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The Influence of Previous Vitamin K Nutrition on Thromboplastic Activity of Brain Extract*
784
RESEARCH NOTES TABLE 2.—Rjject of divalent metal chlorides on BA PN induced mortality in the chick embryo (12-16 fertile eggs/treatment/experiment)
Treatment administered BAPN (0.3 No. of incubation nig./ expts. Divalent salt egg) CaCI2 CaCl2
ZnCl2 ZnCl*
MnCk MnCl 2
MgCk MgCl2 1
+ + + + +
3 3
2 2
5 days 5 days
5 days 5 days
% embryos dead at time of observation related to dose of divalent metal cation injected per egg Ca++ (mgs.XlOO) 0
8
17
5 56
0 59
3 61
0
25
9 55
6 47
0
96
84
126
168
2 0 58 41 Cu (meg.) 127 178 76
3 24
5 28
5 days S days
7 93
7 71
14 79
0
35
106
2 2
22 days 22 days
12 89
9 86
15 83
2 2
22 days 22 days
10
6 87
20 90
++
26 49 20 61 Zn (meg.) 0 480 19 32
1 1
0
42
++
43 (D Mn + + (meg.) 178 248
34 88 mgs.XlOO) Mg 70 40 11 83
74 71 96 14 «
480
\ >
320
37 89
9 79
100
130
13 89
0 98
++
6 90
229
These 3 treatments received 0.2 mg. BAPN/egg.
calcium in the chick. J. Nutr. 78: 207-214. Smiley, J. D., and H. Yeager, 1962. Collagen
metabolism in lathyrism: Site of beta aminopropionitrile (BAPN) action. Fed. Proc. 21: 167c.
THE INFLUENCE OF PREVIOUS VITAMIN K NUTRITION ON THROMBOrLASTIC ACTIVITY OF BRAIN EXTRACT* PAUL GRIMINGER Department of Poultry Science, Rutgers-The Stale University, New Brunswick, New Jersey (Received for publication January 21, 1963)
Due do a partial species specificity of thromboplastin (Hecht, 1955; Didisheim el al., 1959) it is requisite to use a chicken thromboplastin extract for prothrombin determinations on chicken blood by the one-stage method (Quick, 1957). Whether thromboplastin extracts from * Paper of the Journal Series, New Jersey Agricultural Experiment Station, Rutgers, The State University of New Jersey, New Brunswick.
other avian species are equally effective is not perfectly clear (Griminger, 1957; Didisheim el al., 1959). Chicken brain thromboplastin extract is usually employed, although embryo extract and muscle extracts have also been used successfully. Although comparable results may be obtained with various extracts (Griminger, 1962b), the material of choice appears to be a carefully prepared chicken brain extract resulting, with the one-stage
Downloaded from http://ps.oxfordjournals.org/ at University of North Dakota on May 23, 2015
CuCl2 CuCl2
—
Day of incubation at observation
RESEARCH NOTES
EXPERIMENTAL
In the first trial, three thromboplastin extracts were prepared from the brains of chickens that had either received a purified vitamin-K-deficient diet, a conventional starter ration, or the latter ration supplemented with 10 mg./kg. of vitamin Ki. Duplicate prothrombin assays were then carried out with these three preparations on the plasma of 10 chickens of various ages that had received different levels of vitamin K. In the second experiment four thromboplastin extracts were prepared, one again from chickens that had received a purified K-deficient diet, and the other three from chickens that had received the same diet supplemented with either 5 mg./kg. of menadione sodium bisulfite complex (MSBC), 5 mg./kg. of vitamin Ki, or 100 mg./kg. of MSBC. The chickens were killed at 3 weeks of age and their brains excised and cleaned carefully with cotton applicators to remove as many of the blood vessels as possible. The brains were then ground in a mortar with acetone for 5 minutes (using approximately 100 ml. for 10 brains) and the extract filtered. The acetone extraction was repeated 4 times. After the final filtration the brain preparation was left to dry overnight in a partially evacuated desiccator at room temperature. The dried preparation was then ground in a mortar
TABLE 1.—Influence of thromboplastin on plasma prothrombin times of chickens1 Blood sample
Thromboplastin preparations2
Sequence
Expt. 1
A
B
C
1 2 3 4 5 6 7 8 9 10
13.7 13.9 14.7 25.0 25.8 29.3 36.3 38.1 45.6 49.3
13.0 13.7 13.4 21.5 27.4 30.0 34.0 37.5 44.5 52.6
11.6 13.5 13.0 19.4 19.5 25.4 30.5 35.6 44.1 46.6
Expt. 2 11 12 13 14 15 16 17 1
D
E
F
12.3 13.4 14.7 15.2 16.2 51.7 71.2
13.1 12.9 14.7 15.2 15.6 52.8 63.1
12.1 13.1 12.9 16.2 15.6 55.1 61.5
C 10 .6 12..2 • 13.1 13..5 13..9 49. 0 69..7
A A A A
B B B B
C C C C
B A C B A C
A B C A B C ABC
