive vitamin. In long-term experiments in which radiovitamin B,, is injected into man’, 2 or animalsap 4 it has usually been assumed that the labelled compound ultimately mixes uniformly with the nonradioactive tissue vitamin, and there is some circumstantial evidence that this is truest B. Moreover, it has been demonstrated that some, at least, of the radioactivity stored in the livers of such animals persits in the form of vitamin B,,‘. However, final proof of total mixing can only be obtained by demonstrating that the specific activity of the labelled vitamin becomes the same everywhere in the body. For this purpose, rats were injected with radiovitamin B,, and subsequently the ratio of radioactive to microbiological vitamin B,, activity was determined in various organs and subcellular liver fractions at different time intervals. With similar aims in mind, COOPERMAN et aZ.* determined the distribution of radioactive
and non-radioactive
vitamin
B,, in two dogs.
MATERIAL Twelve male rats of the Wistar strain, weighing 160-310 g, were given a subcutaneous injection of 40 mpg of %o-labelled cyanocobalamin in I ml of physiological saline. The radiovitamin was purchased from Philips-Duphar, the Netherlands, and had a specific activity of 18 mC/mg at the start of the experiment. The animals were kept on a normal stock diet throughout the experiment and housed in metabolic cages. Three rats at a time were killed at 3, IO, 17, and 31 days. For three days before killing, their excreta were collected three times daily. After killing, the organs were removed immediately, cleaned, weighed, and homogenized as described below. * Aided by grants from the Sigrid JusClius Foundation. Reported briefly in a discussion 7th European Congress of Haematology, London, September 7-12, 1959.
at the
SPECIFIC ACTIVITYOF RADIOVITAMIN B,,
57
METHODS Tissue preparation
The livers were homogenized in 0.25 M sucrose containing 0.1 M K,HPO,, in a Potter-Elvehjem type homogenizer with a Teflon pestle. The homogenates were fractionated essentially according to the technique described by SCHNEIDERANDHOGEBOOMS.The nuclei were separated by centrifuging at 700 x g, the mitochondria at 6,500 x g, and the microsomes at 105,000 x g, the two last-mentioned centrifugations being performed in the Spinco model L centrifuge. All the particulate fractions were washed once with 0.25 M sucrose containing phosphate buffer of pH 7.4 in a final concentration of 0.1 M, and finally suspended in this medium and diluted to a suitable volume. All operations were performed at o to 4’. The other organs were homogenized in distilled water. 7 ml aliquots of these and the unfractionated liver homogenates and the subcellular liver fractions were pipetted into test-tubes of equal size, fitting into a well-type scintillation crystal having the internal dimensions I g/16” x 11/16”. The remainder of each preparation was frozen and stored at -15’ until subjected to microbiological assay. The feces samples and the combined carcasses and skins were ashed in porcelain cups in a muffle furnace kept at 600’. The urines were first dried by evaporation on a steam bath and then treated in the same way. The ashes were packed into counting tubes and made up to 7-ml volume with finely ground rat food. Radioactivity
counting
The radioactivity of the samples was measured in an Ekco model N550 A scintillation counter having a model N597 2” x 2 5116” thallium-activated sodium iodide crystal. The counter was equipped with extra lead shielding which reduced the background considerably. When counted in this manner the samples having the lowest radioactivity gave a count about 1.5 x that of the background. For these, zo-min counting times were used. Each sample was counted twice, and the mean count used in calculating the result. Standards containing radioactive vitamin in carrier solution were counted after every 10th sample. The radioactivity of the samples was converted into micrograms of radiovitamin B,,. Microbiological
assay
The tissue suspensions were diluted with distilled water to contain between 0.1 and 0.02 g of tissue per ml. To one volume of the dilution was added one volume of papain (Merck), suspended in 0.1 M sodium acetate buffer containing a trace of sodium cyanide and with a pH of 4.6. The amount of papain used was 30 mg per g of tissue. The mixture was incubated for three hours at 57”, half of the enzyme suspension being added at the beginning and the remainder half-way through the incubation period. The digest was then adjusted to the original volume with distilled water, and finally appropriately diluted and assayed for vitamin B,, after heating in steam for 30 min lo. The microbiological vitamin B,, assay was performed by the method of HUTNER et al.ll without modification, Euglena gracilis of the z strain being employed. Papain suspensions in the dilutions used in the digestions had no effect on the growth of the assay organism. Cl&. Chim.
