Nutrient Metabolism
Comparative Metabolism and Requirement K in Chicks and Rats1
of Vitamin
BIRGIT H. WILL, YÃœJIUSUI* ANDJ. W. SÃœTTIE Departments of Biochemistry and Nutritional Sciences, College of Agricultural and Life Sciences, University of Wisconsin-Madison, Madison, Wl 53706 and *Second Department of Surgery, Kyoto University, 54-Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606, Japan enzyme that converts specific glutamyl residues in intracellular precursors of vitamin K-dependent pro teins to y-carboxyglutamyl (Gla) residues in the mature protein. These Gla residues are essential for full biological activity of this class of proteins. A vitamin K deficiency is most commonly defined by an impairment of blood coagulation resulting from decreased prothrombin, factor VII, factor IX and factor X activity, and is usually measured as a prolongation of a one-stage prothrombin time. Such a deficiency is easily produced in poultry by dietary restriction of the vitamin, but is not easily accomplished in most other species, including rats. The dietary vitamin K re quirement of most species is not well defined and depends to a large extent on the sensitivity of the clotting-based assay used, the route of vitamin ad ministration, and the form of the vitamin used. However, the requirement of chicks is usually indi cated as being 5-10 times that of rats (Suttie 1991). The basis for this species difference in requirement has not been established and is usually attributed to poor absorption of dietary vitamin by the short digestive tract of chicks, and to the importance of coprophagy in rats to furnish menaquinones. Other possible explanations would include alteration in the rate or route of metabolism of the vitamin in the two species, or alterations in the activity or sensitivity of enzymes involved in vitamin K action between chicks and rats. This report describes studies directed at determining the important factors responsible for the different vitamin K requirements of rats and chicks.
ABSTRACT The metabolic basis for the high vitamin K requirement of chicks compared with rats was inves tigated. When chicks and rats were fed the same diet, containing 500 ug phylloquinone/kg, the total amounts of phylloquinone and its epoxide metabolite found in the liver and plasma were similar in both species. However, phylloquinone 2,3-epoxide was present in high concen trations in chick liver and serum but not in rat liver and serum. This metabolite of the vitamin is normally reduced by a hepatic vitamin K epoxide reducÃ-ase. The activity of this enzyme in chicks was -10% of that in rats, and the inability of chicks to effectively recycle the epoxide of vitamin K seems to be the major factor in its high requirement. Other species differences in vitamin K metabolism were observed. Much higher concentrations of bacterial menaquinones were present in rat feces compared with chick fèces, but neither species had ap preciable hepatic concentrations of menaquinones. Chicks, but not rats, were found to have a liver concen tration of menaquinone-4 that exceeded that of phylloq uinone. This vitamer was present even when its recog nized precursor, menadione, was not present in the diet, and the data indicate that chicks convert phylloquinone to menaquinone-4 under the conditions of these experi ments. The mechanism of this conversion was not es tablished. J. Nutr. 122: 2354-2360, 1992. INDEXING KEY WORDS:
•vitamin K •phylloquinone •rats •menaquinone-4 •chicks
Vitamin K was discovered by Dam (1935) as a dietary factor needed to cure a hemorrhagic condition that developed in chicks fed a diet low in lipids. Vitamin activity was eventually shown to be a property of 2-Me-l,4-naphthoquinones substituted at the 3 position with a phytyl group in the case of the plant vitamin, phylloquinone, or a polyisoprenyl group in the case of bacterial menaquinones (Suttie 1991). Much later the biochemical role of the vitamin was shown to be that of a substrate for a hepatic 0022-3166/92
$3.00 ©1992 American
Institute
of Nutrition.
'Supported by the College of Agricultural and Life Sciences of the University of Wisconsin-Madison, and in part by grants DK14881 and HL-29586 from the National Institutes of Health, Bethesda, MD, and CRCR-1-1415 from the U.S. Department of Agriculture, Washington, DC.
