James W. Peterson

Effect of Vitamin K Supplementation on Bone Loss in Elderly Men and Women

CONTEXT Vitamin K has been implicated in bone health, primarily in observational studies. However, little is known about the role of phylloquinone supplementation on prevention of bone loss in men and women. OBJECTIVE The objective of this study was to determine the effect of 3-yr phylloquinone supplementation on change in bone mineral density (BMD) of the femoral neck bone in older men and women who were calcium and vitamin D replete. DESIGN, PARTICIPANTS, AND INTERVENTION In this 3-yr, double-blind, controlled trial, 452 men and women (60-80 yr) were randomized equally to receive a multivitamin that contained either 500 mug/d or no phylloquinone plus a daily calcium (600 mg elemental calcium) and vitamin D (400 IU) supplement. MAIN OUTCOME MEASURES Measurements of the femoral neck, spine (L2-L4), and total-body BMD, bone turnover, and vitamins K and D status were measured every 6-12 months. Intent-to-treat analysis was used to compare change in measures in 401 participants who completed the trial. RESULTS There were no differences in changes in BMD measurements at any of the anatomical sites measured between the two groups. The group that received the phylloquinone supplement had significantly higher phylloquinone and significantly lower percent undercarboxylated osteocalcin concentrations compared with the group that did not receive phylloquinone. No other biochemical measures differed between the two groups. CONCLUSIONS Phylloquinone supplementation in a dose attainable in the diet does not confer any additional benefit for bone health at the spine or hip when taken with recommended amounts of calcium and vitamin D.

HPLC and GC/MS determination of deuterated vitamin K (phylloquinone) in human serum after ingestion of deuterium-labeled broccoli

The ability to intrinsically label plant constituents with stable isotopes has the potential to advance the study of vitamin K-absorption and metabolism in humans. Broccoli, a primary food source of phylloquinone (VK-1), was grown hydroponically using 31 atom % deuterium oxide in order to label VK-1 within the food matrix. Deuterium-labeled broccoli (115 g; 168 &mgr;g VK-1) was fed to one male subject in a single serving. Multiple serum samples were drawn throughout the subsequent 24-hr period. Reversed-phase HPLC was used to extract and purify VK-1 in both broccoli and serum. Ion abundances of the deuterium-labeled and unlabeled (endogenous) VK-1 were determined using GC/MS in negative chemical ionization mode. No sample derivatization was required. Endogenous VK-1 produced isotopomers from m/z 450 to m/z 453. The labeled VK-1 isotopomers in the broccoli were from m/z 452 to m/z 467, with the most abundant isotopomer being m/z 458 (14.1% of total labeled VK-1). The GC/MS chromatograms from serum revealed both endogenous VK-1 and VK-1 derived from the deuterium-labeled broccoli. The profile of labeled VK-1 isotopomers in serum was identical to the VK-1 isotopomer profile in labeled broccoli, indicating that no deuterium was lost due to exchange either in the body or in sample preparation. At 4 hr following broccoli intake, there was an 81.1% enrichment of phylloquinone in serum; labeled VK-1 was no longer detectable in serum at 24 hr. Use of isotope labeled vegetables enables one to discriminate exogenous intake of VK-1 from endogenous pools and ultimately to determine bioavailability of VK-1 from foods.

Adulthood Obesity Is Positively Associated with Adipose Tissue Concentrations of Vitamin K and Inversely Associated with Circulating Indicators of Vitamin K Status in Men and Women

Increased adiposity is associated with increased storage of several fat-soluble nutrients. However, the extent to which vitamin K is stored in fat and the association between vitamin K status and adiposity are unknown. Our objectives in this study were to determine whether vitamin K is stored in human adipose tissue and the association between vitamin K status and body fat in older men and women. In study A, the vitamin K concentration of subcutaneous and visceral adipose tissue was quantified in samples taken from 16 gastric bypass patients [13 women, 3 men, age 40 +/- 10 y (mean +/- SD)] using HPLC. In study B, cross-sectional associations between percent body fat (%BF) and circulating measures of vitamin K status were examined in 260 women and 183 men [age = 68 +/- 5 y]. The phylloquinone (K(1)) concentrations in subcutaneous and visceral adipose tissue were 148.2 +/- 71.8 and 175 +/- 112 nmol/kg, respectively, which is higher than the reported concentrations of other organs known to store vitamin K. There was an inverse association between %BF and plasma K(1) in women (P-trend < 0.001). Higher %BF was associated with greater circulating concentrations of uncarboxylated prothrombin, indicative of lower hepatic utilization of vitamin K in both men (P-trend = 0.02) and women (P-trend = 0.002) but not with the percentage of undercarboxylated osteocalcin. Adipose tissue contained high concentrations of vitamin K, and increased adiposity was associated with poorer vitamin K status in older adults. Additional studies are needed to further explore the relationships among body fat, storage of vitamin K in adipose tissue, and implications for vitamin K status and function.

