Vitamin D, Alzheimer's disease and related dementia.

Vitamin D has been proposed as a potential strategy to mitigate age-related cognitive decline and dementia, including Alzheimer's dementia, the predominant type of dementia. Rodent studies have provided insight into the potential mechanisms underlying the role of vitamin D in Alzheimer's disease and dementia. However, inconsistencies with respect to age, sex, and genetic background of the rodent models used poses some limitations regarding scientific rigor and translation. Several human observational studies have evaluated the association of vitamin D status with cognitive decline and dementia, and the results are conflicting. Randomized clinical trials of vitamin D supplementation have included cognitive outcomes. However, most of the available trials have not been designed specifically to test the effect of vitamin D on age-related cognitive decline and dementia, so it remains questionable how much additional vitamin D will improve cognitive performance. Here we evaluate the strengths and limitations of the available evidence regarding the role of vitamin D in AD, cognitive decline, dementia.

Perspective: Promoting Healthy Aging through Nutrition: A Research Centers Collaborative Network Workshop Report.

Within 20 y, the number of adults in the United States over the age of 65 y is expected to more than double and the number over age 85 y is expected to more than triple. The risk for most chronic diseases and disabilities increases with age, so this demographic shift carries significant implications for the individual, health care providers, and population health. Strategies that delay or prevent the onset of age-related diseases are becoming increasingly important. Although considerable progress has been made in understanding the contribution of nutrition to healthy aging, it has become increasingly apparent that much remains to be learned, especially because the aging process is highly variable. Most federal nutrition programs and nutrition research studies define all adults over age 65 y as "older" and do not account for physiological and metabolic changes that occur throughout older adulthood that influence nutritional needs. Moreover, the older adult population is becoming more racially and ethnically diverse, so cultural preferences and other social determinants of health need to be considered. The Research Centers Collaborative Network sponsored a 1.5-d multidisciplinary workshop that included sessions on dietary patterns in health and disease, timing and targeting interventions, and health disparities and the social context of diet and food choice. The agenda and presentations can be found at https://www.rccn-aging.org/nutrition-2023-rccn-workshop. Here we summarize the workshop's themes and discussions and highlight research gaps that if filled will considerably advance our understanding of the role of nutrition in healthy aging.

Matrix Gla protein and the long-term incidence and progression of coronary artery and aortic calcification in the Multi-Ethnic Study of Atherosclerosis.

BACKGROUND AND AIMS: Matrix Gla protein (MGP) is an inhibitor of calcification that requires carboxylation by vitamin K for activity. The inactive form of MGP, dephosphorylated-uncarboxylated matrix Gla protein (dp-ucMGP), has been associated with increased calcification. However, it is not known whether there is a longitudinal relationship between dephosphorylated-uncarboxylated matrix Gla protein levels and coronary and aortic calcification in large population cohorts. METHODS: The Multi-Ethnic Study of Atherosclerosis (MESA) followed participants with serial cardiac computed tomography (CT) measures of vascular calcification. Dp-ucMGP was measured at baseline in a subset of participants who completed baseline and follow-up CTs approximately 10 years later and had available plasma specimens (n = 2663). Linear mixed effects models (LMMs) were used to determine the association of dp-ucMGP with the simultaneous incidence and progression of coronary artery, ascending thoracic aortic, or descending thoracic aortic calcification (CAC, ATAC, DTAC)]. RESULTS: For every one standard deviation (SD, 178 pmol/L) increment in dp-ucMGP, CAC increased by 3.44 ([95% CI = 1.68, 5.21], p < 0.001) Agatston units/year (AU/year), ATAC increased by 0.63 ([95% CI = 0.27, 0.98], p = 0.001) AU/year, and DTAC increased by 8.61 ([95% CI = 4.55, 12.67], p < 0.001) AU/year. The association was stronger for DTAC in those ≥65 years and with diabetes. CONCLUSIONS: We found a positive association of the inactive form of matrix Gla protein, dp-ucMGP, and long-term incidence/progression of CAC, ATAC, and DTAC. Future studies should investigate dp-ucMGP as a calcification regulator and MGP as a possible therapeutic target to slow progression of calcification in the vasculature.

