Hindawi Publishing Corporation
International Journal of Endocrinology
Volume 2010, Article ID 631971, 8 pages
AnalyzingAdherenceto Prenatal Supplement:
DoesPill CountMeasure Up?
Kristie E.Appelgren,1Paul J. Nietert,2Thomas C.Hulsey,3Bruce W.Hollis,3
1Department of Pediatrics, Duke University, Durham, NC 27710, USA
2Department of Biostatistics, Bioinformatics, and Epidemiology, Medical University of South Carolina, Charleston, SC 29425, USA
3Division of Neonatology, Department of Pediatrics, Medical University of South Carolina, Charleston, SC 29425, USA
Correspondence should be addressed to Kristie E. Appelgren, email@example.com
Received 22 April 2009; Revised 3 August 2009; Accepted 20 October 2009
Academic Editor: Suzanne E. Judd
Copyright © 2010 Kristie E. Appelgren et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
Objective. To determine if adherence as measured by pill count would show a significant association with serum-based measures
of adherence. Methods. Data were obtained from a prenatal vitamin D supplementation trial where subjects were stratified by race
and randomized into three dosing groups: 400 (control), 2000, or 4000 IU vitamin D3/day. One measurement of adherence was
obtained via pill counts remaining compared to a novel definition for adherence using serum 25-hydroxy-vitamin D (25-OH-
D) levels (absolute change in 25(OH)D over the study period and the subject’s steady-state variation in their 25(OH)D levels). A
adherence measure by serum metabolite levels. Results. Subjects’ mean percentage of adherence by pill count was not a significant
predictor of adherence by serum metabolite levels. This finding was robust across a series of sensitivity analyses. Conclusions. Based
on our novel definition of adherence, pill count was not a reliable predictor of adherence to protocol, and calls into question how
adherence is measured in clinical research. Our findings have implications regarding the determination of efficacy of medications
under study and offer an alternative approach to measuring adherence of long half-life supplements/medications.
Vitamin D is an important nutrient that is widely known
to be vital to bone health and development, although it
has recently been linked to other systems such as immune
function [1, 2]. The two main human sources of vitamin D
are sunlight exposure, which converts 7-dehydrocholesterol
in the skin to vitamin D3, and oral intake. Due to the limited
dietary sources of vitamin D, serum vitamin D levels are
primarily determined by sunlight exposure. The amount
of vitamin D produced by a given amount of exposure
is modified by skin pigmentation, with darkly-pigmented
populations producing significantly less vitamin D than fair-
skinned populations after exposure to similar conditions [3–
There is a strong relationship between maternal and fetal
(cord blood) circulating 25(OH)D levels [8–11]. At the time
of birth, cord blood as a direct reflection of fetal vitamin D
status will contain approximately 50–60% of the maternal
circulating levels of 25(OH)D. This relationship appears to
be linear even at pharmacological intakes of vitamin D
. With respect to the more polar metabolites of vitamin
D, a similar (but lesser) relationship is observed between
mother and fetus . Interestingly, there appears to be
little, if any, relationship with respect to the parent vitamin,
vitamin D . This lack of placental transfer of the parent
vitamin D from mother to fetus also has been observed
in a porcine experimental animal model . Thus, in the
human fetus, vitamin D metabolism in all likelihood begins
with 25(OH)D. As a result, the nutritional vitamin D status
of the human fetus/neonate is totally dependent on the
vitamin D stores of the mother ; thus, if the mother
is hypovitaminotic D, her fetus will experience depleted
vitamin D exposure throughout gestation .
