This study analyzed the relationship between baseline serum 25-hydroxyvitamin D (25-(OH)D) concentration and risk of major clinical disease events in 1621 older white adults over a median 11-year follow up period. The composite primary outcome included hip fracture, myocardial infarction, incident cancer, and death. Results showed the association between low 25-(OH)D and risk of the composite outcome varied by season. A 25-(OH)D concentration lower than season-specific Z-scores of -0.54 was associated with a 24% higher risk and corresponded to concentrations of 43, 50, 61, and 55 nmol/L in winter, spring, summer and autumn respectively. The study suggests season-specific 25

1. Serum 25-Hydroxyvitamin D Concentration and Risk for Major Clinical
Disease Events in a Community-Based Population of Older Adults
A Cohort Study
Ian H. de Boer, MD, MS; Gregory Levin, MS; Cassianne Robinson-Cohen, MS; Mary L. Biggs, PhD; Andy N. Hoofnagle, MD, PhD;
David S. Siscovick, MD, MPH; and Bryan Kestenbaum, MD, MS
Background: Circulating concentrations of 25-hydroxyvitamin D
[25-(OH)D] are used to define vitamin D deficiency. Current clinical
25-(OH)D targets based on associations with intermediate markers
of bone metabolism may not reflect optimal levels for other chronic
diseases and do not account for known seasonal variation in 25-
(OH)D concentration.
Objective: To evaluate the relationship of 25-(OH)D concentration
with the incidence of major clinical disease events that are patho-
physiologically relevant to vitamin D.
Design: Cohort study.
Setting: The Cardiovascular Health Study conducted in 4 U.S. com-
munities. Data from 1992 to 2006 were included in this analysis.
Participants: 1621 white older adults.
Measurements: Serum 25-(OH)D concentration (using a high-
performance liquid chromatography–tandem mass spectrometry as-
say that conforms to National Institute of Standards and Technol-
ogy reference standards) and associations with time to a composite
outcome of incident hip fracture, myocardial infarction, cancer, or
death.
Results: Over a median 11-year follow-up, the composite outcome
occurred in 1018 participants (63%). Defining events included 137
hip fractures, 186 myocardial infarctions, 335 incidences of cancer,
and 360 deaths. The association of low 25-(OH)D concentration
with risk for the composite outcome varied by season (P ϭ 0.057).
A concentration lower than a season-specific Z score of Ϫ0.54 best
discriminated risk for the composite outcome and was associated
with a 24% higher risk in adjusted analyses (95% CI, 9% to 42%).
Corresponding season-specific 25-(OH)D concentrations were 43,
50, 61, and 55 nmol/L (17, 20, 24, and 22 ng/mL) in winter,
spring, summer, and autumn, respectively.
Limitation: The observational study was restricted to white
participants.
Conclusion: Threshold concentrations of 25-(OH)D associated with
increased risk for relevant clinical disease events center near 50
nmol/L (20 ng/mL). Season-specific targets for 25-(OH)D concen-
tration may be more appropriate than static targets when evaluat-
ing health risk.
Primary Funding Source: National Institutes of Health.
Ann Intern Med. 2012;156:627-634. www.annals.org
For author affiliations, see end of text.
Vitamin D has attracted increasing attention in clinical
medicine and research, in part because of its pleiotro-
pic effects on biological processes other than calcium and
bone homeostasis (1–3). Animal experimental studies dem-
onstrate that 1,25-dihydroxyvitamin D, the active vitamin
D hormone, suppresses the renin–angiotensin–aldosterone
system, modulates immune cell function, and suppresses
abnormal cell proliferation (4). Epidemiologic studies sug-
gest that these actions may have clinical relevance, demon-
strating that, in addition to fracture, vitamin D deficiency
is associated with increased risks for coronary heart disease,
cancer, and all-cause mortality (5–11).
Circulating concentrations of 25-hydroxyvitamin D
[25-(OH)D], which reflect total vitamin D intake from
cutaneous synthesis and dietary consumption, are used to
define vitamin D deficiency (1–3). Biological 25-(OH)D
thresholds below which adequate conversion to 1,25-
dihydroxyvitamin D cannot be maintained may exist.