Dietary protein intake in midlife in relation to healthy aging - results from the prospective Nurses' Health Study cohort.

BACKGROUND: Protein intake plays an important role in maintaining the health status of older adults. However, few epidemiologic studies examined midlife protein intake in relation to healthy aging. OBJECTIVES: The objective of this study was to evaluate the long-term role of dietary protein intake in healthy aging among female participants in the prospective Nurses' Health Study (NHS) cohort. METHODS: We included 48,762 NHS participants aged <60 y in 1984. Total protein, animal protein, dairy protein (a subset of animal protein), and plant protein were derived from validated food frequency questionnaires. Healthy aging was defined as being free from 11 major chronic diseases, having good mental health, and not having impairments in either cognitive or physical function, as assessed in the 2014 or 2016 NHS participant questionnaires. We used multivariate logistic regression adjusted for lifestyle, demographics, and health status to estimate the odds ratios (ORs) and 95% confidence intervals for protein intake in relation to healthy aging. RESULTS: A total of 3721 (7.6%) NHS participants met our healthy aging definition. Protein intake was significantly associated with higher odds of healthy aging. The ORs (95% confidence intervals) per 3%-energy increment with healthy aging were 1.05 (1.01, 1.10) for total protein, 1.07 (1.02, 1.11) for animal protein, 1.14 (1.06, 1.23) for dairy protein, and 1.38 (1.24, 1.54) for plant protein. Plant protein was also associated with higher odds of absence of physical function limitations and good mental status. In substitution analyses, we observed significant positive associations for the isocaloric replacement of animal or dairy protein, carbohydrate, or fat with plant protein (ORs for healthy aging: 1.22-1.58 for 3% energy replacement with plant protein). CONCLUSIONS: Dietary protein intake, especially plant protein, in midlife, is associated with higher odds of healthy aging and with several domains of positive health status in a large cohort of female nurses.

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.

Lack of Consensus Between Measurements of Plasma Phylloquinone by Enzyme-Linked Immunosorbent assays and a Well-Validated High-Performance Liquid Chromatographic Method.

Enzyme-linked i2mmunosorbent assays (ELISAs) that measure circulating phylloquinone have become commercially available, but their validity is uncertain. The objective of this study was to compare plasma phylloquinone concentrations measured using two commercially available ELISAs with concentrations measured using a validated high-performance liquid chromatography (HPLC) assay in 108 samples obtained from participants in a depletion (∼10 mcg phylloquinone/d)-supplementation (∼500 mcg phylloquinone/d) study. The geometric mean of plasma phylloquinone measured with ELISA A was 0.70 nmol/L, 37% lower than that measured with HPLC. The mean of the ELISA B measures was 12.4 nmol/L, >700% higher than the HPLC measures. Plasma phylloquinone measured using HPLC was significantly lower during phylloquinone depletion than supplementation (0.4 ± 0.1 compared with 1.2 ± 0.2 nmol/L; P < 0.001). Neither of the two ELISAs detected any significant difference in plasma phylloquinone concentrations between depletion and supplementation (ELISA A, P = 0.76; ELISA B, P = 0.29). These findings reinforce the need to validate plasma phylloquinone assays as they become available. Curr Dev Nutr 2023;x:xx.

Association of Vitamin K Status with Arterial Calcification and Stiffness in Chronic Kidney Disease: The Chronic Renal Insufficiency Cohort.

