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Relative Vitamin K Potency of Two Water-Soluble Menadione Analogues1
210
W. 0. BROWN AND H. G. BADMAN
ACKNOWLEDGEMENT One of the authors (H.G.B.) thanks the British Egg Marketing Board for the award of a Research Studentship to enable him to carry out this work. REFERENCES Bolton, W., 1952. Observations on the vitamin metabolism of the common fowl. 2. The effects of estrogen and progesterone injections in immature pullets on the riboflavin content of the magnum. Brit. J. Nutrit. 6: 386-392. Brant, J. W. A., and A. V. Nalbandov, 1956. Role of
sex hormones in albumen secretion by the oviduct of chickens. Poultry Sci. 35: 692-700. Brown, W. O., 1954. Nitrogen metabolism in the folic acid-deficient chick studies by a balance technique. Tenth World Poultry Congress, Edinburgh. page 149-152. Durrum, E. L., 1951. Paper ionophoresis. Science, 113: 66-69. Evans, R. J., and S. L. Bandemer, 1956. Separation of egg white protein by paper electrophoresis. Agric. Food Chem. 4: 802-810. Hertz, R., 1950. Endocrine and vitamin factors in hormone-induced tissue growth. Texas. Rep. Biol. Med. 8: 154-158. Lodge, P. D. B., and C. L. Smith, 1960. Hormonal control of secretion in the oviduct of the Amphibia. Nature, 185: 774-775. Mandeles, S., and E. D. Ducay, 1962. Site of egg white protein formation. J. Biol. Chem. 237: 3196-3199. Markham, R., 1942. A steam distillation apparatus suitable for micro-Kjeldahl analysis. Biochem. J. 36: 790-791. Mulgaonkar, A. G., and A. Sreenivasan, 1957. Alteration in rat serum proteins in folic acid and vitamin B12 deficiency. Proc. Soc. Exp. Biol. Med. 94: 44-46. Smith, A. H., C. M. Winget and T. J. Hague, 1957. Effect of local x-irradiation on albumen formation by the hen's oviduct. Am. J. Physiol. 191: 605-609. Wolff, E., 1936. L'action de l'oestrone sur l'oviducte et la cloaque des poussins femelle intersexues et males et sur les reaction des cannaux de Muller des oiseaux aux hormones sexuelles. Compt. Rend. Soc. Biol. 123: 235-236.
Relative Vitamin K Potency of Two Water-Soluble Menadione Analogues 1 PAUL GRIMINGER
Department of Animal Sciences, Rutgers, The State University, New Brunswick, New Jersey (Received for publication July 2. 1964)
M
ENADIONE sodium bisulfite, a used as a source of vitamin K in poultry water-soluble form of the pro- feeds. This form differs in its vitamin K vitamin 2-methyl-l,4-naphthoquinone, is activity from the fat-soluble menadione (Frost and Spruth, 1955). The data pre1 Paper of the Journal Series, New Jersey Agrisented in this report indicate that this cultural Experiment Station, Rutgers, The State difference is not necessarily a function, or, University of New Jersey, Department of Animal at least, not only a function of the change Sciences, New Brunswick.
Downloaded from http://ps.oxfordjournals.org/ at University of Nebraska-Lincoln Libraries on June 10, 2016
estradiol, estradiol plus progesterone and estradiol plus testosterone were separated by a paper electrophoretic technique. Evidence is presented to show that albumins and conalbumins of similar mobility to those from egg-white proteins are present after estradiol treatment but that there is an increase in the quantity of water-soluble proteins when both hormones are administered. An experiment is also described in which it is shown that in folic acid deficiency there is a large reduction in the quantity of albumins in the water-soluble proteins in the oviducts of immature female chicks treated with estradiol and estradiol plus testosterone.
211
V I T A M I N K. P O T E N C Y
in solubility status, since another watersoluble form of menadione, namely, menadione dimethyl pyrimidinol bisulfite, 2 differs in its activity from menadione sodium bisulfite. EXPERIMENTAL
* Hetrazeen, kindly supplied by the Heterochemical Corporation, Valley Stream, New York.
