Associations of inflammatory and metabolic biomarkers with incident erosive hand osteoarthritis in the osteoarthritis initiative cohort.

OBJECTIVE: Erosive hand osteoarthritis (eHOA) is a subtype of hand osteoarthritis (OA) that develops in finger joints with pre-existing OA and is differentiated by clinical characteristics (hand pain/disability, inflammation, and erosions) that suggest inflammatory or metabolic processes. METHOD: This was a longitudinal nested case-cohort design among Osteoarthritis Initiative participants who had hand radiographs at baseline and 48-months, and biospecimens collected at baseline. We classified incident radiographic eHOA in individuals with ≥1 joint with Kellgren-Lawrence ≥2 and a central erosion present at 48-months but not at baseline. We used a random representative sample (n = 1282) for comparison. We measured serum biomarkers of inflammation, insulin resistance and dysglycemia, and adipokines using immunoassays and enzymatic colorimetric procedures, blinded to case status. RESULTS: Eighty-six participants developed incident radiographic eHOA. In the multivariate analyses adjusted for age, gender, race, smoking, and body mass index, and after adjustment for multiple analyses, incident radiographic eHOA was associated with elevated levels of interleukin-7 (risk ratio (RR) per SD = 1.30 [95% confidence interval (CI) 1.09, 1.55] p trend 0.01). CONCLUSION: This exploratory study suggests an association of elevated interleukin-7, an inflammatory cytokine, with incident eHOA, while other cytokines or biomarkers of metabolic inflammation were not associated. Interleukin-7 may mediate inflammation and tissue damage in susceptible osteoarthritic finger joints and participate in erosive progression.

Dietary Intakes of Community-Dwelling Adults in the United States across Older Adulthood: National Health and Nutrition Examination Survey 2015-March 2020.

BACKGROUND: Dietary guidance is set on the basis of age and life stage and defines older adults as ≥60 y. Yet, little is known about if and/or how diet quality differs beyond the age of 60. OBJECTIVE: The objective of this study was to compare the dietary intakes of 60-69 (n = 2079), 70-79 (n = 1181), and 80+ y old (n = 644) noninstitutionalized men and women in the United States using the Healthy Eating Index 2015 (HEI) and the What We Eat in America food categories. METHODS: Data were obtained from National Health and Nutrition Examination Survey 2015-2016 and 2017-March 2020. HEI and component scores were calculated using the population ratio method. Population estimates for dietary intake were calculated as the average reported over 2 separate nonconsecutive 24-h dietary recalls. RESULTS: In men and women, the reported energy intake was lower among the 80+ y olds (kcal/d men-80+: 1884 ± 30, 70-79: 2022 ± 33, 60-69: 2142 ± 39; women-80+: 1523 ± 36; 70-79: 1525 ± 33, 60-69: 1650 ± 25; P-trend < 0.001). Total HEI scores did not differ significantly across the 3 age categories, but the 80+ y olds had significantly lower scores for the green vegetables and beans component than the 60-69 y olds [men-mean (95% confidence interval): 2.0 (1.5, 2.5) compared with 3.4 (2.6, 4.1); women-2.3 (1.8, 2.8) compared with 4.4 (3.7, 5.0)]. In women, the percentage of daily calories from protein was significantly lower in the 80+ y olds than in the 60-69 and 70-79 y olds (12.9% ± 0.6%, compared with 17.0% ± 0.9% and 15.6% ± 0.6%, respectively). Protein intake did not differ significantly among the 3 age groups in men. The 80+ y old men and women reported consuming a significantly higher percentage of calories from snacks and sweets compared with the 60-69 y olds (men-80+: 18.1% ± 0.8%, 60-69: 15.4% ± 0.7%; women-80+: 19.6% ± 0.8%, 60-69: 15.5% ± 0.7%). CONCLUSION: The diet of 80+ y olds differed from that of 60-69 y olds in some key components, including energy, snacks and sweets, protein, and green vegetables. Future research is needed to determine if there are health-related consequences to these differences.