Background: Arterial calcification and stiffness are common in people with chronic kidney disease (CKD). Higher vitamin K status has been associated with less arterial calcification and stiffness in CKD in cross-sectional studies. Objectives: To determine the association of vitamin K status with coronary artery calcium (CAC) and arterial stiffness [pulse wave velocity (PWV)] at baseline and over 2-4 follow-up years in adults with mild-to-moderate CKD. Methods: Participants (n = 2722) were drawn from the well-characterized Chronic Renal Insufficiency Cohort. Two vitamin K status biomarkers, plasma phylloquinone and plasma dephospho-uncarboxylated matrix gla protein [(dp)ucMGP], were measured at baseline. CAC and PWV were measured at baseline and over 2-4 y of follow-up. Differences across vitamin K status categories in CAC prevalence, incidence, and progression (defined as ≥100 Agatston units/y increase) and PWV at baseline and over follow-up were evaluated using multivariable-adjusted generalized linear models. Results: CAC prevalence, incidence, and progression did not differ across plasma phylloquinone categories. Moreover, CAC prevalence and incidence did not differ according to plasma (dp)ucMGP concentration. Compared with participants with the highest (dp)ucMGP (≥450 pmol/L), those in the middle category (300-449 pmol/L) had a 49% lower rate of CAC progression (incidence rate ratio: 0.51; 95% CI: 0.33, 0.78). However, CAC progression did not differ between those with the lowest (<300 pmol/L) and those with the highest plasma (dp)ucMGP concentration (incidence rate ratio: 0.82; 95% CI: 0.56, 1.19). Neither vitamin K status biomarker was associated with PWV at baseline or longitudinally. Conclusions: Vitamin K status was not consistently associated with CAC or PWV in adults with mild-to-moderate CKD.

An Exploratory Case-Control Study on the Associations of Bacterially-Derived Vitamin K Forms with the Intestinal Microbiome and Obesity-Related Osteoarthritis.

Background: Evidence suggests that natural metabolites produced by intestinal microorganisms may have beneficial or harmful effects on osteoarthritis (OA). This could include menaquinones, which are bacterially-synthesized, biologically-active vitamin K forms abundant in the intestinal microbiome. Objectives: The overall goal of this study was to evaluate the association of intestinally-derived menaquinones with obesity-related OA. Methods: This case-control study used data and biospecimens derived from a subgroup of Johnston County Osteoarthritis Study participants. Fecal menaquinone concentrations and microbial composition were determined in 52 obese participants with hand and knee OA and 42 age- and sex-matched obese participants without OA. The inter-relationships among fecal menaquinones were evaluated using principal component analysis. The differences in alpha and beta diversities and microbial composition across menaquinone clusters were evaluated using ANOVA. Results: The samples were clustered into the following 3 groups: cluster 1 characterized by higher fecal menaquinone-9 and -10 concentrations, cluster 2 characterized by lower overall menaquinone concentrations, and cluster 3 characterized by higher menaquinone-12 and -13 concentrations. Overall, fecal menaquinone clusters did not differ between participants with or without OA (P = 0.707). Microbial diversity did not differ across the fecal menaquinone clusters (all F-test P > 0.12). However, the relative abundance of bacterial taxa differed among clusters, with higher abundance of Coprococcus, Prevotella, and Eggerthella in cluster 2 than in cluster 1; higher abundance of Oscillospira, Dorea, Eubacterium, and Bacteroides in cluster 3 than in cluster 1; and higher abundance of Prevotella, Sutterella, and Dorea in cluster 3 than in cluster 2 (all P < 0.001). Conclusion: Menaquinones were variable and abundant in the human gut, but the fecal menaquinone clusters did not differ with OA status. Although the relative abundance of specific bacterial taxa differed among fecal menaquinone clusters, the relevance of these differences with respect to vitamin K status and human health is uncertain.

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.

Vitamin K Status and Cognitive Function in Adults with Chronic Kidney Disease: The Chronic Renal Insufficiency Cohort.