Received 27 April 1992. Accepted 28 July 1992. 2354
Downloaded from https://academic.oup.com/jn/article-abstract/122/12/2354/4754665 by Denise Hannibal user on 08 August 2018
VITAMIN K METABOLISM AND REQUIREMENT
2355
MATERIALS AND METHODS TABLE
Animals and diets. Tissue and fecal phylloquinone and menaquinone concentrations were studied in 250- to 270-g male rats obtained from SpragueDawley (Madison, WI) and 4- to 5-wk-old LeghornNuhamp chicks obtained from the University of Wis consin-Madison Poultry Science Department. All animal protocols were approved by the University of Wisconsin-Madison Animal Care Committee. Both species were fed a soybean protein, sucrose and cornstarch, vegetable oil-based vitamin K-deficient diet (#81053) obtained from Teklad (Madison, WI), con taining 3 ug phylloquinone/kg by direct analysis (Kindberg and Suttie 1989). Animals were fed the vitamin K-deficient diet for 3 d, then fed the same diet supplemented with 500 ug/kg of phylloquinone (Sigma Chemical, St. Louis, MO) for an additional 8 d. Animals were housed in standard wire-bottomed cages under conditions of 12 h light: 12 h dark and were fed the diet with ad libitum access. They were killed by decapitation, and plasma, serum, livers and fecal material from the colon were obtained, frozen and stored at -70°C until analysis. Comparative me tabolism of menadione and phylloquinone was studied in similar rats and chicks fed phylloquinone or menadione. Phylloquinone or menadione, as pure menadione, were dissolved in a small amount of ethanol and mixed into the vitamin K-deficient diet the day before the experiments were started. Diets were stored at 4°C.Synthesis of menaquinone-4 (MK4) was studied in 4- to 8-wk-old Leghorn-Nuhamp chicks that were fed the vitamin K-deficient diet and injected with 50 uL of ethanol containing 50 ug/kg body wt of phylloquinone or menadione in the wing vein each day. Activities of the hepatic vitamin Kdependent carboxylase and vitamin K epoxide reducÃ-ase were studied in livers obtained from 7-wk-old Cornish-White Rock broilers or 250-g male rats maintained for 8 d on diets containing 500 ug phylloquinone/kg. Analytical procedures. One-stage prothrombin times were determined using a fibrometer (BBL, Bal timore, MD) and a commercial rabbit brain and lung thromboplastin preparation (Simplastin, Organon Teknika Corp., Durham, NC). Plasma prothrombin concentration was measured using a chromogenic assay for thrombin following thromboplastin acti vation of prothrombin as described by Shah et al. (1984). Total plasma prothrombin, which measures both biologically active and undercarboxylated forms, was measured following snake venom activation as described by Allison et al. (1987). Under-y-carboxylated prothrombin was measured using the total pro thrombin assay after biologically active prothrombin was absorbed by barium citrate as described by Francis (1988). Microsomal pellets for enzyme assays were pre pared as described by Cheung et al. (1989). Vitamin Downloaded from https://academic.oup.com/jn/article-abstract/122/12/2354/4754665 by Denise Hannibal user on 08 August 2018
1
Measurements of vitamin K status in plasma of rats and chicks fed a diet containing 500 ug phylloquinone/kg1 Assay Prothrombin time, s Prothrombin concentration, % Total prothrombin concentration, % Under-y-carboxylated prothrombin, % 'Prothrombin
Chick
Rat 10.7 (10.4-11.4)
36.4 (29.4-45.7)
87
±2
22
±2
110
±3
42
±2
1.7 ±0.2
3.6 ±0.4
times are means and ranges for eight animals per
group. Other values are means ±SEMfor eight animals per group and are expressed as the percentage of a control normal rat plasma pool. Prothrombin times and prothrombin concentration assays utilized a commercial rabbit thromboplastin preparation optimized for human plasma assays, and plasma from different species may not be activated at the same rate.