2International Journal of Endocrinology
While the demands of the fetus on the maternal system
for calcium increase throughout pregnancy, the demands for
vitamin D do not appear to change. The main determinants
of calcium homeostasis during pregnancy are parathyroid
hormone, calcitonin, and the active form of vitamin D—
1,25(OH)2D (whose synthesis is preferentially maintained
even during times of vitamin D deficiency by upregulation
of enzymes for improved utilization of whatever 25(OH)D
is available). The principal maternal adjustment to the
greater calcium requirements during pregnancy appears to
be increasing PTH, which maintains the serum calcium
concentration in the face of a falling albumin level, an
expanding extracellular fluid volume, and increasing renal
excretion and placental calcium transfer [15, 16]. The
placenta itself is responsible for active transport of calcium
ions, making the fetus hypercalcemic relative to the mother,
thereby stimulating fetal calcitonin and suppressing fetal
PTH secretion .
With respect to vitamin D, the placenta and/or deciduas
are active extrarenal sites of conversion of 25(OH)D as they
contain 1α-hydroxylase, providing a source of 1,25(OH)2D
for the fetus. Despite an expanding intravascular volume
during pregnancy, maternal vitamin D status shows seasonal
variation but beyond the typical factors that influence
vitamin D status in adults (such as sunlight exposure and
BMI), vitamin D status does not fluctuate during pregnancy
and once supplemented, maternal blood levels reach steady-
state within 2 months . These aspects of calcium and
vitamin D metabolism are important in the understanding
of vitamin D sufficiency during pregnancy and the premise
that the requirements of vitamin D are stable throughout
Existing guidelines recommend that pregnant women
receive 400IU of vitamin D per day via an oral supplement
[17, 18]. Among African-American women who receive
supplementation of this amount, however, the rate of
hypovitaminosis D (defined conservatively as the circulat-
ing 25-hydroxy-vitamin D [25(OH)D] level <15ng/mL or
<37.5nmol/L) is still 28% . The incidence of rickets, a
pathology which can develop among children with hypovita-
minosis D, increased during the past two decades, especially
among populations with higher levels of skin pigmentation
[20–22]. While rickets has been traditionally thought to be
the main sequelae of vitamin D deficiency, numerous other
long-latency disease states (such as multiple sclerosis [23–
25], rheumatoid arthritis [26–29], type 1 and 2 diabetes [30–
34], cardiovascular disease [35, 36], various cancers [37–43],
and systemic lupus erythematosus , to name but a few)
as well as acute infections such as upper respiratory illnesses
 and tuberculosis [46, 47] are now linked with vitamin
D deficiency that are explained by alterations in immune
function [47–49]. Those specific to the pregnant woman
and her fetus include impaired fetal growth [50–53], altered
development of dentition manifested in early childhood,
hypocalcemia—and rarely—neonatal seizures [54–56], and
maternal preeclampsia [57, 58]. Studies are currently under-
way to determine the optimum supplementation dose
of vitamin D among pregnant women (clinicaltrials.gov:
NCT00412087 and NCT00292591). Once the optimum dose
is determined, the treatment effect of this intervention will
be modified by the level of nonadherence among the patient
population. The clinical implications of nonadherence are
numerous and well recognized [59–61].
Adherence to study medication is often measured by a
calculation of pill count. This is an inexpensive measure of
compliance, but the data may be unreliable and often miss-
ing. For example, in a 2005 study of prenatal supplements
56% of subjects remembered to return only one monthly
pill bottle during the duration of the 2-3 month study .
With this method, researchers are not only depending on
patients to return bottles, but not to alter the number of
remaining doses as well. In a 2001 study of protease inhibitor
regimen adherence among HIV patients, pill count measures
estimated adherence at 83%, but electronic monitors on the
pill bottles revealed that true adherence was only 63% .
Adherence affects outcomes in all treatments and among
all groups, having an impact on public health. The purposes
of this study were to define parameters for measuring adher-
ence to vitamin D supplementation by measures of serum
metabolite, which could then be applied to other medication
regimens, and to examine the association between adherence
as determined by serum metabolites and adherence as
determined by patients’ pill counts.