Inhibit progression of coronary artery calcification with vitamin K in hemodialysis patients (the iPACK-HD study): a randomized, placebo-controlled multi-center, pilot trial.

BACKGROUND: Vitamin K activates matrix Gla protein (MGP), a key inhibitor of vascular calcification. There is a high prevalence of sub-clinical vitamin K deficiency in patients with end-stage kidney disease. METHODS: A parallel randomized placebo-controlled pilot trial was designed to determine whether 10 mg of phylloquinone thrice weekly versus placebo modifies coronary artery calcification progression over 12 months in patients requiring hemodialysis with a coronary artery calcium score (CAC) ≥30 Agatston Units (ClinicalTrials.gov identifier NCT01528800). The primary outcome was feasibility (recruitment rate, compliance with study medication, study completion and adherence overall to study protocol). CAC score was used to assess calcification at baseline and 12 months. Secondary objectives were to explore the impact of phylloquinone on vitamin K-related biomarkers (phylloquinone, dephospho-uncarboxylated MGP and the Gla-osteocalcin to Glu-osteocalcin ratio) and events of clinical interest. RESULTS: A total of 86 patients with a CAC score ≥30 Agatston Units were randomized to either 10 mg of phylloquinone or a matching placebo three times per week. In all, 69 participants (80%) completed the trial. Recruitment rate (4.4 participants/month) and medication compliance (96%) met pre-defined feasibility criteria of ≥4.17 and ≥90%, respectively. Patients randomized to phylloquinone for 12 months had significantly reduced levels of dephospho-uncarboxylated MGP (86% reduction) and increased levels of phylloquinone and Gla-osteocalcin to Glu-osteocalcin ratio compared with placebo. There was no difference in the absolute or relative progression of coronary artery calcification between groups. CONCLUSION: We demonstrated that phylloquinone treatment improves vitamin K status and that a fully powered randomized trial may be feasible.

Brain vitamin D forms, cognitive decline, and neuropathology in community-dwelling older adults.

INTRODUCTION: Vitamin D purportedly protects against cognitive decline and dementia based on observational data using circulating 25-hydroxyvitamin D (25(OH)D). Little is known about vitamin D in the human brain and the association with dementia or neuropathology. METHODS: Decedents of the Rush Memory and Aging Project (n = 290) had vitamin D concentrations measured in four brain regions. Associations with cognitive and neuropathological outcomes were estimated using linear and logistic regression. RESULTS: The main form of vitamin D in all brain regions measured was 25(OH)D3 . Higher brain 25(OH)D3 concentrations were associated with a 25% to 33% lower odds of dementia or mild cognitive impairment (MCI) at the last visit before death (all P ≤ .031). However, brain 25(OH)D concentrations were not associated with any post-mortem neuropathology outcome studied. DISCUSSION: Higher brain 25(OH)D3 concentrations were associated with better cognitive function prior to death. Additional research is needed to clarify the specific mechanisms underlying this potentially protective relationship.

Multiple Dietary Vitamin K Forms Are Converted to Tissue Menaquinone-4 in Mice.

BACKGROUND: Vitamin K is a term that comprises a family of structurally related quinones, phylloquinone (PK) and the menaquinones (MKn), that share a common naphthoquinone ring but vary in sidechain length (n) and saturation. Dietary PK is a biosynthetic precursor to tissue menaquinone-4 (MK4), but little is known about the absorption and metabolism of dietary MKn. OBJECTIVE: To characterize the absorption and metabolism of dietary MKn relative to PK. METHODS: In the 4-week diet study, 10-week-old male and female C57BL/6 mice were pair-fed a vitamin K deficient diet (control) or a diet supplemented with 5.0 µmol/kg total PK, MK4, and/or MK9 (separately and in combination). In the 1-week stable isotope study, 12-week-old mice were pair-fed diets containing 2.2 µmol/kg PK (unlabeled control), 2H7PK, 13C11MK4, 2H7MK7, or 2H7MK9. Vitamin K tissue content was quantified by HPLC and/or LC-MS, and concentrations were compared by sex and diet group using 2-factor ANOVA. RESULTS: Regardless of the form(s) of vitamin K provided in the diet, tissue MK4 concentrations did not differ across equimolar supplemented groups in the kidney, adipose, reproductive organ, bone, or pancreas in either males or females in the diet study (all P values > 0.05). Isotopic labeling confirmed the naphthoquinone ring of MK4 in tissues originated from the administered dietary PK or MKn. Despite equimolar supplementation, accumulation of the administered dietary form differed across diet groups in small intestinal segments (all P values < 0.002) and the liver (P < 0.001). Female mice had greater total vitamin K than males in every tissue examined (P < 0.05). CONCLUSIONS: Dietary PK, MK4, MK7, and MK9 all served as precursors to tissue MK4 in mice. This study expands our understanding of vitamin K metabolism and supports a common conversion mechanism of all dietary vitamin K forms to MK4. Further investigation of the metabolism and physiological roles of MK4 that may be independent of classical vitamin K function is warranted.

