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Articles

Tissue Phylloquinone and Menaquinones in Rats Are Affected by Age and Gender¹

Vitamin K Laboratory, USDA Human Nutrition Research Center on Aging, Tufts University, Boston MA 02111

²To whom correspondence should be addressed.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Phylloquinone and ten menaquinones (MK-1–MK-10) were measured in liver and eight extrahepatic tissues from male and female rats at 3, 12 and 24 mo of age. Phylloquinone and menaquinones showed characteristic tissue distribution. In liver, all 11 vitamers of vitamin K assayed were present in varying concentrations with phylloquinone and MK-6 the major forms. The only forms of vitamin K found in extrahepatic tissues were phylloquinone, MK-4 and MK-6. Brain contained only MK-4 and traces of phylloquinone. No significant gender difference was observed for phylloquinone except in heart at 3 mo of age (P 0.05). In heart, kidney and brain, MK-4 was significantly higher in females than in males (P 0.05). A similar gender effect was seen in kidney and lung for MK-6 (P 0.05). With age, hepatic phylloquinone and MK-6 significantly increased (P 0.05), whereas MK-4 was unchanged. In extrahepatic tissues, MK-4 decreased with age in heart and kidney of males and females, and in lung and cerebellum of males (P 0.05). MK-6 decreased with age in all extrahepatic tissued tested (P 0.05). The results suggest that in extrahepatic tissues, certain menaquinones may be the predominant form of vitamin K. The specific tissue distribution and the general decline of MK-4 and MK-6 in extrahepatic tissues during aging suggest a vitamin K tissue dynamic that is affected not only by diet, but also by gender, age and the specific roles of phylloquinone, MK-4 and MK-6 in metabolism. All of these factors must be taken into account in establishing the nutrient requirement for vitamin K.

KEY WORDS: • phylloquinone • menaquinones • gender • age • rats

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Vitamin K is a cofactor for the post-translational carboxylation of glutamic acid residues to -carboxyglutamic acid (Gla)³ to form Gla-containing proteins (Suttie 1996 ). The Gla domains in these proteins are required for calcium-mediated conformational changes to interact with phospholipid membranes (Furie and Furie 1990 and 1997 ). In dietary phylloquinone deficiency, Gla proteins may be undercarboxylated with loss of their activity (Sokoll and Sadowski 1996 ). Prothrombin, factor VII, IX and X are Gla proteins that originate in the liver and are involved in blood coagulation (Sadowski et al. 1991 ). Examples of extrahepatic Gla proteins are osteocalcin, matrix Gla protein and protein S; the last mentioned is secreted by osteoblasts in addition to other tissues (Shearer 1995 , Vermeer et al. 1995 ). A protein with Gla domains is encoded by the Gas 6 gene and appears to function as a ligand in signal transduction in certain neuronal cells (Varnum et al. 1995 ) as well as in osteoclasts (Nakamura et al. 1998 ). In addition, a novel role for vitamin K in the metabolism of sulfatides has been suggested (Sundaram and Meir Lev 1990 , Sundaram et al. 1996 ). Such studies and evidence from the carboxylase distribution in tissues suggest that vitamin K has a wider function in biological systems than formerly assumed.

In addition to phylloquinone (K₁,2-Me-3-phytyl-1,4-naphthoquinone), menaquinones (2-Me-3-polyisoprenyl homologues), which differ from phylloquinone in their side chain, may have vitamin K activity (Suttie 1995 ). Menaquinones with up to 13 isoprenyl units in their side chain have been identified. These can originate from bacterial synthesis in the gut (Conly et al. 1994 , Suttie 1995 ). Menadione (2-methyl naphthoquinone), commonly added to rat diets, is another source of menaquinones. Menaquinone-4 is thought to be synthesized from activated menadione by alkylation with geranyl-geranyl pyrophosphate (Dialameh 1978 , Dialameh et al. 1971 , Taggart and Matchiner 1969 ). Menadione supplementation in germ-free rats led to high levels of MK-4, particularly in extrahepatic tissues (Ronden et al. 1998a ). Another synthetic pathway for MK-4 has been described recently by several researchers. Davidson et al. (1998) observed MK-4 synthesis from phylloquinone in germ-free animals as well as in cultured kidney cells. Similar synthesis of MK-4 has been shown in studies by Thijssen and Drittij-Reijnders (1994 and 1996) , Thijssen et al. (1996) , and Ronden et al. (1998a) .