Acta,
6 (1961)
56-62
R. GRiiSBECK et al.
58
RESULTS
The data for the distribution of microbiological activity are given in Table I. In this table all the rats are treated as one group, though the animals killed soon after the TARLE MEAN
MICROBIOLOGICAL
(ranges
Kidneys
ACTIVITY
tissue
1137 (483-1690) 202.3
Liver
(115.0-391.0)
Intestine Spleen
42.7 (21.3-63.5)
Testes
36.7 (14.4-54.0) 28.8 (‘0.1-44.0)
Lungs
.
Psoas muscle (piece) _~ _____~
2064 (900-3100) 1438 100.9 (52.5-138.5) ‘I54 (466-2642) 34.4 (13.2-63.0) 74.3 (38.9-112.5) 35.2 (17.2-52.2)
17.3 (6.1-27.0)
__~
Kidneys
mpg zn whole organ
(575-2972)
150.6 (87.5-277.0) 111.2 (35.6254.0)
Heart
OF RAT ORGANS
in parentheses)
mpg lg
OYpZ
I
B,,
VITAMIN
_
1
\
100
\ \ \ Lungs
Liver Psocs Heart
Days
after
injection
of
rodiovitomin
8,~
Fig. I. The specific activity in organs at different times following the injection B,,. Each point represents the average of three observations.
of radiovitamin
injection may not be strictly comparable to those killed later. The lowest concentration of microbiological activity was found in the muscle. If the mean concentration in muscle is denoted with the figure I, the corresponding figures for the other organs are: lungs 1.7, testes 2.1, spleen 2.5, stomach and intestine 6.4, heart 8.7, liver 11.7, kidneys 65.7. Of the microbiological activity contained in the unfractionated liver C/in.Chim. Acta,
G (1961)
jh-61
SPECIFIC ACTIVITY OF RADIOVITAMIN
B,,
59
homogenate an average of 71% was recovered in the washed subcellular particles and the supernatant liquid remaining after the sedimentation of the particulate elements. In the subcellular fractions the microbiological activity had the following average distribution: microsomes 2.3%, mitochondria 14.6%, supernatant liquid 15.6%, nuclei and debris 67.5%. The relatively high activity found in the nuclear fraction is best explained on the basis of incomplete rupture of cells. The ratios of radioactivity to microbiological activity “B,,*/B,, ratio”, are given in Table II. Figs. I and 2 describe this ratio as a function of time. For the sake of simplicity the B,,*/B,, ratio is used to express the specific activity; the conventional units, mC/mg, extrapolated to the time of the injection, may be obtained by multiplying by the specific activity of the injected vitamin. TABLE II THE
B,,* IB,,
RATIO
( X
Id)
AT DIFFERENT
TIMES
AFTER
THE
INJECTION
OF RADIOVITAMIN
B,,
Mean values for three rats (ranges in parentheses) Days after injection of radiovitamin B,, _____ Kidneys
Organs
3
10
(55-180) Lungs
17
31
26 (22-28)
108
(67%2)
27 (20-35)
23 (11-33)
(16?zh)
(52%
(245-594)
(24%
(rG3)
Spleen
(,7,,
18 (‘4-22)
(334-047)
(27%
(2z531
(&o)
25 (20-29)
25 (11-43)
Intestine (&O)
(&:6)
Testes (3Z54) Liver (193-‘2, Psoas muscle (17%
(4%7)
Heart (1527) Liver fractions Supernatant
(13:5,
(r4::8) 18 (15-22)
37 (11-82)
(*&;I,
Microsomes (&o) Nuclei and debris
68 (28-106)
Mitochondria (2830,
In the rats killed three days after the injection, the specific activities of the tissues and the subcellular fractions varied over a considerable range, being highest in the kidneys, lungs, and spleen, and among the liver fractions, in the supernatant liquid remaining when the particulate elements had been removed by centrifugation. Later the variation in the B,,*/B,, ratio tended to decrease. At 17 and 31 days there was no clearcut difference in the specific activities of different organs, or in those of the liver fractions if compared with each other. Clin. Chim. Acta, 6 (1961) 56-62
R. GRiiSBECK et al.
60
Since the B,,*/B,, ratio tended to become equal in different parts of the body, it was suspected that the slight differences observed in the specific activities could be explained on the basis of normal errors of observation. Therefore, the data for the rats killed at 17 and 31 days were subjected to statistical analysis. The statistical method was two-dimensional variance analysis, one argument being the variation from one organ to another. The possible disturbance caused by the specific activity attaining a different value in different rats was eliminated by taking the variation from one rat to another as the second argument. At 17 days the specific activities of
Supernatant
Microsomes Nuclei L debris Mitochondria
Days oftcr
injection
of radiovitamin
Bt2
Fig. z. The specific activity in subcellular liver fractions following the injection of radiovitamin B,,. Each point represents the average of three observations.