Optimal concentrations of 25-(OH)D have been pro-
posed on the basis of cross-sectional correlations with in-
termediate measures of bone and mineral metabolism, such
as parathyroid hormone concentration, bone mineral den-
sity, and intestinal calcium absorption (1, 12–15). This
approach relates biomarker levels to biological function, an
important strength, but it also has several limitations. First,
25-(OH)D concentrations that are optimal for bone and
mineral metabolism may not equal those for nonbone vi-
tamin D activities. Second, current recommendations for
target 25-(OH)D concentrations do not account for
known seasonal variation in 25-(OH)D concentration
(16–19). Third, existing recommendations are based on
divergent 25-(OH)D assays, and Standard Reference Ma-
terials released by the National Institute of Standards and
Technology (NIST) now permit reproducible 25-(OH)D
testing to enhance external validity (20). In addition, 25-
See also:
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Annals of Internal Medicine Original Research
© 2012 American College of Physicians 627

2. (OH)D targets are highly controversial—the Institute of
Medicine (IOM) recently recommended a threshold of 50
nmol/L (20 ng/mL), substantially less than the 75-nmol/L
(30-ng/mL) threshold recommended by other professional
societies and expert panels (1, 12–15).
The goal of this study was to examine the relationship
of serum 25-(OH)D concentration to vitamin D in terms
of risk for major clinical disease events of global pathophys-
iologic relevance, focusing on threshold concentrations as-
sociated with disease risk.
METHODS
Study Population
The CHS (Cardiovascular Health Study) is a prospec-
tive, community-based cohort study designed to examine
risk factors for the development and progression of cardio-
vascular disease in people aged 65 years or older (21). Par-
ticipants were recruited from 4 U.S. communities: Forsyth
County, North Carolina; Sacramento County, California;
Washington County, Maryland; and Pittsburgh, Pennsyl-
vania. Eligible participants were sampled by using Medi-
care eligibility lists, were not institutionalized, and were
expected to remain in the area for at least 3 years. Persons
who were wheelchair-bound in the home or receiving hos-
pice treatment, radiation therapy, or chemotherapy were
excluded. The original CHS cohort of 5201 participants
was enrolled between 1989 and 1990, with an additional
687 predominantly black participants enrolled between
1992 and 1993.
We measured serum 25-(OH)D concentration at the
1992–1993 study visit for 2312 CHS participants who had
no clinical evidence of cardiovascular disease at that time
and who had available frozen serum (11). To expand our
focus to incident cancer and hip fracture for this study, we
additionally excluded 328 participants with a history of
cancer and 13 participants with a previous hip fracture. We
also excluded 45 participants with missing data on smok-
ing and physical activity (Supplement 1, available at www
.annals.org). Because 25-(OH)D concentrations and
possibly their associations with health outcomes vary by
race, we focused on the 1621 white persons meeting
these criteria.
25-(OH)D Concentration
Fasting serum was collected from CHS participants at
the 1992–1993 study visit and stored at Ϫ70 °C. We mea-
sured total 25-(OH)D [25-(OH)D2 and 25-(OH)D3] by
using high-performance liquid chromatography–tandem
mass spectrometry on a Waters Quattro micro mass spec-
trometer (Waters Corporation, Milford, Massachusetts) in
2008. The interassay coefficient of variation was less than
3.4%. The assay was validated by using NIST Standard
Reference Material 972 (accuracy within 5%) (20). 25-
Hydroxyvitamin D is known to be stable for long periods
at Ϫ70 °C (22).
Composite Clinical Outcome
The primary study outcome was time to first occur-
rence of incident hip fracture, incident myocardial infarc-
tion (MI), incident cancer, or death from any cause. This
composite outcome was chosen before analysis to capture
previously described associations of 25-(OH)D with dis-
ease outcomes. We defined hip fracture by the Interna-
tional Classification of Diseases, Ninth Revision, codes
820.xx without a concomitant code for motor vehicle ac-
cident (E810–E819) or pathologic fracture (733.1x) (6).
The CHS Events Committee adjudicated cases of MI by
using available hospital discharge summaries, diagnostic
test records, and consultation reports (23). Investigators for
the CHS identified incident cancer cases by linking CHS
records with population-based cancer registries serving the
4 CHS regions (24). We omitted outcomes that may be
causally related to low 25-(OH)D concentration but have a
diagnosis that is imprecisely ascertained or is made largely
on the basis of physical measurements, such as diabetes,
hypertension, and impaired muscle function. We defined
time to composite outcome as the time elapsed between
the 1992–1993 examination, when serum 25-(OH)D con-
centrations were measured (baseline), and either the earliest
event or the end of follow-up for cancer ascertainment
(31 December 2005 for the California, Pennsylvania,
and North Carolina sites, and 31 December 2006 for the
Maryland site).
