Metabolism of Vitamin K and Influence on Prothrombin Time in Milk-Fed Preruminant Calves 1 K. E. NESTOR, JR., and H. R. CONRAD Department of Dairy Science Ohio Agricultural Research and Development Center The Ohio State University Wooster 44691 ABSTRACT
loquinone and has equal biological activity (15). Menadione sodium bisulfite complex (MSBq is a salt of menadione used in many feed fonnulations. The metabolism of dietary vitamin K has been studied in chickens and rats (7, 8, 14). Chemical interconversion of vitamin K by intestinal microorganisms was observed (8). Not much research has been done on vitamin K in cattle, and little is known of the metabolism of vitamin K in the ruminant or preruminant animal. The objective of this experiment was to study the metabolism of dietary MSBC and phylloquinone in preruminant veal and young female calves fed milk replacers to detennine if the milk-fed calves have an additional dietary requirement for vitamin K.
The metabolism of vitamin K was studied in 66 preruminant veal calves that were fed supplemental menadione sodium bisulfite complex or phylloquinone. Menadione sodium bisulfite complex was converted by intestinal microorganisms to menaquinone-4 and absorbed and stored in the liver as menaquinone-4. Phylloquinone was absorbed unchanged. Production of menaquinones 6, 7, 8, and 10 by intestinal microorganisms also was observed, but was not dependent upon dietary vitamin K. No difference was noted in prothrombin time among the groups. Intestinal microorganisms provide sufficient vitamin K to meet the physiological needs of calves fed milk replacers. Menaquinone-4 was the fonn of vitamin K used to meet the calf's requirement. (Key words: vitamin K, menadione, prothrombin time)
MATERIALS AND METHODS
INTRODUCTION
Vitamin K is a generic tenn that refers to several compounds exhibiting vitamin K activity. These include phylloquinone (Kl), found naturally in green plants; menaquinones (K2), a series of vitamins produced by microorganisms; and menadione (K3), the parent compound. Nomenclature used for the menaquinones is MK-n, where n refers to the number of isoprenoid groups in the side chain of the molecule. Menaquinone-4 is similar in structure to phyl-
Received October 12, 1989. Accepted lune 11, 1990. ISaIaries and research support provided by stale and federal fonds appropriated to the Ohio Agricultural Research and Development Center, The Ohio Stale University. Manuscript Number 304-89. 1990 1 Daily Sci 73:3291-3296
Sixty-six animals were used in a randomized block experiment to study vitamin K in the preruminant calf. Two trials were conducted. Eighteen male Holstein calves were used in trial 1 and 48 female Holstein calves were used in trial 2. Bull calves remained with their dams to 3 d of age, and then were housed in individual elevated wooden crates until slaughter at approximately 13 wk of age. In trials 1 and 2, humidity and temperature were controlled by exchanging the air in the nursery barn (210 cubic m, 20 stalls) with outside air. Time and humidistat-eontrolled fans and two space heaters were used to limit minimum temperature to 7°C and humidity approaching outside air. The estimated mean air exchange was 13.3 times/h, but varied widely with environmental humidity. The 12-yr mortality rate in the calf bam was less than .5%/yr. Animals were fed exclusively an all-milk component milk replacer (Milk Specialties, Dundee, IL) that lacked vitamin K supplement.
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The replacer was fed twice daily at approximately 0500 and 1300 h in equal amounts according to the manufacturer's recommendations. A starter formula (20% CP, 16% fat) was fed during the first 3 wk, and a finisher formula (16% CP, 18% fat) was fed from wk 8 until slaughter. Animals were acclimated to the finisher formula by increasing fmisher and decreasing starter by 20%/wk between wk 4 and 7 and 10% between wk 7 and 8. All nutrients were balanced to meet the nutritional needs of veal calves. The starter and fmisher were medicated with oxytetracycline and chlortetracycline, respectively. Animals in trial 1 were randomly assigned to a treatment group at 4 d of age. Treatments were: control-no supplemental MSBC, MSBC supplemented at 8 mg/kg diet OM per day, and MSBC supplemented at 16 mg/kg diet OM per day. The MSBC was given daily during the a.m. feeding after being dissolved in distilled deionized water and added to the milk replacer solution. The replacer was mixed according to the manufacturer's recommendations. In trial 2, 48 female Holstein calves were managed as in trial 1 with the following differences. Animals were housed in calf tie stalls (1.35 m 2 of space per calf) from 4 to 56 d of age. Animals were weaned at d 57 and entered into normal herd practices to be raised as herd replacements. Treatments used in this trial were: control - no supplemental vitamin K, MSBC supplemented at 4 mg/kg diet OM, MSBC supplemented at 8 mg/kg diet OM, MSBC supplemented at 16 mg/kg diet OM, MSBC supplemented at 32 mg/kg diet OM, and vitamin K 1 (2-methyl-3-phytl-l,4-naphthoquinone; 3-phytylmenadione) supplemented at 4 mg/kg diet OM. All treatments were given daily. The MSBC was given as described in trial 1. Vitamin K 1 was diluted in com oil and was given to calves in gelatin capsules. Bull calves were slaughtered (Kosher) at 89 ± 8.2 d of age. The digestive tract, liver, and spleen of each animal were collected. Subsamples of liver, spleen, and intestine (jejunum) were collected and frozen until later analysis. Blood and fecal samples were collected from each animal twice weekly (every 3 to 4 d) for trials 1 and 2. Samples were collected approximately 2 to 3 h postfeeding. Blood was collected by venipuncture of the jugular vein using a 20-gauge needle. Two samples of approxiJournal of Dairy Science Vol. 73,