Vitamin D and Vitamin K Concentrations in Human Brain Tissue Are Influenced by Freezer Storage Time: The Memory and Aging Project.

BACKGROUND: Vitamins D and K, which are present in human brain, may have a role in neurodegenerative disease. OBJECTIVES: Given the interest in measuring nutrient concentrations in archived brain samples, it is important to evaluate whether freezer storage time affects these concentrations. Therefore, we evaluated differences in vitamin D and vitamin K concentrations in human brain samples stored for various lengths of time. METHODS: Postmortem brain samples were obtained from 499 participants in the Rush Memory and Aging Project (mean age 92 y, 72% female). Concentrations of vitamins D and K and their metabolites were measured in 4 regions (midtemporal cortex, midfrontal cortex, cerebellum, anterior watershed white matter) using LC-MS/MS and HPLC, respectively. The predominant forms were 25-hydroxycholecalciferol [25(OH)D3] and menaquinone-4 (MK4). ANOVA was used to determine if concentrations differed according to storage time. RESULTS: The geometric mean of the mean 25(OH)D3 concentration (across 4 regions) in brains stored for 1.1 to 6.0 y did not differ from that in brains stored ≤1.0 y (all P ≥ 0.37), whereas 25(OH)D3 in brains stored >6.0 y was 31-40% lower (P ≤ 0.003). MK4 had similar results, with the geometric mean MK4 concentration in the brains stored ≥9.0 y being 48-52% lower than those in brains stored ≤1.0 y (P ≤ 0.012). The 25(OH)D3 and MK4 concentrations were positively correlated across all 4 regions (all Spearman ρ ≥ 0.79, P < 0.001). CONCLUSIONS: 25(OH)D3 and MK4 appear to be stable in brain tissue from older adults stored at -80°C for up to 6 and 9 y, respectively, but not longer. Freezer storage time should be considered in the design and interpretation of studies using archived brain tissue.

Relationship Between Chronic Kidney Disease, Glucose Homeostasis, and Plasma Osteocalcin Carboxylation and Fragmentation.

OBJECTIVE: Chronic kidney disease (CKD) is associated with reduced insulin sensitivity, through mechanisms that are not well understood. Low vitamin K intake and incomplete carboxylation of the vitamin K-dependent protein osteocalcin may promote insulin resistance. We assessed relationships of osteocalcin concentration, carboxylation, and fragmentation with CKD and glucose homeostasis in a cross-sectional study. METHODS: We included 87 participants without diabetes: 50 (27 female) with moderate to severe CKD (estimated glomerular filtration rate <60 mL/min/1.73 m2 not treated with dialysis) and 37 (17 female) healthy controls. Total osteocalcin was measured by immunoassay, and osteocalcin carboxylation and fragmentation status by liquid chromatography-electrospray ionization-based mass spectrometric immunoassay. Endpoints included glucose tolerance (based on 2-hour oral glucose tolerance test), insulin sensitivity (hyperinsulinemic-euglycemic clamp), and pancreatic beta-cell function (intravenous glucose tolerance test). RESULTS: The total plasma osteocalcin concentration was higher in the CKD group (mean [standard deviation] 102.9 [147.5]) than that in the control group (53.6 [51.1] ng/mL, P = .03), and more osteocalcin was circulating as fragments. The extent of osteocalcin carbocylation did not differ between individuals with and without CKD. Osteocalcin concentration, carboxylation, and fragmentation were not associated with any measure of glucose homeostasis in multivariable-adjusted analyses. CONCLUSIONS: In CKD, circulating osteocalcin concentrations are elevated, in part due to larger proportions of fragmented forms. However, osteocalcin carboxylation status is not significantly different between individuals with and without CKD. Our data also do not provide support for the hypothesis that differences in osteocalcin carboxylation may explain reduced insulin sensitivity in individuals with CKD.

