Vitamin B12 Binding Proteins in Bovine Serum 1 D. M. POLAK, J. M. ELLIOT 2, and M. H A L U S K A Department of Animal Science Cornell University Ithaca, N ¥ 14853
ABSTRACT
Examination of bovine serum by the diethylaminoethyl cellulose small column method revealed three proteins binding vitamin B12. The elution pattern suggested that they are similar to the three transcobalamins recognized in human serum. Distribution of u n b o u n d binding capacity among serum binders was assessed in serum from normal, ketotic, and B12- and Factor B-supplemented cows in early lactation. No major differences were observed among groups; however, cow serum displayed a pattern different from human serum. Mean total binding capacity of bovine serum for B12 as well as mean u n b o u n d binding capacity were lower than the corresponding means for human serum. INTRODUCTION
Although proteins binding vitamin B12 in serum have been studied extensively in the human (3, 4, 6, 9, 10, 11, 13, 16), little attention has been given to the ruminant. Three binders have been identified in normal human serum by DEAE-cellulose column chromatography (6, 10). These have been designated transcobalamin (TC) I, TC II, and main protein peak binder (MPPB) or TC III. The individual roles of these binders still are not elucidated completely, but a large percentage of the endogenous vitamin B12 in human serum appears to be carried by TC I (4, 9, 11). Tan and Blaisdell (18)examined the substances binding B12 in serum of 19 vertebrates including human and bovine. The methods detected one binder in cow serum, but since they did not separate the three binders usually detected in
Received July 19, 1978. 1Supported in part by a grant from Agway, Inc. 2Address reprint requests to this author. 1979 J Dairy Sci 62:697-701
human serum, a meaningful comparison of the cow and the human was impossible. Our study was to determine whether the cow, like the human, has three binders of vitamin B12 in serum and to examine the binding capacity for B1 ~ of cow sera. In view of our interest in the vitamin B12 status of the ketotic cow, samples from cows in this and other physiological states were examined. MATERIALS AND METHODS
Samples of serum, prepared from venous blood and stored frozen, were available from normal cows at approximately 8 wk postpartum, ketotic cows in early lactation, cows at 8 wk postpartum that had been injected (i.m.) with supplementary vitamin B12 (20 mg/wk) for 12 wk, and cows at 8 wk postpartum that had been injected (i.m.) with Factor B (35 or 175 mg/wk) for 8 wk. Factor B (cobinamide) is one of the analogues of vitamin B 12- All cows were Holsteins from the Cornell University herd. Samples of human blood were from healthy individuals working in our laboratory. The small DEAE-cellulose column method (7) was used to separate the B12-binding proteins. The DEAE-cellulose (Cellex D, .85 meq/g, Bio-Rad Laboratories)was packed, after preparation, into .6 × 25 cm columns. The [s 7Co ] B12 (15 uCi/ug, Amersham/Searle, Arlington Heights, IL) was diluted to contain 3.33 ng/ml. To .5 ml of serum, 1.0 ml of labeled B12 solution was added. The mixture was allowed to stand at 37 C for 1 h. Albumincoated charcoal (2 ml) then was added to remove u n b o u n d B12. The tube was centrifuged, and the radioactivity in the supernatant was counted in a well-type counter (Nuclear Chicago). The supernatant subsequently was applied to a DEAE cellulose column. Any free B12 was eluted with .175 M sodium phosphate buffer, pH 6.3. The following sodium phosphate buffers then were used in sequence: .04 M, pH 5.9; .1 M, pH 5.8; and ~4 M, pH 5.2. Quantities were usually 20, 36, 20, and 14 ml, 697
698
POLAK ET AL.