B A C E DF G DF E G D EG F F DE G DE F G F ED G DGE F
Averages of twodeterminations; all calculations are base
2
Thromboplastin preparations from brains of chicks on the following dietary regimes: A and D, vit. K-deficient diet; B, conventional starter; C, as B+10 mg.Ki/kg. diet; E, as A + 5 mg. menadione sodium bisulfite complex (MSBC)/kg. diet; F, as A + 5 mg. Ki/kg. diet; G, as A+100 mg. MSBC/kg. diet.
and stored in small vials under nitrogen below 0°C. For the prothrombin determinations the brain powder was activated for use as discussed in an earlier publication (Griminger, 1962b). RESULTS AND DISCUSSION
The values in Table 1 show a tendency for shorter prothrombin times with the use of the thromboplastin originating from chickens which had been fed the higher levels of vitamin K active compounds. In the first experiment, preparation C (from chicks fed a conventional starter ration supplemented with 10 mg.Ki/kg.), resulted in shorter prothrombin times with all blood samples than either of the other two preparations, and an analysis of variance showed the differences between treatments to be highly significant (P<0.1%). In the second experiment the ranking of the preparations was less well defined, although the preparation from the chicks receiving the highest level of vitamin K (G) resulted in the shortest prothrombin
Downloaded from http://ps.oxfordjournals.org/ at University of North Dakota on May 23, 2015
method, in prothrombin times of 12 seconds or less. For many years we have been preparing brain thromboplastin extracts from chickens that had previously been fed a vitamin-K-deficient diet. Since we felt some question as to the influence of the level of vitamin K nutriture on the thromboplastic activity of the extract, we decided to investigate this influence experimentally.
785
786
RESEARCH NOTES
Acetone-dehydrated brain powder is not likely to contain active prothrombin. The cause for the shortened prothrombin times must therefore lie with the thromboplastinogen complex itself, which the brain powder contains. Consequently we would like to suggest t h a t higher levels of vitamin K increase certain components of the thromboplastinogen complex. There is, indeed, considerable evidence (Brinkhous, 1959) t h a t vitamin K nutriture plays a role in the formation of the plasma thromboplastin component ( P T C , factor 9). In the chicken, however, this factor is either lacking entirely (Didisheim et al., 1959) or present only in very small
amounts (Wartelle, 1958). This, on the other hand, does not exclude the possibility of the existence of a similar factor, specific for some or all avian species, which is enhanced by K (S0rbye and Kruse, 1960), and that in this manner the thromboplastin activity in chicken brains from birds fed high levels of vitamin K is increased. If this assumption were correct it would be advantageous to employ the brains of chickens, which had previously received high levels of vitamin K, in the preparation of thromboplastin extracts. SUMMARY
Vitamin K supplementation of the ration of chickens used as a source of brain thromboplastin extract shortened plasma prothrombin times determined by the one-stage method with this extract. REFERENCES Brinkhous, K. M., 1959. Blood clotting: the plasma procoagulants. Am. Rev. Physiol. 21: 271-298. Didisheim, P., K. Hattori and J. H. Lewis. 1959. Hematologic and coagulation studies in various animal species. J. Lab. Clin. Med. 53: 866-875. Griminger, P., 1957. On the vitamin K requirement of turkey poults. Poultry Sci. 36: 1227-1235. Griminger, P., 1962a. Arsanilic acid and blood coagulation. Poultry Sci. 41: 982-985. Griminger, P., 1962b. Prothrombin bioassay for vitamin K with different thromboplastin preparations. Internat. J. Vit. Res. 32: 405-412. Hecht, E., 1955. Zur Kenntnis der Thrombokinasen. Biochem. Zeitschr. 326: 325-342. Quick, A. J., 1957 (1959). Hemorrhagic Diseases. Lea & Febiger, Philadelphia. S0rbye, 0., and I. Kruse, 1960. Studies on the coagulation of chicken blood. I. Factors which limit plasma coagulation rate during vitamin K deficiency and treatment with coumarin drugs. Acta chem. Scand. 14: 2177-2190. Wartelle, O., 1957. Mecanisme de la coagulation chez la poule. I.-Etude des elements «du complex prothrombique» et de la thromboplastino-formation. Revue d'hemat. 12: 351-387.