TABLE MEAN
RADIOACTIVE
III
AND MICROBIOLOGICAL VITAMIN B,, ACTIVITIESIN TISSUES AND EXCRETA (ranges in parentheses) Days after injection 3
Radioactivity
IO
of radiovitamin B,, =7
31
11.4
11.1 (9.6-12.4)
fm,ug)
A. Organs
14.8
19.2
(15.5-22.3) IO.1 (9.2-10.9)
B. Carcasses C. Total body (A + B)
29.3 (25.7-31.6)
(12.6-16.1)
(9.5-12.5)
8.9 (7.4-10.4) 23.6 (21.5-26.5)
7.3 (6.3-7.9) 18.8
0.25 (0.20-0.30)
3.5% (2.9-4.0)
0.5 (0.5-0.5) 2.5% (2.3-2.7)
4.9 (3.2-6.7) -
5.9 (5.7-6.2) -
5.4 (4.0-6.3) 8.2
D. Daily excretion*
1.9 (1.7-2.0)
0.8 (OS0.9)
E. D as % of C
6.5% (5.5-7.5) 3.6 (2.2-4.9)
Microbiological
activzty (yg)
F. Organs G. Total body
(17.4-20.3)
1.5% (1.0-1.7)
(5.8-10.1)
(A + B) x F A I [ H. Estimated daily excretion (E x G) * The radioactivity
5.5 (5.1-5.8) 16.6 (15.4-18.2)
-
in the three-day
-
-
0.11 (0.10-0.14)
colleckion of excreta divided by 3. Cl&. Chim. Acta, 6 (1~61) 56-62
SPECIFIC ACTIVITY
the different
B,,
OF RADIOVITAMIN
organs still differed from each other significantly
at 31 days no significant
difference
could be detected
61 at the 97.5%
[F(7,13)
= 0.761.
level, but When
the
liver fractions were compared with each other no significant differences in the specific activities could be detected in rats killed at 17 and 31 days [F(3,6) = 1.9 at 17 days, and = 2.5 at 31 days]. The radioactivity found in the organs, carcasses, and three-day collections of excreta is reported in Table III. In calculating the percentage of the total body radioactivity excreted per day during the period when excreta were collected (line E in the table) the assumption was made that at the time when the excretion occurred the total body radioactivity was the same as that measured after the killing of the rat. The mathematical error caused by this simplification is negligible, at least in the rats killed at 31 days. The table demonstrates that the percentage of the total body radioactivity excreted per day decreases with time. This is apparently a phenomenon related to the mixing of the isotope with the tissue vitamin. In the rats killed at 31 days this value figure correvaried between 1.0 and 1.7% with a mean of 1.50/b. The last-mentioned sponds to a biological half-life of about 46 days. Since there is evidence of total mixing of the isotope with the body vitamin B,, pool, the value 1.5%/day should represent a fairly good estimate of the turnover rate of this pool. An estimate of the corresponding loss of microbiological activity is therefore included in the table. The microbiological vitamin B, 2 activity contained in the part of the body which was not assayed, was estimated by assuming that the distribution was the same as that of the radioactive vitamin. The calculations indicate that the rats lost an average of 0.11 pug of microbiological vitamin B,, activity per day. DISCUSSION
The distribution of microbiological activity observed in the present series resembles that described by othersr21 13, and the same applies to the subcellular distribution in the liver14. However, it is evident that the vitamin B,, level in the tissues is subject to considerable individual variation. The fact that after a sufficient lapse of time injected radiovitamin B,, appears to have mixed uniformly with the microbiological vitamin B,, activity of the tissues, indicates that radiovitamin B,, behaves as a true tracer. It should perhaps be mentioned that the results of a preliminary series consisting of IO rats are in complete agreement with the present data, but owing to the low specific activity (I mC/mg) of the injected vitamin, the methodological variations were too great to permit determination of the time required for total mixing with any accuracy. The fact that total mixing appears to occur should be a welcome observation to those who have tried to study the behavior of vitamin B,, in the tissues with the radioisotope technique. However, the time required for equilibration is relatively long. Also, the reservation must be made that in the present study the number of organs and tissues examined was limited, and that if the experimental error could have been diminished, slight differences in the specific activities might have been detected. Our present results agree with those obtained by COOPERMANet al.8 in studies on dogs, though in that species the time required for equilibration appears to be 14 days or less. This slight discrepancy may naturally be due to a species difference, but also Clin. Chim.