Although hepatic vitamin K has been the focus of studies in relation to blood coagulation (Kindberg and Suttie 1989 , Sadowski et al. 1991 ), the discovery of extrahepatic Gla-proteins suggested extrahepatic vitamin K functions. Davidson et al. (1998) measured phylloquinone and MK-4 in heart, brain, kidney and salivary gland of germ-free rats and found specific phylloquinone and MK-4 distribution in these tissues. Ronden et al. (1998b) measured phylloquinone and MK-4 in extrahepatic tissues in relation to low and high dietary phylloquinone or MK-4 intakes. Synthesis of MK-4 from phylloquinone and menadione suggested a specific distribution of this menaquinone in extrahepatic tissues (Ronden et al. 1998a ).

Male rats were used in all of the above studies on extrahepatic vitamin K distribution, and it is not known whether the greater resistance of female rats to vitamin K deficiency is related, at least in part, to increased vitamin K tissue levels (Jolly et al. 1977 , Metta and Johnson 1960 ) as has been suggested by Olsen (1984) . In addition to the gender difference in resistance to vitamin K deficiency in animal experiments, there are data from human studies to suggest that vitamin K may be affected by age. In a review by Booth and Suttie (1998) , human dietary phylloquinone intake increased with age, and the elderly appeared more resistant to vitamin K deficiency. Although undercarboxylated prothrombin is rarely seen in the elderly, elevated undercarboxylated osteocalcin is more frequent (Vermeer 1995 ), suggesting that extrahepatic vitamin K status may be affected by aging.

The purpose of this study was to further investigate tissue distribution of phylloquinone and menaquinones in both male and female rats at three stages of maturation (3, 12 and 24 mo of age). The animals were fed the same nonpurified diet from weaning until they were killed to study the effect of gender and age on hepatic and eight extrahepatic tissues including ovaries, testes and brain.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Animals and diets.

All procedures involving animals were reviewed and approved by the Animal Care and Use Committee of the USDA Human Nutrition Research Center on Aging at Tufts University.

Male and female Brown Norway rats were obtained from Charles River Laboratories (Stone Ridge, NY). They were housed in individual metabolic cages and maintained in a controlled atmosphere (temperature, 23°C, 45% relative humidity) with 12-h light:dark cycles. They were fed a nonpurified (NIH 31 M) rodent diet.⁴ At 3, 12 and 24 mo of age, food was withheld overnight after which five male and five female rats in each group were anesthetized with fenfluor and bled by heart puncture. Tissue samples were quickly excised, washed in cold saline, weighed and frozen immediately in liquid nitrogen. Plasma and tissue samples were stored at -70°C until they were extracted for phylloquinone and menaquinone analysis.

Assays.

Phylloquinone and menaquinones were either purchased commercially (Sigma Chemical, St Louis, MO) or received as gifts (Hoffman-LaRoche, Basel, Switzerland). The 2,3-epoxide of MK-4 and dihydro-phylloquinone were synthesized (Langenberg and Tjaden 1984 , Tishler et al. 1940 ). Solvents used for tissue extraction and chromatography were purchased from Fisher Scientific (Springfield, NJ) and were HPLC grade. Anhydrous sodium sulfate was obtained from Sigma Chemical. Working standards were prepared in HPLC-grade methanol and were characterized spectrophotochemically and chromatographically before use. All standards were stored at 4°C and protected from light. Because vitamin K compounds are UV sensitive, all operations were performed under yellow lighting. To prevent contamination of samples with fluorescent material, all glassware was washed in a solution of chromic and sulfuric acid (Fisher Scientific).