Covariates
Covariates were ascertained at the 1992–1993 CHS
study visit and were selected on the basis of their suspected
confounding influence on associations of 25-(OH)D with
study outcomes. Total physical activity was estimated by
using the Minnesota Leisure Time Physical Activity Ques-
tionnaire, which assesses a range of common activities,
Context
Vitamin D deficiency is defined by its association with
markers of bone metabolism, not by its association with
clinical outcomes.
Contribution
This study followed elderly people and found that base-
line levels of 25-hydroxyvitamin D less than 50 nmol/L
(20 ng/mL) were associated with a composite outcome
that included hip fracture, myocardial infarction, incident
cancer, and death.
Caution
This was an observational study of white persons.
Implication
The threshold identified in this study is closer to the value
recently recommended by the Institute of Medicine than
to the value recommended by most other professional
societies and expert panels (75 nmol/L [30 ng/mL]).
—The Editors
Original Research Serum 25-(OH)D and Risk for Major Clinical Disease Events
628 1 May 2012 Annals of Internal Medicine Volume 156 • Number 9 www.annals.org

3. such as walking for exercise, jogging, biking, aerobics, golf,
tennis, swimming, weight training, mowing the lawn,
strenuous household chores, and use of a treadmill or aer-
obic machine (25, 26). Current smoking was ascertained
by questionnaire. Time of blood collection was categorized
in 3-month blocks to reflect the 4 seasons and the observed
pattern of seasonal variation in 25-(OH)D concentration
in our population.
Statistical Analysis
We tested associations of 25-(OH)D concentration
with study outcomes by using Cox proportional hazards
models with robust SEs, adjusted for age, sex, clinical site,
smoking (current or not current), body mass index (in
categories), and physical activity (kilocalories per week in
categories). This set of covariates was chosen before anal-
ysis to include important demographic characteristics
and potential strong confounders while also maintaining
a relatively parsimonious model. We censored partici-
pants at the time of death in analyses of nonfatal
outcomes.
We evaluated 25-(OH)D as a dichotomous variable to
address the clinical utility of a 25-(OH)D threshold for
risk assessment and because we and others have seen
threshold associations of 25-(OH)D concentration with
risks for fracture, MI, and death (6–10). Given known
seasonal variability in 25-(OH)D concentration (16–19)
and its associated effect on modeling (27), we planned
before analysis to assess the effect of season on the relation-
ship of 25-(OH)D concentration and the composite out-
come. We compared nested models with and without in-
teraction terms for 25-(OH)D concentration by season by
using a multivariate Wald test. When we saw significant
heterogeneity, we examined season-specific 25-(OH)D
concentrations as exposures. This approach has been ap-
plied previously and reduces bias (27, 28).
To describe the functional form of the association of
25-(OH)D concentration with the composite outcome, we
first calculated unadjusted incidence rates by season-
specific decile of 25-(OH)D concentration. Second, we
created an adjusted penalized spline model with season-
specific 25-(OH)D Z score as the flexibly modeled expo-
sure variable and graphically displayed the spline at the
mean values of adjustment covariates (29). The penalized
spline was computed by using the default algorithm of the
survival package in R 2.12.1 (R Foundation for Statis-
tical Computing, Vienna, Austria), which uses evenly
spaced knots, cubic polynomials, and a penalty to re-
strict the overall flexibility of the fitted curve (30).
Third, we used a simple statistical approach similar to the
Contal–O’Quigley method to estimate an “optimal” season-
specific 25-(OH)D Z score cut-point (31). For each possi-
ble threshold from the inner 90% of the season-specific
25-(OH)D Z score distribution (0.01 unit increments), we
computed the Wald statistic with robust SE to describe the
strength of the adjusted association between 25-(OH)D
deficiency (using that candidate cut-point) and rates of the
composite outcome. The Z score cut-point that produced
the largest Wald statistic was defined as “optimal” in the
sense that it best discriminated between low- and high-risk
participants with these statistical criteria. We quantified the
uncertainty in our estimated optimal threshold by comput-
ing approximate CIs based on the observed quantiles of the
distribution of estimates across 2000 nonparametric boot-
strap samples (32). Because an optimal cut-point with high
statistical precision is difficult to estimate, we present
both standard 95% CIs and the narrower 75% CIs. We
computed net reclassification improvement to assess
whether season-specific 25-(OH)D concentration im-
proved prediction of the primary composite outcome
(cumulative incidence at 10 years, through which
follow-up was 100% complete) compared with static
25-(OH)D concentration (50 nmol/L [20 ng/mL] re-
gardless of season) (33).
All P values are 2-sided. Statistical analyses were com-
pleted using R 2.12.1 and STATA 10.1 (Stata Corp, Col-
lege Station, Texas).