Vitamin K is linked to cognitive function, but studies in individuals with chronic kidney disease (CKD), who are at risk for vitamin K insufficiency and cognitive impairment, are lacking. The cross-sectional association of vitamin K status biomarkers with cognitive performance was evaluated in ≥55-y-old adults with CKD (N = 714, 49% female, 44% black). A composite score of a cognitive performance test battery, calculated by averaging the z scores of the individual tests, was the primary outcome. Vitamin K status was measured using plasma phylloquinone and dephospho-uncarboxylated matrix Gla protein [(dp)ucMGP]. Participants with low plasma (dp)ucMGP, reflecting higher vitamin K status, had better cognitive performance than those in the two higher (dp)ucMGP categories based on the composite outcome (P = 0.03), whereas it did not significantly differ according to plasma phylloquinone categories (P = 0.08). Neither biomarker was significantly associated with performance on individual tests (all P > 0.05). The importance of vitamin K to cognitive performance in adults with CKD remains to be clarified.

Association of vitamin K with cognitive decline and neuropathology in community-dwelling older persons.

Higher vitamin K intakes have been associated with better cognitive function, suggestive of a vitamin K mechanistic effect or simply reflective of a healthy diet. To test the hypothesis that brain vitamin K is linked to cognitive decline and dementia, vitamin K concentrations were measured in four brain regions, and their associations with cognitive and neuropathological outcomes were estimated in 325 decedents of the Rush Memory and Aging Project. Menaquinone-4 (MK4) was the main vitamin K form in the brain regions evaluated. Higher brain MK4 concentrations were associated with a 17% to 20% lower odds of dementia or mild cognitive impairment (MCI) (P-value < .014), with a 14% to 16% lower odds of Braak stage ≥IV (P-value < 0.045), with lower Alzheimer's disease global pathology scores and fewer neuronal neurofibrillary tangles (P-value < 0.012). These findings provide new and compelling evidence implicating vitamin K in neuropathology underlying cognitive decline and dementia.

Leveraging Observational Cohorts to Study Diet and Nutrition in Older Adults: Opportunities and Obstacles.

By 2060, the number of adults aged ≥65 y is expected to double, and the ≥85 y segment of the population is expected to triple in the United States. US federal nutrition guidance is based on the premise that healthy diets contribute to delaying the onset and progression of many age-related diseases and disability. Yet, little is known about the dietary intakes or nutritional needs across the older adulthood age span. This review aims to identify community-based cohorts that collected information on dietary intake of adults ≥65 y in the United States. Thirty-two cohorts met all inclusion criteria. We summarized information on the cohorts' design, demographics, and diet assessment. We also identified key gaps in the existing databases that, if filled, could enhance their utility to address certain research questions. This review serves as a valuable inventory of cohorts that can be leveraged to answer key questions about the diet and nutritional needs of the oldest old, who represent the fastest growing segment of the population in the United States.

Vitamin K status, all-cause mortality, and cardiovascular disease in adults with chronic kidney disease: the Chronic Renal Insufficiency Cohort.

BACKGROUND: Vascular calcification contributes to cardiovascular disease (CVD) and mortality in individuals with chronic kidney disease (CKD). Vitamin K-dependent proteins function as calcification inhibitors in vascular tissue. OBJECTIVES: We sought to determine the association of vitamin K status with mortality and CVD events in adults with CKD. METHODS: Plasma dephospho-uncarboxylated matrix gla protein ((dp)ucMGP), which increases when vitamin K status is low, and plasma phylloquinone (vitamin K1), which decreases when vitamin K status is low, were measured in 3066 Chronic Renal Insufficiency Cohort participants (median age = 61 y, 45% female, 41% non-Hispanic black, median estimated glomerular filtration rate [eGFR] = 41 mL/min/1.73m2). The association of vitamin K status biomarkers with all-cause mortality and atherosclerotic-related CVD was determined using multivariable Cox proportional hazards regression. RESULTS: There were 1122 deaths and 599 atherosclerotic CVD events over the median 12.8 follow-up years. All-cause mortality risk was 21-29% lower among participants with plasma (dp)ucMGP <450 pmol/L (n = 2361) compared with those with plasma (dp)ucMGP ≥450 pmol/L (adjusted HRs [95% CIs]: <300 pmol/L = 0.71 [0.61, 0.83], 300-449 pmol/L = 0.77 [0.66, 0.90]) and 16-19% lower among participants with plasma phylloquinone ≥0.50 nmol/L (n = 2421) compared to those with plasma phylloquinone <0.50 nmol/L (adjusted HRs: 0.50, 0.99 nmol/L = 0.84 [0.72, 0.99], ≥1.00 nmol/L = 0.81 [0.70, 0.95]). The risk of atherosclerotic CVD events did not significantly differ across plasma (dp)ucMGP or phylloquinone categories. CONCLUSIONS: Two biomarkers of vitamin K status were associated with a lower all-cause mortality risk but not atherosclerotic CVD events. Additional studies are needed to clarify the mechanism underlying this association and evaluate the impact of improving vitamin K status in people with CKD.