No. 11, 1990
mately 10 ml blood were collected. The blood was immediately placed on ice until further laboratory analysis. Fecal samples (50 to 250 g) were collected into a seal-top plastic bag and placed on ice until transported to the laboratory. Care was taken to minimize exposure of the samples to light by storing in darkrooms and performing analysis under yellow incandescent light (100 W). Fecal samples were frozen (-20"C) until further analysis. An aliquot of approximately 2 ml of whole blood was collected into a 5-ml polypropylene tube and frozen (-20°C) until analysis. From the same sample tube, whole blood was drawn into a capillary tube and centrifuged (13,000 x g) with a microhematocrit centrifuge for 5 min. The hematocrit was determined using a micro-capillary reader. The remaining blood was centrifuged at 1500 x g for 15 min, and the plasma was collected. All of the laboratory sample preparations and procedures were done in a laboratory illuminated by one 40-W yellow light bulb. Prothrombin time was determined on the subsample of plasma (11). The prothrombin time test was performed within 2 h of sample collection. The K vitamins (phylloquinone and menaquinones) were determined in plasma with a HPLC using a fluorescence detector with a deuterium light source according to Harron et al. (3). This procedure, with slight modifications, was also used to determine the K vitamins in fecal and tissue samples. The K vitamins were identified by comparing peak retention times in solvent solutions and samples to standards of the individual vitamins. Oata were analyzed by least squares ANDVA (4). Main effects tested were diet, week, and diet x week interaction. Individual means were tested by independent contrasts when significant differences were observed in the variables. Orthogonal polynomials were determined for week. Significant difference was P
loquinone and has equal biological activity (15). Menadione sodium bisulfite complex (MSBq is a salt of menadione used in many feed fonnulations. The metabolism of dietary vitamin K has been studied in chickens and rats (7, 8, 14). Chemical interconversion of vitamin K by intestinal microorganisms was observed (8). Not much research has been done on vitamin K in cattle, and little is known of the metabolism of vitamin K in the ruminant or preruminant animal. The objective of this experiment was to study the metabolism of dietary MSBC and phylloquinone in preruminant veal and young female calves fed milk replacers to detennine if the milk-fed calves have an additional dietary requirement for vitamin K.
The metabolism of vitamin K was studied in 66 preruminant veal calves that were fed supplemental menadione sodium bisulfite complex or phylloquinone. Menadione sodium bisulfite complex was converted by intestinal microorganisms to menaquinone-4 and absorbed and stored in the liver as menaquinone-4. Phylloquinone was absorbed unchanged. Production of menaquinones 6, 7, 8, and 10 by intestinal microorganisms also was observed, but was not dependent upon dietary vitamin K. No difference was noted in prothrombin time among the groups. Intestinal microorganisms provide sufficient vitamin K to meet the physiological needs of calves fed milk replacers. Menaquinone-4 was the fonn of vitamin K used to meet the calf's requirement. (Key words: vitamin K, menadione, prothrombin time)
MATERIALS AND METHODS
INTRODUCTION
Vitamin K is a generic tenn that refers to several compounds exhibiting vitamin K activity. These include phylloquinone (Kl), found naturally in green plants; menaquinones (K2), a series of vitamins produced by microorganisms; and menadione (K3), the parent compound. Nomenclature used for the menaquinones is MK-n, where n refers to the number of isoprenoid groups in the side chain of the molecule. Menaquinone-4 is similar in structure to phyl-
Received October 12, 1989. Accepted lune 11, 1990. ISaIaries and research support provided by stale and federal fonds appropriated to the Ohio Agricultural Research and Development Center, The Ohio Stale University. Manuscript Number 304-89. 1990 1 Daily Sci 73:3291-3296
Sixty-six animals were used in a randomized block experiment to study vitamin K in the preruminant calf. Two trials were conducted. Eighteen male Holstein calves were used in trial 1 and 48 female Holstein calves were used in trial 2. Bull calves remained with their dams to 3 d of age, and then were housed in individual elevated wooden crates until slaughter at approximately 13 wk of age. In trials 1 and 2, humidity and temperature were controlled by exchanging the air in the nursery barn (210 cubic m, 20 stalls) with outside air. Time and humidistat-eontrolled fans and two space heaters were used to limit minimum temperature to 7°C and humidity approaching outside air. The estimated mean air exchange was 13.3 times/h, but varied widely with environmental humidity. The 12-yr mortality rate in the calf bam was less than .5%/yr. Animals were fed exclusively an all-milk component milk replacer (Milk Specialties, Dundee, IL) that lacked vitamin K supplement.