Role of the Funding Source
The National Institutes of Health provided funding
for this study. The funding source had no role in the de-
sign, conduct, or analysis of this study or the decision to
submit the manuscript for publication.
Figure 1. Box plot of 25-(OH)D concentration by season,
showing the 25th, 50th, and 75th percentiles of
distributions, with outliers not shown.
25-(OH)DConcentration,nmol/L
20
40
60
80
100
120
Winter Spring Summer Autumn
Mean 25-(OH)D was 56 nmol/L (SD, 24), 63 nmol/L (SD, 24), 74
nmol/L (SD, 25), and 69 nmol/L (SD, 26) (22 ng/mL [SD, 10], 25
ng/mL [SD, 10], 30 ng/mL [SD, 10], and 28 ng/mL [SD, 11]) in winter
(January–March), spring (April–June), summer (July–September),
and autumn (October–December), respectively. 25-(OH)D ϭ 25-
hydroxyvitamin D.
Original ResearchSerum 25-(OH)D and Risk for Major Clinical Disease Events
www.annals.org 1 May 2012 Annals of Internal Medicine Volume 156 • Number 9 629

4. RESULTS
Baseline Characteristics
Baseline 25-(OH)D concentration varied strongly by
season (Figure 1). It was lowest in January through March
(“winter”), highest in July through September (“summer”),
and intermediate in April through June (“spring”) and Oc-
tober through December (“autumn”). Low season-specific
25-(OH)D concentration, defined as less than the season-
specific 29th percentile, was more common among women
and participants at more northerly study sites and was as-
sociated with higher body mass index, hypertension, re-
duced physical activity, and higher circulating concentra-
tions of parathyroid hormone (Table 1).
Events
Median follow-up for the 1621 participants was 11
years (interquartile range, 6 to 13 years). The composite
clinical outcome occurred in 1018 participants (63%)
(Supplement 2, available at www.annals.org). The qualify-
ing event was hip fracture for 137 participants (8%), MI
for 186 participants (11%), cancer for 335 participants
(21%), and death for 360 participants (22%). Qualifying
events are tabulated by cause in Supplement 3 available at
www.annals.org).
Associations of 25-(OH)D With Events
We first evaluated the association of 25-(OH)D with
the composite outcome by using the previously published
25-(OH)D threshold of 50 nmol/L (20 ng/mL) (1, 3).
Using this approach, we saw borderline statistical evidence
of heterogeneity by season (P ϭ 0.057). Deviations from
strong associations occurred in winter and summer, the
extremes of seasonal variation in 25-(OH)D (Supplement
4, available at www.annals.org).
We next evaluated the associations of 25-(OH)D with
the composite outcome by season. Participants in the low-
est 2 to 3 deciles of 25-(OH)D concentration (lowest 20%
to 30%) tended to have increased risk for the composite
clinical outcome, compared with those in the highest 7 to
8 deciles (Supplement 5, available at www.annals.org).
Evaluation of a season-based 25-(OH)D Z score similarly
suggested that elevated risk for the composite outcome was
Table 1. Characteristics of Participants in 1992 to 1993*
Characteristic Overall (n ‫؍‬ 1621) Normal 25-(OH)D (n ‫؍‬ 1126) Low 25-(OH)D (n ‫؍‬ 495)†
Demographic data
Age, y 74.0 (4.6) 73.7 (4.5) 74.5 (4.7)
Men 491 (30) 406 (36) 85 (17)
Site
Forsythe County, North Carolina 450 (28) 330 (29) 120 (24)
Sacramento County, California 370 (23) 277 (25) 93 (19)
Washington County, Maryland 457 (28) 295 (26) 162 (33)
Pittsburgh, Pennsylvania 344 (21) 224 (20) 120 (24)
Medical history and lifestyle
Diabetes‡ 162 (10) 90 (8) 72 (15)
Hypertension‡ 906 (56) 609 (54) 297 (60)
Current smoking 151 (9) 93 (8) 58 (12)
Current alcohol use 740 (46) 521 (46) 219 (44)
Physical activity category
Ͻ500 kcal/wk 453 (28) 261 (23) 192 (39)
500–1000 kcal/wk 329 (20) 214 (19) 115 (23)
1000–2000 kcal/wk 393 (24) 298 (26) 95 (19)
Ͼ2000 kcal/wk 446 (28) 353 (31) 93 (19)
Physical examination
BMI category
Ͻ25 kg/m2
664 (41) 484 (43) 180 (36)
25–30 kg/m2
660 (41) 466 (41) 194 (39)
30–35 kg/m2
229 (14) 143 (13) 86 (17)
Ͼ35 kg/m2
68 (4) 33 (3) 35 (7)
Laboratory data
Estimated GFR, mL/min per 1.73 m2
‡ 75.3 (17.8) 75.9 (17.7) 74.0 (17.8)
Parathyroid hormone, ng/L 54.7 (27.3) 50.9 (25.4) 63.1 (31.1)
Bone alkaline phosphate, ␮g/L 14.6 (6.9) 14.1 (6.7) 15.8 (7.1)
Calcium
mg/dL 9.5 (0.4) 9.5 (0.4) 9.5 (0.4)
mmol/L 2.4 (0.1) 2.4 (0.1) 2.4 (0.1)
Phosphate, mmol/L 1.2 (0.2) 1.2 (0.2) 1.2 (0.2)
Total 25-(OH)D
nmol/L 66.2 (25.8) 77.8 (21.5) 39.8 (11.1)
ng/mL 26.5 (10.3) 31.2 (8.6) 15.9 (4.5)
25-(OH)D ϭ 25-hydroxyvitamin D; BMI ϭ body mass index; GFR ϭ glomerular filtration rate.