K-dependent carboxylase activity in microsomes was measured by a modification of the method described by de Boer-van den Berg et al. (1987), which used 0.5 mmol/L Boc-EEL-methyl ester (Bachern, Terranee, CA) instead of Boc-FLEEL-methyl ester as a substrate. The buffer used contained 0.25 mol/L sucrose, 0.025 mol/L imidazole, pH 7.3, and 0.4 g/L phosphatidyl choline, 0.4 g/L CHAPS and 0.4 g/L sodium cholate. Microsomal epoxide reducÃ-ase activily was measured as described by Misenheimer and Suttie (1990). Phylloquinone, MK-4 and the corresponding 2,3-epoxides in serum were measured by HPLC ana lyses as previously described (Usui et al. 1990) utilizing fluorometric detection (Nagaoko et al. 1989, Nishimura et al. 1990). Vitamin K was extracted from l g of liver tissue as previously described (Usui et al. 1989), and phylloquinone, phylloquinone epoxide, MK-4 and MK-4 epoxide were separated and detected as described above. Long-chain menaquinones were separated by gradient HPLC solution as described by Usui et al. (1989) with omission of the thin-layerchromatographic step because of the low level of contaminants in rat and chick liver compared with human liver. Fluorometric detection was preceded by reduction of menaquinones on a Platinum-Black column (Nagaoko et al. 1989). Rat and chick feces (0.1-0.2 g) were analyzed for phylloquinone and menaquinones as described for liver.
RESULTS When rats and chicks were fed the same diet con taining 500 ug phylloquinone/kg diet for 8 d, the apparent concentration of prothrombin in rat plasma was about four times that observed in chick plasma (Table 1). This difference in apparent prothrombin
WILL ET AL.
2356 40
O
Chicken
Rat
Chicken
Chicken
FIGURE1 Liverand serumphylloquinone(solidbar)and phylloquinone 2,3-epoxide(openbar) concentrations in rats and chicks fed a vitamin K-deficient diet for 3 d and then fed a diet supplemented with 500 ug phylloquinone/kgdiet for 8 d. Valuesare means ± SEMfor eight animals per group.
FIGURE 2 Liver and serum menaquinone-4 (solid bar) and menaquinone-4 2,3-epoxide (open bar) concentrations in rats and chicks fed a vitamin K-deficient diet for 3 d and then fed a diet supplemented with 500 ug phylloquinone/kg diet for 8 d. Values are means ±SEMfor eight animals per group.
concentration was not altered when the vitamin K concentration of the diet was increased to 2500 ug phylloquinone/kg diet (data not shown). The ability of the two species to absorb and retain dietary vitamin K was assessed by comparing the serum and liver phyl loquinone concentrations in both species (Fig. 1). The phylloquinone concentration in both serum and liver of the rats was -50% higher than in the chicks. The concentration of the major vitamin K metabolite, its 2,3-epoxide, was also measured. Phylloquinone epoxide was present in high concentrations in both chick liver and serum, but present in only low con centrations in rat liver and serum. The total concen tration of phylloquinone and its epoxide in both serum and liver was similar in chicks and rats. Menaquinones produced by gut bacteria are a second potential source of vitamin K, and the concen tration of these vitamers (MK-5 through MK-13) was measured in rat and chick liver and in feces obtained from the colon when the animals were killed (Table 2). Both rat and chick liver contained only low con centrations of menaquinones. The concentrations of most long-chain menaquinones in rat liver were below the limit of detection, whereas measurable menaquinone concentrations, particularly of MK-8 and MK-10, were found in chick liver. However, the total amount of MK-5 through MK-13 was only 4 nmol/kg chick liver, or -20% of the phylloquinone concentration of chick liver. Rat fecal contents, in contrast to those of chicks, contained large amounts of long-chain menaquinones. The nearly 14 umol/kg dry wt observed in rat feces was about 50 times that found in chick feces. A major difference in liver vitamin content in the two species was a high concentration of MK-4 in chick liver (Fig. 2). This vitamin is not a major bac terial metabolite (Fernandez and Collins 1987, Ramotar et al. 1984) but is known to be readily