Deuterium-Labeled Phylloquinone Has Tissue-Specific Conversion to Menaquinone-4 among Fischer 344 Male Rats

Phylloquinone (PK) is converted into menaquinone-4 (MK-4) via side chain removal-addition. Stable isotope use is an effective approach to identify the tissue location of this conversion, which is currently unknown. Following a 14-d PK-deficient diet, male Fischer 344 rats (8 mo; n = 15) were fed 1.6 mg deuterium-labeled PK (L-PK) per kg diet for 0 (control), 1 d (PK-1d), and 7 d (PK-7d). Both L-PK and deuterium-labeled MK-4 (L-MK-4) were detected in tissues in PK-1d and PK-7d, although the results varied. Whereas some tissues had an overall increase in MK-4 in response to L-PK, total brain, testes, and fat MK-4 concentrations did not. In contrast, L-MK-4 concentrations increased in all 3 tissues. The deuterium label was found only on the L-MK-4 naphthoquinone ring, confirming the need for side chain removal for the formation of MK-4. Labeled menadione (MD) was detected in urine and serum in PK-1d and PK-7d, confirming its role as an intermediate. A Caco-2 cell monolayer model was used to study the role of the enterocytes in the conversion process. Neither MK-4 nor MD was detected in Caco-2 cells treated with PK. However, when Caco-2 cells were treated with MD, MK-4 was formed. Similarly, MK-4 was formed in response to MD-treated 293T kidney cells, but not HuH7 liver cells. These data demonstrate that MK-4 is the predominant form of vitamin K in multiple tissues, but there appears to be a tissue-specific regulation for the conversion of PK to MK-4.

Measurement of Deuterium-Labeled Phylloquinone in Plasma by High-Performance Liquid Chromatography/Mass Spectrometry

Phylloquinone (vitamin K(1)) is a lipophilic compound present in plasma at low concentrations, which presents technical challenges for determining its bioavailability or metabolic fate using stable isotopes. We developed a method to simultaneously measure unlabeled and deuterium-labeled phylloquinone concentrations in plasma specimens using high-performance liquid chromatography/mass spectrometry with atmospheric pressure chemical ionization (LC-APCI/MS). Phylloquinone was extracted from plasma using hexane, further purified by solid-phase extraction, and then quantified using high-performance liquid chromatography with an APCI/MS as a detector. Plotting the expected versus the measured amount of serial dilutions of either unlabeled or labeled phylloquinone gave correlation coefficients (R) of 0.999 for both compounds. The minimum detectable concentrations of unlabeled and labeled phylloquinone were 0.05 and 0.08 pmol/injection, respectively. Pooled plasma samples spiked with between 0.5 and 32 nmol phylloquinone/L gave average recoveries of 96.7% with 5.4% relative standard deviation (RSD) for unlabeled phylloquinone and 96.2% with 6.6% RSD for labeled phylloquinone. Plasma phylloquinone concentrations determined by LC-fluorescence and LC-APCI/MS methods from healthy subjects (n = 17) were not statistically different (P = 0.13). The LC-APCI/MS method is a sensitive technique for simultaneous determination of both unlabeled and labeled phylloquinone and can be applied to bioavailability studies.