* Values are means (SDs) for continuous variables or numbers (percentages) for categorical variables.
† Defined as less than the lowest season-specific 29th percentile (43, 50, 61, and 55 nmol/L [17, 20, 24, and 22 ng/mL] in winter, spring, summer, and fall, respectively).
‡ Diabetes was defined as use of insulin or oral hypoglycemic agents or fasting blood glucose level Ն6.99 mmol/L (Ն126 mg/dL). Hypertension was defined as systolic blood
pressure Ն140 mm Hg, diastolic blood pressure Ն90 mm Hg, or use of an antihypertensive medication. Serum cystatin C was measured by using a BNII nephelometer (N
Latex Cystatin C; Dade Behring, Deerfield, Illinois) and used to estimate GFR with the following equation: GFR ϭ 76.7 ϫ [cystatin C]Ϫ1.18
.
Original Research Serum 25-(OH)D and Risk for Major Clinical Disease Events
630 1 May 2012 Annals of Internal Medicine Volume 156 • Number 9 www.annals.org

5. greatest below a Z score of approximately Ϫ0.5 (near 30%
of the normal distribution) (Figure 2).
Based on a simple statistical approach, the season-
specific 25-(OH)D Z score that best separated low- and
high-risk 25-(OH)D groups with respect to the composite
outcome was Ϫ0.54 (29th percentile of the normal distri-
bution). This threshold corresponded to season-specific
cut-points of 43, 50, 61, and 55 nmol/L (17, 20, 24, and
22 ng/mL) under the normal approximation to the distri-
bution of 25-(OH)D concentration for winter, spring,
summer, and autumn, respectively (mean threshold of 52
nmol/L [21 ng/mL]). The observed season-specific distri-
butions of 25-(OH)D concentrations in this population,
although slightly right-skewed, suggested that the normal
approximation was reasonable. A 25-(OH)D concentration
below the season-specific 29th percentile was associated
with a 24% increased risk for the composite outcome in
the adjusted model (95% CI, 9% to 42%) and with simi-
larly increased risks for each component of the composite
outcome (Table 2).
We did several analyses to evaluate whether the iden-
tified optimal threshold was robust. Statistical significance
of the association of low season-specific 25-(OH)D con-
centration with risk for the composite outcomes decreased
markedly when a threshold Z score above Ϫ0.44 was used;
this corresponds to season-specific cut-points of 45, 52, 63,
and 57 nmol/L (18, 21, 24, and 23 ng/mL) for winter,
spring, summer, and autumn, respectively (mean threshold
of 54 nmol/L [22 ng/mL]) (Supplement 6, available at
www.annals.org). In bootstrap analyses, 95% of optimal
25-(OH)D Z score thresholds fell between Ϫ1.48 and
0.13 (mean season-specific thresholds of 29 and 69 nmol/L
[12 and 28 ng/mL], respectively), whereas 75% fell be-
tween Ϫ1.38 and Ϫ0.40 (mean thresholds of 31 and 55
nmol/L [12 and 22 ng/mL], respectively).
Reclassification
Nine percent of participants were reclassified compar-
ing low 25-(OH)D concentration defined by season-
specific thresholds (29th percentile) versus the static
threshold of 50 nmol/L (20 ng/mL) (Table 3). When low
season-specific 25-(OH)D concentration was compared
with a concentration less than 50 nmol/L (20 ng/mL), net
reclassification improvement was 2.4% (95% CI, Ϫ0.6%
to 5.3%; P ϭ 0.118).