formed by menadione alkylation in the liver (Dialameh et al. 1971, Martius and Esser 1958, Taggart and Matschiner 1969). In rat liver, MK-4 was detected at a concentration of -10% that of phylloquinone, whereas in chick liver the concentration of MK-4 was more than twice that of phylloquinone. High concen trations of MK-4 were also found in chick serum, but not rat serum. As was found for phylloquinone, the 2,3-epoxide of MK-4 was present in chick liver at -50% the concentration of MK-4; however, only low concentrations of MK-4 epoxide were found in chick serum. The increased dietary vitamin K requirement for chicks compared with rats could result from a decreased affinity of the chick vitamin K-dependent y-glutamyl carboxylase for its substrate, the reduced form of vitamin K. The increased concentrations of vitamin K epoxides observed in chick liver and serum (Fig. 1 and 2) could result from an inability of chicks to metabolize the epoxide to excretory products or to reduce the epoxide to vitamin K via the well-demon strated epoxide reducÃ-ase pathway. Assay of these enzymes (Table 3) indicates that there are species differences in both the activity and substrate Km values for these enzymes. The chick carboxylase had a higher Km for reduced vitamin K than the rat enzyme, and this difference was more pronounced when reduced MK-4 rather than phylloquinone was utilized as a substrate. The hepatic carboxylase ac tivity (Vmax) of chicks was -50% that of rats when phylloquinone was used as a substrate, but similar in the presence of saturating MK-4. A much more pro nounced difference in the properties of the rat and chick enzymes was seen in the activity of the hepatic epoxide reducÃ-ase. Although the Km values measured did not differ appreciably, the rate of either phylloq uinone epoxide or MK-4 epoxide reduction by the
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VITAMIN K METABOLISM AND REQUIREMENT
2357
TABLE 2 Concentration of bacterial menaquinones in rat and chick liver and feces1 Feces menaquinones
Liver menaquinones Vitamer
Rat
Chick
Rat
Chick
wet wt0.06CPND2.00.41.40.5NDND4±±±±±0.060.80.1.06.0529153031080070083302170 wt450CP180186NDNDND258± MK-5MK-6MK-7MK-8MK-9MK-10MK-11MK-12MK-13Total
MKNDCPNDNDCPNDNDNDND
Comparative Metabolism and Requirement K in Chicks and Rats1
of Vitamin
BIRGIT H. WILL, YÃœJIUSUI* ANDJ. W. SÃœTTIE Departments of Biochemistry and Nutritional Sciences, College of Agricultural and Life Sciences, University of Wisconsin-Madison, Madison, Wl 53706 and *Second Department of Surgery, Kyoto University, 54-Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606, Japan enzyme that converts specific glutamyl residues in intracellular precursors of vitamin K-dependent pro teins to y-carboxyglutamyl (Gla) residues in the mature protein. These Gla residues are essential for full biological activity of this class of proteins. A vitamin K deficiency is most commonly defined by an impairment of blood coagulation resulting from decreased prothrombin, factor VII, factor IX and factor X activity, and is usually measured as a prolongation of a one-stage prothrombin time. Such a deficiency is easily produced in poultry by dietary restriction of the vitamin, but is not easily accomplished in most other species, including rats. The dietary vitamin K re quirement of most species is not well defined and depends to a large extent on the sensitivity of the clotting-based assay used, the route of vitamin ad ministration, and the form of the vitamin used. However, the requirement of chicks is usually indi cated as being 5-10 times that of rats (Suttie 1991). The basis for this species difference in requirement has not been established and is usually attributed to poor absorption of dietary vitamin by the short digestive tract of chicks, and to the importance of coprophagy in rats to furnish menaquinones. Other possible explanations would include alteration in the rate or route of metabolism of the vitamin in the two species, or alterations in the activity or sensitivity of enzymes involved in vitamin K action between chicks and rats. This report describes studies directed at determining the important factors responsible for the different vitamin K requirements of rats and chicks.