Components of the Gut Microbiome That Influence Bone Tissue-Level Strength.

Modifications to the constituents of the gut microbiome influence bone density and tissue-level strength, but the specific microbial components that influence tissue-level strength in bone are not known. Here, we selectively modify constituents of the gut microbiota using narrow-spectrum antibiotics to identify components of the microbiome associated with changes in bone mechanical and material properties. Male C57BL/6J mice (4 weeks) were divided into seven groups (n = 7-10/group) and had taxa within the gut microbiome removed through dosing with: (i) ampicillin; (ii) neomycin; (iii) vancomycin; (iv) metronidazole; (v) a cocktail of all four antibiotics together (with zero-calorie sweetener to ensure intake); (vi) zero-calorie sweetener only; or (vii) no additive (untreated) for 12 weeks. Individual antibiotics remove only some taxa from the gut, while the cocktail of all four removes almost all microbes. After accounting for differences in geometry, whole bone strength was reduced in animals with gut microbiome modified by neomycin (-28%, p = 0.002) and was increased in the group in which the gut microbiome was altered by sweetener alone (+39%, p < 0.001). Analysis of the fecal microbiota detected seven lower-ranked taxa differentially abundant in animals with impaired tissue-level strength and 14 differentially abundant taxa associated with increased tissue-level strength. Histological and serum markers of bone turnover and trabecular bone volume per tissue volume (BV/TV) did not differ among groups. These findings demonstrate that modifications to the taxonomic components of the gut microbiome have the potential to decrease or increase tissue-level strength of bone independent of bone quantity and without noticeable changes in bone turnover. © 2021 American Society for Bone and Mineral Research (ASBMR).

Perspective: Evidence before Enthusiasm-A Critical Review of the Potential Cardiovascular Benefits of Vitamin K.

A protective role for vitamin K in cardiovascular disease (CVD), a leading cause of morbidity and mortality, has been proposed because vitamin K-dependent proteins, such as matrix Gla (γ-carboxyglutamic acid) protein (MGP), are present in vascular tissue. MGP functions as a vascular calcification inhibitor-but only when it is carboxylated, which requires vitamin K. There is more than one naturally occurring form of vitamin K. Phylloquinone (vitamin K1) is found in plant-based foods, whereas menaquinones (vitamin K2) are a class of vitamin K compounds found in animal-based and fermented foods. Phylloquinone and menaquinones are capable of carboxylating MGP and other vitamin K-dependent proteins. In rodent models, high intakes of either phylloquinone or menaquinone reduced vascular calcification. Evidence of the relative importance of phylloquinone and menaquinone to CVD in humans is limited and controversial. In some observational studies, higher dietary menaquinone intake, but not phylloquinone intake, was associated with less coronary artery calcification (a subclinical manifestation of CVD) and a lower risk for clinical CVD events. These findings have led to claims that menaquinones have unique cardiovascular health benefits compared with phylloquinone. However, this claim is not supported by the results of the limited number of intervention trials conducted to date. The purpose of this review is to evaluate the strengths and limitations of the available evidence regarding the role of vitamin K in vascular calcification, CVD, and mortality.

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.