All About Vitamimin D

Sun Exposure, Skin Reflectance and
Vitamin D Intake to Predict Vitamin D Status
By:
LAURA MARIE HALL

R.D. (University of California, San Francisco Medical Center) 2004
B.S. (California Polytechnic State University, San Luis Obispo) 2003
DISSERTATION
Submitted in partial satisfaction of the requirements for the degree of
DOCTOR OF PHILOSOPHY
in
Nutritional Biology
in the
OFFICE OF GRADUATE STUDIES
of the
UNIVERSITY OF CALIFORNIA
DAVIS
Approved:
Committee in Charge
2008
I
UMI Number: 3350731
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Acknowledgements
I would like to take this opportunity to thank the many people who have helped
me achieve this degree. First, I would like to thank Dr. Charles Stephensen for taking a
chance on me two years into my graduate school career and for helping to come up with
a study that matched both of our interests. Because of his continual guidance and
support, I know that we completed a study that we can both be very proud of. I look
forward to future collaborations with him. I would also like to thank the Western
Human Nutrition Research Center (WHNRC) staff and the undergraduate nutrition
student volunteers for their help. I am very thankful for my funding including the
NCMHD Center of Excellence in Nutritional Genomics Grant, the Bristol-Myers Squibb
Foundation, Inc. Freedom to Discover Grant, the Gustavus and Louise Pfeiffer Research
Foundation Grant and for WHNRC funds. I would like to thank our collaborators, Dr.
Michael Kimlin, Dr. Pavel Aronov, Dr. Bruce Hammock, Dr. James Slusser, and Dr.
John Davis, for adding their expertise to our study. Thank you to my Dissertation
Committee, Dr. Francene Steinberg, Dr. Jiming Jiang and Dr. Charles Stephensen, for
their advice and insight.
On top of those who had direct contract with the study, I would like to thank my
parents, Elden and Vivian Zercher, for their love and support which started long before
graduate school and won't end here. Thank you to my husband, Jon Hall, who has
encouraged greatness from me and has helped endure the not so great times. To all my
family and friends, who wondered how long I would be a student for, thank you for your
patience and encouragement. This is just the beginning!
ii
Dedication
To my loving family and
my husband, Jon- You are my sunshine

Contents
Acknowledgements ii
Dedication iii
Abstract 1
1 Vitamin D: A Review of the Literature 3
1.1 The History of Vitamin D 4
1.1a The Discovery of Vitamin D 4
L i b Vitamin D is not a Vitamin 5
1.1c Identification of Vitamin 5
L i d The Hormonal Form of Vitamin D 6
L i e The Physiological Functions of Vitamin D 6
1.1 f Discovery of the Vitamin D Receptor 8
1.2 Non-Calcemic Functions of Vitamin D 9
1.3 The Metabolism of Vitamin D 12
1.3 a Overview 12
1.3 b The Photobiology of Vitamin D and its' Conversion in the Skin 13
1.3 c 25-Hydroxylase in the Liver 14
1.3 d la-Hydroxylase in the Kidney and Other Sites 15
1.4 Vitamin D Deficiency 16
1.4 a Prevalence of Vitamin D Deficiency 17
1.4 b Consequences of Vitamin D Deficiency 20
IV
1.4 c Economic Burden of Vitamin D Deficiency 21
1.4 d Current Recommendations to Alleviate Vitamin D Deficiency 22
1.5 Factors Affecting Vitamin D Status (250HD) 22
1.5 a Seasonal Effects on Vitamin D Status 22
1.5 b Sun Exposure Effects on Vitamin D Status 23
1.5 c Sun Exposure and Dietary Vitamin D Contributions to Vitamin D 24
Status
1.5 d Obesity Influencing Vitamin D Status 33
1.5 e Skin Pigment and Ethnicity Related to Vitamin D Status 33
1.5 f Sun Exposure and Dietary Vitamin D Contributions to Vitamin D 34
Status in High Sun Exposure Locations
1.5 g Conclusion 37
1.6 Factors Affecting the Synthesis of Vitamin D in the Skin: Environmental 37
1.6 a The Influence of Season on Vitamin D Synthesis 37
1.6 b The Influence of Latitude on Vitamin D Synthesis 38
1.6 c The Influence of Time of Day on Vitamin D Synthesis 39
1.6 d The Influence of Altitude on Vitamin D Synthesis 40
1.6 e The Influence of Ozone and Aerosol on Vitamin D Synthesis 40
1.6 f The Influence of Clouds on Vitamin D Synthesis 41
1.6 g The Influence of Reflectance on Vitamin D Synthesis 42
1.7 Factors Affecting the Synthesis of Vitamin D in the Skin: 42
Non-Environmental
1.7 a The Effect of Melanin on Vitamin D Synthesis 43
v
1.7 b The Influence of Sunscreen on Vitamin D Synthesis 44
1.7 c The Influence of Clothing, Glass and Plastics on Vitamin D 45
Synthesis
1.7 d The Effect of Aging on Vitamin D Synthesis 46
1.8 How Much Sun Exposure Do We Need? 47
1.8 a Risk and Benefit Considerations 49
1.9 Assessing Sun Exposure 54
1.9 a Overview 54
1.9 b In Vitro Models of Vitamin D3 Synthesis and Human Skin Samples 56
1.9 c Sun Exposure Questionnaires 58
1.9 d Polysulphone (PS) Badges and Validations against Questionnaires 60
1.9 e Electronic Wrist Watch Dosimeters 63
1.9 f Ground-Based/On Ground UV Measurements 64
1.9 g The UV Index 66
1.9 h Web-Based Tools 67
1.9 i Simulated Solar Radiation 67
1.10 UV Doses Measured 70
1.10 a UV Doses in the United States 70
1.10b Personal UV Doses 71
1.10c UV Doses to the Body and UV Doses with Different Activities 73
1.11 Skin Pigmentation 78
1.11a Studies with Skin Pigmentation 78
1.11b Measuring Skin Pigmentation 79
vi
1.12 Dietary Vitamin D 82
1.12a Dietary Vitamin D Compared to Photosynthesized Vitamin D 82
1.12 b Sources 83
1.12c Vitamin D Intakes 84
1.12 d Intake Recommendations 86
1.12 e Risk Assessment for Vitamin D Intake 88
1.12 f Measuring Intake and Validating Measurement Tools 88
1.13 Setting Recommended Daily Allowances (RDA's) for Vitamin D Intake 89
1.13 a Supplementation Studies and Dose-Response Finding Studies 89
1.14 Vitamin D2 versus Vitamin D3 94
1.15 Determining Vitamin D Status using Serum 25-Hydroxyvitamin D (250HD) 95
1.15 a Overview 95
1.15 b Cutoffs 95
1.15c Measuring 250HD using a Radioimmunoassay (RIA) 97
1.15 d Measuring 250HD using a Liquid Chromatography-Tandem 98
Mass Spectrometry Analysis (LC-MS)
1.16 Conclusion 98
1.17 References 99
2 Vitamin D Intake, Skin Reflectance, and Sun Exposure, using 117
Polysulphone (PS) Dosimeter Badges, to Predict Serum 250HD
2.1 Abstract 118
vii
2.2 Introduction 119
2.3 Subjects and Methods 121
2.3 a Subjects 121
2.3 b Study Design 122
2.3 c Sun Exposure Assessment 123
2.3 d USDA UV-B Monitoring Station at the UC Davis Climate Center 124
2.3 e Dietary Assessment 125
2.3 f Skin Reflectance (Pigment) Assessment 125
2.3 g Covariates 126
2.3 h Serum 25-HydroxyvitaminD (250HD) 127
2.3 i Parathyroid Hormone (PTH) 128
2.4 Statistical Analysis 128
2.5 Results 129
2.5 a Sun Exposure 129
2.5 b Dietary Vitamin D Intake 132
2.5 c Skin Reflectance (Pigment) 133
2.5 d Vitamin D Status 134
2.5 e Parathyroid Hormone (PTH) 136
2.5 f Model Predicting Vitamin D Status 136
2.5 g Model Estimating Vitamin D Intake Needed To Maintain a Healthy 140
Vitamin D Status
2.6 Discussion 140
2.7 Acknowledgements 150
viii
2.8 References 151
2.9 Figures 155
2.10 Tables 180
3 Vitamin D Intake, Skin Reflectance, and Sun Exposure, using Daily Sun 189
Exposure Logs, to Predict Serum 250HD
3.1 Abstract 190
3.3 a Subjects 195
3.3 c USDA UV-B Monitoring and Research Program (UVMRP) 197
3.3 d Daily Sun Exposure Assessment 198
3.3 e Dietary Assessment 201
3.3 f Skin Reflectance (Pigment) Assessment 201
3.3 g Covariates 202
3.3 h Serum 25-HydroxyvitaminD (250HD) 202
3.5 Results 204
3.5 a Daily Sun Exposure Logs 205
3.5 b Other Measures of Sun Exposure 209
3.5 c Other Information Collected From the Daily Logs 213
ix
3.5 d Dietary Vitamin D Intake 214
3.5 e Skin Reflectance (Pigment) 215
3.5 f Vitamin D Status 215
3.5 g Models Predicting Vitamin D Status 216
3.5 h Model Estimating Vitamin D Intake Needed To Maintain a Healthy 220
Vitamin D Status
3.5 i Model Estimating Joules of Sun Exposure Needed To Maintain a 221
Healthy Vitamin D Status
3.6 Discussion 222
3.8 References 232
3.9 Figures 236
3.10 Tables 268
4 Validation of Sun Exposure and Vitamin D Intake Recall 280
Questionnaires for Estimating Vitamin D Status
4.1 Abstract 281
4.3 a Subjects 285
4.3 c USDA UV-B Monitoring and Research Program (UVMRP) 287
x
4.3 d Daily Sun Exposure Logs 288
4.3 e Sun Exposure Recall Questionnaires 290
4.3 f Dietary Assessment using Food Records 291
4.3 g Vitamin D-Specific Food Frequency Questionnaire (FFQ) to 292
Assess Vitamin D Intake
4.3 h Skin Reflectance (Pigment) Assessment 293
4.3 i Covariates 293
4.3 j Serum 25-HydroxyvitaminD (250HD) 294
4.4 a The Method of Triads 296
4.4 b Multiple Linear Regression Analysis 297
4.5 Results 297
4.5 a Sun Exposure Recall Versus Daily Sun Exposure Logs 298
4.5 b Sun Exposure Recall Associations with Serum 250HD 300
4.5 c The Method of Triads for Sun Exposure Recall 302
4.5 d Vitamin D-Specific Food Frequency Questionnaire (FFQ) Versus 303
Food Records for Vitamin D Intake
4.5 e Vitamin D-Specific FFQ Associations with Serum 250HD 304
4.5 f The Method of Triads for the Vitamin D-Specific FFQ 306
4.5 g Model Predicting Vitamin D Status using Sun Exposure Recall 306
Methods
4.5 h Model Predicting Vitamin D Status using Vitamin D Intake Recall 308
Methods (Vitamin D-Specific FFQ)
xi
4.5 i Model Predicting Vitamin D Status using Both Sun Exposure 309
Recall and Vitamin D Intake Recall Methods
4.6 Discussion 310
4.8 References 315
4.9 Figures 318
4.10 Tables 335
Appendices 351
A Study Consent Form 351
B Polysulphone (PS) Badge Instructions and Blood Draw 357
Instructions
C Sun Exposure Log Instructions and Key 359
D Sun Exposure Recall Questionnaires 363
E Food Record Form 367
F Vitamin D-Specific Food Frequency Questionnaires (FFQs) 369
xu
1
Laura Marie Hall

December 2008
Nutritional Biology
Sun Exposure, Skin Reflectance and

Vitamin D Intake to Predict Vitamin D Status
Abstract
The contribution of sun exposure to vitamin D status is rarely assessed in
community studies using validated methods. Our first objective was to measure the
contribution of sun exposure, using different methods, in conjunction with vitamin D
intake and skin reflectance, to vitamin D status (serum 25-hydroxyvitamin D (250HD))
and estimate intake needed to maintain a healthy status. Our second objective was to
validate a sun exposure recall questionnaire and a vitamin D-specific food frequency
questionnaire (FFQ).
Our study was conducted with 4 cohorts of healthy young adults each followed
for 7-8 weeks in the Fall, Winter, Spring and Summer (2006-07) in Davis, CA (n = 72).
Skin reflectance was measured using a reflectance spectrophotometer. Dietary vitamin
D intake was assessed by food records and a vitamin D-specific FFQ. Sun exposure was
assessed using polysulphone badges, daily sun exposure logs and recall questionnaires.
Serum 250HD was measured by ultra-performance liquid-chromatography-tandem mass
spectrometry (LC-MS) and by radioimmunoassay (RIA). Multiple linear regression
analysis was used to predict serum 250HD using continuous variables for sun exposure,
skin reflectance and vitamin D intake, and dummy variables for cohort. Spearman's
correlation coefficients were used examine the association between serum 250HD and
different measures of sun exposure including time outside, a sun exposure index (SEI)
2
and joules. Spearman's correlation coefficients were also used to correlate the recalled
measurements to the reference measurements.
Sun exposure, skin reflectance and vitamin D intake were all significant
independent predictors of serum 250HD (p < 0.05), however, sun exposure contributed
the most to status. Consequently, joules and the SEI were significantly better predictors
of status than time outside. The recall measurements were highly correlated with the
reference measurements and the models using the reference measurements predicted
status as well as the reference models (p > 0.05).
These results show that the contribution of sun exposure, skin reflectance and
vitamin D intake to vitamin D status can be assessed using simple methods in free-living
adults. These methods may help nutritionists make more accurate individualized
recommendations for vitamin D intake based on skin reflectance and sun behavior.
3
Chapter 1:
Vitamin D: A Review of the Literature

4
1.1 The History of Vitamin D
1.1 a The Discovery of Vitamin D
At the end of the 19th century, the industrial revolution had begun and
urbanization of the population forced people into crowed cities where smoke from the
industrial plants polluted the air (1). Countries of higher latitude and low sunlight, such
as England, saw an epidemic of rickets appear after the industrial revolution. Rickets
was first described by Whistler and is a disease found in young children in which the
skeleton is poorly mineralized and deformed (1). Rickets was known as "the English
disease" because of its' overwhelmingly high prevalence in England.
Sir Edward Mellanby of Great Britain thought that rickets could be due to a
dietary deficiency and fed dogs a diet primarily of oatmeal and kept them indoors in
Scotland where the incidence of rickets was also high (2). The dogs indeed developed
rickets (2). Mellanby provided cod liver oil to the dogs which cured their rickets and he
first attributed the healing power to vitamin A, a known nutrient in cod liver oil (2).
McCollum, who had discovered vitamin A in butter fat, decided to destroy the
vitamin A in the cod liver oil and see if it could still cure rickets (3). The vitamin A-
deficient cod liver oil was still able to cure rickets and McCollum termed the unknown
vitamin "vitamin D" (3). From the work of Mellanby and McCollum, vitamin D became
known as an essential nutrient (1).

5
1.1 b Vitamin D is not a Vitamin
While Mellanby was conducting his experiments on dogs, Huldshinsky and
Chick et al. independently found that sunlight or artificial light could cure rickets in
children (4). This finding raised questions about vitamin D and sunlight. Meanwhile,
Steenbock and Hart discovered the importance of sunlight on calcium balance in
lactating goats (5). Then in 1924, Steenbock and Black found that rickets could be cured
by irradiating either their diets or the animals themselves (6). Hess and Weinstock also
reported a similar finding of the antirachitic properties of irradiated food with ultraviolet
light (7). Inducing vitamin D activity in foods as a way of fortifying them helped to
eliminate rickets as a major medical problem (1).
1.1 c Identification of Vitamin D
From irradiated plants sterols, Askew et al. isolated and determined the structure
of vitamin D2 or ergocalciferol (8). Windaus and Block isolated 7-dehydrocholesterol
from the skin, synthesized it and converted it to vitamin D3 or cholecalciferol (9).
Holick et al. discovered another step in which previtamin D3 is produced in the skin
from 7-dehydrocholesterol and then converted to vitamin D3 (10). The structures of the
nutritional forms of vitamin D, ergocalciferol and cholecalciferol, became known and
synthetically produced vitamin D replaced irradiated foods as a way of fortifying foods
with vitamin D (1).