Atorvastatin Decreases Renal Menaquinone-4 Formation in C57BL/6 Male Mice.

BACKGROUND: Menaquinone-4 (MK4), a vitamin K metabolite, is converted from phylloquinone through a process that requires intermediates of endogenous cholesterol production. Recent evidence suggests that MK4 is involved in kidney function. OBJECTIVE: The purpose of this study was to determine the effect of atorvastatin treatment on MK4 formation in young and old male mice. METHODS: C57BL/6 male mice (4-mo-old and 20-mo-old) were randomly assigned to either a diet containing 300 mg atorvastatin/kg with 3 mg phylloquinone/kg or a control diet containing 3 mg phylloquinone/kg for 8 wk. During week 8, all mice received deuterium-labeled phylloquinone in the diet. Labeled and unlabeled phylloquinone and MK4 in liver, kidney, brain, and intestine were measured by atmospheric pressure chemical ionization LC/MS. 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase gene expression was quantified by reverse transcriptase-PCR. Tissue MK4 and phylloquinone concentrations were compared between atorvastatin treatment groups with use of general linear models. RESULTS: There was no age-treatment interaction on MK4 tissue concentrations. In atorvastatin-treated mice, total MK4 and percentage of deuterium-labeled MK4 in kidney were both approximately 45% lower compared to values in mice not given atorvastatin (all P < 0.05). MK4 concentrations did not differ between groups in any other tissue measured. CONCLUSION: In male mice, atorvastatin reduced endogenous MK4 formation in the kidney, but not other organs. These observations are consistent with our hypothesis that cholesterol metabolism is involved in the generation of MK4. Further research is needed to understand potential regulatory mechanisms and the unique functions of MK4 in the kidney.

Plasma Response to Deuterium-Labeled Vitamin K Intake Varies by TG Response, but Not Age or Vitamin K Status, in Older and Younger Adults.

Background: Phylloquinone is the primary form of vitamin K in the diet and circulation. Large intra- and interindividual variances in circulating phylloquinone have been partially attributed to age. However, little is known about the nondietary factors that influence phylloquinone absorption and metabolism. Similarly, it is not known if phylloquinone absorption is altered by the individual's existing vitamin K status. Objective: The purpose of this secondary substudy was to compare plasma response with deuterium-labeled phylloquinone intake in older and younger adults after dietary phylloquinone depletion and repletion. Methods: Forty-two older [mean ± SD age: 67.2 ± 8.0 y; body mass index (BMI; in kg/m2): 25.4 ± 4.6; n = 12 men, 9 women] and younger (mean ± SEM age: 31.8 ± 6.6 y; BMI: 25.5 ± 3.3; n = 9 men, 12 women) adults were maintained on sequential 28-d phylloquinone depletion (∼10 µg phylloquinone/d) and 28-d phylloquinone repletion (∼500 µg phylloquinone/d) diets. On the 23rd d of each diet phase, participants consumed deuterated phylloquinone-rich collard greens (2H-phylloquinone). Plasma and urinary outcome measures over 72 h were compared by age group, sex, and dietary phase via 2-factor repeated-measures ANOVA. Results: The plasma 2H-phylloquinone area under the curve (AUC) did not differ in response to phylloquinone depletion or repletion, but was 34% higher in older than in younger adults (P = 0.02). However, plasma 2H-phylloquinone AUC was highly correlated with the serum triglyceride (TG) AUC (r2 = 0.45). After adjustment for serum TG response, the age effect on the plasma 2H-phylloquinone AUC was no longer significant. Conclusions: Plasma 2H-phylloquinone response did not differ between phylloquinone depletion and repletion in older and younger adults. The age effect observed was explained by the serum TG response and was completely attenuated after adjustment. Plasma response to phylloquinone intake, therefore, seems to be a predominantly lipid-driven effect and not dependent on existing vitamin K status. More research is required to differentiate the effect of endogenous compared with exogenous lipids on phylloquinone absorption. This trial was registered at clinicaltrials.gov as NCT00336232.