O C T O B E R 8-10. A N N U A L E X P O S I T I O N A N D C O N V E N T I O N , N O R T H E A S T E R N POULTRY PRODUCERS COUNCIL, H A R R I S B U R G , PA.
Downloaded from http://ps.oxfordjournals.org/ at University of North Dakota on May 23, 2015
times in 5 out of 7 cases. In one of the other two cases (bird 13) the difference between prothrombin times with preparations G and F was very small indeed (0.2 s e c ) . Again an analysis of variance showed the difference for the treatment effect to be significant at the 0 . 1 % level. In the analyses of both experiments the interactions between treatments and birds were also significant; the reason for this can be visualized by plotting the reciprocals of prothrombin times obtained with B and C relative to those obtained with A, and those obtained with E, F, and G relative to D . Regression lines for the values obtained with the different preparations are not parallel, but increase in distance from each other at higher numerical values (shorter prothrombin times). Reciprocals of prothrombin times were used for all calculations, the results being reconverted into seconds; this method of calculation is superior to the use of the unconverted data, since reciprocals of prothrombin times are more normally distributed t h a n unconverted prothrombin times (Griminger, 1962a).
RESEARCH NOTES TABLE 2.—Rjject of divalent metal chlorides on BA PN induced mortality in the chick embryo (12-16 fertile eggs/treatment/experiment)
Treatment administered BAPN (0.3 No. of incubation nig./ expts. Divalent salt egg) CaCI2 CaCl2
ZnCl2 ZnCl*
MnCk MnCl 2
MgCk MgCl2 1
+ + + + +
3 3
2 2
5 days 5 days
5 days 5 days
% embryos dead at time of observation related to dose of divalent metal cation injected per egg Ca++ (mgs.XlOO) 0
8
17
5 56
0 59
3 61
0
25
9 55
6 47
0
96
84
126
168
2 0 58 41 Cu (meg.) 127 178 76
3 24
5 28
5 days S days
7 93
7 71
14 79
0
35
106
2 2
22 days 22 days
12 89
9 86
15 83
2 2
22 days 22 days
10
6 87
20 90
++
26 49 20 61 Zn (meg.) 0 480 19 32
1 1
0
42
++
43 (D Mn + + (meg.) 178 248
34 88 mgs.XlOO) Mg 70 40 11 83
74 71 96 14 «
480
\ >
320
37 89
9 79
100
130
13 89
0 98
++
6 90
229
These 3 treatments received 0.2 mg. BAPN/egg.
calcium in the chick. J. Nutr. 78: 207-214. Smiley, J. D., and H. Yeager, 1962. Collagen
metabolism in lathyrism: Site of beta aminopropionitrile (BAPN) action. Fed. Proc. 21: 167c.