6
1.1 d The Hormonal Form of Vitamin D
Kodicek used a bioassay to study the fate of the vitamin D and discovered that
much of vitamin D is converted to biologically inactive products (11). After years of
work, Kodicek concluded that vitamin D is inactive without metabolic modification (12).
In 1968, the first active metabolite was isolated and chemically identified as 25-
hydroxyvitamin D3 (25OHD3) and this was thought to be the active form of vitamin D
but when 25OHD3 was radiolabeled it was rapidly metabolized to more polar
metabolites (13). DeLuca et al. finally isolated the active metabolite from the intestine
of chickens and the structure was shown to be l,25(OH)2D3 (14). The final proof was in
experiments which used anephric animals that responded to l,25(OH)2D3 by increasing
intestinal absorption of calcium while those receiving 25OHD3 did not (15). Studies that
looked at animals on low-calcium and high-calcium diets showed that when calcium is
needed there is an increase in l,25(OH)2D3 but when it is not then 24R,25(OH)2D3 is
produced (16). Fraser and Kodicek injected parathyroid hormone (PTH) into chickens
and increased lot-hydroxylation to l,25(OH)2D3 showing that PTH activates this step in
the kidneys (17). In the early 1970s the basic vitamin D endocrine system was
discovered and through the late 1970s and early 1980s, over 30 metabolites of vitamin D
were identified (1).
1.1 e The Physiological Functions of Vitamin D

7
After the discovery that vitamin D is important for bone mineralization,
Nicolaysen clearly demonstrated the role of vitamin D in calcium and phosphorus
absorption from the diet in the gut (18). Researchers tried to show that vitamin D played
a direct role in bone mineralization but it has since been shown that the elevation of
calcium and phosphorus in the plasma by vitamin D is what mineralizes bone (19).
Carlsson also discovered that vitamin D was needed to induce the mobilization of
calcium from the skeleton (20). DeLuca et al. showed that the rise in serum calcium
induced by vitamin D in animals on low-calcium diets was from bone demineralization
(21). It has been discovered that l,25(OH)2D3 stimulates osteoblasts to produce receptor
activator nuclear factor-KP ligand (RANKL) which activates osteoclasts for bone
resportion (22). Both vitamin D and the parathyroid hormone (PTH) are required for
this osteoclastic-mediated bone resorption to occur and this event is an essential step in
the bone remodeling process (23). The parathyroid gland contains calcium-sensing
proteins that sense plasma calcium levels and when levels decrease these transmembrane
proteins, coupled to a G protein system, stimulate the secretion of the parathyroid
hormone (24).
Yamamoto et al. first showed the vitamin D was able to elevate plasma calcium
through its' action in the distal renal tubule (25). Reabsorption of the last 1% of calcium
requires both vitamin D and PTH. Thus, vitamin D increases plasma calcium through
its' action on the gut, the skeleton, and the kidney and these functions are important to
meet the essential needs of calcium in the body.
Heaney stresses that calcium and vitamin D are both necessary for the full
expression of each others' effects and where their actions are independent, their effects
8
on skeletal health are complementary (26). Both nutrients enhance bone gain during
growth, reduce age-related bone loss and reduce fragility fractures (26). A high calcium
intake is needed to offset corresponding outputs and vitamin D is needed for efficient
absorption of dietary calcium (26). Vitamin D facilitates bone remodeling with active
calcium transport from typical calcium intakes, whereas high calcium intakes would be
needed for passive absorption (26).
In this endocrine system, when there is not enough dietary calcium available,
vitamin D helps with calcium absorption from the gut, mobilization from the bones and
reabsorption in the kidneys to support the needs of the individual. The resulting loss of
calcium from the bones could lead to osteomalacia and ultimately to osteoporosis (27).
1.1 f Discovery of the Vitamin D Receptor
Zull et al. showed that the function of vitamin D is blocked by transcription and
protein inhibitors and that nuclear activity is required for vitamin D to function (28).
However, Brumbaugh and Haussler were the first researchers to clearly demonstrate the
existence of a vitamin D receptor (VDR) (29). It was discovered that the VDR was
localized in the distal renal tubule cells, enterocytes of the small intestine, bone lining
cells, and osteoblasts as it relates to vitamin D's role in calcium metabolism (1). The
VDR acts through vitamin D-responsive elements (VDRE) (27). The VDR forms a
heterodimer on the VDRE with the retinoid-X receptor (RXR) protein on the 5' arm of
the responsive element and on the 3' segment of the VDR (30). At the same time, it
9
binds several other proteins required in the transcription complex and binds an activator
(31). At least 3 coactivators has been identified (27). Other important discoveries
include the discovery that VDREs are important in regulating expression of the
osteopontin, osteocalcin, preproparathyroid and 24-OHase genes (1).
Soon after the discovery of the VDR, other tissues not earlier known as targets of
vitamin D's action, were discovered to express VDR. Tissues expressing VDR include
the parathyroid gland, islet cells of the pancreas, cells in bone marrow, lymphocytes and
specific neural cells (32). It has also been recognized that macrophages, placenta and
skin, colon, breast, prostate and lung cells express the 1-OHase enzyme and therefore,
produce l,25(OH)2D locally (1).
1.2 Non-Calcemic Functions of Vitamin D
The discovery of the VDR in other tissues raised questions about other functions
of vitamin D not related to calcium metabolism and these functions have since been
found. Vitamin D causes differentiation of promonocytes to monocytes and coalescing
of monocytes into multinuclear osteoclast precursors and then into osteoclasts (33).
Vitamin D suppresses parathyroid cell growth and parathyroid hormone gene expression
(34). Vitamin D also appears to play a role in the suppression of growth and in cellular
differentiation of keratinocytes in the skin (35).
An important area of study has been with the immune system and it was
discovered that Multiple Sclerosis, an autoimmune disorder, can be suppressed or
eliminated with adequate amounts of vitamin D given daily in animal models (36). It
10
has been known for over 100 years that sunlight helps in the treatment of tuberculosis
(37). Vitamin D could possibly suppress type 1 diabetes mellitus and prevent the
destruction of islet cells (38). It is likely that the suppression of autoimmune diseases
involves l,25(OH)2D3 interacting with T helper lymphocytes which suppresses their
inflammatory response (27). Chiu et al. found that serum 250HD was positively related
to insulin sensitivity and negatively related to first- and second-phase insulin response
(39). Recently, there has been strong observational evidence in Europe that vitamin D is
beneficial for reducing the incidence of type 1 diabetes (40). A case-controlled study
with 7 European countries found an odds ratio of 0.65 for onset of type 1 diabetes by the
age of 15 in children who were supplemented versus those that were not supplemented
with vitamin D (41). The Diabetes Autoimmunity Study in the Young (DAISY) found
that the presence of islet auto-antibodies in offspring was inversely correlated with
maternal dietary vitamin D intake during pregnancy (42). Vitamin D deficiency in
pregnancy has also been associated with preeclampsia (43). Therefore, vitamin D
deficiency has detrimental effects on immune function and in pregnancy.
It has been shown that people with vitamin D deficiency are more prone to
developing schizophrenia (44). Vitamin D deficiency has also been associated with
depression (45). In the elderly, vitamin D deficiency has been associated with higher
admission to nursing homes (46). Thus, vitamin D deficiency can affect the mental
health and well-being of individuals.
Muscle cells also contain vitamin D receptors and in the National Health and
Nutrition Evaluation Survey (NHANES III) the walking test and the chair stand test,
which reflect muscle strength, took less time when 250HD was higher (47). Studies
11
have shown that vitamin D supplementation decreases the number of falls by improving
muscle strength (48). Bischoff et al. supplemented elderly women (mean serum 30
nmol/L) with 800 IU/day and reduced falls by 49% within 12 weeks of starting the
treatment (49). In another study by Bischoff-Ferrari et al. vitamin D decreased falls by
56% in all of the women and by 76% in the sedentary women (50). Therefore, vitamin
D deficiency affects muscle strength and ability.
Li et al. found that l,25(OH)2D3 is an effective regulator of renin production,
which controls blood pressure (51). Krause et al. exposed hypertensive adults to
simulated sunlight 3 times a week for 3 months and found an increase in 250HD by
more than 150% (52). They also found a significant decrease in both systolic and
diastolic blood pressure (52). Thus, vitamin D deficiency is associated with
hypertension and cardiovascular disease, along with living at higher latitudes (53-55).
Wang et al. measured serum 250HD in peritoneal dialysis patients and followed
them prospectively for 3 years (56). They found that a lower serum 250HD
concentration was associated with an increased risk of cardiovascular events (56).
Specifically, after adjusting for clinical and demographic factors, every 1 unit increase in
log-transformed serum 250HD was associated with a 44% reduction in the hazard of
cardiovascular events, fatal and nonfatal (56). Two recent epidemiological studies
showed improved survival and reduced cardiovascular mortality with l,25(OH)2D
therapy in patients receiving hemodialysis (57, 58). Low vitamin D status has also been
shown to contribute to the pathogenesis of heart failure in the general population even
though the exact mechanism is not currently known (55). A recent study showed that
low 250HD and l,25(OH)2D levels are independently associated with all-cause
12
mortality and cardiovascular mortality in women and men (59). They found elevated
levels of C-reactive protein and interleukin 6 in patients with lower 250HD values
suggesting that 250HD has anti-inflammatory properties (59). Consequently, vitamin D
deficiency has negative effects on heart health.
Ecological studies have shown a relationship between lower sunshine exposure
(higher latitude) and higher cancer prevalence and mortality. Boscoe and Schymura
looked specifically at UV-B exposure and cancer incidence and mortality in the US from
1993-2002 and after adjusting for cofounding variables in non-Hispanic whites found an
inverse relationship for ten sites: bladder, colon, Hodgkin lymphoma, myeloma, other
biliary, prostate, rectum, stomach, uterus and vulva (60). Weaker evidence of an inverse
relationship was found for breast, kidney, leukemia, non-Hodgkin lymphoma, pancreas
and small intestine (60). In a randomized trial, although not designed to examine cancer,
found that postmenopausal women who increased their vitamin D3 intake by 1,100 IU,
reduced their relative risk (RR) of cancer by 60-77% (61). Accordingly, l,25(OH)aD is
known to down-regulate hyperproliferative cell growth (62).
Many genes in prostate, colon and breast cells are regulated either positively or
negatively through the VDR (63). It is now estimated that over 200 genes are either
directly or indirectly influenced by l,25(OH)2D (63). Thus, l,25(OH)2D has a multitude
of biological effects.
1.3 The Metabolism of Vitamin D
1.3 a Overview
13
Vitamin D in general refers to both vitamin D2 and vitamin D3, however vitamin
D2 (ergocalciferol) is the naturally occurring form in plants and vitamin D3
(cholecalciferol) is the form synthesized by vertebrates (1). Vitamin D in general also
refers to a group of compounds that possess antirachitic activity but technically vitamin
D is a classified as secosteroid in which one of its' rings has been broken (9,10 carbon-
carbon bond of the B ring) (1).
Vitamin D is technically a prosteriod hormone rather than a vitamin because it is
made in the skin and converted to 25-hydroxyvitamin D (250HD) in the liver and then
to 1,25-dihyroxyvitamin D (l,25(OH)2D) in the kidneys (1). This simplified view is
complicated by the fact that vitamin D3 can be oxidatively metabolized to a variety of
metabolites but many of these have no identifiable biological function and were likely
identified in animals fed extremely high amounts of vitamin D3 (1).
1.3 b The Photobiology of Vitamin D and its' Conversion in the Skin
When solar ultraviolet (UV)- B photons with energies between 290 and 315 nm
from sunlight strike the skin, photolysis of 7-dehydrocholesterol (provitamin D3) to
previtamin D3 occurs in the plasma membrane of skin cells (64). No more than 15% of
the initial provitamin D3 concentration ends up as previtamin D3 (65). Previtamin D3 in
its' cis,cis isomer is thermodynamically unstable and is rapidly transformed to vitamin
D3 by rearrangement of its' double bonds (66). Approximately, 50% of previtamin D3 is
converted to vitamin D3 in 2 hours and as vitamin D3 is formed in the membrane, its'

14
open flexible structure is thought to escape into the extracellular space (1). Once
vitamin D3 enters the extracellular space, it is attracted to the vitamin D binding protein
(DBP) in the circulation and together they enter the dermal capillary bed (1). Vitamin D
can be continuously synthesized from the skin for three days after a single exposure to
sunlight (10).
Loomis had first speculated that human skin evolved with increased melanin
production in those living near the equator to prevent excessive production of vitamin D3
(67). However, we now know that previtamin D3 and vitamin D3 can be broken down
with further UV-B radiation to inert photoproducts (68, 69). This prevents intoxication
of vitamin D3 from the sun. Once previtamin D3 is produced in the skin, it can be
converted to vitamin D3 or into biologically inert photoisomers, lumisterol and
tachysterol upon further UV-B exposure (68). Once vitamin D3 is formed it can either
enter circulation or isomerize to at least 3 photoproducts, suprasterol I, suprasterol II,
and/or 5,6-trans-vitamin D3 (69). Therefore, sunlight can act as a regulator of vitamin
D3 production and prevent against intoxication.
1.3 c 25-Hydroxylase in the Liver
The initial step in vitamin D activation is the 25-hydroxylation by the 25-
hydroxylase enzyme in the liver which converts vitamin D to 25-hydroxyvitamin D
(250HD). Four enzymes have this activity. Other sites of 25-hydroxylation have been
discovered but the liver remains the major site (70, 71). Liver production of 250HD is
not tightly regulated and is primarily dependent on substrate concentration (vitamin D)

15
reflecting vitamin D intake and production (1). It has been shown that animals can use
vitamin D2 as a sole source of vitamin D as well as vitamin D3. Data from studies that
looked at pig liver 25-hydroxylation of vitamin D3 found an enzyme designated
CYP2D25 which is found equally in males and females and can equally hydroxylate
vitamin D2 and vitamin D3 (72). However, the 25-hydroxylation is not completely
understood and much still needs to be sorted out.
1.3 d lo-Hydroxylase in the Kidney and Other Sites
Originally 250HD was thought to be the active form but studies showed a more
polar metabolite was formed from 250HD administration (73). Studies by Lawson et al.
led them to suggest that the new metabolite had an oxygen function inserted at the C-l
position in addition to the hydroxyl group at the C-25 position (74). Fraser and Kodicek
showed that this new metabolite was synthesized by kidney mitochondria (75). In 1971,
it was determined that the active form of vitamin D is 1,25-dihyroxyvitamin D
(l,25(OH)2D) (76). Since then extrarenal sites of production have been identified in the
liver, bone, placenta, macrophages and skin (77).
The enzyme capable of la-hydroxylation is designated CYP27B1 and is located
on the inner mitochondrial membrane of the proximal convoluted tubule cells of the
kidney (78). Regulation of l,25(OH)2D production is increased by PTH (17) and
reciprocally regulated with respect to 24R,25(OH)2D (79). There is an indirect role of
plasma calcium, through PTH, in regulating l,25(OH)2D production and what appears to
be a direct role of plasma phosphorus (80). As plasma phosphorus declines, animals