DISCUSSION
We characterized associations of NIST-verified serum
25-(OH)D concentration with risk for adverse clinical
events that are pathophysiologically relevant to pleiotropic
vitamin D actions in a community-based population. The
association of 25-(OH)D with a composite clinical out-
come of hip fracture, MI, cancer, and death varied by sea-
son, supporting use of season-specific 25-(OH)D thresh-
olds. In our study population, threshold 25-(OH)D
concentrations optimally associated with risk for the com-
posite outcome were 43 nmol/L (17 ng/mL) in winter
months, 50 nmol/L (20 ng/mL) in spring months, 61
nmol/L (24 ng/mL) in summer months, and 55 nmol/L
(22 ng/mL) in autumn months.
Figure 2. Association of season-specific 25-(OH)D Z score
with the risk for incident myocardial infarction, cancer,
hip fracture, or death (composite outcome) among 1621
participants in the Cardiovascular Health Study, evaluated
using a penalized spline.
LogHazardRatio
–0.05
0.05
0.10
0.15
0.20
0.25
0.00
–1.5 –0.5
25 50 75
755025
50 75 100
100
1007550
–1 0.5 1.50 1
Winter
Spring
Summer
Autumn
Season-Specific 25-(OH)D Z Score
25-(OH)D Concentration, nmol/L
Proportional hazards model adjusts for age, sex, clinical site, body mass
index, physical activity, and smoking. The shaded area represents Z score
less than Ϫ0.54 (29th percentile of the normal distribution), which best
discriminated risk for the composite outcome. The x-axis is displayed as
season-specific Z score (uppermost x-axis, reflecting the primary method
of analysis) and as corresponding season-specific absolute 25-(OH)D
concentrations (lower 4 axes). 25-(OH)D ϭ 25-hydroxyvitamin D.
Table 2. Associations of Low Season-Specific 25-(OH)D
Concentration With Rates of the Composite Outcome of MI,
Cancer, Hip Fracture, or Death Among Participants
Outcome Events (Incidence Rate), n* Hazard Ratio
(95% CI)†
Normal
25-(OH)D
Low
25-(OH)D‡
Composite 681 (6.4) 337 (7.7) 1.24 (1.09–1.42)
MI 154 (1.2) 67 (1.3) 1.24 (0.91–1.70)
Cancer 259 (2.3) 111 (2.3) 1.13 (0.90–1.42)
Hip fracture 118 (0.9) 72 (1.4) 1.34 (0.97–1.84)
Death 539 (4.0) 287 (5.3) 1.32 (1.14–1.53)
25-(OH)D ϭ 25-hydroxyvitamin D; MI ϭ myocardial infarction.
* Participants may be included in more than 1 event category, but only the first
event for each participant was used in analysis of the composite outcome. Inci-
dence rates are unadjusted event rates per 100 person-years of follow-up.
† Adjusted for age, sex, clinical site, smoking, body mass index, and physical
activity.
‡ Defined as less than the lowest season-specific 29th percentile (43, 50, 61, and
55 nmol/L [17, 20, 24, and 22 ng/mL] in winter, spring, summer, and autumn,
respectively).
Original ResearchSerum 25-(OH)D and Risk for Major Clinical Disease Events
www.annals.org 1 May 2012 Annals of Internal Medicine Volume 156 • Number 9 631

6. The IOM recently evaluated the clinical application of
25-(OH)D testing in the context of vitamin D supplemen-
tation (1). In reviewing available data, it concluded that
inadequate vitamin D can contribute to bone disease, vita-
min D supplementation can decrease risk for bone disease
in at-risk populations, and 25-(OH)D concentration less
than 50 nmol/L (20 ng/mL) identifies persons at increased
risk. The proposed threshold of 50 nmol/L (20 ng/mL)
was lower than that of 75 nmol/L (30 ng/mL) recom-
mended by many professional societies and vitamin D re-
searchers (12–15). The IOM noted a lack of high-quality
data about the effects of vitamin D supplementation on
risk for nonbone health outcomes, including MI, cancer,
and death, and it did not, therefore, base its estimate of
target 25-(OH)D concentration on these outcomes. These
findings were echoed in an updated clinical practice sum-
mary on vitamin D deficiency (3).
In comparison with existing literature and recommen-
dations, we have 2 principal findings. First, 25-(OH)D
thresholds associated with risk for diverse major clinical
disease events in our work center close to the 50 nmol/L
(20 ng/mL) recommended by the IOM for bone health.