THE INFLUENCE OF PREVIOUS VITAMIN K NUTRITION ON THROMBOrLASTIC ACTIVITY OF BRAIN EXTRACT* PAUL GRIMINGER Department of Poultry Science, Rutgers-The Stale University, New Brunswick, New Jersey (Received for publication January 21, 1963)
Due do a partial species specificity of thromboplastin (Hecht, 1955; Didisheim el al., 1959) it is requisite to use a chicken thromboplastin extract for prothrombin determinations on chicken blood by the one-stage method (Quick, 1957). Whether thromboplastin extracts from * Paper of the Journal Series, New Jersey Agricultural Experiment Station, Rutgers, The State University of New Jersey, New Brunswick.
other avian species are equally effective is not perfectly clear (Griminger, 1957; Didisheim el al., 1959). Chicken brain thromboplastin extract is usually employed, although embryo extract and muscle extracts have also been used successfully. Although comparable results may be obtained with various extracts (Griminger, 1962b), the material of choice appears to be a carefully prepared chicken brain extract resulting, with the one-stage
Downloaded from http://ps.oxfordjournals.org/ at University of North Dakota on May 23, 2015
CuCl2 CuCl2
—
Day of incubation at observation
RESEARCH NOTES
EXPERIMENTAL
In the first trial, three thromboplastin extracts were prepared from the brains of chickens that had either received a purified vitamin-K-deficient diet, a conventional starter ration, or the latter ration supplemented with 10 mg./kg. of vitamin Ki. Duplicate prothrombin assays were then carried out with these three preparations on the plasma of 10 chickens of various ages that had received different levels of vitamin K. In the second experiment four thromboplastin extracts were prepared, one again from chickens that had received a purified K-deficient diet, and the other three from chickens that had received the same diet supplemented with either 5 mg./kg. of menadione sodium bisulfite complex (MSBC), 5 mg./kg. of vitamin Ki, or 100 mg./kg. of MSBC. The chickens were killed at 3 weeks of age and their brains excised and cleaned carefully with cotton applicators to remove as many of the blood vessels as possible. The brains were then ground in a mortar with acetone for 5 minutes (using approximately 100 ml. for 10 brains) and the extract filtered. The acetone extraction was repeated 4 times. After the final filtration the brain preparation was left to dry overnight in a partially evacuated desiccator at room temperature. The dried preparation was then ground in a mortar
TABLE 1.—Influence of thromboplastin on plasma prothrombin times of chickens1 Blood sample
Thromboplastin preparations2
Sequence
Expt. 1
A
B
C
1 2 3 4 5 6 7 8 9 10
13.7 13.9 14.7 25.0 25.8 29.3 36.3 38.1 45.6 49.3
13.0 13.7 13.4 21.5 27.4 30.0 34.0 37.5 44.5 52.6
11.6 13.5 13.0 19.4 19.5 25.4 30.5 35.6 44.1 46.6
Expt. 2 11 12 13 14 15 16 17 1
D
E
F
12.3 13.4 14.7 15.2 16.2 51.7 71.2
13.1 12.9 14.7 15.2 15.6 52.8 63.1
12.1 13.1 12.9 16.2 15.6 55.1 61.5
C 10 .6 12..2 • 13.1 13..5 13..9 49. 0 69..7
A A A A
B B B B
C C C C
B A C B A C
A B C A B C ABC
B A C E DF G DF E G D EG F F DE G DE F G F ED G DGE F
Averages of twodeterminations; all calculations are base
2
Thromboplastin preparations from brains of chicks on the following dietary regimes: A and D, vit. K-deficient diet; B, conventional starter; C, as B+10 mg.Ki/kg. diet; E, as A + 5 mg. menadione sodium bisulfite complex (MSBC)/kg. diet; F, as A + 5 mg. Ki/kg. diet; G, as A+100 mg. MSBC/kg. diet.
and stored in small vials under nitrogen below 0°C. For the prothrombin determinations the brain powder was activated for use as discussed in an earlier publication (Griminger, 1962b). RESULTS AND DISCUSSION
The values in Table 1 show a tendency for shorter prothrombin times with the use of the thromboplastin originating from chickens which had been fed the higher levels of vitamin K active compounds. In the first experiment, preparation C (from chicks fed a conventional starter ration supplemented with 10 mg.Ki/kg.), resulted in shorter prothrombin times with all blood samples than either of the other two preparations, and an analysis of variance showed the differences between treatments to be highly significant (P<0.1%). In the second experiment the ranking of the preparations was less well defined, although the preparation from the chicks receiving the highest level of vitamin K (G) resulted in the shortest prothrombin
Downloaded from http://ps.oxfordjournals.org/ at University of North Dakota on May 23, 2015
method, in prothrombin times of 12 seconds or less. For many years we have been preparing brain thromboplastin extracts from chickens that had previously been fed a vitamin-K-deficient diet. Since we felt some question as to the influence of the level of vitamin K nutriture on the thromboplastic activity of the extract, we decided to investigate this influence experimentally.