16
shift from 24R,25(OH)2D production to enhanced l,25(OH)2D production (80). A direct
negative effect of l,25(OH)2D on CYP27B1 activity has been reported both in vivo and
in vitro and is likely indirect (81). Other factors have been reported as potential
regulators of l,25(OH)2D production, such as, calcitonin, acidosis, sex steroids, growth
hormone, glucocorticoids, thyroid hormone, and pregnancy (1). Since cloning of the
renal CYP27B1 has been achieved, knockout models will likely provide more insight
into the functions and regulation of l,25(OH)2D.
It has since been discovered that the kidney is not the only site for la-
hydroxylation. Extrarenal synthesis of l,25(OH)2D occurs under the influence of
cytokines and is important for the paracine regulation of cell differentiation and function
(82).
1.4 Vitamin D Deficiency
There are many causes and contributors to vitamin D deficiency including
reduced skin synthesis from sunscreen over-use, dark skin pigment, increased age and
the dependence on season, latitude and time of day in which vitamin D production
occurs (83). There could be decreased bioavailability of vitamin D from malabsorption
syndromes or medications and obesity (83). There could also be increased catabolism
from certain medications and increased urinary loss of 250HD from Nephrotic
syndrome (83). Liver failure decreases synthesis of 250HD and chronic kidney disease
decreases synthesis of l,25(OH)2D (83). There are heritable disorders that cause vitamin
D deficiency, such as, vitamin D-resistant rickets types 1-3, and acquired disorders, such
17
as, tumor-induced osteomalacia and hyperthyroidism (83). Even breastfeeding is
associated with an increased risk because of the poor vitamin D content in human milk
(83). Thus, there are many causes of deficiency.
1.4 a Prevalence of Vitamin D Deficiency
Nesby-O'Dell et al. found that 42% of African American women (15-49 years
old) had serum 250HD levels < 37.5 nmol/L at the end of winter throughout the United
States (84). Tangpricha et al. found the 36% of healthy young men and women (18-29
years old) were vitamin D deficient (< 50 nmol/L) at the end of winter in Boston (42°N)
(85). Holick found that 30%, 42% and 84% of free-living white, Hispanic and Black
elderly, respectively, were deficient (< 50 nmol/L) at the end of August in Boston (62).
Also in Boston, 57% of medical inpatients were vitamin D deficient (> 37.5 nmol/L) and
22% were severely deficient (< 20 nmol/L) (86). For the patients that didn't consume
the recommended intake level, 66% were deficient and for those that did, 37% were
deficient anyhow (86). If 75 nmol/L was used as the cutoff then 93% of the inpatients
would have been considered deficient (86).
Harkness and Cromer in a cross-sectional study found that 17% of adolescent
girls (12-18 years old) were extremely vitamin D deficient (< 27.5 nmol/L) and 54%
were deficient (< 50 nmol/L) from the greater Cleveland, Ohio area (87). African
American girls (43.0 nmol/L) had serum 250HD levels significantly lower than non-
African American girls (72.2 nmol/L) (p < 0.0001) (87). Serum levels were also higher
18
in the spring/summer months (60.2 nmol/L) compared to the fall/winter months (52.8
nmol/L) (p < 0.01) (87).
Looker et al. examined the prevalence of deficiency in the 18,875 individuals
from the Third National Health and Nutrition Survey (NHANES III 1988-1994) by
stratifying the sample into two seasonal subpopulations to address the logistical flaw in
the seasonal data collection (88). They found in the winter/lower latitude subpopulation,
1-5% were < 25 nmol/L and 25-57% were < 62.5 nmol/L (88). With the exception of
elderly women, prevalence of insufficiency was lower in the summer/higher latitude
group with 1-3% < 25 nmol/L and 21-49% < 62.5 nmol/L (88). Mean 250HD levels
were highest among non-Hispanic whites, then Mexican Americans and lowest in non-
Hispanic blacks (88).
In a multiethnic population (18-84 years old) in Boston (42°N), 60% of the
subjects were vitamin D deficient (< 50 nmol/L) and 87% were insufficient (< 75
nmol/L) in February even though -29% took a multivitamin (400IU) and ~47% drank
around 1.2 glasses of milk per day (89). Therefore, vitamin D intake did not prevent
against deficiency.
A study in Turkey (40°N) looked at adults in nursing homes and in their own
homes and found that 40.1% of those living in nursing homes and 24.4% of those in
their own homes were vitamin D deficient (< 37.5 nmol/L) (90). A cross-sectional study
looked at vitamin D deficiency in Turkish immigrants living in Germany, Germans and
Turkish residents of Turkey (91). They found that more than 78% of Turkish nationals
(immigrants and residents) had 250HD levels < 50 nmol/L (91). Turkish females had
19
the lowest 250HD levels (91). In comparison, only 29% of Germans had levels < 50
nmol/L (91). Thus, vitamin D deficiency varied by ethnicity and gender.
Brot et al. found that 39.7% of healthy perimenopausal Danish (54-58°N) women
were vitamin D insufficient (< 50 nmol/L) (92). Of the women who reported avoiding
sunlight and not taking vitamins, 32.8% were deficient (< 25 nmol/L) and 79.7% were
insufficient (< 50 nmol/L) (92). As a result, sun exposure may contribute more than
dietary intake.
In the Netherlands (52°N), van der Meer et al. assessed the prevalence of vitamin
D deficiency (< 25 nmol/L) in a multiethnic population (93). The prevalence of
deficiency was higher in Surinam South Asian (51.4%), Surinam Creole (45.3%),
Turkish (41.3%), Moroccan (36.5%), sub-Saharan African (19.3%) and other (29.1%)
compared to the indigenous Dutch (5.9%) (93). Also in the Netherlands, van Dam found
that 51% of older adults (60-87 years old) were deficient (< 50nmol/L) in the winter and
34% in the summer (94). In the Norwegian arctic (65-71°N), a quarter of the
participants had 250HD levels < 37.5 nmol/L between January and February when the
prevalence of hypovitaminosis D was highest (95). Almost two-thirds were below 50
nmol/L (95). Thus, serum levels have varied by ethnicity and season.
Hypovitaminosis D is also a widespread problem in middle-aged whites in Great
Britain (50-60°N) (96). Hypponen and Power found that nearly half of the population
had 250HD levels < 40 nmol/L during the winter and spring (96). They showed that for
winter and spring, the problem is not limited to high-risk groups. Almost 90% of the
population had levels < 75 nmol/L during the winter and spring, demonstrating a
seasonal effect, and 60% had suboptimal concentrations year round (96).
20
Even in high-sun areas, vitamin D deficiency is a problem. For instance, in
Australia (37°S), Pasco et al. found that over the winter months 43% of females were
vitamin D insufficient and 8% were deficient (< 25 nmol/L) (97). In Honolulu, Hawaii
(21°N), Binkley et al. found that 51% of their study population was deficient (< 75
nmol/L) in late March and the highest 250HD concentration was 155 nmol/L (98).
They also found that whites had higher serum levels than Asians and multiracial
individuals (p < 0.01) (98). In Arizona (32-33°N), 25.4% of the study population had
serum 250HD levels < 50 nmol/L and 2% were less than 25 nmol/L (99). Fifty-five
percent of Blacks and 37.6% of Hispanics were more likely to have serum 250HD levels
< 50nmol/L than were non-Hispanic whites (22.7%) (99). In south Florida (25°N), Levis
et al. found that 39% of the study participants were insufficient (< 50nmol/L) in March
(100). Thus, living in low latitudes and sunny climates does not prevent against vitamin
D deficiency.
Similarly, in high sun areas, where people extensively cover up with clothes,
vitamin D deficiency is also a concern. In Saudi Arabia, 59% of mothers and 70% of
neonates were deficient (101). Extensive coverage has also resulted in rickets and
osteomalacia in the Bedouins in the Negev Desert, Isreal (102) and in American Muslins
(103). Thus, covering the skin does not allow dermal synthesis of vitamin D and can
result in deficiency.
1.4 b Consequences of Vitamin D Deficiency

21
Severe vitamin D deficiency causes rickets in children and osteomalacia in adults
(104). Rickets causes deformities to the legs that makes it difficult to walk (105).
Osteomalacia, known as the softening of the bones, is characterized by surfaces of
trabecular and cortical bone covered with thick osteoid streams (104). Vitamin D
deficiency with low l,25(OH)2D and low calcium causes higher secretion of PTH
(secondary hyperparathyroidism) which results in higher bone turnover and resorption
(104). Cortical bone loss results from vitamin D deficiency and can contribute hip
fractures and osteoporosis later in life (106). Jacobsen et al. found that most of the loss
of bone mineral density (BMD) in the lumbar spine occurred during the fall and winter
when there was less sunlight and a decline in 250HD and an increase in PTH levels
(107). Not only are there the consequences of vitamin D deficiency, such as, poor bone
health but now it appears there may be other consequences, such as, autoimmune
diseases, certain cancers and cardiovascular disease (108).
1.4 c Economic Burden of Vitamin D Deficiency
A recent review estimated that vitamin D insufficiency in the United States costs
$40-56 billion per year in regards to cancer, multiple sclerosis and osteoporotic fractures
for the year 2004 (109). This doesn't include the several other diseases now associated
with vitamin D insufficiency. The economic burden due to excess UV radiation
(cataracts, melanoma and non-melanoma skin cancers) is $5-7 billion in comparison
(109). The estimated ratio of benefit to harm is between 6 and 11 to 1 (109). Increased
22
vitamin D through sun exposure, fortification or supplements could possibly reduce the
health care burden in the US (109).
1.4 d Current Recommendations to Alleviate Vitamin D Deficiency
Vitamin D deficiency is currently addressed through oral supplementation.
Those that are deficient are either given a dose of 2,000 IU/day (27) or a single dose of
50,000 IU/week for 8 weeks which has been proven safe and effective (62). Vitamin D
sufficiency has been maintained in nursing-home residents by giving them 50,000
IU/month (62). Vitamin D3 intoxication has only been seen when daily doses in excess
of 10,000 IU vitamin D3 are ingested (62).
1.5 Factors Affecting Vitamin D Status (250HD)
1.5 a Seasonal Effects on Vitamin D Status
Many studies have shown a seasonal fluctuation with serum 250HD, with higher
levels in the summer versus the winter. This seasonal variation indicates the importance
of sunlight as a source of vitamin D. Webb et al. found that free-living elderly and
nursing home-bound elderly who were not supplemented with vitamin D had lower
250HD levels and a greater seasonal variation (110). The seasonal variation was more
pronounced in individuals with low dietary vitamin D intakes (110).