2.1. Study Design. Data were obtained from a large NIH-
supported (#R01 HD043921) randomized, double-blind,
placebo-control trial of vitamin D supplements in preg-
nant women that took place at the Medical University of
South Carolina (MUSC). This paper, however, describes
an observational analysis of a subset of subjects in the
original trial. This research protocol was approved by the
MUSC Institutional Review Board for Human Research
Women entered the study at or before 14 weeks gestation
The women participants had been stratified by race and
randomized into three groups, each of which received a
400IU (control), 2000IU, or 4000IU dose of vitamin D3
once daily. Only the women in the 2000IU and 4000IU
dose groups were included in these analyses, since only these
doses were expected to have significant influence on subjects’
serum 20(OH)D levels.
Subjects (self-identified as Caucasian, African-American,
Hispanic/Latina, Asian, or American Indian) who carried
singleton pregnancies were eligible for participation in the
study. All subjects were patients at obstetric clinics at
MUSC and participated in a monthly study visit as an
extension of their regular prenatal checkup until delivery.
They were also asked to return to the clinic three times for
study visits after the birth of their infants. At each study
visit, as a marker of vitamin D status, circulating levels of
25(OH)D and vitamin D3were measured in patient serum.
In addition, subjects were asked to bring their supplement
bottle with them to their monthly visits, containing all
International Journal of Endocrinology3
2.2. Measures. The key variables of interest in this study
were measured as (1) mean percent adherence by pill count
and (2) adherence by serum metabolite levels. These values
were derived for each subject based on data obtained across
multiple time points.
2.2.1. Mean Percent Adherence by Pill Count. Subjects were
asked to bring their supplement containers, containing all
unused doses, with them to each monthly study visit. The
percentage adherence at each time point for each subject was
determined by the following formula:
(no. of pills dispensed – no. of pills returned)/(no. of
elapsed days between dispense date and return date).
Using the formula above, some subjects’ adherence levels
were evaluated to be more than 100%. This could occur if
women returned the pill bottle containing fewer doses than
were expected in the given time interval. In such instances,
we assigned each of these subjects a 100% adherence for the
pill count for each subject was then calculated by taking the
mean of percent adherence for all visits for which pill count
data were available for that subject.
2.2.2. Adherence by Serum Metabolite Levels. Defining adher-
ence by serum metabolite levels was not a straightforward
process. While there is a model in existence which calculates
the predicted change in serum 25(OH)D for different doses
of vitamin D supplement, this model was developed using
data from men , and its applicability to a population
of pregnant females is debatable. Therefore, we were faced
with the challenge of establishing novel criteria for vitamin
D supplement adherence by serum 25(OH)D levels; how we
arrived at this definition is described below.
The 25(OH)D level for each patient had been obtained at
each monthly visit. In developing a definition of adherence
by serum 25(OH)D levels, several assumptions were made a
priori about what types of patterns of 25(OH)D levels would
be exhibited by adherent and nonadherent patients based on
current knowledge about the metabolism of vitamin D.
to rise over time, eventually reaching a steady state after 2
months of therapy; nonadherent patients’ 25(OH)D levels
would be expected to be erratic and to exhibit variable pat-
terns that would differ significantly from adherent patients’
25(OH)D levels over the same dosing interval. Therefore,the
average of each subject’s steady-state 25(OH)D levels (i.e.,
levels obtained at least 2 months after initiation of vitamin D
therapy) was calculated, and the subject’s baseline 25(OH)D
level was subtracted from that value to obtain a value for
the change in 25(OH)D level during the study (Change
Assumption 2. Change 25(OH)D would be greater among
adherent patients when compared to nonadherent patients.
Assumption 3. Individual subjects who were adherent would
tend to exhibit less variability in their 25(OH)D measures
over time (i.e., our “steady-state” assumption). In other
words, we assumed that relative fluctuations in adherent
subjects’ 25(OH)D levels would be lower than those of
nonadherent subjects. The coefficient of variation (CV),
defined as the standard deviation of a set of measurements
divided by the arithmetic mean of the measurements [65,
66], was selected as a tool for use in comparing subjects with
respect to the degree of variation observed in their serum
of each subject’s measures relative to her mean, it provides,
in effect, a way of standardizing the degree of variability
A lower CV represents less relative variability and indicates
consistency in level of supplementation received.