785
786
RESEARCH NOTES
Acetone-dehydrated brain powder is not likely to contain active prothrombin. The cause for the shortened prothrombin times must therefore lie with the thromboplastinogen complex itself, which the brain powder contains. Consequently we would like to suggest t h a t higher levels of vitamin K increase certain components of the thromboplastinogen complex. There is, indeed, considerable evidence (Brinkhous, 1959) t h a t vitamin K nutriture plays a role in the formation of the plasma thromboplastin component ( P T C , factor 9). In the chicken, however, this factor is either lacking entirely (Didisheim et al., 1959) or present only in very small
amounts (Wartelle, 1958). This, on the other hand, does not exclude the possibility of the existence of a similar factor, specific for some or all avian species, which is enhanced by K (S0rbye and Kruse, 1960), and that in this manner the thromboplastin activity in chicken brains from birds fed high levels of vitamin K is increased. If this assumption were correct it would be advantageous to employ the brains of chickens, which had previously received high levels of vitamin K, in the preparation of thromboplastin extracts. SUMMARY
Vitamin K supplementation of the ration of chickens used as a source of brain thromboplastin extract shortened plasma prothrombin times determined by the one-stage method with this extract. REFERENCES Brinkhous, K. M., 1959. Blood clotting: the plasma procoagulants. Am. Rev. Physiol. 21: 271-298. Didisheim, P., K. Hattori and J. H. Lewis. 1959. Hematologic and coagulation studies in various animal species. J. Lab. Clin. Med. 53: 866-875. Griminger, P., 1957. On the vitamin K requirement of turkey poults. Poultry Sci. 36: 1227-1235. Griminger, P., 1962a. Arsanilic acid and blood coagulation. Poultry Sci. 41: 982-985. Griminger, P., 1962b. Prothrombin bioassay for vitamin K with different thromboplastin preparations. Internat. J. Vit. Res. 32: 405-412. Hecht, E., 1955. Zur Kenntnis der Thrombokinasen. Biochem. Zeitschr. 326: 325-342. Quick, A. J., 1957 (1959). Hemorrhagic Diseases. Lea & Febiger, Philadelphia. S0rbye, 0., and I. Kruse, 1960. Studies on the coagulation of chicken blood. I. Factors which limit plasma coagulation rate during vitamin K deficiency and treatment with coumarin drugs. Acta chem. Scand. 14: 2177-2190. Wartelle, O., 1957. Mecanisme de la coagulation chez la poule. I.-Etude des elements «du complex prothrombique» et de la thromboplastino-formation. Revue d'hemat. 12: 351-387.
O C T O B E R 8-10. A N N U A L E X P O S I T I O N A N D C O N V E N T I O N , N O R T H E A S T E R N POULTRY PRODUCERS COUNCIL, H A R R I S B U R G , PA.
Downloaded from http://ps.oxfordjournals.org/ at University of North Dakota on May 23, 2015
times in 5 out of 7 cases. In one of the other two cases (bird 13) the difference between prothrombin times with preparations G and F was very small indeed (0.2 s e c ) . Again an analysis of variance showed the difference for the treatment effect to be significant at the 0 . 1 % level. In the analyses of both experiments the interactions between treatments and birds were also significant; the reason for this can be visualized by plotting the reciprocals of prothrombin times obtained with B and C relative to those obtained with A, and those obtained with E, F, and G relative to D . Regression lines for the values obtained with the different preparations are not parallel, but increase in distance from each other at higher numerical values (shorter prothrombin times). Reciprocals of prothrombin times were used for all calculations, the results being reconverted into seconds; this method of calculation is superior to the use of the unconverted data, since reciprocals of prothrombin times are more normally distributed t h a n unconverted prothrombin times (Griminger, 1962a).