In a study by Rockell et al. ethnicity and season were major determinants of
serum 250HD in a cross-sectional study in New Zealand children (111). However, age,
sex, latitude and obesity were not significant predictors of having vitamin D
insufficiency (111). Serum 250HD declined by 30 nmol/L, or 50%, between March
(Summer) and August (Winter) with a total 31% of all children and 59% of Pacific
children below 37.5 nmol/L (111). Thus, there was a significant seasonal decline in
250HD.
1.5 b Sun Exposure Effects on Vitamin D Status
Dawson-Hughes et al. showed that tropical sun exposure from vacations can also
attenuate the seasonal variation of 250HD (112). They found that in the participants
who had traveled to lower latitudes (< 35°N) for at least one day in the previous month
had a lower seasonal decrease than those who didn't travel (112). Tangpricha et al. also
found that in participants who used a tanning bed (at least once a week) had higher
250HD levels and higher BMD at the total hip area than for non-tanners (113). On the
other hand, chronic sunscreen users have shown to have lower 250HD levels than
controls (114). Sun exposure and tanning bed use are important contributors of vitamin
D but sunscreen use can minimize the effect.
Villareal et al. found a significant correlation between time spent outdoors and
serum 250HD (r = 0.23) (115). The time between also 12-2 pm showed the highest
correlation to serum 250HD (r = 0.33) (115). Dlugos et al. looked at serum 250HD in
submariners and found that without sun exposure for two months their serum levels
24
declined by half emphasizing the importance of sun exposure in maintaining serum
250HD levels (116).
1.5 c Sun Exposure and Dietary Vitamin D Contributions to Vitamin D
Status
Studies have shown that dietary vitamin D correlates poorly with 250HD
concentrations (86, 117) and that 250HD remains near what is considered a insufficient
level (-38 nmol/L) despite consumption of the AI for vitamin D (86). Sun exposure and
time spent outdoors are better predictors of 250HD than is dietary vitamin D intake (86).
Sowers et al. found that 250HD levels were more closely correlated with estimated sun
exposure (r = 0.26) than estimated dietary intake (119 ± 148 IU) (r = 0.11) and
supplement use (319 ± 463 IU) (r = 0.21) in women aged 20 to 80 (118). However,
dietary intake was assessed from one 24-hour recall and sun exposure was also recalled
based on time outdoors, clothing worn and sunscreen use (118).
A study in Korean women found that there was a seasonal variation to 250HD
from the group that was enrolled in September and re-measured 6 months later (119).
Serum 250HD correlated with sun exposure especially between 12pm-2pm as assessed
by time spent outdoors (r = 0.33, p < 0.0001) (119). There was a significant correlation
between dietary intake and 250HD (r = 0.34, p < 0.0001) (119). In their regression
model, they found dietary intake explained 33.6% of the variation in 250HD and time
spent outdoors explained 19.7% of the variation (119). Age also had a significant effect
on 250HD (119). However, consumption of vitamin D was not calculated exactly and
25
had to be assumed which was ~90-128 IU (119). This explained more of the variation in
250HD, however, average time spent outdoors (76.5 ± 52 mins.) may not represent
actual sun exposure since clothing and sunscreen use was not taken into account.
Studies in Demark and Britain showed a correlation between serum 250HD
levels and dietary vitamin D intake and levels were higher in Danish subjects taking
supplements (120). However, Poskitt et al. found that in Britain the most important
long-term source of vitamin D in healthy individuals was from sun exposure (121).
Giving vitamin D orally at twice the recommended level only caused a slight increase in
250HD which suggested that the average intake of 100 IU/day of vitamin D is an
insufficient source to meet the requirements for vitamin D (121).
Salamone et al. evaluated the contribution of vitamin D intake and sunlight
exposure to plasma 250HD in free-living elderly women during both the summer and
the winter (122). Subjects were categorized into high and low vitamin D intakes and
then within each intake group they were categorized into high and low sunlight exposure
groups based on summer exposure (122). Significant seasonal variation was seen in 3 of
the groups but not in the group with the high intake and low sun exposure (122). In
subjects with low sun exposure there was a strong correlation between vitamin D intake
and 250HD in the summer (r = 0.71, p < 0.01) and the winter (r = 0.80, p < 0.01) (122).
However, in the high sun exposure group the correlations weren't as strong (122).
Lamberg-Allardt observed four populations in Finland which included long-stay
geriatrics, residents of a nursing home, healthy elderly adults and healthy young adults
and found that all four groups showed seasonal variations in 250HD (123). However,
the variation in 250HD decreased with decreasing activity (123). The only group that
26
showed a correlation between diet and 250HD were the long-stay geriatrics who have
the lowest levels of 250HD, the least plasma variation and the smallest outdoor
exposure during the summer (123). Even the healthy elderly had higher levels of
250HD then the institutionalized elderly.
A study in Turkey looked at adults in nursing homes and in their own homes and
found that vitamin D deficiency (< 37.5 nmol/L) was higher in those in nursing homes (p
= 0.001), females (p < 0.001), those with lower sun exposure (p < 0.001) and those that
were older (p < 0.001) (90). They found a positive correlation between 250HD and
their sunlight exposure questionnaire (p < 0.001) (90). A cross-sectional study looked at
predictors of vitamin D status in Turkish immigrants living in Germany, Germans and
Turkish residents of Turkey (91). They filled out a detailed questionnaire that included
questions about wearing a scarf, sun exposure/sports activities and nutritional habits
(91). Using a multivariate regression analysis they found that women wearing a scarf
had a five-fold higher risk of being insufficient (< 50 nmol/L) and women with multiple
children had a two-fold higher risk (91). Use of vitamin-D rich foods did not predict low
250HD levels (91). An unconditional logistic regression identified the most important
predictors for low 250HD as sex, BMI, lack of sun exposure and living at a higher
latitude (91).
Lawson et al. assessed the relative contributions of diet and sunlight to vitamin D
status in the elderly (72-86 years old) over 16 months and blood was drawn every 3
months (124). Dietary vitamin D was estimated from daily food records and sun
exposure was estimated using a 14-point scale assigned to the number of times a week
that the subject went shopping, visiting, gardening or on holiday (124). The scores were
used to assess the relative variation in potential UV exposure not an actual amount of
time spent outdoors (124). The outdoor score and 250HD was significantly correlated
in August-September (r = 0.62, p < 0.01) (124). There was not a seasonal variation in
vitamin D intake and average vitamin D intake was significantly related to 250HD in
the winter (r = 0.55, p < 0.02) but not the summer (r - 0.17, NS) (124). They also found
that vitamin D intake in the week preceding the blood draw had a higher correlation (r =
0.30) than the preceding day (r = 0.15) or month (r = 0.21) (124). They conclude that
UV exposure is more important than dietary intake in the elderly (124).
Brot et al. assessed the relationship of status to sun exposure, dietary vitamin D
and PTH in healthy Danish perimenopausal women using the baseline data from a large
cross-sectional study called the Danish Osteoporosis Prevention Study (DOPS) (92).
The degree of sun exposure was based on a question surrounding sun behavior and
participants were categorized as never, occasionally or regularly go out into the sun. A
multiple linear regression analysis was used to predict 250HD with a continuous
variable for dietary vitamin D and categorical variables for tanning bed use (no = 0 or
yes =1), supplement use (no = 0 or yes =1), and whole body sun exposure (never = 0
and occasionally/regularly =1) (92). Dietary vitamin D intake, vitamin D
supplementation (200IU), sun exposure, and tanning bed use were all significantly
related to serum 250HD (92). In the whole population sun exposure
(occasionally/regularly) was associated with 27.6% higher serum 250HD (92). Those
reporting regular sun exposure had the highest 250HD year round, then those reporting
occasional sun exposure and lastly those reporting no sun exposure had the lowest
250HD (92). They saw a seasonal fluctuation in serum 250HD in all 3 categories
(highest in regular sun exposure group) with a rise in June and maintenance until
October and they related the fluctuation to the number of hours in the sun per month
with a 2 month lag time (92). They found that the women were prone to insufficiency
during the winter and spring if they avoided sun exposure and vitamin D
supplementation (92). They also found that mean PTH increased with decreasing
250HD (inverse relationship, r = -0.14, p < 0.001) which is consistent with other studies
(92). In Denmark (54-58°N) no vitamin D synthesis occurs for 6-7 months of the year
and if there is not enough sun exposure during the summer or supplementation year
round then vitamin D insufficiency is likely to occur during the winter and spring (92).
In the Norwegian arctic, Brustad et al. looked at the vitamin D status of a cross-
sectional sub-set of middle aged women from a larger cohort from November 2001 to
June 2002 (95). Traditionally in the coastal areas of northern Norway, fish was
consumed in high amounts and this prevented rickets (95). Each participant completed a
two-page questionnaire covering various questions about sun exposure, tanning bed use
and dietary intake of vitamin D (95). Specifically, the sun exposure questions asked
participants to recall the number of hours they has spent in the sun over the last week
(prior to the blood draw), residency and vacations over the summer prior to the blood
collection and tanning bed use in the past month (95). They devised a biological
effective UV dose rate (BED) for the photoconversion of 7-DHC to previtamin D3 in the
skin based on previous work in the field and by using the FastRT UV simulation tool
(95). They used a multiple linear regression model to predict plasma 250HD with diet,
season and a variable named 'UV-hours' representing hours per day of sun exposure
above the threshold for vitamin D3 production (BED > 0.472) (95). There was no
difference in vitamin D intake by calendar month and for 45.7% of the participants their
'UV-hours' was zero (95). The adjusted mean values of 250HD in relation to 'UV-
hours' increased up to 1.1-2.0 UV-hours per day and then seemingly leveled off (95).
Significant predictors of 250HD included vitamin D intake (p = 0.0003), time of year of
blood draw (p = 0.005), sun holiday (Y/N) (p = 0.02), tanning bed use (YZN) (p <
0.0001), and UV hours (p = 0.0002) while residing in Norway during the summer prior
to the blood draw was negatively associated (p = 0.001) (95). Their model with vitamin
D intake, age, BMI, use of dietary supplements, tanning bed use, sun holiday, 'UV-
hours' and summer residency in Norway, explained 25% (R2adj) of the variance in
plasma 250HD (p < 0.0001) (95). They found that 'UV-hours' was a better
measurement of UV exposure than 'hours in daylight last week prior to blood sampling',
'time of year when the blood sample was collected' and 'latitude' (95). They conclude
that at these latitudes, skin synthesis of vitamin D cannot compensate for low vitamin D
intakes during the winter and that eating traditional foods (fish) and traveling south
during the summer could protect against deficiency (95).
Hypponen and Power looked at a nationwide cohort of white British adults at the
age of 45 to evaluate the dietary and lifestyle factors that influence vitamin D (96). The
season of the blood draw was classified as Winter (Dec-Feb), Spring (March-May),
Summer (June-Aug), and Fall (Sept-Nov) (96). Vitamin D intake was assessed using a
food frequency questionnaire with consumption offish classified as weekly, less than
weekly and never, and consumption of margarine (mandatory fortification) as daily,
weekly and less than weekly (96). Fish oil and supplement intake was also assessed
(96). Usual time outdoors per day during daylight hours was assessed for the previous
30
month and reported as 0, <15 mins, 15-30 mins, 30-60 mins, 1-2 hrs, 3-4 hrs and > 4 hrs
(96). Sunscreen and clothing used during sunny weather was categorized as often,
sometimes, rarely and never, however, there was no information on vacations outside of
Great Britain (96). Skin color on the inner arm was reported as light, medium or dark
(96). Time per day spent watching tv or using a computer was categorized as 1-2 hrs, 2-
3 hrs, 3-4 hrs and > 4 hrs (96). Serum 250HD peaked in September and was lowest
from January through April (96). Month of blood sampling was the strongest predictor
of 250HD levels and it explained 21.5% of the variation in 250HD (p < 0.0001) (96).
Time spent outdoors was strongly associated with 250HD during the summer and fall
but there was no apparent association during the winter months (p < 0.0001) (96).
Vitamin D supplements and the consumption of oily fish were associated with 250HD
but consumption of fortified margarine was not strongly associated (96). The use of sun
protection was associated with slightly higher 250HD, suggesting the use partly reflects
levels of sun exposure (96). Hypovitaminosis D was more common in women, in the
north and in the obese (96). Thus, vitamin D deficiency varied by gender, BMI and
latitude with a seasonal effect.
Tangpricha et al. found that in Boston drinking milk (~160 IU) was not
associated with 250HD but taking a multivitamin was (400 IU) (85). Participants that
took a multivitamin had a 30% higher serum 250HD than those who did not (p < 0.01)
(85). Those who took a multivitamin during the winter had higher 250HD levels at the
end of winter (p = 0.0002) (85). Similarly, those who took a multivitamin during the
summer had 23% higher 250HD levels at the end of summer than those who did not
take a multivitamin (85). They also found a weak but positive relationship between time
31
spent outdoors during the summer and 250HD levels (r = 0.20, p = 0.001) (85). Thus,
time spent outside and taking a multivitamin are important contributors to vitamin D
status.
In the Netherlands (52°N), van der Meer et al. in a cross-sectional study assessed
the relative contribution of vitamin D consumption and sun exposure to vitamin D status
in a multiethnic population (93). A general regression model was used to analyze the
determinants of serum 250HD and they found that season and then ethnic group had the
highest regression coefficients (93). They found significant associations between some
of the sun exposure determinants, such as, season, area of uncovered skin and preference
for the sun but not for time spent outdoors (93). They reasoned that the effect of
clothing and skin pigmentation might overrule the effect of time spent outdoors (93). Of
the modifiable determinants, fatty fish and supplements were the greatest contributors to
serum 250HD (93). Also in the Netherlands, van Dam et al. looked at potentially
modifiable determinants of vitamin D status in an older population using a multivariable
model (94). They determined usual food consumption, supplement use and physical
activity using validated questionnaires (94). After multivariate adjustment, including
season, they found that older age, higher educational level and higher body fat was
significantly associated with worse vitamin D status, while more time outdoors, use of
supplements, consumption of fortified margarine, fatty fish and red meat were associated
with better vitamin D status (94). They found that the greater body fatness of women
explained the sex difference in 250HD (94). They concluded that individually
supplements, fatty fish and margarine were not sufficient to achieve adequate vitamin D
32
status in their population and that fortification of widely used foods should be considered
(94).
Barger-Lux and Heaney looked serum 250HD in outdoor workers (n = 26) who
had just completed a summer season of extended outdoor activity and -175 days later
after the winter sun deprivation in Omaha, Nebraska (125). They interviewed each
subject to determine summer sun exposure in terms of locale, length of outdoor work,
usual weekly schedule, sunscreen use and usual clothing worn (125). They used an
adaption of the rule of nines to determine BSA exposed according to each subjects
combination of shirt, pants and hat worn (125). Then they generated a sun exposure
index using hours of sun exposure per week multiplied by BSA exposed (125). They
also measured skin color of sun exposed areas using a cosmetic color chart (125).
Median 250HD decreased from 122 nmol/L in late summer to 74 nmol/L in late winter
with a seasonal difference of 49 nmol/L which was highly significant (p < 0.0001) (125).
The median daily decrease in 250HD was 0.274 nmol/L (125). They examined the
relationship between the three measures of sun exposure and the summer increment in
250HD and found that for hours of sun exposure per week r = 0.39 (p < 0.05), for
fraction of BSA exposed r - 0.66 (p < 0.001) and for the sun index r = 0.67 (p < 0.001)
(125). Limitations include recalled sun exposure data, no consideration of sunny versus
overcast days and no incorporation of skin pigment, age, time of day or time per
exposure (125). They estimated that the participants' sun exposure was equivalent in
250HD production, using their dose-response finding study results (126), to a daily oral
vitamin D3 dose of 2,780 IU (125). They concluded that even intense sun exposure
during the summer may not protect against a winter deficit and that a late summer
33
250HD level of-127 nmol/L is needed to avoid falling below 75 nmol/L by late winter
(125).
1.5 d Obesity Influencing Vitamin D Status
Wortsman et al. found that obese individuals are only able to increase their blood
levels of vitamin D -50% compared to normal weight individuals (127). BMI was
inversely correlated with serum vitamin D3 after irradiation (r = -0.55, p = 0.003) and
with peak serum vitamin D2 after vitamin D2 supplementation (r = -0.56, p - 0.007)
(127). The percent conversion of previtamin D3 to vitamin D3 was similar in the obese
and non-obese, therefore, obesity did not affect the skin production of vitamin D3 but
may have altered its' release from the skin into circulation (127). They claim that there
is decreased bioavailability of vitamin D in the obese because of its' deposition in body
fat (127). They also suggest that higher doses of vitamin D may be needed to correct
deficiency in obese people (127). Thus, vitamin D deficiency is higher in obese
individuals.
1.5 e Skin Pigment and Ethnicity Related to Vitamin D Status
Skin pigment is an important factor because melanin absorbs UV-B radiation and
people with darker skin need more time in the sun to make the same amount of vitamin
D as someone with lighter skin (128). Harris and Dawson-Hughes measured serum
250HD in young black and white women in Boston four times over one year (129).
They found that the black women had significantly lower 250HD levels throughout the
year and a smaller seasonal change between winter and summer (129) demonstrating the
ability of melanin to block dermal vitamin D synthesis.
Kant and Graubard used data from NHANES III and NHANES 1999-2002 to
compare vitamin D intake and vitamin D status in Mexican Americans, non-Hispanic
whites and non-Hispanic Blacks (130). They found that marginal concentrations of
250HD (< 37.5 nmol/L) were more likely among non-Hispanic blacks and Mexican
Americans than non-Hispanic whites (130). Non-Hispanic whites also had the highest
intakes (130). Using a logistic regression model, they found that ethnicity was a strong
independent predictor of dietary vitamin D intake and serum 250HD after accounting
for income and education (130). Not only is skin pigmentation an important factor to
consider but ethnicity is also important because of differences in behaviors and genetics.
1.5 f Sun Exposure and Dietary Vitamin D Contributions to Vitamin D
Status in High Sun Exposure Locations
It has been assumed the vitamin D deficiency was not a problem in all areas of
the United States because of fortification of milk and abundant available sunlight.
However, studies are now showing that even with vitamin D fortification and a sunny
climate, vitamin D deficiency is a problem. In Honolulu, Hawaii, Binkley et al.
recruited participants with self-reported sun exposure of 3 or more hours per day on 5 or
more days per week for at least the 3 preceding months (98). They used a non-validated,
self-administered questionnaire that included questions about ethnicity, sun exposure,

35
sunscreen use and dietary vitamin D intake (98). They measured skin color by
reflectance colorimetry and used a sun exposure index which multiplies usual body
surface area exposed by the reported average sun exposure per week without sunscreen
(98). The lightest skin color (mean L*) was 63.7 and the darkest was 50.5 (98). On
average, they reported being outside 22.4 hr/wk without sunscreen and 28.9 hr/wk total
and had an average sun exposure index of 11.1 (98). They didn't find a correlation
between 250HD and age, lightest or darkest skin color, change in skin color, hours/week
of sun exposure without sunscreen, the sun exposure index, total hours/week of sun
exposure or BMI (98). The lowest quartile of 250HD was compared to the rest of the
group and they had a significantly lower sun exposure index score and change in skin
color (p < 0.05) (98). Limitations include the cross-sectional design and self-report of
sun exposure (98). The study was also conducted at the end of winter in late March
which could reflect the lowest sun exposure (98). However, they found that 51% of their
participants had 250HD levels < 75 nmol/L and argue against the common clinical
advice to allow sun exposure to the hands and face for 15 minutes because this may not
ensure vitamin D adequacy (98).
Jacobs et al. looked at what correlated to 250HD in a sun-replete location,
Arizona, and constructed a model to determine which variables contributed most to
circulating 250HD (99). Dietary data was collected using the Arizona Food Frequency
Questionnaire (AFFQ) which was completed at baseline to capture usual food intake
over the prior 12 months (99). Sun exposure was evaluated using the Arizona Physical
Activity (APAQ) which assesses activity over the past month (99). Indoor activities
were coded "0" and outdoor activities were coded " 1 " then the time spent performing
each activity was multiplied by the sun exposure variable to get an average amount of
sun exposure per week (99). The participants who had serum 250HD levels > 75
nmol/L were more likely to be male, white, have a lower BMI, spend more time in the
sun and consume more energy, calcium and vitamin D than those < 25 nmol/L (99). In
their predictive model, sun exposure and dietary vitamin D had a larger effect on
250HD in whites than among blacks and Hispanics and there was a significant race x
sex x season interaction (p = 0.03) (99). They considered ethnicity in their model but
did not actually measure skin pigment (99). Sun exposure ranged from 0-100 min/wk
with an average of 6.6 min/wk which is lower than what would be expected for Arizona,
however, significant differences were still observed in 250HD relative to sun exposure
(99). There were differences in 250HD by race-ethnicity, by sex and by season (p <
0.05) (99). Despite high sunshine, deficiency was more common in the winter months
(99). Thus, living at lower latitudes does not prevent against deficiency.
Levis et al. looked at the vitamin D status of adults, mainly Hispanic, aged 18-88
residing in Miami, Florida at the end of winter (March) and summer (September) (100).
Sun exposure in the previous week was determined using a questionnaire (100). In the
winter study, sun exposure was categorized as mild (55.2%), moderate (14.2%) and
extensive (30.6%) and diet was determined using a validated FFQ for both calcium and
vitamin D (100). Vitamin supplements were used by 97.6% of the participants and the
average vitamin D intake per day was 326 IU including supplements (100). Serum
250HD was 8.3% higher in those with extensive sun exposure compared to those with
mild sun exposure and 17% higher in those consuming more than 800 IU/d compared to
those consuming 400 IU/d (100). A subset of participants returned for a second visit in
37
September and the seasonal increase was 14.8% in men and 13% in women (100). Thus,
both sun exposure and diet were important at this lower latitude.
1.5 g Conclusion
The correlations between UV-B radiation and blood levels of 250HD suggest
that sun exposure and cutaneous synthesis of vitamin remain very important in the US,
despite the availability of vitamin-D fortified foods and multivitamins.
1.6 Factors Affecting the Synthesis of Vitamin D in the Skin: Environmental
1.6 a The Influence of Season on Vitamin D Synthesis
Vitamin D production in the skin occurs only when the incident UV radiation
exceeds a certain threshold and the number of photons that reach the earth's surface are
dependent on many factors, including the length of the path that the photons have to
travel through the ozone layer known as the solar zenith angle (SZA). The SZA
describes the difference in degrees of the actual position of the sun to the earth compared
to when the sun is directly overhead. For example, during the winter the SZA is larger
and with the sun lower in the sky there is more atmosphere for the UV-B photons to
travel through. The solar spectrum can change by up to two orders of magnitude from
summer to winter with the shorter wavelengths (UVB) also being cut off by changes in
season and latitude (131). At higher latitudes and during the wintertime, the UV-B
38
photons may not even reach the earth to contribute to vitamin D synthesis in the skin.
Therefore, large SZA are a feature of winter, early morning/late afternoon and high
latitude while small SZA are a feature of summer, noon and low latitudes (132). Thus,
the SZA changes with the season, latitude and time of day.
At the beginning of the 20 century, it was recognized that the incidence of
rickets was much higher during the winter and early spring months and less during the
summer and fall months demonstrating a seasonal effect (1). The SZA increases in the
fall and winter so that the amount of solar radiation reaching the earth's surface is
reduced compared to spring and summer when the SZA is smaller (133). Not only is the
solar zenith angle larger in the winter but people wear more clothes and go outside less
because of cold weather. Webb et al. showed that in Boston during the summer months
of June and July, 7-dehyrdocholestrol was most efficiently converted to previtamin D3
using ampoules of human skin (134). They found that there was a gradual decline after
August and essentially no previtamin D3 after November and into the winter months
until mid-March (134). This pronounced annual cycle is driven primarily by the tilt in
the earth's axis, resulting in maximal irradiance in the summer and minimal in the winter
(135).
1.6 b The Influence of Latitude on Vitamin D Synthesis
Webb et al. also conducted similar studies to look at the effect of latitude on
previtamin D3 production in Edmonton, Canada (52°N), Los Angeles, California (34°N),
and San Juan, Puerto Rico (18°N) (134). They found that in Los Angeles and San Juan
39
previtamin D3 synthesis occurred throughout the year but in Edmonton synthesis ceased
by mid-October and did not resume until mid-April (134). The SZA is larger for higher
latitudes and therefore, UV radiation has to travel further. Recently, Engelsen et al. used
a simulation model to study UV irradiances throughout the year and at different latitudes
and found that at 51°N there was no vitamin D production for part of the year and at
70°N there was no production for 5 months (136). The period when adequate sun
exposure is not available and there is no vitamin D production in the skin is termed the
"vitamin D winter" (136). This occurs above 37°N during the months of November-
February with an -80-100% decrease in the number of photons reaching the earth's
surface (108). Thus, there is no "vitamin D winter" at the equator with the highest
irradiances year around and decreasing radiation away from the equator (135).
1.6 c The Influence of Time of Day on Vitamin D Synthesis
Webb et al. also looked at the effect of time of day in Boston by placing the
ampoules of human skin outside every hour from sunrise to sunset in the middle of every
month for one full year (134). During the summer, previtamin D 3 synthesis occurred
from 7am to as late as 5pm (Eastern Standard Time (EST)) but during the winter and fall
synthesis began around 9am and ceased by 4pm (EST) (134). There is maximum
irradiance at noon with 75% of total UV radiation between 9am-3pm and 20-30%
between 1 lam-lpm (135). For instance, when the sun is directly overhead midday, there
is the least amount of UV-B scattering and atmospheric absorption and thus a greater
amount of UV-B radiation reaches the earth. Even in the summer in the early morning
40
and late afternoon little vitamin D is produced in the skin because the sun's rays are
more oblique (137).
1.6 d The Influence of Altitude on Vitamin D Synthesis
UV radiation increases with altitude because of the decreasing amount of
scattering and absorbance that occurs (138). Holick et al. demonstrated that altitude has
a significant effect on vitamin D3 synthesis (139). They placed 7-DCH ampoules and
found that at 27°N in November less than 0.5% previtamin D3 was detected in Agra (169
m) and Katmandu (1400 m), however, there was a 2-4 fold increase in previtamin D3
synthesis at -3,400 m and 5,300 m (Everest base camp), respectively (139). Webb and
Engelsen found that by using their simulation model exposure times to produce one
standard vitamin D dose (SDD) decreased by ~7% per kilometer increase in altitude
(140). The resulting increase in UV radiation is described as the altitude effect (AE)
(138). Pfeifer et al. analyzed the AE due to variations in solar elevation, albedo and
aerosol properties on UV radiation (138). Their model showed that depending on the
solar elevation and albedo the AE ranges between 3-7% per km (138).
1.6 e The Influence of Ozone and Aerosol on Vitamin D Synthesis
The total amount of ozone that solar radiation encounters before reaching the
earth's surface is referred to as the "column ozone" and is expressed in Dubson Units
(DU) (141). The stratospheric ozone layer absorbs solar radiation < 290 nm (133). As
41
the distance that the radiation has to travel through the ozone increases so does the
absorption which attenuates the shortest wavelengths more than the longer ones (142).
There is concern about chlorofluorohydrocarbons depleting this ozone layer and
resulting in a larger number of higher-energy photons reaching the earth (133). On the
other hand, air pollution that contains ozone can be detrimental by efficiently absorbing
the UV-B photons and potentially reducing the cutaneous synthesis of vitamin D. It has
been shown that air pollution can cause urban areas to have 20-50% lower UV-B
readings than nearby rural sites (143). This could lead to increased prevalence of
vitamin D deficiency in polluted areas in the United States and Northern Europe (133).
Even in urban areas, tall buildings that block the sun could likely contribute to poor
vitamin D status in the residents living in those cities. In addition, awnings, umbrellas,
porches, and other structures also decrease individual UV exposure.
There is also scattering of UV radiation by air molecules/particles, otherwise
known as Rayleigh scattering (142). A major part of UV radiation is actually scattered
back to space (142). Aerosols in the atmosphere also attenuate surface UV-B radiation
(136). Pollutants and aerosols tend to be concentrated in the lowest layers of the
atmosphere, therefore, being at a higher altitude will significantly reduce the effect of
aerosols (132).
1.6 f The Influence of Clouds on Vitamin D Synthesis
Clouds in the atmosphere also affect the UV-B exposure that reaches the earth
and thus, human skin. Clouds are also very unpredictable, especially in the mid-latitude
regions characterized by the passage of low pressure systems interspersed with regions
of high pressure (132). Sullivan et al. showed that in vivo previtamin D3 synthesis at
midday was reduced by 50% on a cloudy day compared to a sunny day (144). Using the
FastRT simulation took, Engelsen et al. found that thick clouds could reduce surface
radiation to as much as 1% of clear sky levels and even scattered clouds could reduce
available radiation (136). They found that even at the equator a thick cloud cover could
halt cutaneous vitamin D production (136). Although thick clouds scatter UV radiation
back to space, thin clouds actually scatter UV radiation toward the earth (141).
Therefore, depending on the type of clouds overhead UV radiation can change
instantaneously.
1.6 g The Influence of Reflectance on Vitamin D Synthesis
On the other hand, the albedo or the fraction of radiation striking a surface that is
reflected by that surface, can enhance the UV-B radiation available for vitamin D
production (135,136). Most ground surfaces have albedos of < 10% with water
reflecting < 5% of incident UV radiation (135). However, snow on the ground is very
reflective with an albedo of 30-80% and sand has a albedo of 15-30% (135). Using the
FastRT simulation tool, Engelsen et al. showed that snow cover can change the lower
latitude occurrence of the vitamin D winter by a couple of degrees and can alter the
duration of vitamin D synthesis by about 1-2 weeks (136).
1.7 Factors Affecting the Synthesis of Vitamin D in the Skin: Non-Environmental