respectively, b u t occasionally varied as experience determined what was necessary to achieve dear separation of peaks. Buffer solutions were made up in distilled water containing .02% NAN3. Flow rate was 6 to 10 ml per h, and all chromatography was at 4 C. An automatic fractionator allowed collection of eluate in approximately 2 ml fractions. Radioactivity of these fractions was counted, and protein was estimated by absorbance at 280 nm. Extracts of serum were assayed for vitamin B12 with both Ochromonas malbamensis (5) and a nonspecific radiodilution method (1) which employs toadfish serum as a binder. Extracts were prepared at pH 5.0 by steaming a mixture of 5.0 ml of serum, .5 ml of fresh KCN solution (40 mg/100 ml), and approximately 5 ml redistilled water in an autoclave at atmospheric pressure for 30 min. After cooling, the samples were brought to volume (25 ml), well mixed, and centrifuged to remove the protein. The supernatant was stored frozen until assayed. Vitamin B12 u n b o u n d binding capacity (UBBC) was measured by the rapid charcoal method (8) with modifications. The [S TCo] B12 was diluted with unlabeled cyanocobalamin to contain 6.06 ng/ml. One drop of KCN solution (2 mg/ml) was added to each bottle (50 ml) of standardized labeled B12 solution. In a 10-ml test tube, .1 ml of serum and 1.0 ml of the B x2 solution were allowed to react for 24 h at 4 C. At this temperature, exchange with endogenous bound B12 is avoided, and saturation, at least with human plasma, is achieved in about 4 h (2). Preliminary studies suggested a similar time for cow serum with no subsequent change over 3 days. Therefore, a 24-h incubation was used for convenience. After 24 h, 2 ml of albumin-coated charcoal were added to each tube which, after thorough mixing, was centrifuged. The radioactivity in the supernatant was counted to permit calculation of u n b o u n d binding capacity (8). Total binding capacity was calculated as the sum of UBBC and vitamin B12. Comparisons among physiological states and between the bovine and the human were made by analysis of variance for unbalanced data by methods outlined by Searle (15). RESULTS AND DISCUSSION
Bovine
serum
contained
three
vitamin
Journal of Dairy Science VoL 62, No. 5, 1979
•
,
CPM
H U M A N (D.M P) {9/1/76)
O,D.
.,o1t 4/~~
TC I
20
~0
30 FRACTION
40
50
FIG. l. Radioactive and protein elution profiles for a typical human serum. Arrows indicate buffer changes.
B] 2-binding proteins which eluted from DEAE cellulose with the same buffers as the three human transcobalamins. Profiles of s 7Co and protein elution of a human serum and a typical bovine serum are illustrated in Figures 1 and 2. In a preliminary test, with a gradient of nine buffers, no significant additional peaks were found. Samples from four each normal, ketotic, B1 2-supplemented, and Factor B-supplemented cows were examined. The distribution of radioactive label among the three peaks was calculated, and mean data are in Table 1. There was little difference in mean distribution of u n b o u n d binding capacity among the animals in different physiological states although
COW
~--~
E" b
=z.
NO. IIII
(2/27/76)
CPM O,D
.04M
.40M
,LOM pH ~ 8
pfl 5.9
pH52
o
ABSTRACT
Examination of bovine serum by the diethylaminoethyl cellulose small column method revealed three proteins binding vitamin B12. The elution pattern suggested that they are similar to the three transcobalamins recognized in human serum. Distribution of u n b o u n d binding capacity among serum binders was assessed in serum from normal, ketotic, and B12- and Factor B-supplemented cows in early lactation. No major differences were observed among groups; however, cow serum displayed a pattern different from human serum. Mean total binding capacity of bovine serum for B12 as well as mean u n b o u n d binding capacity were lower than the corresponding means for human serum. INTRODUCTION
Although proteins binding vitamin B12 in serum have been studied extensively in the human (3, 4, 6, 9, 10, 11, 13, 16), little attention has been given to the ruminant. Three binders have been identified in normal human serum by DEAE-cellulose column chromatography (6, 10). These have been designated transcobalamin (TC) I, TC II, and main protein peak binder (MPPB) or TC III. The individual roles of these binders still are not elucidated completely, but a large percentage of the endogenous vitamin B12 in human serum appears to be carried by TC I (4, 9, 11). Tan and Blaisdell (18)examined the substances binding B12 in serum of 19 vertebrates including human and bovine. The methods detected one binder in cow serum, but since they did not separate the three binders usually detected in
Received July 19, 1978. 1Supported in part by a grant from Agway, Inc. 2Address reprint requests to this author. 1979 J Dairy Sci 62:697-701
human serum, a meaningful comparison of the cow and the human was impossible. Our study was to determine whether the cow, like the human, has three binders of vitamin B12 in serum and to examine the binding capacity for B1 ~ of cow sera. In view of our interest in the vitamin B12 status of the ketotic cow, samples from cows in this and other physiological states were examined. MATERIALS AND METHODS
Samples of serum, prepared from venous blood and stored frozen, were available from normal cows at approximately 8 wk postpartum, ketotic cows in early lactation, cows at 8 wk postpartum that had been injected (i.m.) with supplementary vitamin B12 (20 mg/wk) for 12 wk, and cows at 8 wk postpartum that had been injected (i.m.) with Factor B (35 or 175 mg/wk) for 8 wk. Factor B (cobinamide) is one of the analogues of vitamin B 12- All cows were Holsteins from the Cornell University herd. Samples of human blood were from healthy individuals working in our laboratory. The small DEAE-cellulose column method (7) was used to separate the B12-binding proteins. The DEAE-cellulose (Cellex D, .85 meq/g, Bio-Rad Laboratories)was packed, after preparation, into .6 × 25 cm columns. The [s 7Co ] B12 (15 uCi/ug, Amersham/Searle, Arlington Heights, IL) was diluted to contain 3.33 ng/ml. To .5 ml of serum, 1.0 ml of labeled B12 solution was added. The mixture was allowed to stand at 37 C for 1 h. Albumincoated charcoal (2 ml) then was added to remove u n b o u n d B12. The tube was centrifuged, and the radioactivity in the supernatant was counted in a well-type counter (Nuclear Chicago). The supernatant subsequently was applied to a DEAE cellulose column. Any free B12 was eluted with .175 M sodium phosphate buffer, pH 6.3. The following sodium phosphate buffers then were used in sequence: .04 M, pH 5.9; .1 M, pH 5.8; and ~4 M, pH 5.2. Quantities were usually 20, 36, 20, and 14 ml, 697
698
POLAK ET AL.