ABSTRACT The metabolic basis for the high vitamin K requirement of chicks compared with rats was inves tigated. When chicks and rats were fed the same diet, containing 500 ug phylloquinone/kg, the total amounts of phylloquinone and its epoxide metabolite found in the liver and plasma were similar in both species. However, phylloquinone 2,3-epoxide was present in high concen trations in chick liver and serum but not in rat liver and serum. This metabolite of the vitamin is normally reduced by a hepatic vitamin K epoxide reducÃ-ase. The activity of this enzyme in chicks was -10% of that in rats, and the inability of chicks to effectively recycle the epoxide of vitamin K seems to be the major factor in its high requirement. Other species differences in vitamin K metabolism were observed. Much higher concentrations of bacterial menaquinones were present in rat feces compared with chick fèces, but neither species had ap preciable hepatic concentrations of menaquinones. Chicks, but not rats, were found to have a liver concen tration of menaquinone-4 that exceeded that of phylloq uinone. This vitamer was present even when its recog nized precursor, menadione, was not present in the diet, and the data indicate that chicks convert phylloquinone to menaquinone-4 under the conditions of these experi ments. The mechanism of this conversion was not es tablished. J. Nutr. 122: 2354-2360, 1992. INDEXING KEY WORDS:
•vitamin K •phylloquinone •rats •menaquinone-4 •chicks
Vitamin K was discovered by Dam (1935) as a dietary factor needed to cure a hemorrhagic condition that developed in chicks fed a diet low in lipids. Vitamin activity was eventually shown to be a property of 2-Me-l,4-naphthoquinones substituted at the 3 position with a phytyl group in the case of the plant vitamin, phylloquinone, or a polyisoprenyl group in the case of bacterial menaquinones (Suttie 1991). Much later the biochemical role of the vitamin was shown to be that of a substrate for a hepatic 0022-3166/92
$3.00 ©1992 American
Institute
of Nutrition.
'Supported by the College of Agricultural and Life Sciences of the University of Wisconsin-Madison, and in part by grants DK14881 and HL-29586 from the National Institutes of Health, Bethesda, MD, and CRCR-1-1415 from the U.S. Department of Agriculture, Washington, DC.
Received 27 April 1992. Accepted 28 July 1992. 2354
Downloaded from https://academic.oup.com/jn/article-abstract/122/12/2354/4754665 by Denise Hannibal user on 08 August 2018
VITAMIN K METABOLISM AND REQUIREMENT
2355
MATERIALS AND METHODS TABLE
Animals and diets. Tissue and fecal phylloquinone and menaquinone concentrations were studied in 250- to 270-g male rats obtained from SpragueDawley (Madison, WI) and 4- to 5-wk-old LeghornNuhamp chicks obtained from the University of Wis consin-Madison Poultry Science Department. All animal protocols were approved by the University of Wisconsin-Madison Animal Care Committee. Both species were fed a soybean protein, sucrose and cornstarch, vegetable oil-based vitamin K-deficient diet (#81053) obtained from Teklad (Madison, WI), con taining 3 ug phylloquinone/kg by direct analysis (Kindberg and Suttie 1989). Animals were fed the vitamin K-deficient diet for 3 d, then fed the same diet supplemented with 500 ug/kg of phylloquinone (Sigma Chemical, St. Louis, MO) for an additional 8 d. Animals were housed in standard wire-bottomed cages under conditions of 12 h light: 12 h dark and were fed the diet with ad libitum access. They were killed by decapitation, and plasma, serum, livers and fecal material from the colon were obtained, frozen and stored at -70°C until analysis. Comparative me tabolism of menadione and phylloquinone was studied in similar rats and chicks fed phylloquinone or menadione. Phylloquinone or menadione, as pure menadione, were dissolved in a small amount of ethanol and mixed into the vitamin K-deficient diet the day before the experiments were started. Diets were stored at 4°C.Synthesis of menaquinone-4 (MK4) was studied in 4- to 8-wk-old Leghorn-Nuhamp chicks that were fed the vitamin