To summarize the previous assumptions, an adherent
subject’s level of serum 25(OH)D was expected not only to
increase over the study period, but also to be fairly consistent
after 2 months (time to achieve steady state).
Finally, a definition of adherence by serum metabolite
levels was proposed based upon the above assumptions.
A subject who met the following criteria was classified as
adherent by her serum metabolite levels.
(1) high increase from baseline: the subject’s overall
change in 25(OH)D level over the course of the study
(baseline value to steady state concentration) was
at or above the median among subjects taking that
(2) low relative variability: the subject’s CV in her
25(OH)D level was less than or equal to the median
across all study subjects.
Other Variables. Assigned dose was categorized by treat-
ment group. Maternal race (classified as African Ameri-
can, Hispanic, and Caucasian), marital status, and highest
education level were determined by self-identification on
a questionnaire administered at enrollment. Maternal age
was calculated as self-reported date of birth subtracted from
date of enrollment. Season at enrollment was determined by
season (Winter, Spring, Summer, Fall) at first visit date. The
median value was determined, and a dichotomous variable
was created defining women as darker or lighter than the
median subject skin pigmentation.
2.3. Statistical Analyses. The sample of subjects included in
our analyses provided approximately 80% power to detect
an odds ratio as small as 1.55, assuming 2-sided hypothesis
testing and an alpha level of 0.05. Since the only way to assess
whether mothers achieved steady state during the study was
to examine mothers with repeated pill counts and serum
measurements, an inclusion requisite established a priori for
the study was inclusion of mothers who were seen monthly
throughout their pregnancy. Thus, for the subjects included
in our study, response rates were all approximately 100%.
To create the model, descriptive statistics pertaining to
the adherence measures were first calculated for all patient
characteristic subgroups. Next, a logistic regression model
was created to examine whether mean percent adherence
4 International Journal of Endocrinology
Table 1: Subject characteristics by serum 25(OH)D and adherence measurements.
Change in serum
CV for steady-state
adherent by pill
count data, mean
Percent adherent by
the novel serum
18.5 (10.3) +22.4 (17.4)18.9% (9.0%) 74.3% (16.6%)33.3%
23.2 (7.9) +23.2 (7.9)17.5% (8.4%) 81.4% (15.3%)46.0%
23.5 (8.7) +23.5 (8.7)18.4% (8.2%)83.4% (12.8%)47.7%
26.6 (7.9)+26.6 (7.9) 17.9% (10.1%)84.7% (12.6%)47.5%
17.3 (7.1)+21.1 (13.3) 20.4% (11.6%) 74.8% (13.4%)53.1%
24.5 (7.6) +18.1 (11.0)16.7% (8.3%)86.1% (13.4%) 43.4%
28.6 (7.2) +18.9 (10.6)18.2% (7.8%)83.2% (12.7%)47.2%
Body mass index
29.5 (7.9)+17.5 (9.6) 16.8% (8.5%) 90.0% (6.1%)33.3%
25.4 (9.3) +18.0 (12.5) 17.7% (9.8%) 82.6% (11.5%)44.6%
23.0 (7.8) +22.4 (11.9)17.2% (7.6%)86.0% (10.5%)53.8%
23.3 (7.5) +18.2 (9.8)19.2% (8.1%) 80.3% (16.0%) 47.5%
Season at enrollment
24.3 (8.4) +20.2 (12.2) 16.9% (9.4%)83.4% (12.6%)50.0%
22.6 (7.8) +20.1 (10.9)18.8% (10.7%) 83.7% (15.9%)43.4%
25.2 (7.3)+17.2 (11.2)17.7% (7.3%)84.5% (12.3%)40.0%
26.5 (9.9)+18.2 (11.5)17.9% (7.4%)79.4% (12.7%) 55.9%
24.5 (8.7) +15.5 (10.7)18.5% (8.9%) 82.3% (13.3%)37.6%
24.4 (8.0)+22.9 (10.8) 17.2% (9.1%)83.6% (14.3%)56.6%
Adherence by pill count
25.7 (9.8)+13.8 (10.1) 17.4% (9.0%) 57.8% (12.0%)40.0%
24.2 (8.1)+20.0 (11.4)18.0% (9.0%)87.5% (7.8%)47.8%
24.4 (8.4)+19.0 (11.4)17.9% (9.0%)82.9% (13.7%)46.6%
by pill count was significantly associated with adherence
by serum metabolite levels, while controlling for age, race,
season at enrollment, and baseline body mass index (BMI).