Plasma extractions of phylloquinone and menaquinones were carried out with hexane as described by Haroon et al. (1986) . For tissue analysis of phylloquinone and menaquinones, three 100-mg samples were obtained from each tissue with the exception of the ovaries, which were pooled for each rat. The tissue samples were pulverized with a mortar and pestle in anhydrous Na₂SO₄ (10 times tissue weight). The fine powder was transferred quantitatively to a teflon-lined, screw-capped borosilicate glass tube. An internal standard consisting of 2.5 pmol/50 µL dihydro-phylloquinone or MK-5 was added to tissue samples with a Hamilton syringe followed by 10 mL of acetone. The mixture was then extracted by gently mixing on a rotary mixer overnight. After centrifugation at 1000 x g at 4°C for 5 min, the acetone extract was separated with a pasteur pipette and placed into a fresh tube. In a centrifugational evaporator (Savant Instruments, Farmingdale, NY), the acetone was evaporated; the solid residue was reextracted and partitioned with 6 mL hexane and 2 mL H₂O by shaking vigorously for 2 min. The water and hexane layers were separated by centrifugation at 1000 x g at 4°C for 5 min and the hexane layer transferred to a 16 x 100 mm culture tube. The residue, obtained after centrifugational evaporation of the hexane, was redissolved in 1.0 mL of hexane for further processing by solid phase extraction.

For solid phase extraction, 3-mL silica gel columns (JT Baker, Phillipsburg, NJ) were preconditioned by washing with 8 mL of hexane-diethyl ether (97:3, v/v) followed by 8 mL of hexane. The 1.0-mL hexane extract was then transferred quantitatively to the silica column, the adsorbed band washed with 8 mL of hexane and eluted with 8 mL of hexane-diethyl ether (97:3, v/v). The eluent was collected and the solvent evaporated by centrifugational evaporation. The final residue was then dissolved for HPLC.

Detection of phylloquinone and menaquinones by HPLC.

The chromatographic system consisted of a model 231–401 automated sample injector (Gilson Medical Electronics, Middleton, WI), model 510 reciprocating pump (Waters, Milford, MA) and model 980 fluorescence detector (Kratos Analytical, Ramsey, NJ) with excitation and emission at 244 and 418 nm, respectively. The analytical column (150 x 4.6 mm) was packed with 3 µm ODS-Hypersil (Keystone Scientific, Belfonte, PA). Fluorescent derivatives of the injected quinones were produced on-line after separation on the analytical column using a post-column, solid-phase reactor (2.0 mm x 50 mm) packed with zinc metal (200 mesh; Alpha Products, Danvers, MA). An 860 Vax-based data station with Expert-Ease software (Waters) was used for integration and quantification.

The dry samples were dissolved for HPLC in 0.01 mL of methylene chloride and 0.09 mL methanol containing aqueous phase (10 mmol/L zinc chloride, 10 mmol/L acetic acid and 5 mmol/L sodium acetate; 5 mL aqueous phase was added to 1 L methanol). A 50-µL sample was used for analysis. The standard solution contained phylloquinone, ten menaquinones (MK-1–MK-10) and the 2,3 epoxide of MK-4 as well as dihydro-phylloquinone or MK-5 in methanol to give a final known concentration of ~2.5 pmol of each, per 0.1 mL. Separation of phylloquinone and menaquinones was achieved by concave gradient elution (curve 7; Waters) at 1.0 mL/min, starting with 100% solvent A (100% methanol and 10 mmol/L zinc chloride, 10 mmol/L acetic acid and 5 mmol/L sodium acetate) going to 100% solvent B (40% methylene chloride in methanol with 10 mmol/L zinc chloride, 10 mmol/L acetic acid and 5 mmol/L sodium acetate) by 35 min. At this point, the composition of the mobile phase was switched back to solvent A for 10 min to equilibrate the column for the next injection. The chromatographic peaks obtained were quantified using peak area ratios to the authentic standards of phylloquinone, menaquinones and the epoxide of MK-4; these were included after every five tissue samples. The percent recovery for the various tissues were: means ± SD, n = 30, liver 78 ± 5.6; spleen, 78 ± 18.9; cerebellum, 83 ± 13.3; brain cortex, 87 ± 6.6; lung, 81 ± 7; kidney, 80 ± 9.6; testis, 77 ± 9; ovaries, 75 ± 16.