K-deficient diet and injected with 50 uL of ethanol containing 50 ug/kg body wt of phylloquinone or menadione in the wing vein each day. Activities of the hepatic vitamin Kdependent carboxylase and vitamin K epoxide reducÃ-ase were studied in livers obtained from 7-wk-old Cornish-White Rock broilers or 250-g male rats maintained for 8 d on diets containing 500 ug phylloquinone/kg. Analytical procedures. One-stage prothrombin times were determined using a fibrometer (BBL, Bal timore, MD) and a commercial rabbit brain and lung thromboplastin preparation (Simplastin, Organon Teknika Corp., Durham, NC). Plasma prothrombin concentration was measured using a chromogenic assay for thrombin following thromboplastin acti vation of prothrombin as described by Shah et al. (1984). Total plasma prothrombin, which measures both biologically active and undercarboxylated forms, was measured following snake venom activation as described by Allison et al. (1987). Under-y-carboxylated prothrombin was measured using the total pro thrombin assay after biologically active prothrombin was absorbed by barium citrate as described by Francis (1988). Microsomal pellets for enzyme assays were pre pared as described by Cheung et al. (1989). Vitamin Downloaded from https://academic.oup.com/jn/article-abstract/122/12/2354/4754665 by Denise Hannibal user on 08 August 2018
1
Measurements of vitamin K status in plasma of rats and chicks fed a diet containing 500 ug phylloquinone/kg1 Assay Prothrombin time, s Prothrombin concentration, % Total prothrombin concentration, % Under-y-carboxylated prothrombin, % 'Prothrombin
Chick
Rat 10.7 (10.4-11.4)
36.4 (29.4-45.7)
87
±2
22
±2
110
±3
42
±2
1.7 ±0.2
3.6 ±0.4
times are means and ranges for eight animals per
group. Other values are means ±SEMfor eight animals per group and are expressed as the percentage of a control normal rat plasma pool. Prothrombin times and prothrombin concentration assays utilized a commercial rabbit thromboplastin preparation optimized for human plasma assays, and plasma from different species may not be activated at the same rate.
K-dependent carboxylase activity in microsomes was measured by a modification of the method described by de Boer-van den Berg et al. (1987), which used 0.5 mmol/L Boc-EEL-methyl ester (Bachern, Terranee, CA) instead of Boc-FLEEL-methyl ester as a substrate. The buffer used contained 0.25 mol/L sucrose, 0.025 mol/L imidazole, pH 7.3, and 0.4 g/L phosphatidyl choline, 0.4 g/L CHAPS and 0.4 g/L sodium cholate. Microsomal epoxide reducÃ-ase activily was measured as described by Misenheimer and Suttie (1990). Phylloquinone, MK-4 and the corresponding 2,3-epoxides in serum were measured by HPLC ana lyses as previously described (Usui et al. 1990) utilizing fluorometric detection (Nagaoko et al. 1989, Nishimura et al. 1990). Vitamin K was extracted from l g of liver tissue as previously described (Usui et al. 1989), and phylloquinone, phylloquinone epoxide, MK-4 and MK-4 epoxide were separated and detected as described above. Long-chain menaquinones were separated by gradient HPLC solution as described by Usui et al. (1989) with omission of the thin-layerchromatographic step because of the low level of contaminants in rat and chick liver compared with human liver. Fluorometric detection was preceded by reduction of menaquinones on a Platinum-Black column (Nagaoko et al. 1989). Rat and chick feces (0.1-0.2 g) were analyzed for phylloquinone and menaquinones as described for liver.
RESULTS When rats and chicks were fed the same diet con taining 500 ug phylloquinone/kg diet for 8 d, the apparent concentration of prothrombin in rat plasma was about four times that observed in chick plasma (Table 1). This difference in apparent prothrombin
WILL ET AL.
2356 40
O
Chicken
Rat
Chicken
Chicken
FIGURE1 Liverand serumphylloquinone(solidbar)and phylloquinone 2,3-epoxide(openbar) concentrations in rats and chicks fed a vitamin K-deficient diet for 3 d and then fed a diet supplemented with 500 ug phylloquinone/kgdiet for 8 d. Valuesare means ± SEMfor eight animals per group.