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The replacer was fed twice daily at approximately 0500 and 1300 h in equal amounts according to the manufacturer's recommendations. A starter formula (20% CP, 16% fat) was fed during the first 3 wk, and a finisher formula (16% CP, 18% fat) was fed from wk 8 until slaughter. Animals were acclimated to the finisher formula by increasing fmisher and decreasing starter by 20%/wk between wk 4 and 7 and 10% between wk 7 and 8. All nutrients were balanced to meet the nutritional needs of veal calves. The starter and fmisher were medicated with oxytetracycline and chlortetracycline, respectively. Animals in trial 1 were randomly assigned to a treatment group at 4 d of age. Treatments were: control-no supplemental MSBC, MSBC supplemented at 8 mg/kg diet OM per day, and MSBC supplemented at 16 mg/kg diet OM per day. The MSBC was given daily during the a.m. feeding after being dissolved in distilled deionized water and added to the milk replacer solution. The replacer was mixed according to the manufacturer's recommendations. In trial 2, 48 female Holstein calves were managed as in trial 1 with the following differences. Animals were housed in calf tie stalls (1.35 m 2 of space per calf) from 4 to 56 d of age. Animals were weaned at d 57 and entered into normal herd practices to be raised as herd replacements. Treatments used in this trial were: control - no supplemental vitamin K, MSBC supplemented at 4 mg/kg diet OM, MSBC supplemented at 8 mg/kg diet OM, MSBC supplemented at 16 mg/kg diet OM, MSBC supplemented at 32 mg/kg diet OM, and vitamin K 1 (2-methyl-3-phytl-l,4-naphthoquinone; 3-phytylmenadione) supplemented at 4 mg/kg diet OM. All treatments were given daily. The MSBC was given as described in trial 1. Vitamin K 1 was diluted in com oil and was given to calves in gelatin capsules. Bull calves were slaughtered (Kosher) at 89 ± 8.2 d of age. The digestive tract, liver, and spleen of each animal were collected. Subsamples of liver, spleen, and intestine (jejunum) were collected and frozen until later analysis. Blood and fecal samples were collected from each animal twice weekly (every 3 to 4 d) for trials 1 and 2. Samples were collected approximately 2 to 3 h postfeeding. Blood was collected by venipuncture of the jugular vein using a 20-gauge needle. Two samples of approxiJournal of Dairy Science Vol. 73,
No. 11, 1990
mately 10 ml blood were collected. The blood was immediately placed on ice until further laboratory analysis. Fecal samples (50 to 250 g) were collected into a seal-top plastic bag and placed on ice until transported to the laboratory. Care was taken to minimize exposure of the samples to light by storing in darkrooms and performing analysis under yellow incandescent light (100 W). Fecal samples were frozen (-20"C) until further analysis. An aliquot of approximately 2 ml of whole blood was collected into a 5-ml polypropylene tube and frozen (-20°C) until analysis. From the same sample tube, whole blood was drawn into a capillary tube and centrifuged (13,000 x g) with a microhematocrit centrifuge for 5 min. The hematocrit was determined using a micro-capillary reader. The remaining blood was centrifuged at 1500 x g for 15 min, and the plasma was collected. All of the laboratory sample preparations and procedures were done in a laboratory illuminated by one 40-W yellow light bulb. Prothrombin time was determined on the subsample of plasma (11). The prothrombin time test was performed within 2 h of sample collection. The K vitamins (phylloquinone and menaquinones) were determined in plasma with a HPLC using a fluorescence detector with a deuterium light source according to Harron et al. (3). This procedure, with slight modifications, was also used to determine the K vitamins in fecal and tissue samples. The K vitamins were identified by comparing peak retention times in solvent solutions and samples to standards of the individual vitamins. Oata were analyzed by least squares ANDVA (4). Main effects tested were diet, week, and diet x week interaction. Individual means were tested by independent contrasts when significant differences were observed in the variables. Orthogonal polynomials were determined for week. Significant difference was P