RESULTS
The levels of supplementation and the corresponding average prothrombin times are shown in Table 1. Since the relative response to the two forms of menadione at 2 and 3 weeks of age was essentially simiTABLE 1.—Prothrombin times of chickens fed 0.2% sulfaquinoxaline, and graded levels of 2 water-soluble menadione analogues1 2-weeks2 Level of supplemenMSBC 4 MPB 1 tation mg./kg. 0.1 0.2 0.4 0.8 1.6 3.2 6.4
sec. 119.8 148.4 48.9 32.9 29.7 23.0 20.4
sec. 104.3 67.7 37.2 33.4 22.0 21.3 18.1
3-weeks3 MSBC
MPB
sec. 180.0[4] 54.1 [2] 38.0 26.7 23.9 17.9 18.8
sec. 98.0 35.2 27.7 20.3 19.6 17.8 14.3
1 All averages were calculated on the basis of the reciprocals of the prothrombin times (Griminger, 1962b). Samples not clotted within 180 sec. were treated as if they had clotted at 180 sec. The use of reciprocals minimizes the error thus incurred. 2 Averages of 8 chicks each. 3 Averages of 7 chicks each, except where shown differently (in brackets). 4 MSBC—menadione sodium bisulfite complex, containing 33% menadione; MPB—Menadione dimethyl pyrimidinol bisulfite, containing 45% menadione.
Downloaded from http://ps.oxfordjournals.org/ at University of Nebraska-Lincoln Libraries on June 10, 2016
Fourteen groups of 20 cross-bred, female chicks received a purified vitamin K-deficient diet (Griminger, 1962a) from hatching to 1 week of age. At t h a t time group uniformity was increased by eliminating 4 chicks with extreme weights. From 1 to 3 weeks of age the chicks received the K-deficient diet supplemented with 0.2% sulfaquinoxaline and with seven graded levels of each of the two water-soluble menadione derivatives, menadione sodium bisulfite complex (MSBC), containing 3 3 % menadione, and menadione dimethyl pyrimidinol bisulfite ( M P B ) , containing 4 5 % menadione. At 2 weeks of age, 1 ml. of blood was drawn by heart puncture from each of 8 chicks picked at random from each of the 14 groups (a total of 112 chicks), and plasma prothrombin times determined b y the method of Quick (1957), using sodium citrate as an anticoagulant, and acetonedehydrated chicken brain powder as a source of thromboplastin (Griminger, 1962a). At 3 weeks of age the procedure was repeated, using 7 chicks from each group, or fewer where necessitated b y mortality. During the entire experiment the chicks were kept on raised wire floors in electrically heated battery brooders; the stainless steel water containers, the feed containers, and the wire screens underwent frequent cleaning to prevent the chicks from obtaining vitamin K synthesized by microorganisms in the water, the feed, or the droppings adhering to the wire screen.
T h e data were averaged on the basis of the reciprocals of the individual prothrombin times, and the mean values thus obtained reconverted to seconds. I n the graphic presentation the averages of the reciprocals were plotted against the logarithms (decimal) of the dose (vitamin K supplement), as suggested b y Almquist (1954). Good comparisons between different forms of vitamin K are difficult to obtain even when a purified diet is fed. Increasing the vitamin K requirement through the use of sulfaquinoxaline as a stress agent to improve evaluation of different vitamin K forms was proposed b y Perdue and Frost (1961), and was successfully employed for the present comparisons.
212
P. GRIMINGER
600
MPB-
MSBC^
500
z
400
oci 5
i t-
o a.
300
<
2 «>
200
100,
__1 0.1
I
0.4
J_ 0.6
-J_ 1.6
3.2
0.2 SUPPLEMENT
(IN
MG/KG
6.4
DIET)
VITAMIN
FIG. 1. Prothrombin time response to graded levels of menadione dimethyl pyrimidinol bisulfite (MPB) and menadione sodium bisulfite complex (MSBC).