FIGURE 2 Liver and serum menaquinone-4 (solid bar) and menaquinone-4 2,3-epoxide (open bar) concentrations in rats and chicks fed a vitamin K-deficient diet for 3 d and then fed a diet supplemented with 500 ug phylloquinone/kg diet for 8 d. Values are means ±SEMfor eight animals per group.
concentration was not altered when the vitamin K concentration of the diet was increased to 2500 ug phylloquinone/kg diet (data not shown). The ability of the two species to absorb and retain dietary vitamin K was assessed by comparing the serum and liver phyl loquinone concentrations in both species (Fig. 1). The phylloquinone concentration in both serum and liver of the rats was -50% higher than in the chicks. The concentration of the major vitamin K metabolite, its 2,3-epoxide, was also measured. Phylloquinone epoxide was present in high concentrations in both chick liver and serum, but present in only low con centrations in rat liver and serum. The total concen tration of phylloquinone and its epoxide in both serum and liver was similar in chicks and rats. Menaquinones produced by gut bacteria are a second potential source of vitamin K, and the concen tration of these vitamers (MK-5 through MK-13) was measured in rat and chick liver and in feces obtained from the colon when the animals were killed (Table 2). Both rat and chick liver contained only low con centrations of menaquinones. The concentrations of most long-chain menaquinones in rat liver were below the limit of detection, whereas measurable menaquinone concentrations, particularly of MK-8 and MK-10, were found in chick liver. However, the total amount of MK-5 through MK-13 was only 4 nmol/kg chick liver, or -20% of the phylloquinone concentration of chick liver. Rat fecal contents, in contrast to those of chicks, contained large amounts of long-chain menaquinones. The nearly 14 umol/kg dry wt observed in rat feces was about 50 times that found in chick feces. A major difference in liver vitamin content in the two species was a high concentration of MK-4 in chick liver (Fig. 2). This vitamin is not a major bac terial metabolite (Fernandez and Collins 1987, Ramotar et al. 1984) but is known to be readily
formed by menadione alkylation in the liver (Dialameh et al. 1971, Martius and Esser 1958, Taggart and Matschiner 1969). In rat liver, MK-4 was detected at a concentration of -10% that of phylloquinone, whereas in chick liver the concentration of MK-4 was more than twice that of phylloquinone. High concen trations of MK-4 were also found in chick serum, but not rat serum. As was found for phylloquinone, the 2,3-epoxide of MK-4 was present in chick liver at -50% the concentration of MK-4; however, only low concentrations of MK-4 epoxide were found in chick serum. The increased dietary vitamin K requirement for chicks compared with rats could result from a decreased affinity of the chick vitamin K-dependent y-glutamyl carboxylase for its substrate, the reduced form of vitamin K. The increased concentrations of vitamin K epoxides observed in chick liver and serum (Fig. 1 and 2) could result from an inability of chicks to metabolize the epoxide to excretory products or to reduce the epoxide to vitamin K via the well-demon strated epoxide reducÃ-ase pathway. Assay of these enzymes (Table 3) indicates that there are species differences in both the activity and substrate Km values for these enzymes. The chick carboxylase had a higher Km for reduced vitamin K than the rat enzyme, and this difference was more pronounced when reduced MK-4 rather than phylloquinone was utilized as a substrate. The hepatic carboxylase ac tivity (Vmax) of chicks was -50% that of rats when phylloquinone was used as a substrate, but similar in the presence of saturating MK-4. A much more pro nounced difference in the properties of the rat and chick enzymes was seen in the activity of the hepatic epoxide reducÃ-ase. Although the Km values measured did not differ appreciably, the rate of either phylloq uinone epoxide or MK-4 epoxide reduction by the
Downloaded from https://academic.oup.com/jn/article-abstract/122/12/2354/4754665 by Denise Hannibal user on 08 August 2018
VITAMIN K METABOLISM AND REQUIREMENT
2357
TABLE 2 Concentration of bacterial menaquinones in rat and chick liver and feces1 Feces menaquinones
Liver menaquinones Vitamer
Rat
Chick
Rat
Chick
wet wt0.06CPND2.00.41.40.5NDND4±±±±±0.060.80.1.06.0529153031080070083302170 wt450CP180186NDNDND258± MK-5MK-6MK-7MK-8MK-9MK-10MK-11MK-12MK-13Total
MKNDCPNDNDCPNDNDNDND