43
1.7 a The Effect of Melanin on Vitamin D Synthesis
Melanin, or skin pigment, essentially provides the body with an effective
sunscreen and increased melanin will reduce the efficiency of sun-mediated
photosynthesis of previtamin D3 in the skin (145). In people with lighter skin, 20-30%
of the UV-B radiation is transmitted through the epidermis and absorbed in the stratum
spinosum and basale (65). However, in darker skin the melanin in the epidermis absorbs
UV radiation and less than 5% of the UVB radiation is transmitted (65). Therefore,
production in darker skin is predicted to be 40% of Caucasian skin (146).
Holick et al. first showed that skin with more melanin (darker skin) requires
longer exposure or higher intensity UV radiation to maximize previtamin D3 formation
(147). They showed that given sufficient UV radiation skin pigmentation (melanin) does
not prevent previtamin D3 formation (147). Lo et al. showed that Indian and Pakistani
immigrants increased their serum 250HD levels the same as Caucasians but with more
total irradiation because of their need for more exposure to produce 1 MED (148).
African Americans have lower circulating concentration of 250HD and are more
prone to developing vitamin D deficiency likely because they have more melanin and
darker skin pigment which attenuates vitamin D synthesis in the skin (149). This is
particularly important in African Americans living away from the equator in more
northern latitudes. It is also important to note that a reemergence of rickets in US infants
is virtually restricted to dark-skinned infants exclusively fed human milk (a poor source
ofvitaminD)(150).
44
1.7 b The Influence of Sunscreen on Vitamin D Synthesis
Sunscreens are used because they protect against sunburn, skin cancer and skin
damage that is associated with sunlight exposure. However, the UV-B solar radiation
that is responsible for causing damage to the skin is also responsible for vitamin D
synthesis. Sunscreens absorb the solar UV-B radiation on the surface of the skin before
they can reach the deeper layers of the skin and therefore, diminishes the total number of
UV-B photons that reach the 7-dehydrocholesterol in the epidermis to form previtamin
D3 (151). Studies have looked at application of sunscreen with a sun protection factor
(SPF) of 8 and then whole-body exposure to one minimal erythemal dose (MED) of
simulated sunlight and found that this prevented any significant increase in vitamin D3
(151). For the subjects that didn't apply any sunscreen there was a 10-20 fold increase
in vitamin D3 after exposure to one MED (151). Matsuoka et al. looked at the effect of
chronic sunscreen use on vitamin D status and found that serum 250HD was
significantly lower in participants who always wore sunscreen (114).
However, most people still burn or tan after sunscreen application because they
do not apply the correct amount or apply an uneven distribution on their bodies. The sun
protection factor (SPF) is defined by the FDA based on applying 2 mg/cm of sunscreen
(152) while studies have shown that users apply insufficient amounts and likely only
receive 50% of the SPF written on the package (153). The relationship between SPF and
sunscreen quantity follows an exponential growth and an application of 1 mg/cm or 0.5
mg/cm2 makes the SPF fall as the square or fourth root, respectively (154). When only
0.5 mg/cm is applied for a declared SPF of 4, 8, or 16 would yield an effective SPF on
the skin of only 1.4,1.7, or 2.0, respectively (154). Therefore, an increase in SPF can't
adequately compensate for too thin a layer of sunscreen (154).
Thieden et al. actually found higher UV doses on days that participants wore
sunscreen (p < 0.001) and that sunscreen use was highly correlated with risk behavior (r
= 0.39, p < 0.001) during a prospective study (155). They found that 10% of females
and 40% of males never used sunscreens (155). Sunscreen was not correlated to age and
there was no significant difference between sunscreen use and skin type (155). In their
study the median sun protection factor was 10.5 with the sun-protecting effect
corresponding to an application density of 0.5 mg/cm2 (155). They concluded that
sunscreens were used as tanning aids to avoid sunburn (155). In comparison to
retrospective studies, people tend to overestimate their sunscreen use especially in with
regard to children likely because sunscreen use is thought to demonstrate good parental
care (155). Nonetheless, for efficient cutaneous vitamin D synthesis, unprotected skin
must be exposed to the sunlight (132).
1.7 c The Influence of Clothing, Glass and Plastics on Vitamin D Synthesis
Clothing absorbs UV-B radiation and prevents the cutaneous production of
vitamin D3 in the covered areas (156). Matsuoka et al. found that cotton, wool and
polyester in black and white colors prevented the formation of vitamin D3 after
photostimulation with up to 6 MEDS of UV-B radiation (156). They also looked at
subjects who wore no clothing, summer-type clothing, fall-type clothing, or summer-

46
type clothing with sunscreen 24 hours after a whole-body exposure to one MED (27
mJ/cm ) and found the lowest circulating vitamin D3 levels in the subjects with summer-
type clothing and sunscreen (156). The highest vitamin D3 levels were in the subjects
wearing no clothing, then summer-type clothing and then fall-type clothing
demonstrating the at the more body surface area exposed to the sun, the more vitamin D 3
that will be made (156). A study by Sedrani et al. found that in Saudi Arabia where the
culture requires covering almost the entire body with clothing, that even despite intense
sunlight exposure, the Bedouin women had an increased risk of vitamin D deficiency
and osteomalacia (101). They also found that their children were more prone to
developing rickets (101). The skin area exposed to sunlight is highly variable depending
on what someone is wearing and therefore, any association between UV dose and
vitamin D status must be qualified by the amount of surface area exposed (132).
Holick et al. found the prevention of provitamin D3 formation with the protection
from glass, plexiglass, or plastic placed between a simulated sunlight source and
participants versus no shield (133). It has been found that only UV-A, not UV-B
radiation, can pass through the glass in car windows and building windows (157).
1.7 d The Effect of Aging on Vitamin D Synthesis
Aging significantly decreases the amount of 7-dehydrocholesterol (7-DHC) in
the epidermis and thus, the ability to produce vitamin D3 (158). There is a linear
decrease in 7-DHC concentration in the skin with age and an 80 year old has
approximately half the concentration as a 20 year old (158). Healthy young adults and
elderly adults were exposed to the same amount of a whole-body dose of UV radiation,
however, within 24 hours the young adults had increased their serum 250HD to a
maximum of 78.1 nmol/L while the older adults (62-80 years old) only reached a
maximum level of 20.8 nmol/L (159). Even skin samples from older subjects exposed
to solar light had only half the amount of previtamin D3 as the younger skin samples
(158).
Chronic sunscreen use in the elderly adds to their risk of vitamin D deficiency
(151). Elderly people are also more likely to avoid the sun and wear clothing that covers
more of their bodies which also contributes to their risk of deficiency (110). Elderly
people are more likely to be lactose-intolerant and therefore, avoid vitamin D fortified
milk (133). Elderly people are less likely to take a multivitamin on top of all the other
possible medications that they have to take (133).
A study by Webb et al. showed that 56% nursing home residents were deficient
by the end of summer and 83% were deficient by the middle of winter (110). Elderly
people that are home-bound or institution-bound are at risk for deficiency because their
diets are low in vitamin D and they do not go outside enough to produce vitamin D in
their skin (110). This compounded with lower 7-dehydrocholesterol in their skin puts
them at a higher risk for vitamin D deficiency than the general public.
1.8 How Much Sun Exposure Do We Need?
Casual exposure to sunlight provides most humans with their vitamin D
requirement (133). However, the question of how much sunlight is needed by

48
individuals is complicated by the latitude of where someone lives, season, time of day
and personal factors such as age, skin pigmentation, and sun behavior (clothing and
sunscreen use). Matsuoka et al. first demonstrated in vivo that a UV-B threshold of 18
mJ/cm2 (180 J/m2) is required for vitamin D3 formation and release into circulation
(160). At most central European latitudes it is thought that a very short and unprotected
exposure to solar radiation is needed to achieve adequate vitamin D3 levels, which would
be approximately 7 minutes for someone with skin-type 2 (161). An exposure to solar
radiation of 1 MED of the whole body in a bathing suit is equivalent to ingesting 10,000-
20,000 IU of vitamin D3 (162). Assuming 10,000 IU of vitamin D is produced from full
body exposure to 1 MED, then exposure to 0.25 MED would produce 2,500 IU and with
only 40% of the body exposed to this, 1,000 IU would be produced (163). Holick et al.
reported that an exposure of less than 18% of the body surface, which includes the face,
arms and hands, 2 to 3 times a week to 1/3 to 1/2 MED in the spring, summer or fall is
sufficient for adequate vitamin D3 synthesis (137). For someone in the southern US with
light skin, the exposure time for 1 MED in the summer noonday sun is ~4-10 minutes
but for someone with dark skin the corresponding time is -60-80 minutes (108). An
easy way to think about moderate sun exposure is, for instance, if an elderly person with
light skin goes outside in the afternoon in July and burns after 30 minutes then 5-10
minutes of sun exposure would be adequate (133). On the other hand, caution is
recommended even for short amounts of time because UV-B radiation is a trigger for
nonmelonoma skin cancer (164). At the same time, it has been postulated that cancer
mortality could be reduced by moderate unprotected UV exposure (161). A simple

solution may be that after a 5-15 minute unprotected exposure then sunscreen could be
applied if the person is going to stay outside longer.
1.8 a Risk and Benefit Considerations
The UV action spectrum for vitamin D photosynthesis and for DNA damage
leading to skin cancer are so similar that the harmful and beneficial effects to sun
exposure are hard to separate (165). Wolpowitz and Gilchrest note that cutaneous
vitamin D synthesis is capped after prolonged sun exposure but that DNA damage
continues linearly with increased sun exposure (165). They argue that dietary
supplementation is effective in preventing vitamin D deficiency and that dietary vitamin
D is a completely interchangeable precursor to the active hormone as sun derived
vitamin D is (165). They obviously feel that the risks outweigh the benefits in terms of
sun exposure.
On the other hand, Webb and Engelsen wanted to calculate exposure levels for a
healthy vitamin D status using the FastRT UV simulation tool (166) which is a web-
based tool that enables the user to calculate associated exposure times for any time and
place using either default of selected conditions (140). They wanted to answer the
question, "What is moderate sun exposure?" using a standard vitamin D dose (SDD)
corresponding to the UV equivalent of 1,000 IU of dietary intake (167). Looking at
exposure times needed to gain 1 SDD as a function of latitude, season, time and skin
type, they found that there was little change in exposure times in the hours around noon
50
but approximately double exposure times in the early morning (9 am) and late afternoon
(3 pm) (140).
Webb and Engelsen also calculated how much sun exposure is needed to gain a
number of proposed oral-equivalent doses of vitamin D using minimum erythema doses
(MED) (142). They have taken into account latitude, season, skin type and skin area
exposed with the risk of erythema (skin reddening) (142). UV radiation is a known
carcinogen and exposure increases risk of developing skin cancer (142). Because of this
risk, current public health policies advise against unprotected sun exposure especially
during the middle of the day (142). However, they recognize that sun exposure is
important for the production of vitamin D.
The vitamin D action spectrum is in the UV-B range of 280-315 nm while the
erythema action spectrum (EAS) includes both UV-B and UV-A radiation (315-400 nm)
and each has a biologically effective dose (BED) rate (142). Vitamin D synthesis occurs
on all exposed skin and increasing percent body surface area exposed can increase
vitamin D status (142). Erythema is experienced when UV exposure is greater than or
equal to a personal MED in a single exposure or multiple close exposures (142). The
MED dose that produces skin reddening is individual-dependent and it is broadly related
to skin pigmentation (142). Prolonged sun exposure is of no benefit to produce more
vitamin D, therefore the optimal exposure for maintaining adequate vitamin D status
may be "little and often" (142). This would be a sub-erythemal dose of sufficient UV-B
exposure every day or two and the same recommendation would also be suitable for
avoiding erythema (142).