We agree with the IOM’s conclusions that high-quality
intervention studies are needed to test whether vitamin D
deficiency is causally related to nonbone outcomes in hu-
mans. Until these data are available, the finding of a similar
25-(OH)D threshold for risk for major clinical disease
events to that recommended by the IOM for bone health is
reassuring and supports generally targeting 50 nmol/L (20
ng/mL) over 75 nmol/L (30 ng/mL) when 25-(OH)D test-
ing is clinically indicated.
In our study, 30.5% of participants had a 25-(OH)D
concentration less than the season-specific threshold cen-
tered near 50 nmol/L (20 ng/mL). This proportion is con-
gruent with the prevalence of 25-(OH)D concentrations
less than 50 nmol/L (20 ng/mL) in other populations and
emphasizes the large number of people at risk for potential
complications of low 25-(OH)D concentration (34). How-
ever, the distinction between 50 and 75 nmol/L (20 and
30 ng/mL) is important because more than 40% of the
U.S. population has concentrations between 50 and 75
nmol/L (20 and 30 ng/mL) (34). Our estimate of the 25-
(OH)D threshold that best discriminates risk for clinical
disease events was generated with some statistical uncer-
tainty, but a threshold as high as 75 nmol/L (30 ng/mL)
was unlikely to be congruent with our data.
Second, our data suggest that season-specific targets
are most appropriate for 25-(OH)D concentration. Varia-
tion in 25-(OH)D concentration within persons and pop-
ulations over the calendar year is well-known to be large
relative to mean concentration (16–19). This is probably
due to seasonal variation in exposure to ultraviolet light. As
a result, clinical decisions about initiation and dose of year-
long vitamin D supplementation are likely to be heavily
influenced by time of ascertainment, which is often arbi-
trary. Combined with this background knowledge, our re-
sults that demonstrated heterogeneity of the 25-(OH)D–
composite outcome association by season and a trend
toward improved classification of risk using season-specific
25-(OH)D thresholds suggest that season-specific targets
for 25-(OH)D concentration are more appropriate than
the static targets previously recommended when the need
for year-long vitamin D supplementation is being consid-
ered (1, 12–15).
We examined a composite end point of clinical disease
events that plausibly reflect net pleiotropic vitamin D ac-
tions, are supported by existing literature, and have a quan-
tifiable time of onset, understanding that this may include
1 or more outcomes that are not causally related to 25-
(OH)D and may omit some important vitamin D–related
effects. Associations of low season-specific 25-(OH)D con-
centration with the composite outcome and each of its
components were of similar magnitude, enabling this ap-
proach. Statistical significance using the standard ␣ level of
0.05 was achieved only for the composite outcome and for
death. However, this study was not powered to detect as-
sociations with individual composite outcome compo-
nents, and statistical confidence is likely to be inflated by
testing inference in the same data set in which threshold
25-(OH)D concentrations were derived. Moreover, the
primary goal of this study was to study the pattern of the
25-(OH)D–composite outcome relationship, not its exis-
tence, which has been demonstrated in previous studies
(5–11).
Strengths of this study include the use of a
community-based population of older adults, who are of-
ten targeted for 25-(OH)D testing; the use of NIST-
verified 25-(OH)D concentration, which has not, to our
knowledge, been previously applied to large epidemiologic
studies; and the ascertainment of clinical outcomes directly
relevant to both bone and nonbone vitamin D actions over
Table 3. Classification of 10-Year Risk for Composite
Outcome of MI, Cancer, Hip Fracture, or Death Among
Participants
Static 25-(OH)D* Season-Specific 25-(OH)D
Normal,
n (%)
Low,
n (%)†
Total,
n
Participants who have an event
Ն50 nmol/L (Ն20 ng/mL) 484 (90) 55 (10) 539
Ͻ50 nmol/L (Ͻ20 ng/mL) 18 (8) 199 (92) 217
Total 502 254 756
Participants who do not have an event
Ն50 nmol/L (Ն20 ng/mL) 598 (93) 48 (7) 646
Ͻ50 nmol/L (Ͻ20 ng/mL) 26 (12) 193 (88) 219
Total 624 241 865
25-(OH)D ϭ 25-hydroxyvitamin D.
* Year-long static threshold of 50 nmol/L (20 ng/mL).
† Defined as less than the lowest season-specific 29th percentile (43, 50, 61, and
55 nmol/L [17, 20, 24, and 22 ng/mL] in winter, spring, summer, and autumn,
respectively).