lar, weighted averages were calculated; these averages, and their respective regression lines, calculated by the method of least squares, are shown in Fig. 1. It appears that the response lines to the two water-soluble menadione analogues compared here have a similar slope; however, equivalent amounts of MPB were considerably more active than MSBC in decreasing prothrombin times. If we compare the levels needed to obtain an average prothrombin time of 40 sec. (1/250 X104), requirements of 0.285 mg. of MPB or of 0.52 of MSBC/kg. diet can be read from the graph; this is a ratio of 1:1.8. To obtain an average prothrombin time of 20 sec. (1/500 X104), the corresponding dosages would be 2.25 and 4.55, or a ratio of approximately 1:2. Since the menadione content of MPB is higher than that of MSBC, a ratio of 1:1.36 may be expected
if utilization of menadione from these two compounds were alike. It may be concluded from the appreciably higher ratios observed in the present trial, that the menadione moiety of MPB is more efficiently utilized than that of MSBC in decreasing prothrombin times in chicks. SUMMARY
Two water-soluble menadione analogues were compared for vitamin K activity, using a vitamin K-deficient ration supplemented with 0.2% sulfaquinoxaline. Over the range of dietary supplementation used, menadione dimethyl pyrimidinol bisulfite promoted a higher plasma prothrombin level in growing chicks than did menadione sodium bisulfite. This difference in activity was greater than could be explained on the basis of the menadione content of the two compounds.
Downloaded from http://ps.oxfordjournals.org/ at University of Nebraska-Lincoln Libraries on June 10, 2016
o a.
VITAMIN K POTENCY REFERENCES Almquist, H. J., 1954. The Vitamins. Vol. 2. Academic Press, New York. Frost, D. V., and H. C. Spruth, 1955. Control of hemorrhagic condition in chickens with menadione sodium bisulfite. Poultry Sci. 34: 56-64. Griminger, P., 1962a. Prothrombin bioassay for vitamin K with different thromboplastin prepara-
213
tions. Intern. J. Vit. Res. 32: 405-412. Griminger, P., 1962b. Arsanilic acid and blood coagulation. Poultry Sci. 4 1 : 982-985. Perdue, H. S., and D. V. Frost, 1961. Recent developments on a proposed ANRC vitamin K reference standard. Feedstuffs, January 21, p. 56. Quick, A. J., 1957 (1959). Hemorrhagic Diseases, Lea and Febiger, Philadelphia.
P. W. WALDROUP2, R. H. HARMS 2 AND M. FRIED 8 Florida A gricuitural Experiment Stations and College of Medicine, University of Florida, Gainesville, Florida (Received for publication July 3, 1964)
1
T HAS been reported that lowering the level of protein in the diet of laying hens resulted in a significant drop in egg weight (Harms and Waldroup, 1962). The present experiment was designed to compare the serum and egg proteins electrophoretically to determine if the reduced egg weight was causally related to a variation in any of the electrophoretic components. MATERIALS AND METHODS
Two diets containing 9 and 17 percent protein (Waldroup and Harms, 1964) were prepared. Each diet was fed to a group of ten egg-production type pullets which had been in production for approximately two months and were laying at a rate of about 80 percent when the experiment began. Blood samples were obtained by cardiac puncture from pullets on the second day of each week over an eight week test period. Samples were obtained at 10:00 1
Florida Agri. Exp. Sta. Journal Series No. 1899. Department of Poultry Science 3 Department of Biochemistry Supported in part by a grant (AM-02547) from the National Institute of Health. 2
a.m. from all pullets that had laid an egg on that day. The blood was allowed to clot and then centrifuged to collect the serum. For determination of egg constituents the egg contents were blended with distilled water in an egg: water ratio of 1:4. Serum and egg samples of four pullets from each group were randomly selected for electrophoretic studies. Samples of the blood sera and the diluted egg suspension were analyzed for protein and lipoprotein components using the Beckman Spinco Electrophoresis System. The procedures outlined in the Beckman Paper Electrophoresis System Manual (Anonymous, 1958) were followed in determining the proteins and lipoproteins. Duplicate paper strips spotted with sera and egg suspension were run in parallel. In addition, total protein content was determined by the Biuret method (Gornall et al., 1949). RESULTS AND DISCUSSION