Finally, a sensitivity analysis was performed, in which
the limits on serum 25(OH)D change from baseline (i.e.,
>median) and coefficient of variation (i.e., <median) were
varied to examine whether our findings were sensitive to
the cutoffs selected for our novel definition of adherence by
serum metabolite levels. The additional definitions of adher-
ence incorporated cutoffs of the 25th and 75th percentile
for serum 25(OH)D change from baseline (as opposed to
using the median, or 50th percentile), along with the 75th
analyses, additional multivariate logistic regression models
were created for each combination of cutoffs for change and
coefficient of variation, treating the revised novel definition
of adherence as the dependent variable of interest.
A total of 161 subjects with available data were included in
the study. Descriptive statistics for each subgroup of subjects
is presented in Table 1. The mean (SD) baseline serum
25(OH)D level was 24.4 (8.4)ng/mL across all subjects,
increasing, on average, by 19.0 (11.4)ng/mL to their steady-
state value, with higher changes observed in the higher dose
group, as expected. By pill count, subjects were adherent
82.9% of the time, while only 46.6% were adherent using our
novel definition based on serum 25(OH)D levels.
Table 2 summarizes the results of the logistic regression
model associated with our primary analysis. The analysis
revealed that mean percentage of adherence by pill count
was not significantly associated with adherence as measured
by serum metabolite levels (odds ratio [OR] = 1.2, 95%
confidence interval [CI] = 0.9 to 1.6). Of the other subject
International Journal of Endocrinology5
Table 2: Results of the primary logistic regression model predicting adherence as determined by the novel serum 25(OH)D definition.
Mean percent adherence
(as measured by pill count)
Odd ratio 95% confidence interval
(0.9, 1.6) .29
32 1.8 (0.6, 5.0) .20
761.0 (0.4, 2.3) .41
Body mass index
(1.0, 1.1) .22
85 0.5 (0.2, 0.9) .02
Season at enrollment
53 0.7(0.3, 1.8) .30
40 0.7(0.2, 1.8) .24
34 1.7(0.6, 4.9) .08
∗Mean percent adherence was treated as a continuous measure in the model; however, this variable was coded such that the odds ratio reflects the increase in
odds associated with a 10-percent increase in mean percent adherence.
†Maternal age at baseline was treated as a continuous measure in the model; however, this variable was coded such that the odds ratio reflects the increase in
odds associated with a 1-year increase in age.
‡Body mass index was treated as a continuous measure in the model; however, this variable was coded such that the odds ratio reflects the increase in odds
associated with a 1-unit increase in body mass index.
characteristics included in the model, the only variable
exhibiting a significant association with adherence by serum
adherence among patients in the 2000IU dose group being
about half that of patients in the 4000IU dose group (OR =
0.5, 95% CI = 0.2 to 0.9). The generalized R-square value for
the model was 10.5%, indicating that much of the variation
in adherence (by serum 25(OH)D) was not explained by
pill count or the other variables in the model. Sensitivity
analyses (data not shown) revealed that our findings were
not sensitive to the additional cutoff limits used within our
novel definition of adherence by serum metabolite levels
(i.e., using the 25th and 75th percentiles for the cutoff for
serum 25(OH)D change from baseline, and using the 75th
percentile for the cutoff for the coefficient of variation).