Statistical analysis.

Age and gender main effects and interactions with respect to tissue concentrations of phylloquinone and menaquinones were analyzed using two-way ANOVA. When significant age-by-gender interactions were identified, age groups were compared for males and females separately by using Tukey's honestly significant difference test; males and females were compared within each age group by using Student's t test for independent samples. A two-sided observed significance level (P-value) 0.05 was used to indicate significance. Values in the text are means ± SD.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Liver.

The liver contained phylloquinone and all ten menaquinones (MK-1–MK-10) (Fig. 1) . The mean total vitamin K calculated as phylloquinone plus the sum of all menaquinones varied from 54.0 ± 9.0 pmol/g for 12-mo-old males to 171.4 ± 30.1 pmol/g for females 24 mo of age. At all three ages, the menaquinone/phylloquinone ratios were greater than 1 (1.2–1.9), indicating that liver contained more menaquinones than phylloquinone. The major vitamin K form in liver was phylloquinone, followed by MK-6. The rest of the menaquinones including MK-4 were present in much lower concentrations. Hepatic phylloquinone, MK-4 and MK-6 concentrations at three ages are given in Table 1. A significant gender difference was observed for MK-4 and MK-6 at 3 mo of age (P 0.05). During aging, phylloquinone and MK-6 increased significantly in liver of males and females (P 0.05).

View larger version (22K): [in this window] [in a new window]   Figure 1. Hepatic phylloquinone (K1) and menaquinone distribution (MK-1–MK-10) in male and female rats fed a nonpurified diet for 3, 12 or 24 mo. Each column is the mean ± SD, n = 5.

Extrahepatic tissues differed from liver by the presence of only phylloquinone, MK-4 and MK-6. In brain only, MK-4 and traces of phylloquinone were observed. Because no significant age and gender effects were observed, the phylloquinone values of all age and gender groups were pooled for the following tissues (mean ± SD, n = 30 as pmol/g): spleen, 12.8 ± 3.0; kidney, 6.1 ± 1.3; lung, 11.2 ± 5.7; testis, 3.5 ± 0.8; ovaries, 67.7 ± 35.2; brain cortex, 1.8 ± 0.3; cerebellum, 0.7 ± 0.2. For heart at 3 mo of age, the mean phylloquinone concentrations were 38.9 ± 1.5 pmol/g in males (n = 5) and 46.2 ± 1.9 pmol/g in females (n = 5) with a significant gender difference (P 0.05). During aging, phylloquinone in heart decreased (P 0.05).

Menaquinone-4 (Table 2) concentrations in tissues varied. The highest concentrations were observed in ovaries, testis and brain. For all tissues analyzed, the mean concentration of MK-4 was lower in males than in females. Significant gender effects were observed for heart, kidney, brain cortex and cerebellum (P 0.05). Aging reduced the MK-4 concentrations in heart and kidney of males and females and in lung and cerebellum of the males (P 0.05).

In brain samples, a small companion peak to MK-4 was found; it was identified in brain cortex as the 2,3 epoxide of MK-4. Menaquinone-4 epoxide concentrations in brain cortex at 3 mo were (means ± SD, n = 5) 1.51 ± 0.3 pmol/g for males and 1.96 ± 0.18 for females; at 12 mo, 0.97 ± 0.16 for males and 1.56 ± 0.19 for females; at 24 mo, 1.09 ± 0.12 for males and 1.20 ± 0.10 pmol/g for females. The 2,3 epoxide of MK-4 was significantly correlated with MK-4 (r = 0.738, P < 0.001). When cortex values of the males and females were combined (n = 30).