51
There is spectral dependence on the SZA which means that with a smaller SZA
the proportion of UV-B in the total UV waveband is greater (142). The action spectrum
for vitamin D synthesis and for erythema differ with the ratio between the two biological
doses changing as the SZA changes (142). For example, there is more vitamin D
effective radiation per dose of erythemally effective radiation at smaller SZAs. Thus,
the most efficient time to synthesis vitamin D is around noon on any given day and in
summer months (ie. small SZA) (142).
Another thing to consider is that vitamin D synthesis is increased with more body
surface area (BSA) exposed while surface area does not determine the severity of
erythema (142). Therefore, vitamin D status can be improved by exposing more BSA
for a short period of time rather than a small area for a long amount of time. It has been
estimated using the rule of nines for burn victims that exposure to the face is 3.5% BSA,
exposure to the face, neck and hands is 11.5% BSA and exposure to the face, neck, arms,
hands and legs is 57.5% BSA (168).
Webb and Engelsen calculated the default exposure as a flat surface at sea level
and a cloudless sky which gives the maximum available radiation (142). They also had
to consider realistic exposure times and decided that one hour was the maximum period
for a realistic daily exposure (142). Then they again used the FastRT UV simulation
tool (166) to calculate the UV radiation needed to correspond to the UV equivalent of an
oral vitamin D dose (142). This standard vitamin D dose (SDD) was based on V* MED
exposure to XA skin area (face, hands, arms) being equivalent to 1,000 IU of oral vitamin
D (167). They came up with different scenarios for different vitamin D intakes needed,
different skin types and different amounts of BSA exposed. They determined that
52
doubling the dietary vitamin D requirement would require twice the exposure, that a skin
type requiring twice the UV radiation to burn would require twice the exposure, and
exposing twice the skin area would half the exposure time (142). They concluded that a
recommendation of 400 IU/day is achievable through sun exposure, without risk of
erythema, for all or part of the year (142). At 1,000 IU/day sun exposure of less than
one hour can still serve as a single vitamin D source at low latitudes and middle-high
latitudes with sufficient BSA exposed for all or part of the year (142). Of course,
increases in latitude and skin pigment make the recommendations harder to achieve
without supplementation.
Assumptions were made in the model (ie. linear relationship of BSA exposed to
250HD produced, average skin types) and real life cases would likely need higher
exposure times than produced by the model (ie. horizontal surface). The authors note
that the recommendations are not precise but they do allow for comparison to existing
recommendations (142).
As stated, UV radiation promotes biopositive effects, such as vitamin D3
synthesis, and bionegative effects, such as the development of skin cancer and skin aging
(164). Moan et al. also looked at the risks and benefits involved to obtain the optimal
amount of vitamin D from sun exposure with the least amount of risk of getting
cutaneous malignant melanoma (CMM) (169). They suggested that the recommendation
to avoid sun exposure 3-5 hours around noon may be inappropriate because the action
spectrum for CMM is likely centered around longer wavelengths (UV-A) than the
vitamin D spectrum and that high UV-A fluence rates last twice as long after noon than
53
high UV-B fluence rates (169). They concluded that short exposures around noon would
be better than longer exposures after noon for synthesizing vitamin D (169).
Reichrath also debated the potential positive and negative effects of sunlight in
relation to skin cancer (170). He speculated that public health campaigns did not
consider the positive effects of sun exposure since they generally proposed a strict "no
sun policy" (170). However, Reichrath noted that the need for sun protection may vary
between individuals and that the skin types (darker skin) most resistant to the harmful
effects of UV radiation are also the ones most associated with diseases of vitamin D
deficiency (170). Even though melanoma skin cancer is associated with sun exposure,
particularly burning in childhood, various other cancers (colon, prostate, breast) are
associated with inadequate sun exposure (170). Recently, however, Brewick et al. found
that sun exposure increased survival from melanoma reinforcing the fact that there are
many sides to consider for positive and negative effects (171). Reichrath stresses that
strict sun protection campaigns may induce the severe health risks such as vitamin D
deficiency and should be reevaluated (170).
On July 15,2004 there was an expert meeting hosted by the Cancer Council
Victoria (Australia) and the National Cancer Control Initiative to find a common
understanding between the risks and benefits of sun exposure (172). They wanted to
ensure consistency in messages delivered from the media to the public on the vitamin D
issue (172). The position statement from the meeting included an agreement that sun
protection messages needed to shift away from the notion that people have to protect
themselves against the sun at all times, that sun protection should only be applicable
when the UV index is > 3, and that the balance between harm and benefit needs to be
considered with sun exposure (172). Sinclair stressed that people should be encouraged
to be more physically active outdoors during the wintertime to increase their vitamin D
levels (172). He also argued that with refinements to the sun protection messages, they
can be complementary to the vitamin D message (172). Lucas et al. also argues that sun
protection messages may need to be tailored to different countries, in recognition of the
importance of skin pigment, cultural and behavioral sun-seeking or avoiding practices,
and the diversity in both the susceptibility to adverse and beneficial effects of UV
radiation exposure (173).
1.9 Assessing Sun Exposure
1.9 a Overview
First, it is important to understand what defines solar ultraviolet (UV) radiation.
UV radiation is actually only a small component of a broad electromagnetic spectrum
which is defined by wavelength and frequency (135). Actual UV radiation refers to a
narrow waveband of 100-400 nm while visible light is in the 400-700 nm region (135).
UV radiation is electromagnetic radiation that travels, quantized as photons, in waves
and is measured by wavelength and frequency with shorter wavelengths having a higher
frequency and energy (135). UV radiation is further broken down into 3 spectral regions
based on biological effect: UV-A (320-400), UV-B (290-320) and UV-C (200-290)
(135). Energy can be defined in joules (J) and a radiant exposure in joules per square
meter (J/m2) (135).

55
The UV dose required to elicit an acute sunburn (skin reddening) is known as the
minimum erythemal dose (MED) and varies individually as a function of epidermal
melanin fraction volume (based on ethnicity and tanning ability) and stratum corneum
thickness (135). The biologically effective dose (BED) includes the MED and an action
spectrum as a weighting factor for the given effect (135). Attempts have been made to
measure an action spectrum for erythema using various doses at different wavelengths as
end points and they have been combined to generate a reference erythema action
spectrum (EAS) which has been adopted by La Commission de l'Eclairage (CIE) (135).
A standard erythema dose (SED) was proposed to overcome the individual variability of
the MED and represents a fixed dose of 100 J/m and is weighted by the CIE EAS and
the spectral power distribution of the source (135). A standard vitamin D dose (SDD)
was also devised based on a VA MED exposure to VA skin area (face, hands, arms) being
equivalent to 1,000 IU of oral vitamin D (167).
Assessing the quality and quantity of UV radiation is essential for understanding
the biological effects of sunlight in the skin and should not be ignored in studies that
assess vitamin D status. Sun exposure can be estimated using geographic estimates
and/or personal estimates. Sun exposure is difficult to quantify because there are many
factors that affect sunlight on any given day, these include latitude, day of the year
(season) and time of day. On top of a somewhat predictable seasonal cycle there are
other components in the atmosphere, such as clouds, aerosols and ozone that can change
unpredictably and also affect incident radiation. Personal factors also affect sun
exposure such as skin pigment, clothing worn and sunscreen used (protective behaviors).
All of these factors must be considered when trying to assessment sun exposure on the
56
individual level. From time spent outdoors a personal ambient exposure can be
calculated as the fraction of the total available UV radiation that a person gets relative to
a horizontal plane and then converted into a personal UV dose using terrestrial UV doses
for that time frame (174).
A recent review of the peer-reviewed literature in relation to sunlight exposure
assessment and the validity of using sunlight exposure questionnaires to quantify vitamin
D status concluded that questionnaires currently provide imprecise estimates of vitamin
D status (175). Individual-level data is needed for understanding sun exposure because
personal sun protection and behavior has a greater influence on personal UV exposure
than do ambient UV levels (175). Thus, results differ from studies that include personal-
level data with those that include only environmental-level data (175). Environmental
estimates have used latitude of residence and ambient UV exposure while personal
estimates have used personal UV exposure and estimates about the anatomical
distribution of the UV exposure (175). Personal ambient exposures can be calculated by
knowing the amount of time spent outdoors with respect to specific time of the year and
day, use of sun protection such as sunscreen and clothes, position to the sun (anatomical
distribution of UV exposure) and the available terrestrial UV (175). Both environmental
and personal influences need to be considered.
1.9 b In Vitro Models of Vitamin D3 Synthesis and Human Skin Samples
One in vitro model of vitamin D3 synthesis involves an ethanol solution of 7-
dehyrdocholestrol (7-DHC) and subsequent exposure of UV radiation (176). Using this

57
"D-dosimeter" model, UV exposure causes a multicomponent photoisomer mixture to be
formed from the original 7-DHC (176). The UV absorption spectra of the solution are
recorded by a spectrophotometer before and after UV exposure and the photoisomer
concentrations are derived by a computer program (176). The 'antirachitic' dose is then
determined by measuring the previtamin D3 accumulation at a specific exposure time
(176). The Belgian Institute for Space Aeronomy has preformed radiometric
characterization to link physical and erythemal UV doses with concentrations of
accumulated previtamin D3 (177).
Another model uses a photoisomerization of 7-DHC in a nematic liquid
crystalline (LC) matrix and visual estimation of accumulated previtamin D3 in the LC
cell (176). Upon UV radiation the Cano-Grandjean stripes number (Nc) decreases as a
result of provitamin D3 phototransformations and the Nc is well representative of the
accumulated dose of biologically active "antirachitic" UV radiation (176). Terenetskaya
showed that this model can be used for personal UV biodosimetry and to evaluate UV
exposure in realistic conditions such as changing orientation and surface position (176).
Actual human skin samples have been used to determine vitamin D3 synthesis at
different latitudes and for different seasons and different times of the day (134). Chen et
al. used human skin samples from Caucasian and Black participants to determine the
effect of skin pigmentation of vitamin D3 synthesis (178). The samples (1 cm2) were
placed on gauzes moistened with saline in quartz petri dishes and exposed to noon
sunlight (178). Then they were analyzed for vitamin D3 formation using a high
performance liquid chromatography (HPLC) analysis (10).

58
1.9 c Sun Exposure Questionnaires
Sun Exposure Questionnaires have been developed but many have not been
validated yet. Lips et al. developed a questionnaire using two categories of time
outdoors (little or frequent) and two categories of reported sun exposure level (low or
high) to get a sunshine score of low, intermediate or high to categorize people (179).
Salamone et al. used this categorization with another questionnaire that asked about time
spent outside and sunscreen use over the past week (122). They used this to categorize
the sun exposure levels of healthy elderly women from one to nine and found a
significant difference between summer and winter rankings (122). Lawson et al. used a
14-point scale assigned to the number of times a week that the subject went shopping,
visiting, gardening or on holiday (124). The scores were used to assess the relative
variation in potential UV exposure not an actual amount of time spent outdoors (124).
They found that the outdoor score and 250HD was significantly correlated in August-
September (r = 0.62, p < 0.01) (124).
Brot et al. used never, occasionally and regularly for categorizing sun exposure
and in a multiple linear regression model they found that sun exposure (never = 0,
occasionally/regularly = 1) was a significant predictor of 250HD (92). In the whole
population sun exposure (occasionally/regularly) was associated with 27.6% higher
serum 250HD (92). Sowers et al. used a sun exposure score to rank women based on a
self-reported sunlight of time outside adjusted for percent body surface area exposed
considering sunscreen but not time of day and found that it was correlated to 250HD (r
= 0.26) (118). Barger-Lux and Heaney used a sun exposure index (SEI) by multiplying
59
usual body surface area exposed (using the rule of nines) by the reported average sun
exposure per week without sunscreen (125). Binkley et al. used this sun exposure index
but didn't find a correlation with 250HD, even though the lowest quartile of 250HD
had a lower index than the rest of the cohort (p < 0.05) (98). Similarly, Holick et al. also
used this sun exposure index and found a non-significant relative risk (RR) of
insufficiency (< 75 nmol/L) of 1.10 for zero sun exposure and a RR of 1.04 for 0-8
hrs/wk in postmenopausal North American women (180).
Atli et al. used a questionnaire to collect information about clothing and level of
sunlight exposure times called the "benefiting from ultraviolet index (BFUI)" (90). The
BFUI represents the degree of sunlight exposure calculated as the ratio of sunlight
exposure to clothing using points (90). For instance for sun exposure, 1 point was given
for not being exposed directly to the sun and 4 points was given for being exposed to the
sun all day long (90). For clothing, 1 point was given for the least amount of clothing
and 4 points for being covered up (90). The BFUI = points for sun exposure / points for
sunshine prevention capacity of the outfit (90). They found a positive correlation
between 250HD and BFUI (r = 0.34, p < 0.001) (90). They concluded that simple
questions about sun exposure and clothing habits can identity elderly at risk of vitamin D
insufficiency (90).
The reproducibility of sun exposure questionnaires has been tested in several
studies, from 6 months to 5 years after the initial questionnaire was given (181,182).
Reproducibility studies showed similar results in which outdoor exposure that was
occupational was more reproducible than recreational exposure, sunburns were
moderately reproducible, and residential history was most reproducible (181,182).

In Australia, a case-control study of multiple sclerosis found highly significant
correlations between various measures of self-reported sun exposure and serum 250HD
concentrations with the largest correlation (r = 0.39) for activities outside in the past 3
years (183). However, > 85% of the variation in 250HD was not explained by self-
reported sun exposure (183).
Both observations and personal UV dosimetry have been used to validate self-
reported sunlight exposure questionnaires (175). However, relatively low correlations
have been found between questionnaires and dosimeter measurements (175).
1.9 d Polysulphone (PS) Badges and Validations against Questionnaires
A more objective way to measure sun exposure is with polysulphone (PS) film
which is a photosensitive polymer film that is used to measure broadband UV-B
exposure and is sensitive to the same wavelengths that affect human skin (184). PS film
has a response to UV radiation that peaks at 300 nm and drops to 1% at 330 nm. UV
radiation causes deterioration of the film and increases the films absorption of the UV
rays. The change in optical absorbance is analyzed through the measurement of
transmission at 330 nm in a spectrophotometer, where the change in absorbance is
proportional to the erythemal UV exposure. There is a 10% error rate associated with
the PS film (185). The PS film is calibrated to MED in which one MED = 250 J/m2 of
erythemal action spectrum (EAS)-weighted UV energy (1 MED causes slight pinkness
to the skin). To obtain personal ambient exposures, a badge is left in direct sunlight all
day to get the total available UV exposure and then the UV exposure that the person gets
61
is expressed as a fraction of the total (174). This personal ambient exposure can be
converted into a personal dose by multiplying by the available terrestrial UV dose in
J/m2 or MED (200-250 J/m2 for skin type II) or SED (100 J/m2) (174).
Webb et al. monitored an elderly Caucasian population in a long-term-care
facility for a year and used PS badges to determine their UV exposure (110). They wore
the badges on their lapel everyday for a week and then a new badge was issued (110).
The weekly UVB doses from each badge were used to calculate a weekly average
representative of each month of the year for every resident (110). Blood was drawn on
the 15th of each month (110). They looked at the PS badge measurements in 3 groups of
elderly participants with differing levels of independence and found that UV exposure
increased in residents who were mobile and independent and they showed the most
seasonal variation (110). The PS badges helped to distinguish between the sun exposure
received by the groups.
Correlations between exposure diaries or observers and PS badge estimates have
been good depending on the activity. Herlihy et al. looked at personal UV exposures in
Hobart, Tasmania for six different outdoor activities using PS badges attached to seven
anatomical sites over two consecutive days (186). At the same time, environmental and
behavioral data were collected. The measurements from the film badges were compared
to personal diaries and ambient UV-B levels from a monitoring station (186). The
horizontal badge which measured ambient exposure was closely correlated to the
monitoring station measurement (slope 1.08 ± 0.10) (186). Correlations between the
badge erythemal effective dose (EED) and the EED from the diary times with the
monitoring station measurements were good for activities in the open and direct sun and
62
less correlated for partially shaded activities (lower badge values) (186). Thus, they
found good correlations between the PS badges, ambient UV measurementsfromthe
monitoring station and personal diaries (186). They concluded that the high correlations
seen in this study compared to other studies was because of the close supervision by the
research staff and immediate entry of sun exposure into the sun diaries upon completion
of the activities (186).
Chodick et al. assessed how well a 7-day record of time spent outdoors compared
to PS badges worn each of the seven days as an objective measure of ultraviolet
radiation (UVR) (187). In a linear regression model, time spent outdoors was associated
with an increase of 8.2% in the badge exposure with every hour spent outdoors (187).
They showed that there was a significant correlation between records with personal
UVR measurements. Chodick et al. also compared the 7-day records to recalls mailed
out six months later which asked the participants to recall the same 7 days (188). They
found that agreement was significantly higher for reported time outdoors during
weekdays than for weekends (p < 0.05).
Sullivan et al. had young girls keep track of their outdoor activities greater than 5
minutes for one day and wear a PS badge on the same day (144). Their questionnaire
assessment was novel because they took into account the changing strength of UVB
radiation throughout the day by using average UVB numbers each hour (7am-5pm) from
the USDA UV-B Monitoring Program in Presque Isle, Maine (144). In vivo
measurements were also taken to estimate the amount of vitamin D3 synthesis associated
with a given sun exposure (144). The participants also wore PS badges fastened to their
front upper left-hand corner of their shoulders. They showed a correlation between PS
63
badges and self-reported minutes outdoors adjusted for the time of day (r = 0.64) in
adolescents (144). They also showed that UV-B levels were strongly associated with
percent vitamin D3 production in vivo (r = 0.96) and PS badge readings (r = 0.96) (144).
Dwyer et al. compared estimates of erythemally effective doses (EED) obtained
from PS badges worn on 4 consecutive weekend days in late spring to several questions
on habitual sun exposure in 14-15 year olds (189). The Pearson correlation coefficients
for the association with EED were 0.36 for girls and 0.23 for boys for the question
"During weekends and school holidays, how much time do you spend in the sun each
day" (189). They concluded that habitual sun exposure in teens was reported with an
acceptable degree of reliability and validity (189).
In a study in Australia, O'Riordan et al. compared UV exposures from diaries to
dosimeter readings (190). They found statistically significant yet modest correlations for
mothers (r = 0.32) and children (r = 0.34) (190). Therefore, sun exposure diaries are
useful tools to measure UV-B exposure.
1.9 e Electronic Wrist Watch Dosimeters
The wrist watch dosimeter, "SunSaver", contains a sensor and a data logger
housed together with a digital watch (191). The sensor is a silicon carbide photodiode
which is sensitive in the 200-400 nm range and has a built in diffuser and has a cosine
response (191). The spectral response of the sensor is similar to the CIE erythema
spectrum. The data logger controls the sensor which measure UV exposure every 8
seconds and stores an average of the last 75 measurements every 10 minutes along with
the corresponding time (192). The watch is battery driven, can run for 145 days without
maintenance, and the data can be transferred to a computer (192). Subjects are suppose
to wear the watch uncovered on the dorsal aspect of their wrist or place it on a towel,
sensor side up, if they are going swimming (192). Thieden et al. looked at the
compliance and data reliability with the SunSaver watches and sun diaries (193). They
found that the watches were worn less during the weekends than on workdays and more
in the beginning of each season (193). Children had significantly lower SunSaver record
rates than adults (p < 0.046) and males had significantly lower SunSaver record rates
than females (p = 0.02) (193). The compliance rate within the subjects' control was
85.5%, with incomplete logs or non-worn SunSavers in 13.6% of the days and lost
SunSavers in 0.9% of the days (193). However, there were SunSaver malfunctions in
10.7% of the days lost (193). Comparing the SunSaver measurements to the diaries
there was a 3.3% error rate associated with the diaries (193). Overall, compliance to the
SunSavers was good and the SunSavers are a useful tool for measuring sun exposure.
1.9 f Ground-Based/On Ground UV Measurements
There are different ground-based UV monitoring stations around the world. The
USDA UV-B Monitoring Station Network has 34 climatologic sites around the United
States and uses instruments, such as the UV-B Pyranometer which measures global
irradiance on a continual basis in the UV-B spectral range of 280-330 nm (194). The
UV-B radiation is converted to visible light and this signal is measured in W/m2 every
15 seconds and given in 3 minute averages (194). Measurements can be downloaded