Meta-analysis across Cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE) consortium provides evidence for an association of serum vitamin D with pulmonary function.

The role that vitamin D plays in pulmonary function remains uncertain. Epidemiological studies reported mixed findings for serum 25-hydroxyvitamin D (25(OH)D)-pulmonary function association. We conducted the largest cross-sectional meta-analysis of the 25(OH)D-pulmonary function association to date, based on nine European ancestry (EA) cohorts (n 22 838) and five African ancestry (AA) cohorts (n 4290) in the Cohorts for Heart and Aging Research in Genomic Epidemiology Consortium. Data were analysed using linear models by cohort and ancestry. Effect modification by smoking status (current/former/never) was tested. Results were combined using fixed-effects meta-analysis. Mean serum 25(OH)D was 68 (sd 29) nmol/l for EA and 49 (sd 21) nmol/l for AA. For each 1 nmol/l higher 25(OH)D, forced expiratory volume in the 1st second (FEV1) was higher by 1·1 ml in EA (95 % CI 0·9, 1·3; P<0·0001) and 1·8 ml (95 % CI 1·1, 2·5; P<0·0001) in AA (P race difference=0·06), and forced vital capacity (FVC) was higher by 1·3 ml in EA (95 % CI 1·0, 1·6; P<0·0001) and 1·5 ml (95 % CI 0·8, 2·3; P=0·0001) in AA (P race difference=0·56). Among EA, the 25(OH)D-FVC association was stronger in smokers: per 1 nmol/l higher 25(OH)D, FVC was higher by 1·7 ml (95 % CI 1·1, 2·3) for current smokers and 1·7 ml (95 % CI 1·2, 2·1) for former smokers, compared with 0·8 ml (95 % CI 0·4, 1·2) for never smokers. In summary, the 25(OH)D associations with FEV1 and FVC were positive in both ancestries. In EA, a stronger association was observed for smokers compared with never smokers, which supports the importance of vitamin D in vulnerable populations.

Vitamin K-Dependent Carboxylation of Matrix Gla Protein Influences the Risk of Calciphylaxis.

Matrix Gla protein (MGP) is a potent inhibitor of vascular calcification. The ability of MGP to inhibit calcification requires the activity of a vitamin K-dependent enzyme, which mediates MGP carboxylation. We investigated how MGP carboxylation influences the risk of calciphylaxis in adult patients receiving dialysis and examined the effects of vitamin K deficiency on MGP carboxylation. Our study included 20 patients receiving hemodialysis with calciphylaxis (cases) and 20 patients receiving hemodialysis without calciphylaxis (controls) matched for age, sex, race, and warfarin use. Cases had higher plasma levels of uncarboxylated MGP (ucMGP) and carboxylated MGP (cMGP) than controls. However, the fraction of total MGP that was carboxylated (relative cMGP concentration = cMGP/[cMGP + uncarboxylated MGP]) was lower in cases than in controls (0.58±0.02 versus 0.69±0.03, respectively; P=0.003). In patients not taking warfarin, cases had a similarly lower relative cMGP concentration. Each 0.1 unit reduction in relative cMGP concentration associated with a more than two-fold increase in calciphylaxis risk. Vitamin K deficiency associated with lower relative cMGP concentration in multivariable adjusted analyses (ß=-8.99; P=0.04). In conclusion, vitamin K deficiency-mediated reduction in relative cMGP concentration may have a role in the pathogenesis of calciphylaxis. Whether vitamin K supplementation can prevent and/or treat calciphylaxis requires further study.