Plasma.

Phylloquionone, MK-4 and MK-6 were the only forms of the vitamin that were found in measurable quantities in plasma (Fig. 2). Significant gender differences were observed for MK-4 at 3 and 12 mo (P 0.05).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Interest in the study of extrahepatic functions of vitamin K stems from the discovery of Gla-proteins in bone (Shearer 1995 , Shearer et al. 1996 , Vermeer et al. 1995 ) and the discovery of the growth arrest specific gene product (Gas 6), a ligand to a receptor tyrosine kinase involved in the signaling cascade of cell growth (Varnum et al. 1995 ). Furthermore, certain menaquinones, in particular MK-4, with a side chain comparable in length to that of phylloquinone, may have specific functions in some extrahepatic tissues (Suttie 1995 , Thijssen et al. 1996 ).

Several recently published rat studies have reported investigations of phylloquinone and MK-4 in extrahepatic tissues. Thijssen and Drittij-Reijnders (1994) , Thijssen et al. (1996) , and Ronden et al. (1998a and 1998b) investigated phylloquinone and MK-4 distribution in male rat tissues in relation to different dietary intakes, some under germ-free conditions to exclude gut-derived menaquinones. Davidson et al. (1998) measured both phylloquinone and MK-4 in germ-free male rat tissues. These data suggest both specific tissue distribution of phylloquinone and MK-4, and the synthesis of MK-4 from phylloquinone and from menadione.

Our study was designed to determine the vitamin K tissue distribution in relation to gender and age of the animals. Males and female rats were fed a nonpurified diet that contained menadione (0.97 µg/g) and phylloquinone (173 ng/g). All ten menaquinones (MK-1–MK-10) in addition to phylloquinone were found in liver, whereas extrahepatic tissues contained only MK-4 and MK-6 in addition to phylloquinone. An exception was brain, which contained only MK-4 with traces of phylloquinone. Another difference between hepatic and extrahepatic vitamin K occurred during aging. In liver, phylloquinone and MK-6 (but not MK-4) increased with age, whereas in extrahepatic tissues, MK-6, and to a lesser degree MK-4, decreased. In -carboxyglutamate excretion studies Craciun et al. (1997) also showed differences in hepatic and extrahepatic vitamin K metabolism. These findings suggest that liver vitamin K concentrations may not be representative of extrahepatic vitamin K, especially during aging. Therefore, vitamin K assessment that is based solely on hepatic variables may be insufficient to establish vitamin K adequacy.

As in the studies by Davidson et al. (1998) and Ronden et al. (1998b) , MK-4 in our study seems to be the predominant form of vitamin K in some extrahepatic tissues. Although the dietary research protocols varied considerably between this study and those cited above, some striking similarities in the tissue distribution of phylloquinone and MK-4 were observed. In all three studies, liver was high in phylloquinone and low in MK-4. The phylloquinone to MK-4 ratios in this study and that of Davidson et al. (1998) were as follows: in liver, 26 and 24; in heart, 5.5 and 9; in brain, 0.068 and 0.14. In kidney, we found a ratio of 0.54, whereas Davidson's was ten times higher at 5. However, our ratio was similar to the 0.65 calculated from Ronden's data for the low phylloquinone intake groups.

The consistent presence of MK-6 in hepatic and extrahepatic tissue (except in brain) requires further investigation. The studies with germ-free animals by Ronden et al. (1998a) and Davidson et al. (1998) have provided evidence that tissue MK-4 was derived by de novo synthesis from phylloquinone and menadione and not from gut bacteria. On the basis of these findings, it is likely that synthesis from menadione is the source of MK-4 in our study. However, whether MK-6 is derived from gut bacterial synthesis or is synthesized from menadiol by geranylation, as suggested by Taggart and Matchiner (1969) and Dialameh (1978) for MK-4, is not known.