Original Research Serum 25-(OH)D and Risk for Major Clinical Disease Events
632 1 May 2012 Annals of Internal Medicine Volume 156 • Number 9 www.annals.org

7. long-term follow-up. Limitations include the inclusion of
only older adults; the availability of only white participants
in sufficient numbers to rigorously evaluate relationships of
25-(OH)D with study outcomes; the availability of only
one 25-(OH)D measurement per participant, which may
bias magnitudes of association toward the null; and the
inability of available statistical methods to precisely deter-
mine optimal threshold concentrations with statistical confi-
dence. Most important, this study is observational. Ulti-
mately, optimal 25-(OH)D concentrations should be defined
as the baseline 25-(OH)D concentrations above which vita-
min D supplementation does not improve relevant clinical
outcomes in large, diverse, randomized clinical trials.
In conclusion, we found that “optimal” concentrations
of 25-(OH)D, gauged by associations with major clinical
disease events, centered near 50 nmol/L (20 ng/mL), the
level recently recommended by the IOM for bone health.
We further report that the association of 25-(OH)D with
clinical health events varies by season and suggest that
season-specific targets for 25-(OH)D concentration are
more appropriate than static targets when considering po-
tential implications for long-term health.
From the University of Washington, Seattle, Washington.
Grant Support: By the National Heart, Lung, and Blood Institute (con-
tracts N01-HC-85239, N01-HC-85079 through N01-HC-85086, N01-
HC-35129, N01 HC-15103, N01 HC-55222, N01-HC-75150, and N01-
HC-45133 and grant HL080295), with additional contribution from the
National Institute of Neurologic Disorders and Stroke. Additional sup-
port was provided by the National Institute on Aging (AG-023629,
AG-15928, AG-20098, and AG-027058); the National Heart, Lung,
and Blood Institute (grants R01HL084443 and R01HL096875); and
the National Institute of Diabetes and Digestive and Kidney Diseases
(grant R01DK088762). A full list of principal CHS investigators and
institutions can be found at www.chs-nhlbi.org/pi.htm.
Potential Conflicts of Interest: Disclosures can be viewed at www
.acponline.org/authors/icmje/ConflictOfInterestForms.do?msNumϭM11
-2074.
Reproducible Research Statement: Study protocol and statistical code:
Available from Dr. de Boer (e-mail, deboer@u.washington.edu). Data
set: Not available.
Requests for Single Reprints: Ian H. de Boer, MD, MS, Box 359606,
325 9th Avenue, Seattle, WA 98104; e-mail, deboer@u.washington.edu.
Current author addresses and author contributions are available at www
.annals.org.
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Original Research Serum 25-(OH)D and Risk for Major Clinical Disease Events
634 1 May 2012 Annals of Internal Medicine Volume 156 • Number 9 www.annals.org

9. Current Author Addresses: Drs. de Boer and Kestenbaum and Ms.
Robinson-Cohen: Kidney Research Institute, Box 359606, 325 9th Av-
enue, Seattle, WA 98104.
Mr. Levin: Department of Biostatistics, Box 357232, 1959 Northeast
Pacific Street, Seattle, WA 98195.
Dr. Biggs: Collaborative Health Studies Coordinating Center, Building
29, Suite 310, 6200 Northeast 74th Street, Seattle, WA 98115.
Dr. Hoofnagle: Department of Laboratory Medicine, Box 357110, 1959
Northeast Pacific Street, Seattle, WA 98195.
Dr. Siscovick: Cardiovascular Health Research Unit, 1730 Minor Ave-
nue, Suite 1360, Seattle, WA 98101.
Author Contributions: Conception and design: I.H. de Boer, G. Levin,
B. Kestenbaum.
Analysis and interpretation of the data: I.H. de Boer, G. Levin,
C. Robinson-Cohen, A.N. Hoofnagle, D.S. Siscovick, B. Kestenbaum.
Drafting of the article: I.H. de Boer, G. Levin, C. Robinson-Cohen.
Critical revision of the article for important intellectual content:
G. Levin, C. Robinson-Cohen, M.L. Biggs, A.N. Hoofnagle,
D.S. Siscovick, B. Kestenbaum.
Final approval of the article: I.H. de Boer, C. Robinson-Cohen,
M.L. Biggs, A.N. Hoofnagle, D.S. Siscovick, B. Kestenbaum.
Statistical expertise: G. Levin, C. Robinson-Cohen, A.N. Hoofnagle.
Obtaining of funding: I.H. de Boer, B. Kestenbaum.
Collection and assembly of data: G. Levin, M.L. Biggs, A.N. Hoofnagle,
D.S. Siscovick.
Annals of Internal Medicine
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