Vegetables and Mixed Dishes Are Top Contributors to Phylloquinone Intake in US Adults: Data from the 2011-2012 NHANES.

Background: Phylloquinone is the most abundant form of vitamin K in US diets. Green vegetables are considered the predominant dietary source of phylloquinone. As our food supply diversifies and expands, the food groups that contribute to phylloquinone intake are also changing, which may change absolute intakes. Thus, it is important to identify the contributors to dietary vitamin K estimates to guide recommendations on intakes and food sources.Objective: The purpose of this study was to estimate 1) the amount of phylloquinone consumed in the diet of US adults, 2) to estimate the contribution of different food groups to phylloquinone intake in individuals with a high or low vegetable intake (≥2 or <2 cups vegetables/d), and 3) to characterize the contribution of different mixed dishes to phylloquinone intake.Methods: Usual phylloquinone intake was determined from NHANES 2011-2012 (≥20 y old; 2092 men and 2214 women) and the National Cancer Institute Method by utilizing a complex, stratified, multistage probability-cluster sampling design.Results: On average, 43.0% of men and 62.5% of women met the adequate intake (120 and 90 µg/d, respectively) for phylloquinone, with the lowest self-reported intakes noted among men, especially in the older age groups (51-70 and ≥71 y). Vegetables were the highest contributor to phylloquinone intake, contributing 60.0% in the high-vegetable-intake group and 36.1% in the low-vegetable-intake group. Mixed dishes were the second-highest contributor to phylloquinone intake, contributing 16.0% in the high-vegetable-intake group and 28.0% in the low-vegetable-intake group.Conclusion: Self-reported phylloquinone intakes from updated food composition data applied to NHANES 2011-2012 reveal that fewer men than women are meeting the current adequate intake. Application of current food composition data confirms that vegetables continue to be the primary dietary source of phylloquinone in the US diet. However, mixed dishes and convenience foods have emerged as previously unrecognized but important contributors to phylloquinone intake in the United States, which challenges the assumption that phylloquinone intake is a marker of a healthy diet. These findings emphasize the need for the expansion of food composition databases that consider how mixed dishes are compiled and defined.

Circulating Vitamin K Is Inversely Associated with Incident Cardiovascular Disease Risk among Those Treated for Hypertension in the Health, Aging, and Body Composition Study (Health ABC).

Background: A role for vitamin K in coronary artery calcification (CAC), a subclinical manifestation of cardiovascular disease (CVD), has been proposed because vitamin K-dependent proteins, including the calcification inhibitor matrix Gla protein (MGP), are present in vascular tissue. Observational studies found that low circulating phylloquinone (vitamin K-1) was associated with increased CAC progression, especially in persons treated for hypertension. It is unknown whether hypertension treatment modifies this putative role of vitamin K in clinical CVD risk.Objective: We determined the association between vitamin K status and incident clinical CVD in older adults in the Health ABC (Health, Aging, and Body Composition Study) and whether the association differed by hypertension treatment status.Methods: Plasma phylloquinone was measured in 1061 participants free of CVD (70-79 y of age, 58% women, 39% black). Plasma uncarboxylated MGP [(dp)ucMGP] was measured in a subset of 635 participants. Multivariate Cox models estimated the HR for incident CVD over 12.1 follow-up years. Effect modification by hypertension was tested with the use of interaction terms.Results: Neither low plasma phylloquinone (<0.2 nmol/L) nor elevated (dp)ucMGP (≥574 pmol/L) was significantly associated with incident CVD [respective HRs (95% CIs): 1.27 (0.75, 2.13) and 1.02 (0.72, 1.45)]. In participants treated for hypertension (n = 489; 135 events), low plasma phylloquinone was associated with higher CVD risk overall (HR: 2.94; 95% CI: 1.41, 6.13). In those with untreated hypertension (n = 153; 48 events) and without hypertension (n = 418; 92 events), low plasma phylloquinone was not associated with incident CVD. The association between high (dp)ucMGP did not differ by hypertension treatment status (P-interaction = 0.72).Conclusions: Vitamin K status was not significantly associated with CVD risk overall, but low plasma phylloquinone was associated with a higher CVD risk in older adults treated for hypertension. Additional evidence from larger clinical studies is needed to clarify the importance of vitamin K to CVD in persons treated for hypertension, a segment of the population at high risk of clinical CVD events.