On the paper strips on which sera were separated, three components were observed following staining for protein with bromphenol blue. In order of decreasing mobility these were albumin, a .mixed
Downloaded from http://ps.oxfordjournals.org/ at University of Nebraska-Lincoln Libraries on June 10, 2016
Alterations in the Serum Protein Components of Laying Hens on Low Protein Diets1
W. 0. BROWN AND H. G. BADMAN
ACKNOWLEDGEMENT One of the authors (H.G.B.) thanks the British Egg Marketing Board for the award of a Research Studentship to enable him to carry out this work. REFERENCES Bolton, W., 1952. Observations on the vitamin metabolism of the common fowl. 2. The effects of estrogen and progesterone injections in immature pullets on the riboflavin content of the magnum. Brit. J. Nutrit. 6: 386-392. Brant, J. W. A., and A. V. Nalbandov, 1956. Role of
sex hormones in albumen secretion by the oviduct of chickens. Poultry Sci. 35: 692-700. Brown, W. O., 1954. Nitrogen metabolism in the folic acid-deficient chick studies by a balance technique. Tenth World Poultry Congress, Edinburgh. page 149-152. Durrum, E. L., 1951. Paper ionophoresis. Science, 113: 66-69. Evans, R. J., and S. L. Bandemer, 1956. Separation of egg white protein by paper electrophoresis. Agric. Food Chem. 4: 802-810. Hertz, R., 1950. Endocrine and vitamin factors in hormone-induced tissue growth. Texas. Rep. Biol. Med. 8: 154-158. Lodge, P. D. B., and C. L. Smith, 1960. Hormonal control of secretion in the oviduct of the Amphibia. Nature, 185: 774-775. Mandeles, S., and E. D. Ducay, 1962. Site of egg white protein formation. J. Biol. Chem. 237: 3196-3199. Markham, R., 1942. A steam distillation apparatus suitable for micro-Kjeldahl analysis. Biochem. J. 36: 790-791. Mulgaonkar, A. G., and A. Sreenivasan, 1957. Alteration in rat serum proteins in folic acid and vitamin B12 deficiency. Proc. Soc. Exp. Biol. Med. 94: 44-46. Smith, A. H., C. M. Winget and T. J. Hague, 1957. Effect of local x-irradiation on albumen formation by the hen's oviduct. Am. J. Physiol. 191: 605-609. Wolff, E., 1936. L'action de l'oestrone sur l'oviducte et la cloaque des poussins femelle intersexues et males et sur les reaction des cannaux de Muller des oiseaux aux hormones sexuelles. Compt. Rend. Soc. Biol. 123: 235-236.
Relative Vitamin K Potency of Two Water-Soluble Menadione Analogues 1 PAUL GRIMINGER
Department of Animal Sciences, Rutgers, The State University, New Brunswick, New Jersey (Received for publication July 2. 1964)
M
ENADIONE sodium bisulfite, a used as a source of vitamin K in poultry water-soluble form of the pro- feeds. This form differs in its vitamin K vitamin 2-methyl-l,4-naphthoquinone, is activity from the fat-soluble menadione (Frost and Spruth, 1955). The data pre1 Paper of the Journal Series, New Jersey Agrisented in this report indicate that this cultural Experiment Station, Rutgers, The State difference is not necessarily a function, or, University of New Jersey, Department of Animal at least, not only a function of the change Sciences, New Brunswick.
Downloaded from http://ps.oxfordjournals.org/ at University of Nebraska-Lincoln Libraries on June 10, 2016
estradiol, estradiol plus progesterone and estradiol plus testosterone were separated by a paper electrophoretic technique. Evidence is presented to show that albumins and conalbumins of similar mobility to those from egg-white proteins are present after estradiol treatment but that there is an increase in the quantity of water-soluble proteins when both hormones are administered. An experiment is also described in which it is shown that in folic acid deficiency there is a large reduction in the quantity of albumins in the water-soluble proteins in the oviducts of immature female chicks treated with estradiol and estradiol plus testosterone.
211
V I T A M I N K. P O T E N C Y
in solubility status, since another watersoluble form of menadione, namely, menadione dimethyl pyrimidinol bisulfite, 2 differs in its activity from menadione sodium bisulfite. EXPERIMENTAL
* Hetrazeen, kindly supplied by the Heterochemical Corporation, Valley Stream, New York.