The importance of adherence to medication has been
recognized in clinical trials and daily clinical practice
alike. In order to properly evaluate the effects of med-
ications, it is vital to determine whether they are taken
as prescribed. The most frequent manner to evaluate
adherence has been pill count, which offers advantages
such as low cost and simplicity of collection and calcula-
tion, but yet has the disadvantages of frequently missing
data and possible manipulation by subjects [67–73]. In
this trial, we sought to determine whether there was a
significant association between adherence to vitamin D
supplementation as measured by pill count and assess-
ment of a novel measure of adherence based upon serum
levels of vitamin D metabolites in a pregnant popula-
the determination of efficacy in clinical research. Currently,
the efficacy of medications and supplements is determined
primarily via randomized controlled clinical trials, in which
subject adherence is assumed or measured via pill count and
results are analyzed on an intent-to-treat basis as a measure
of a medication’s effectiveness. If there were an objective
laboratory value by which adherence could more accurately
be determined, studies could include the most adherent
patients in the analysis to get a clearer picture of efficacy
without the dilution of the effect by nonadherent patients.
According to our findings, while there was a persistently
positive odds ratio between the pill count measure of
adherence and our novel definition of adherence by serum
metabolite levels, this association was not significant for
any of the proposed definitions (Table 2). This confirms
previous studies which showed that pill count data are prone
to errors [67, 69, 73]. Our analytic technique, however, is
unique in that it utilizes the pharmacokinetics of vitamin
D metabolism to predict adherence, and the data are
then compared to pill counts. Because maternal circulating
25(OH)D is not affected by gestational age per se, the
stability of vitamin D during pregnancy allowed for analysis
of adherence once steady state was achieved throughout
6 International Journal of Endocrinology
gestation [15, 16]. Other markers such as 1,25(OH)2D, PTH,
or calcitonin would not offer such stability as they change
throughout gestation .
Strengths of this study include the variety of both
pill count-based and serum metabolite-based definitions of
compliance. The study is strengthened by the consistency of
findings across multiple definitions on sensitivity analysis. In
addition, the subject population in this study was composed
exclusively of pregnant women, which are generally more
A limitation of the study was missing pill count data,
which limited the sample size. In fact, 10% of the original
subject population was eliminated from this analysis at the
which pill count data was available. The majority of patients
did not have pill count data for all time points, meaning that
their general level of adherence was based on time points
at which data were available. This may not be accurate,
as an interim lack of adherence could have contributed to
not returning the pill bottle at certain time points. There
adherence and the likelihood of remembering to bring the
pill bottle to the next appointment. The lack of data is a
persistent problem in pill count adherence analyses, and one
which could be alleviated in the future by changing the way
adherence is measured in clinical trials.
An unexplained finding in this study was the significant
difference with adherence by subject dose group, with the
odds of adherence among patients in the 2000IU dose group
being about half that of patients in the 4000IU dose group.
This may be a spurious finding that would disappear with
further analysis of a larger sample, or it may be an artifact of
our definition that we cannot otherwise explain. To resolve
in a larger sample of subjects.
In summary, based upon a novel definition of adherence
that incorporates serial measurements of serum metabolite
levels, we found that pill count was not a reliable predictor
of adherence to protocol. These findings call into question
the way that adherence is typically measured in clinical
research, which also has implications in the determination
of efficacy of medications or supplements under study.
This study also offers an alternative approach to measuring
compliance for supplements and medications with long half-
lives. This approach could potentially be used clinically to
monitor patient adherence to prescribed regimens of such
supplements or medications, much as HbA1C levels are
currently used to monitor long-term blood glucose control
in diabetic patients.
The project described was supported by Award Number
UL1RR029882 from the National Center for Research
Resources. The content is solely the responsibility of the
authors and does not necessarily represent the official views
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