Vitamin K Metabolism in a Rat Model of Chronic Kidney Disease.

BACKGROUND: Patients with chronic kidney disease (CKD) have very high levels of uncarboxylated, inactive, extra-hepatic vitamin K-dependent proteins measured in circulation, putting them at risk for complications of vitamin K deficiency. The major form of vitamin K found in the liver is phylloquinone (K1). Menaquinone-4 (MK-4) is the form of vitamin K that is preferentially found in extra-hepatic tissues. METHODS: In the present study, we assessed tissue concentrations of K1 and MK-4 and the expression of vitamin K-related genes in a rat model of adenine-induced CKD. RESULTS: It was found that rats with both mild and severe CKD had significantly lower amounts of K1 measured in liver, spleen and heart and higher levels of MK-4 measured in kidney cortex and medulla. All animals treated with high dietary K1 had an increase in tissue levels of both K1 and MK-4; however, the relative increase in K1 differed suggesting that the conversion of K1 to MK-4 may be a regulated/limiting process in some tissues. There was a decrease in the thoracic aorta expression of vitamin K recycling (Vkor) and utilization (Ggcx) enzymes, and a decrease in the kidney level of vitamin K1 to MK-4 bioconversion enzyme Ubiad1 in CKD. CONCLUSION: Taken together, these findings suggest that CKD impacts vitamin K metabolism, and this occurs early in the disease course. Our findings that vitamin K metabolism is altered in the presence of CKD provides further support that sub-clinical vitamin K deficiency may represent a modifiable risk factor for vascular and bone health in this population.

Gamma-carboxylation and fragmentation of osteocalcin in human serum defined by mass spectrometry.

Serum osteocalcin (Oc) concentration is a highly specific measure of bone turnover, but its circulating proteoform(s) have not been well defined. Based on immunological methods, the major forms are thought to be the intact polypeptide and a large N-terminal-mid molecule fragment for which there is no consensus on the precise sequence. Vitamin K-dependent gamma (γ)-carboxylated variants of Oc are also found in circulation but there have been no methods that can define how many of the three potential γ-carboxyglutamic acid (Gla) residues are γ-carboxylated or provide their relative abundances. Recent reports that uncarboxylated and partially γ-carboxylated Oc forms have hormonal function underscore the need for precise evaluation of Oc at all three potential γ-carboxylation sites. Herein, mass spectrometric immunoassay (MSIA) was used to provide qualitative and semiquantitative (relative percent abundance) information on Oc molecular variants as they exist in individual plasma and serum samples. Following verification that observable Oc proteoforms were accurately assigned and not simply ex vivo artifacts, MALDI-MSIA and ESI-MSIA were used to assess the relative abundance of Oc truncation and γ-carboxylation, respectively, in plasma from 130 patients enrolled in vitamin K supplementation trials. Human Oc was found to circulate in over a dozen truncated forms with each of these displaying anywhere from 0-3 Gla residues. The relative abundance of truncated forms was consistent and unaffected by vitamin K supplementation. In contrast, when compared with placebo, vitamin K supplementation dramatically increased the fractional abundance of Oc with three Gla residues, corresponding to a decrease in the fractional abundance of Oc with zero Gla residues. These findings unequivocally document that increased vitamin K intake reduces the uncarboxylated form of Oc. Several reports of a positive effect of vitamin K intake on insulin sensitivity in humans have shown that un- or undercarboxylation of Oc, unlike in mice, is not associated with insulin resistance. Analyses similar to those described here will be useful to understand the functional significance of Oc γ-carboxylation in human health and disease.