When tissue MK-4 and MK-6 are combined, their sum is higher than phylloquinone (except in heart), indicating that menaquinones constitute a large proportion of total tissue vitamin K and in some tissues, females had significantly higher concentrations of menaquinones than males. This suggests that more menaquinones are synthesized in female rats than in males or that males use menaquinones to a greater extent. In light of the considerable accumulation of phylloquinone in liver of older rats, it is puzzling that the phylloquinone to MK-4 conversion observed by Davidson et al. (1998) and Thijssen and Drittij-Reijnders (1994) was not sufficient to prevent the MK-4 decrease during aging at extrahepatic tissue sites.

In vitro studies by Buitenhuis et al. (1990) have shown that MK-2–MK-6 are substrates that are biologically active in the carboxylase reaction. However, in vivo, the longer-chain menaquinones may have lower biological activity (Craciun et al. 1998 , Suttie 1995 ). In our study, MK-4 in brain cortex was shown to be biologically active by the consistent presence of the 2,3-epoxide of MK-4 in these samples. The presence of 2,3-epoxide of MK-4 in other tissues must be established to show whether MK-4 is active in these tissues. The question regarding the origin of MK-6 in our tissue samples and its possible role in the vitamin K cycle requires further investigation. Several similarities between MK-4 and MK-6 exist; these include higher levels in females and decreased levels during aging. However, the complete absence of MK-6 from brain with concomitant high MK-4 levels, as well as the considerable MK-6 and low MK-4 concentrations in liver, require explanation. Equally unexplained is the site of MK-4 synthesis from menadione and whether MK-6 originates by gut or tissue synthesis. In our study, both MK-4 and MK-6 were measured in plasma, indicating that transport from sites of synthesis or storage may occur.

In summary, we have shown that gender and age affect tissue vitamin K. Our results have confirmed some prior data but raised new questions regarding the tissue distribution of phylloquinone, MK-4 and MK-6 and their possible functions. From this study and others, it is clear that vitamin K tissue dynamics are influenced by dietary factors, the biological availability of the different vitamin K forms, by de novo synthesis of MK-4 and possibly MK-6 from precursors at the tissue level, by gender and by age, all of which may affect the nutritional requirement for vitamin K.

View larger version (19K): [in this window] [in a new window]   Figure 2. Plasma concentrations of phylloquinone (K1), and menaquinone (MK)-4, and MK-6 in male and female rats fed a nonpurified diet for 3, 12 or 24 mo. Each column is the mean ± SD, n = 5. MK-4 at 3 and 12 mo differed between males and females (P 0.05).

	ACKNOWLEDGMENTS

	FOOTNOTES

 
¹ This material is based upon work supported by the U.S. Department of Agriculture, under agreement No. 58-1950-9-001. Any opinions, findings, conclusion, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Dept of Agriculture.

³ Abbreviations used: Gla, -carboxyglutamic acid; K₁, phylloquinone, 2-Me-3-phytyl-1,4-naphthoquinone; MK, menaquinone.

⁴ The Agway diet contained the following: crude protein, 18%; crude fat (minimum), 5%; crude fiber (maximum), 5%; ash, 6.8%. The mineral elements were as follows (%): Ca, 0.9; P, 0.8; K, 0.7; Na, 0.4; Mg, 0.23; Cl, 0.1; and (mg/kg): iron, 280; copper, 18; fluorine, 32; manganese 55.1; seleniun, 0.17; zinc, 50; iodine, 1.0; cobalt, 1.0. The vitamins were as follows (IU/kg): vitamin A, 15,684; cholecalciferol, 1,045; vitamin E, 48.2; and (mg/kg): thiamin, 7.7; riboflavin, 7.64; pantothenic acid, 17.18; niacin, 56,32; pyridoxine, 7.39; folic acid, 0.89; biotin, 0.29; vitamin B-12, 0.04; choline, 1,478; menadione, 0.97; and phylloquinone 0.173 according to our analysis.

Manuscript received October 22, 1998. Initial review completed December 10, 1998. Revision accepted February 1, 1999.

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