Extrahepatic tissue concentrations of vitamin K are lower in rats fed a high vitamin E diet

BackgroundAn adverse hematological interaction between vitamins E and K has been reported, primarily in patients on anticoagulants. However, little is known regarding circulating levels or tissue concentrations of vitamin K in response to vitamin E supplementation. The purpose of this study was to examine the effect of different levels of dietary α-tocopherol on phylloquinone and menaquinone-4 concentrations, while maintaining a constant intake of phylloquinone, in rat tissues.MethodsMale 4-wk old Fischer 344 rats (n = 33) were fed one of 3 diets for 12 wk: control (n = 13) with 30 mg all-rac-α-tocopherol acetate/kg diet; vitamin E-supplemented (n = 10) with 100 mg all-rac-α-tocopherol acetate/kg diet; and vitamin E-restricted (n = 10) with <10 mg total tocopherols/kg diet. All 3 diets contained 470 ± 80 μg phylloquinone/kg diet.ResultsPhylloquinone concentrations were lower (P ≤ 0.05) in the vitamin E-supplemented compared to the vitamin E-restricted group (mean ± SD spleen: 531 ± 58 vs.735 ± 77; kidney: 20 ± 17 vs. 94 ± 31, brain: 53 ± 19 vs.136 ± 97 pmol/g protein respectively); no statistically significant differences between groups were found in plasma, liver or testis. Similar results were noted with menaquinone-4 concentrations in response to vitamin E supplementation.ConclusionThere appears to be a tissue-specific interaction between vitamins E and K when vitamin E is supplemented in rat diets. Future research is required to elucidate the mechanism for this nutrient-nutrient interaction.

Vitamin K Status in Spaceflight and Ground-Based Models of Spaceflight

Bone loss is a well‐documented change during and after long‐duration spaceflight. Many types of countermeasures to bone loss have been proposed, including vitamin K supplementation. The objective of this series of studies was to measure change in vitamin K status in response to microgravity under a variety of spaceflight and spaceflight analog (model) conditions, including long‐duration spaceflight studies (n = 15), three bed rest studies (n = 15, 49, and 24), and a 14‐day saturation dive (n = 6). In crew members who flew 2–6 months on the International Space Station, in‐flight and postflight plasma phylloquinone concentrations were unchanged from the preflight mean. Consistent with this finding, urinary γ‐carboxyglutamic acid (GLA), a measure of vitamin K‐dependent protein turnover, did not change in response to flight. Serum undercarboxylated osteocalcin (%ucOC), a measure of vitamin K function, was generally unchanged in response to flight. Spaceflight findings were corroborated by findings of no changes in phylloquinone, urinary GLA, or %ucOC during or after bed rest in three separate bed rest studies (21–90 days in duration) or after a 14‐day saturation dive. The data presented here do not support either a need for vitamin K supplementation during spaceflight or the suggestion of using vitamin K as a bone loss countermeasure in spaceflight.

Measurement of menadione in urine by HPLC.

Menadione is a metabolite of vitamin K that is excreted in urine. A high performance liquid chromatography (HPLC) method using a C(30) column, post-column zinc reduction and fluorescence detection was developed to measure urinary menadione. The mobile phase was composed of 95% methanol with 0.55% aqueous solution and 5% DI H(2)O. Menaquinone-2 (MK-2) was used as an internal standard. The standard calibration curve was linear with a correlation coefficient (R(2)) of 0.999 for both menadione and MK-2. The lower limit of quantification (LLOQ) was 0.3pmole menadione/mL urine. Sample preparation involved hydrolysis of menadiol conjugates and oxidizing the released menadiol to menadione. Using this method, urinary menadione was shown to increase in response to 3 years of phylloquinone supplementation. This HPLC method is a sensitive and reproducible way to detect menadione in urine.

Phylloquinone and dihydrophylloquinone contents of mixed dishes, processed meats, soups and cheeses

Assessment of dietary intakes of phylloquinone (VK-1) and dihydrophylloquinone (dK) has been limited by an overall deficit of food composition data, especially for mixed dishes and processed foods. Ninety-eight geographically representative food samples, obtained as part of the National Food and Nutrient Analysis Program (NFNAP), were analyzed for VK-1 and dK using reversed phase HPLC with fluorescent detection. The VK-1 concentrations of the mixed dishes, processed meats, soups, and cheeses ranged from zero (non-detectable, ND) to 11.1 μg/100 g; the dK concentrations ranged from zero (ND) to 22.4 μg/100 g. No dK was detected in the cheese samples. Minimal variation in VK-1 content was observed between the cooked and uncooked samples. Mixed dishes, processed meats, soups, and cheeses contain relatively small amounts of phylloquinone and dK when compared with vegetables and certain plant oils. However, since these foods may frequently be consumed in large amounts, they may be important dietary contributors of vitamin K.