65
from their website (194). Grant and Slusser looked at spatial variability between 12
paired locations in the network over two summers (May-Aug. 2000 and 2001) (195).
They found that the daily variability between sites was too large to interpolate between
sites and that more accurate interpolations of UV-B exposure would require either the
incorporation of cloud cover from satellite imagery or the use of longer periods of
accumulated exposures (195). However, other studies have shown that these
measurements have provided the ability to accurately interpolate solar radiation over
distances less than 100 km (196, 197). They found a 0.7-0.8 linear correlation between
locations for 100 km spacing distance (195).
The National Cancer Institute (NCI) and the National Oceanic and Atmospheric
Administration (NOAA) had Robertson-Berger (R-B) Spectrometers placed in areas
with cancer registries (198). NOAA has six stations in the western, central and eastern
United States with Brewer meters in place since late 2006 (198). Their measurements
also provide useful data for comparing vitamin D producing UV radiation at different
sites in the US with different climatology. They also have a website that can be accessed
(199).
In Europe, the European Light Dosimeter Network monitors UV radiation and
photosynthetically active radiation using R-B Spectrometers (200). The network has
around 33 stations with 40 broadband instruments coving all of Europe and some other
location including Argentina, Brazil, Egypt, India, Israel, Japan and New Zealand (200).
Spain has their own network of 16 stations with broadband radiometers and some
spectroradiometers for measuring ozone (201).

The Australian Radiation Laboratory set up a network of modified R-B
Spectrometers in the early 1980s to monitor solar UV radiation at 16 sites in Australia
and four in Antarctica (202).
The National Aeronautics and Space Administration (NASA) has a Total Ozone
Mapping Spectrometer (TOMS) Satellite with data for on-ground estimates (198). The
data provides superior coverage but cannot take into account the cofactors that alter the
ground-based measurements (198). Slusser et al. compared TOMS estimates to ground-
based measurements and found agreement within 12% overall and within 8% for clear
skies (203). There is also a TOMS website to download data from (204). Thus, there
are different UV monitoring stations around the world that monitor UV exposure and
can provide useful environmental UV measurements.
1.9 g The UV Index
The UV Index (UVI) is equal to the EAS-weighted irradiance in W/m2 x 40
(205). In the US, the EPA has categorized the Index values as minimal (0-2), low (3-4),
moderate (5-6), high (7-9) and very high (>10) (205). An Index of 10 is equivalent to
0.25 W/m2 (205). The UVI is used as guide for people as when to avoid going outside
because the index is high. The index is even available on most weather channels.
Olds and Kimlin demonstrate the need for a "Vitamin D Index" by contrasting
how the availability of Vitamin D UV and Erythemal UV vary over one year through
computer simulation (206). The action spectra for erythema and vitamin D synthesis are
noticeably different with vitamin D synthesis confined to the UV-B part and erythema
67
extending into the UV-A band (206). To compare the availability of vitamin D UV to
erythemal UV, the ratios of their daily energy totals were calculated and plotted for each
day of the year (206). During the winter at larger SZAs there is greater attenuation of
vitamin D UV then erythemal UV by ozone and therefore, vitamin D UV reduces faster
than erythemal UV with increasing SZAs (206). They also plotted the ratio of the 15th of
each month against the maximum SZA on that day to serve as an average for that month
and observed the same changes, with regard to changing SZAs, affecting vitamin D UV
more strongly (206). They suggest the need for a separate "Vitamin D Index" that is
analogous to the current UV Index but would provide information on what might be
considered appropriate UV exposure for vitamin D production (206).
1.9 h Web-Based Tools
Using the FastRT web-based tool, Engelsen et al. looked at UV simulations
worldwide and throughout the year to find the daily time period when UV radiation
exceeds the required threshold (136). This FastRT simulation allows for changes in
various atmospheric and surface conditions to compute the duration of cutaneous
vitamin D production at different latitudes and seasons worldwide (136). Of course,
there are limitations to the tool and real exposure times needed likely differ somewhat
from the calculated ones. In fact, the effect of clothes and skin are ignored but it is still a
useful tool to determine ambient exposures.
1.9 i Simulated Solar Radiation

68
Many studies have used simulated sunlight to examine the relationship between
UV-B exposure and serum 250HD. Armas et al. exposed participants to UV-B light
(20-80 mJ/cm2) on 90% of their BSA 3 times a week for 4 weeks to various skin types
and measured 250HD weekly (207). Lighter skinned individuals received either a
constant 20 or 40 mJ/cm2 and darker skinned individuals received either a constant 50,
60 or 80 mJ/cm2 (207). They used a multiple linear regression model to then estimate
the dose rate needed to achieve a desired increase in serum 250HD (207). Northern
Europeans (L*= ~70) would need 39 mJ/cm2 of UV-B, sub-Saharan Africans (L*= -35)
would need 78 mJ/cm2 of UV-B, and African Americans (L*= -50) would need 55
mJ/cm2 of UV-B 3 times a week for 4 weeks to increase 250HD by 30 nmol/L (207).
Adams et al. exposed healthy young Caucasian volunteers to a various doses of
whole-body simulated solar UV radiation (208). Circulating concentrations of vitamin
D increased 5-10 fold within the first 24-48 hours of exposure to one MED and declined
towards baseline by day 7 (208). Increasing the MED of exposure resulted in
proportionally higher vitamin D concentrations at 24 hours (208). They estimated that
the half-life of vitamin D3 is about 48 hours and that at least 30 ug (1,200 IU) is
synthesized in the skin and released into circulation for every square meter of body
surface area exposed to one MED of radiation (208). After whole-body exposure to 3
MED, 250HD increased by 30-50% over one week while l,25(OH)2D did not increase
above baseline (208).
Matsuoka et al. found that at least 18 mJ/cm2 (ie. 180 J/m2) of simulated light
was needed to create a significant increase in vitamin D3 in healthy white participants

69
with a skin type III after testing whole-body graded doses of 3-10 mJ/cm (160). They
also found that each incremental increase in UV-B light proportionally increased vitamin
D 3 (160).
Chen et al. recruited participants with different skin types II, III, IV, and V
(lightest to darkest, respectively) and exposed their whole bodies to simulated sunlight in
a tanning bed (178). Each session they were exposed to 0.75 MED and to achieve that
goal, each skin type II, III, IV and V respectively, received an average of 6, 8,11 and 12
minutes of UV irradiation (178). The sessions were held 3 times a week for 12 weeks
(178). The percent increase at the end of the study was 210 ± 53% (skin type II), 187 ±
64% (skin type III), 125 ± 55% (skin type IV) and 40% (skin type V) (178).
It has been determined that exposure of a healthy adult in a bathing suit to one
MED of sunlight or simulated light is equivalent to taking 10,000-20,000 IU of vitamin
D (209). Holick et al. exposed healthy young adults in bathing suits to -0.75 MED (~28
mJ/cm2 for skin type 2 and -32 mJ/cm2 for skin type 3) three times a week for seven
weeks in a tanning bed (5% UV-B) (139). After one week, there was a 50% increase in
250HD which plateaued after 5 weeks at 150% of baseline and was sustained out to
week 7 (139). They also concluded that 15 min (-30 mJ/cm2) in a tanning bed was
similar to one hour of sun exposure in June (Boston) in terms of previtamin D3
production (139).
Marts, however, cautions that a filtered lamp can never replicate ground-level
solar UV radiation and this should be considered especially when looking at biological
endpoints (135).
1.10 UV Doses Measured
1.10 a UV Doses in the United States
Godar assessed outdoor erythemally-weighted effective UV doses (174). UV
radiation was measured in W/m2 and weighted for the CIE erythemal action spectrum
(174). Once weighted, the area under the curve was integrated to get the total
contribution toward the biological effect (174). To get the effective dose, the W/m2 was
multiplied by the number of seconds of exposure to the particular source (174). At any
given latitude and altitude, the solar zenith angle (SZA) has on the most profound effect
on terrestrial UV readings (210). Because the sun's SZA changes with the seasons and
throughout the day so does the UV intensity (174). UV reading are highest in the
summer then in the spring then in the fall and lowest in the winter (174). Summer doses
can exceed winter doses by 4-6 times (174). This also varies by latitude, as well as
season, for instance, Godar found that the erythemal dose for Boston, Massachusetts
(42°N) from winter to summer was 44,000 J/m2 to 272,000 J/m2 and in Riverside,
California (34°N) was from 102,000 J/m2 to 393,000 J/m2 (174). At midlatitudes (28-
46°) around the world the increase in erythemally-effective UV radiation for every
degree of latitude toward the equator is between 3-3.6% and at higher latitudes (60-68°)
the change is -4.2% (174). The change is greater toward the poles because the ozone
layer is thinner (174).
The National Human Activity Pattern Survery (NHAPS) (-10,000 people)
conducted by the Environmental Protection Agency (EPA) was used by Godar et al. to
71
calculate the UV doses of Americans (211). Godar et al. found that in the United States,
on average, a person receives -30% of the available terrestrial UV radiation when they
are outside (211). It has also been found that an indoor worker in the US goes outside
-10% of the time during daylight hours as an average annual value (211). Of course,
they spend more time outdoors in the spring and summer (13.3% on average) and less
time in the winter (3.3% on average) (211).
People living in the US, on average, receive -25,000 J/m2 of erythemally-
weighted UV radiation per year and -33,000 J/m2 including a conservation continental
US vacation (174). Most people receive -25% of their lifetime UV dose for every two
decades of life, assuming a life span of 80 years (174).
Around the world, excluding vacation exposure, adults that work indoors and
children receive - 3 % (2-4%) of the annual terrestrial UV radiation and adults that work
outdoors receive -10% of the annual terrestrial UV radiation (174).
1.10 b Personal UV Doses
Godar et al. calculated the average annual personal ambient exposure in the US
to be 3.8% (211). The annual personal ambient exposure for people in the north (44°N)
is 3.1% and also 3.1% in the south (34°N) (211). Men over 40 years of age in the north
and south have the highest annual personal ambient exposures (4%), while children have
moderate levels (3.1%) and adolescents (13-19 years old) have the lowest levels (211,
212).
72
Thieden et al. monitored outdoor workers in Dublin, Ireland and Copenhagen,
Denmark continuously over 4 summer months using electronic wrist watch dosimeters
(Sunsavers) which give time-stamped readings, and through sun exposure diaries (192).
They also measured UV radiation in Copenhagen with a UV-Biometer model 501 and in
Dublin with a SunSaver on a roof (192). From the total ambient measurements, the daily
available UV dose was higher in Ireland then in Denmark but the Danes actually
received higher UV radiation than the Irish did every day (192). The Irish took a later
lunch break which resulted in the lower daily exposures because in that time interval
(12pm-3pm), when they would take their break, 44% of the ambient UV radiation was
recorded (192).
The Danish gardeners received a median daily exposure of 1.6 SED per work day and
the Irish gardeners received a median exposure of 0.97 SED (192). In a previous study,
Thieden et al. found that in Denmark during the summer gardeners received a median
daily exposure of 1.3 SED and indoor workers received a median exposure of 0.7 SED
(213). The American Conference of Governmental Industrial Hygienists recommends a
daily UV threshold for outdoor workers that corresponds to 1.1 SED to the face (192).
Gies et al. measured erythemal exposures to the chest and shoulders of
lifeguards, school grounds staff and physical education teachers and found that they had
daily exposures of 3-5 MED (214). At a similar latitude, Kimlin et al. found that the
median daily erythemal exposure to the shoulder was 3 MED for outdoor workers, 1.5
MED for school children and 1.2 MED for home workers during their normal daily
activities (184).
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Thieden et al. looked at the proportion of lifetime doses received by children,
teenagers and adults based on time-stamped personal dosimetry (SunSaver watches)
(215). They found that the annual UV dose did not significantly correlate with age,
except for those under 20 years old (r = 0.23, p = 0.04) (215). Twenty-five percent of
the lifetime dose was received before the age of 20 (215). There were no differences
between male and female adults but there was a significantly higher UV exposure in
females < 20 years old (p = 0.008) (215). They also found that participating in outdoor
sports contributed to significantly higher annual UV doses (177 SED vs. 148 SED, p =
0.02) (215). They showed that the UV variation between individuals is great and that
outdoor activities and risk behavior, not age, differentiate between high and low annual
UV doses (215).
1.10 c UV Doses to the Body and UV Doses with Different Activities
Turnbull et al. looked at six common shade environments in Queensland,
Australia in regards to the larger SZAs encountered during the fall and winter (216).
They found previtamin D3 effective UV wavelengths in the shade were most significant
for tree shade and umbrella shade (216). Tree shade was 55% UV exposure compared to
the full sun, umbrella shade was 52%, a northern facing veranda was 11% and a car with
closed windows was 0% (216). Parisi et al. also looked specifically at different shade
protection from single trees (217). They found that ratio from horizontal for medium
density canopy trees dropped by 47-56% and for sparse density canopy trees it dropped
by 37-42% with the higher reduction near the trunk of the tree (217). Parisi et al. also
74
looked at tree shade for the morning, noon and afternoon exposures during the summer
(218). They found the ratio of erythemal UV from horizontal ranged from 0.42-0.71 for
the morning, 0.29-0.54 for noon, and 0.41-0.63 for the afternoon (218). There was
significant UV exposure in the tree shade of ~4 MED on a horizontal plane over 2 hours
centered around noon (218). They also found that 60% of the erythemal radiation in tree
shade was due to the diffuse component and whereas, the diffuse component decreases
in the full sun, it remains relatively constant in tree shade (219). Parisi et al. compared
the UV exposure under trees during the different seasons and found that even though UV
irradiances are lower during the wintertime, the ratio of horizontal under tree shade is up
to 7% higher than the summertime because of the lower SZA (220). Parisi et al. also
looked at different anatomical sites during the summer under tree shade and found that
the vertex of the head, the shoulders and the forearms received the highest exposures
(221).
Parisi et al. looked at the effects of body size and orientation on UV exposure
(222). They found that height and orientation had minimal effects on exposure to the
body but that body size, time of day and time of year had significant effects (222).
Kimlin and Schallhorn looked at the distribution of UV exposure over the human
form at four different sites throughout the US in the year 2000 (223). They used the
EPA network of MKIV Brewer Spectrophotometers for their UV measurements and
specifically looked at solar noon vitamin D producing UV-B irradiance because of its'
maximal potential for vitamin D production (223). Exposure ratios were calculated
using PS dosimeter badges that had been attached to different anatomical locations on a
manikin in an upright position from a previous study (224). Ambient measurements