RESULTS
The levels of supplementation and the corresponding average prothrombin times are shown in Table 1. Since the relative response to the two forms of menadione at 2 and 3 weeks of age was essentially simiTABLE 1.—Prothrombin times of chickens fed 0.2% sulfaquinoxaline, and graded levels of 2 water-soluble menadione analogues1 2-weeks2 Level of supplemenMSBC 4 MPB 1 tation mg./kg. 0.1 0.2 0.4 0.8 1.6 3.2 6.4
sec. 119.8 148.4 48.9 32.9 29.7 23.0 20.4
sec. 104.3 67.7 37.2 33.4 22.0 21.3 18.1
3-weeks3 MSBC
MPB
sec. 180.0[4] 54.1 [2] 38.0 26.7 23.9 17.9 18.8
sec. 98.0 35.2 27.7 20.3 19.6 17.8 14.3
1 All averages were calculated on the basis of the reciprocals of the prothrombin times (Griminger, 1962b). Samples not clotted within 180 sec. were treated as if they had clotted at 180 sec. The use of reciprocals minimizes the error thus incurred. 2 Averages of 8 chicks each. 3 Averages of 7 chicks each, except where shown differently (in brackets). 4 MSBC—menadione sodium bisulfite complex, containing 33% menadione; MPB—Menadione dimethyl pyrimidinol bisulfite, containing 45% menadione.
Downloaded from http://ps.oxfordjournals.org/ at University of Nebraska-Lincoln Libraries on June 10, 2016
Fourteen groups of 20 cross-bred, female chicks received a purified vitamin K-deficient diet (Griminger, 1962a) from hatching to 1 week of age. At t h a t time group uniformity was increased by eliminating 4 chicks with extreme weights. From 1 to 3 weeks of age the chicks received the K-deficient diet supplemented with 0.2% sulfaquinoxaline and with seven graded levels of each of the two water-soluble menadione derivatives, menadione sodium bisulfite complex (MSBC), containing 3 3 % menadione, and menadione dimethyl pyrimidinol bisulfite ( M P B ) , containing 4 5 % menadione. At 2 weeks of age, 1 ml. of blood was drawn by heart puncture from each of 8 chicks picked at random from each of the 14 groups (a total of 112 chicks), and plasma prothrombin times determined b y the method of Quick (1957), using sodium citrate as an anticoagulant, and acetonedehydrated chicken brain powder as a source of thromboplastin (Griminger, 1962a). At 3 weeks of age the procedure was repeated, using 7 chicks from each group, or fewer where necessitated b y mortality. During the entire experiment the chicks were kept on raised wire floors in electrically heated battery brooders; the stainless steel water containers, the feed containers, and the wire screens underwent frequent cleaning to prevent the chicks from obtaining vitamin K synthesized by microorganisms in the water, the feed, or the droppings adhering to the wire screen.
T h e data were averaged on the basis of the reciprocals of the individual prothrombin times, and the mean values thus obtained reconverted to seconds. I n the graphic presentation the averages of the reciprocals were plotted against the logarithms (decimal) of the dose (vitamin K supplement), as suggested b y Almquist (1954). Good comparisons between different forms of vitamin K are difficult to obtain even when a purified diet is fed. Increasing the vitamin K requirement through the use of sulfaquinoxaline as a stress agent to improve evaluation of different vitamin K forms was proposed b y Perdue and Frost (1961), and was successfully employed for the present comparisons.
212
P. GRIMINGER
600
MPB-
MSBC^
500
z
400
oci 5
i t-
o a.
300
<
2 «>
200
100,
__1 0.1
I
0.4
J_ 0.6
-J_ 1.6
3.2
0.2 SUPPLEMENT
(IN
MG/KG
6.4
DIET)
VITAMIN
FIG. 1. Prothrombin time response to graded levels of menadione dimethyl pyrimidinol bisulfite (MPB) and menadione sodium bisulfite complex (MSBC).