respectively, b u t occasionally varied as experience determined what was necessary to achieve dear separation of peaks. Buffer solutions were made up in distilled water containing .02% NAN3. Flow rate was 6 to 10 ml per h, and all chromatography was at 4 C. An automatic fractionator allowed collection of eluate in approximately 2 ml fractions. Radioactivity of these fractions was counted, and protein was estimated by absorbance at 280 nm. Extracts of serum were assayed for vitamin B12 with both Ochromonas malbamensis (5) and a nonspecific radiodilution method (1) which employs toadfish serum as a binder. Extracts were prepared at pH 5.0 by steaming a mixture of 5.0 ml of serum, .5 ml of fresh KCN solution (40 mg/100 ml), and approximately 5 ml redistilled water in an autoclave at atmospheric pressure for 30 min. After cooling, the samples were brought to volume (25 ml), well mixed, and centrifuged to remove the protein. The supernatant was stored frozen until assayed. Vitamin B12 u n b o u n d binding capacity (UBBC) was measured by the rapid charcoal method (8) with modifications. The [S TCo] B12 was diluted with unlabeled cyanocobalamin to contain 6.06 ng/ml. One drop of KCN solution (2 mg/ml) was added to each bottle (50 ml) of standardized labeled B12 solution. In a 10-ml test tube, .1 ml of serum and 1.0 ml of the B x2 solution were allowed to react for 24 h at 4 C. At this temperature, exchange with endogenous bound B12 is avoided, and saturation, at least with human plasma, is achieved in about 4 h (2). Preliminary studies suggested a similar time for cow serum with no subsequent change over 3 days. Therefore, a 24-h incubation was used for convenience. After 24 h, 2 ml of albumin-coated charcoal were added to each tube which, after thorough mixing, was centrifuged. The radioactivity in the supernatant was counted to permit calculation of u n b o u n d binding capacity (8). Total binding capacity was calculated as the sum of UBBC and vitamin B12. Comparisons among physiological states and between the bovine and the human were made by analysis of variance for unbalanced data by methods outlined by Searle (15). RESULTS AND DISCUSSION
Bovine
serum
contained
three
vitamin
Journal of Dairy Science VoL 62, No. 5, 1979
•
,
CPM
H U M A N (D.M P) {9/1/76)
O,D.
.,o1t 4/~~
TC I
20
~0
30 FRACTION
40
50
FIG. l. Radioactive and protein elution profiles for a typical human serum. Arrows indicate buffer changes.
B] 2-binding proteins which eluted from DEAE cellulose with the same buffers as the three human transcobalamins. Profiles of s 7Co and protein elution of a human serum and a typical bovine serum are illustrated in Figures 1 and 2. In a preliminary test, with a gradient of nine buffers, no significant additional peaks were found. Samples from four each normal, ketotic, B1 2-supplemented, and Factor B-supplemented cows were examined. The distribution of radioactive label among the three peaks was calculated, and mean data are in Table 1. There was little difference in mean distribution of u n b o u n d binding capacity among the animals in different physiological states although
COW
~--~
E" b
=z.
NO. IIII
(2/27/76)
CPM O,D
.04M
.40M
,LOM pH ~ 8
pfl 5.9
pH52
o