Ada,
6 (1961)
gh-1.2
62
R. GR;iSBECK Etd.
to our more rigid, statistical criteria of complete mixing. In view of the finding of complete mixing in two species, the dog and the rat, there is no reason to doubt that mixing occurs in man, though the time required for equilibration still remains to be determined. The calculated mean daily loss of vitamin B,, in the rats, 0.11 ,ug/day, is of the same order of magnitude as the daily requirement, 0.125 fug/day, observed by EMERSONI5 in thyroid-fed growing rats weighing about ISO g. I’c is interesting to note that vitamin B,, app ears to be a very stable compound in the body. At least the cobalt atom seems to be continuously associated with microbiological vita.min B,, activity, and in the event of its dissociation, it must be rapidly excreted. Since EzGglena is relatively selective in its vitamin B,, requirement 16, the radiocobalt introduced in the form of vitamin B,, appears to be still associated with a corphinamide (Factor B)-like structure carrying a nucleotide.
The ratio of radioactive to microbiological vitamin B,, activity was determined in organs and subcellular liver fractions of rats which had been given a subcutaneous injection of 5sC,o-labeIled vitamin B,,. In the rats killed 31 days after the injection there was no statistical difference between these ratios in different parts of the body; but in the animals killed earlier such a difference could be detected. This study establishes that radiocobalt-labelled vitamin B,, may be used as a physiological tracer for tissue vitamin B,,, but that a sufficient equilibration time is needed before the labelled compound mixes uniformly with the body stores of vitamin B,,. Data are given for the distribution of microbiological vitamin B,, activity in rat organs and subcellular liver fractions. The turnover of vitamin B,, in the rat appears to be of the order of 0.11 pglday. REFERENCES 1 R, GR~SBZCI-Z, W. NYBERG AND P. REIZENSTEIN, PYOC. Sac. Exptl. BioE. Med., 97 (1955) 780. 2 L. M. MEYER, N. I. BERLIN, M. JIMINEZ-CASADO AND S. N. ARKUN, Proc. SW. ExptE. Sol. Med., 9’ (1956) 129. 2 C. A. LANG, D. M. GLEYSTEEN AND B. F. CHOW, J. Nutrilion, 50 (1953) 213. 4 W. D. WOODS, W. B. HAWKINS AND G. H. WHIPPLE, J. Exptl. Med., 108 (1958) I. 6 R. GRKSBECK, &and. J. Clin. & Lab. Invest., II (1959) 250. fl P. G. REIZENSTEIN, Acta Med. Scasd., 165 (1959) 467. 7 G. 3. J. GLASS, Haematol. katinn,2 (1959) 279. * J. M. COOPERMAN, A. L. LUHBY, D. ?iT.TELLER AND J. F. MARLEY, J. Riot. Chmz., 235 (rg60)
sg1. Q W. C. SCHNEIDER AND G. H. HOGEBOOBI, J. Biol. Cketiz., 183 (1950) 123. 10 M. E. GREGORY AND E. S. HOLDSWORTH, Biochcm. J., 59 (1955) 335. 11 S. H. HUTNER, M. K. BACH AND G. I. M. Ross, I. Prolozool., 3 (1956) IOT. 12 U. J. LEWIS, W. D. REGISTER AND C. -4. ELVEHJEM, Proc. Sot. Exptl. Viol. Med., 71 (1949) 509. 13 R. WOLFF in H. C. HEINRICH (Ed.), Vitamin B,, und Intrinsic Factor, Ferdinand Enke Verlag, Stuttgart, 1957. p. 519. 14M. RACHMILEWITZ, Y. STEIN, 0. STEIN, J. ARONOVITCH AND N. GROSSOWIC~, H~e~~a~o~. Latin% 2 (‘959) 297. 15 G. A. EMERSON, Proc. Sac. Exptl. Biol. Med., 70 (19.49) 392. 18M. E. COATES AND S. K. KON in 1-i. C. HEINRICH (Ed.), Vitamin B,, und Intrinsic nand Enke Verlag, Stuttgart, 1957, p. 72
Factor, Ferdi-
C&n. China. Acta, 6 (1961) 56-62