lar, weighted averages were calculated; these averages, and their respective regression lines, calculated by the method of least squares, are shown in Fig. 1. It appears that the response lines to the two water-soluble menadione analogues compared here have a similar slope; however, equivalent amounts of MPB were considerably more active than MSBC in decreasing prothrombin times. If we compare the levels needed to obtain an average prothrombin time of 40 sec. (1/250 X104), requirements of 0.285 mg. of MPB or of 0.52 of MSBC/kg. diet can be read from the graph; this is a ratio of 1:1.8. To obtain an average prothrombin time of 20 sec. (1/500 X104), the corresponding dosages would be 2.25 and 4.55, or a ratio of approximately 1:2. Since the menadione content of MPB is higher than that of MSBC, a ratio of 1:1.36 may be expected
if utilization of menadione from these two compounds were alike. It may be concluded from the appreciably higher ratios observed in the present trial, that the menadione moiety of MPB is more efficiently utilized than that of MSBC in decreasing prothrombin times in chicks. SUMMARY
Two water-soluble menadione analogues were compared for vitamin K activity, using a vitamin K-deficient ration supplemented with 0.2% sulfaquinoxaline. Over the range of dietary supplementation used, menadione dimethyl pyrimidinol bisulfite promoted a higher plasma prothrombin level in growing chicks than did menadione sodium bisulfite. This difference in activity was greater than could be explained on the basis of the menadione content of the two compounds.
Downloaded from http://ps.oxfordjournals.org/ at University of Nebraska-Lincoln Libraries on June 10, 2016
o a.
VITAMIN K POTENCY REFERENCES Almquist, H. J., 1954. The Vitamins. Vol. 2. Academic Press, New York. Frost, D. V., and H. C. Spruth, 1955. Control of hemorrhagic condition in chickens with menadione sodium bisulfite. Poultry Sci. 34: 56-64. Griminger, P., 1962a. Prothrombin bioassay for vitamin K with different thromboplastin prepara-
213
tions. Intern. J. Vit. Res. 32: 405-412. Griminger, P., 1962b. Arsanilic acid and blood coagulation. Poultry Sci. 4 1 : 982-985. Perdue, H. S., and D. V. Frost, 1961. Recent developments on a proposed ANRC vitamin K reference standard. Feedstuffs, January 21, p. 56. Quick, A. J., 1957 (1959). Hemorrhagic Diseases, Lea and Febiger, Philadelphia.
P. W. WALDROUP2, R. H. HARMS 2 AND M. FRIED 8 Florida A gricuitural Experiment Stations and College of Medicine, University of Florida, Gainesville, Florida (Received for publication July 3, 1964)
1
T HAS been reported that lowering the level of protein in the diet of laying hens resulted in a significant drop in egg weight (Harms and Waldroup, 1962). The present experiment was designed to compare the serum and egg proteins electrophoretically to determine if the reduced egg weight was causally related to a variation in any of the electrophoretic components. MATERIALS AND METHODS
Two diets containing 9 and 17 percent protein (Waldroup and Harms, 1964) were prepared. Each diet was fed to a group of ten egg-production type pullets which had been in production for approximately two months and were laying at a rate of about 80 percent when the experiment began. Blood samples were obtained by cardiac puncture from pullets on the second day of each week over an eight week test period. Samples were obtained at 10:00 1
Florida Agri. Exp. Sta. Journal Series No. 1899. Department of Poultry Science 3 Department of Biochemistry Supported in part by a grant (AM-02547) from the National Institute of Health. 2
a.m. from all pullets that had laid an egg on that day. The blood was allowed to clot and then centrifuged to collect the serum. For determination of egg constituents the egg contents were blended with distilled water in an egg: water ratio of 1:4. Serum and egg samples of four pullets from each group were randomly selected for electrophoretic studies. Samples of the blood sera and the diluted egg suspension were analyzed for protein and lipoprotein components using the Beckman Spinco Electrophoresis System. The procedures outlined in the Beckman Paper Electrophoresis System Manual (Anonymous, 1958) were followed in determining the proteins and lipoproteins. Duplicate paper strips spotted with sera and egg suspension were run in parallel. In addition, total protein content was determined by the Biuret method (Gornall et al., 1949). RESULTS AND DISCUSSION
On the paper strips on which sera were separated, three components were observed following staining for protein with bromphenol blue. In order of decreasing mobility these were albumin, a .mixed
Downloaded from http://ps.oxfordjournals.org/ at University of Nebraska-Lincoln Libraries on June 10, 2016
Alterations in the Serum Protein Components of Laying Hens on Low Protein Diets1