75
were taken at the same time with PS dosimeter badges and data from the selected
anatomical locations were normalized to the ambient UV to obtain an exposure ratio
(224). The exposure ratio was a fraction of the ambient radiation for that particular 10°
SZA range (224). The vertex of the head was the site with the highest exposure (224).
Other locations on the face received 0.2-0.5 that of the vertex of the head (224). In the
summer months (smaller SZA), the UV exposure is distributed more to the horizontal
surfaces to the face while in the winter (larger SZA), the UV exposure is distributed
more to the vertical surfaces (224). At larger SZAs, the relative protective ability of hats
may be reduced (224). Depending on the location and time of year the ratio changed,
but some things remained constant, such as, the nose always receiving more exposure
than the upper chest in all locations (223). The ratio of the nose to the upper chest
plotted as a function of location for January and July showed for July the ratio decreased
with an increase in latitude (223). For all locations, the ratio of the chin to the shoulder
reached a nadir during the summer months (June, July and August) and a peak in the
winter months (December and January) (223). The variations in latitude and season
cause variations in the scattering of incoming radiation which causes the distribution of
vitamin D producing ability over the human form to vary (223).
Parisi et al. looked at the anatomical distribution of UV radiation to the human
legs in standing and sitting positions on a manikin (225). The exposure ratio to the legs
during the summer, which is the ratio of UV exposure to a particular site compared to
ambient exposure, ranged from 0-0.75 for different anatomical sites in the sitting
position and ranged from 0.14-0.39 for the standing position (225). The exposure ratio
to the legs during the winter ranged from 0.01-0.91 for sitting and 0.17-0.81 for standing
(225). For sitting, the exposure ratios were the highest for the anterior thigh, shin and
ankle and for the anterior thigh and shin, UV exposed increased by a factor of ~3 for
sitting compared to standing (225). They also had a manikin in the shade and found that
irradiances measured in the tree shade were 15% of those in the direct sun and that the
exposure ratios (anterior thighs, shins, and ankles) for sitting were -half of those in the
sun and less dramatic for the standing exposure ratios (225). They took the exposure
ratios for the specific sites and multiplied them by the ambient erythemal UV exposure
for each day to calculate annual exposures and found that from 12pm-lpm (Australia
Eastern Standard Time) for humans standing, the annual exposure over each leg was 436
MED, whereas it was 512 MED for sitting (225). Thus, UV exposure varies depending
on the bodies' position to the sun.
Parisi et al. observed the weekday and weekend UV exposures to different
anatomical sites for different professions and students (226). Weekday exposures
ranged from 1.0-11.0 SED from winter to summer and absolute exposures were
generally higher on the weekdays than weekends except for 40-49 year olds (226).
Weekend exposures as a percent of ambient exposure were always higher for indoor
workers, school staff and students, however, weekends only comprise 29% of the year
(226). Consequently, UV exposure varies by individuals depending on whether it is a
weekday or a weekend day.
Thieden et al. looked at the reliability of measuring UV radiation on the wrist
with personal UV dosimeters (227). They found that the mean wrist dose was ~50% of
the dose received on the head and was significantly correlated over an extended period
77
of time (227). They concluded that using the wrist for personal dosimetry is practical
and reliable (227).
Herlihy et al. looked at personal UV exposures in Hobart, Tasmania for six
different outdoor activities using PS badges attached to seven anatomical sites over two
consecutive days (186). A badge was also placed horizontally during the activity and
next to the monitoring station and then the anatomical badges were expressed as a
fraction of the horizontal (surface) badge (186). The ratio of the surface badge
erythemal effective dose (EED) to the ambient EED from the monitoring station on day
1 was 1.0 for tennis, 0.87 for sailing, 0.49 for swimming, 0.31 for walking, 0.67 for
golfing and 0.26 for gardening (186). Tennis and sailing had the highest exposure rates
because of the lack of shade (186). Golf, although partially in the shade, had high
exposure rates because of the long duration making it comparable to sailing (186). They
compared the anatomical distribution of UV radiation exposure over the seven body sites
for the four activities that did not involve water and found 0.13 for the cheek, 0.29 for
the hand, 0.43 for the shoulder, 0.40 for the back, 0.23 for the chest, 0.25 for the thigh,
0.27 for the calf (186). Their numbers were lower than similar studies conducted on
manikins (228,229). There was generally good correlations with the cheek, shoulder
and back measurements and less with the hand, chest and thigh (186).
Kimlin et al. placed PS badges on the head, back of the hands, and ankles of 22
cyclists during a 7-day charity bike ride in Queensland, Australia (230). The top of the
head received the highest exposure with a mean dose of 1.8 MED, then the back of the
hand with 1.28 MED, then the side of the head with 1.14 MED, and then the ankle with
0.94 MED (230). The ankles received 51% of UV dose that the top of the head received,
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the side of the head received 63% and the hands received 71% (230). They concluded
that even vertically-oriented, potentially shaded areas, such as the ankles, typically
receive no less than half of the highest UV dose (230).
Thus, UV doses differ between occupations, genders, ages, weekdays versus
weekends and different body sites.
1.11 Skin Pigmentation
1.11 a Studies with Skin Pigmentation
Armas et al. conducted a study to define the relationship between UV-B exposure
and 250HD levels as a function of skin pigment (207). They exposed subjects to UV-B
light (20-80 mJ/cm2) on 90% of their BSA 3 times a week for 4 weeks to various skin
types and measured 250HD weekly (207). They measured skin reflectance and used the
L* (lightness) value. They found that 80% of the variation in 250HD was explained by
the UV-B dose and skin pigmentation even through 4 weeks was not long enough to
reach a steady state at the higher doses (207). They also found an association between
unexposed skin color and baseline 250HD with lighter skin color being associated with
higher 250HD levels (207). This was the first study to quantify the contribution of skin
pigment and simulated sunlight to vitamin D status.
Rockell et al. examined the association between quantitative measures of skin
color, using reflectance spectrometry, and plasma 250HD (231). They assessed skin
color in sun-exposed and unexposed sites and found that exposed skin color was more
important than unexposed skin color in determining vitamin D status (231). Using
reflectance spectrometry they found that each 10° lower skin color value at an exposed
site (forearm) was associated with a 5 nmol/L higher 250HD level (p < 0.001) (231).
They concluded that the sun-exposed skin color (adjusted for unexposed skin color) is an
indicator of sun exposure because time spent outdoors was also associated with sun-
exposed skin color (231).
Malvy et al. classified skin phototype according to hair color, complexion,
susceptibility to burning, and tanning ability and found that fairer skinned phototypes
had lower levels of 250HD in France (232). They attributed this finding to the fact that
fairer skinned participants are likely avoiding the sun because of their tendency to burn
quicker (232). They also found that sun exposure, using a global assessment scale on
four levels (none, low, moderate and high), had a significant impact on serum vitamin D
levels (p < 0.001) with darker phototypes reporting more sun exposure (232). Malvy et
al. also looked at a global model with all contributive factors and after adjustment for
blood sampling date, found that vitamin D status was strongly related to geographical
location (latitude) and sun exposure and that skin phototype was no longer significant
(232). Thus, it seems that classifying sun exposure also requires acknowledging sun
behavior, such as sun avoidance or sunscreen use.
1.11 b Measuring Skin Pigmentation
Proxy measurements have been used to estimate skin phenotype such as, eye
color, hair color and skin color and by using the Fitzpatrick's phototype classification
(skin types I-VI) (233). Phototype for an individual, by definition, reflects the extent of
sunburning versus subsequent tanning following an initial moderate level sun exposure
after a long period of little or no sun exposure (234). A skin phototype I will burn easily
and tan minimally (234). Barger-Lux and Heaney used a cosmetic color chart to
determine the skin tone of sun-exposed areas using a nine point ordinal scale from 0-8
(lightest to darkest) with half-point values for intermediate skin tones (125). They found
that the lowest 250HD level was in the person who had a skin tone of 7 and that the
seasonal difference in skin color for the group as a whole was highly significant (p <
0.0001) (125). Edwards and Duntley in the 1930's demonstrated that the reflectance of
light (400-700 nm visible spectrum) is influenced by the presence of melanin,
hemoglobin and carotene which make up the color found in skin (235). Ethnicity has
even been used as a proxy for skin color but the most accurate way to quantify skin
pigment is by using reflectance spectrometry (236).
By measuring skin pigment with a spectrophotometer the results can be
accurately quantified and expressed in terms of the Commission International
d'Eclairage (CIE) system, which includes L* (lightness), a* (the amount of red or green)
or b* (the amount of yellow or blue) (237). The CIE 'tristimulus' L*a*b* system was
developed in 1976 to closely and linearly correlate with the response of the human eye
(237). For instance, on unexposed skin, a typical northern European would have an L*
value of ~70, a sub-Saharan African would have an L* value of ~35 and an African
American with African-European ancestry would have an L* value of ~50 (207).
Skin pigmentation and erythema can be measured using a skin reflectance meter
which calculates the photoprotection given by skin pigmentation, called the pigment
81
protection factor (PPF) (192). The PPF is equivalent to the number of SED needed to
induce a just perceptible erythema by a minimal erythemal dose to the buttocks (238).
Thieden measured the PPF in a group of outdoor workers and found that the PPF on the
upper back significantly correlated with number of days with risk behavior in the sun (r
= 0.49) and total number of vacations taken (r = 0.54) (192). Thus, the PPF likely
reflected sun exposure received by an individual.
A more accurate measurement of skin pigmentation involves measuring
concentrations of skin chromophores and how they contribute to the absorption spectrum
of the skin using diffuse reflectance spectroscopy (237). In addition to the L*a*b*
values spectrometers measure reflectance in the visible spectrum (400-700 nm) with a
measurement every 10 nm (239).
Shriver and Parra compared two instruments, one that measures the CIE system
(L*a*b*) and one that measures melanin and erythema indices, on different ethnic
groups (240). They found that when L* decreased indicating less lightness and less
reflectance, the melanin index (MI) increased indicating more melanin in the skin and
therefore, darker skin (R2 = 0.87) (240). Including all ethnic groups they found a high
correlation between the L* value and the MI (R2 = 0.93) (240). However, they
concluded that the MI would be a better measure of the amount of melanin in the skin
compared to the L* value because the MI is less confounded by hemoglobin and the
degree of vascularization of different body sites (240).
Dwyer et al. used spectrophotometry to estimate melanin density in Caucasians
from reflectance of visible light by the skin in 15 nm wavelengths to melanin measured
in skin biopsies (241). They found that melanin could be estimated by the difference
82
between the reflectance by the skin at 400 nm and 420 run wavelengths (r = -0.68) (241).
They also found that the lightness value, L* was associated with melanin density (r = -
0.46) (241). They looked at the associations used to indicate skin phenotype and found
that melanin density was associated with self-reported skin color and skin type but there
was not a strong association (r < -0.42) (241). Thus, using a spectrophotometer is a
quick and noninvasive way to measure melanin.
Chardon et al. have used a vector representation in the L*a*b* space for the UV-
induced tanning reaction and have shown that increases in skin pigment can be graphed
as a shift on the L*-b* plane and skin reddening can be graphed as a shift on the L*-a*
plane (242). The vector direction in the L*-b* plane or the 'individual typology angle
(ITA0)' has been used to quantify skin pigment, where ITA° is given in degrees. ITA°
values are inversely related to increased skin pigment (243). Skin color has been
classified using ITA0 as very light > 55 > light > 41 > intermediate > 28 > tanned > 10 >
brown > -30 > dark (244). Del Bino et al. found a significant correlation between ITA0
and the biologically effective dose (BED) in 42 ex vivo skin samples (244). They
concluded that ITA° values give a quantifiable, objective, reliable and reproducible
classification of samples to color and UV-sensitivity (244).
1.12 Dietary Vitamin D
1.12 a Dietary Vitamin D Compared to Photosynthesized Vitamin D

83
Vitamin D obtained from the diet and vitamin D made in the skin have the same
biological activity once they are metabolized (245). However, the half-life of vitamin D
produced in the skin is prolonged in circulation because 100% is bound to the vitamin D
binding protein (DBP) (245). Only 60% of the vitamin D from the diet is bound to the
DBP while 40% is rapidly cleared in the lipoprotein bound fraction (245). Thus, vitamin
D from the diet and vitamin D from dermal synthesis have slightly different
bioavailability.
1.12 b Sources
Very few foods naturally contain vitamin D, however, some sources include oily
fish (ie. salmon, mackerel, herring), cod liver oil, and sun dried mushrooms (137).
However, even natural sources of vitamin D can vary. Chen et al. recently conducted a
study looking at the vitamin D content in wild salmon versus farm raised salmon (178).
They found that wild salmon had 500-1,000 IU per 3.5 oz while farm raised salmon had
only -100-120 IU per 3.5 oz (178). Outila et al. found that the vitamin D2 in wild
mushrooms {Cantharellus tubaeformis) is well absorbed and significantly increased
serum 250HD (246). In the United States milk has been fortified with vitamin D since
the 1930's and now some juices are also being fortified with vitamin D. Typically there
is 100 IU of vitamin D per 8 fluid ounce serving (137). However, Holick et al. found
that over 70% of milk samples from all over the United States, British Columbia, and
Canada were underfortified with vitamin D (247, 248). They also found that between
14-21% of skim milk samples contained no detectable vitamin D (247,248). Recently,

84
it was found that 1/3 of the milk (skim, 1% and 2%) samples tested fell below the
minimum required level of 400IU (249). A multivitamin typically contains 400 IU of
vitamin D as either D2 or D3 in the US.
1.12 c Vitamin D Intakes
Meier et al. looked at the vitamin D intake of 28-50 year olds and found that
Caucasians had intakes of 84-139 IU/day and that African Americans had intakes of 73-
145 IU/day (250). However, in countries where milk is not fortified intakes are even
lower. In Saudi Arabians the average intake was 55 IU/day (251) and in Mexicans it
was 100 IU/day (252). In the Norwegian arctic the average vitamin D intake was 324 ±
280 IU (95) and in Finland it was 188 ± 100 IU (253). In Sunderland, UK, 72-86 year
olds had an average intake of 96 ± 52 IU and there wasn't a significant seasonal
difference in intake, however, vitamin D intake in the summer wasn't related to 250HD
but was significantly related in the winter (124).
Moore et al. estimated vitamin D intake in the US using NHANES III data and/or
the Continuing Survey of Food Intakes by Individuals (CSFII) 1994-1996,1998 data
(254). Female teenagers and adults had the lowest intakes from food while male
teenagers had the highest (254). Less than 10% of older adults (51-70 years old) and 2%
of the elderly (> 70 years old) met the Adequate Intake (Al) requirements from food
sources alone (254). They determined that dairy products were the primary sources of
vitamin D in the US (254). Although supplements increased the percent of people
meeting by Al by 10-25%, up to 90% of the older population still did not meet their
85
needs (254). They concluded that the intake of vitamin D from food sources and
supplements in the US was not meeting the recommended levels (254).
Moore et al. also looked at vitamin D intakes in children and adults from
different ethnic groups in the US using the NHANES 1999-2000 data (255). Among
children 1-8 years old, Mexican American children had the highest intakes while non-
Hispanic blacks had the lowest (255). Among adolescent and teenage boys, non-
Hispanic whites had the highest intakes while non-Hispanic blacks had the lowest (255).
Mean daily intake among females 9-18 and 19-50 years of age did not differ by ethnicity
but non-Hispanic white females > 51 years of age were higher (255). Fortified milk was
the top contributor in all groups (255). Vitamin D intakes from food sources and
supplements were highest among non-Hispanic whites (255). The percent of individuals
that meet the AI from food declined with increasing age and only 4% of adults > 51
years of age were meeting or exceeding the AI (255). Half of all adolescent and teenage
girls and 34% of women (19-50 years old) did not consume the AI from food and
supplements (255). Supplements helped to increase the percentage of individuals
meeting or exceeding the AI (255).
Calvo et al. looked globally at vitamin D intake and determined that vitamin D
intake is too low to sustain adequate circulating concentrations of 250HD in countries
without mandatory food fortification (252). They also recognized that even in countries
with fortification, vitamin D can be low from unique dietary patterns, such as loss of
traditional high fish intakes, low milk consumption, vegetarian diets, and limited use of
supplements (252). In the US there is mandatory fortification of milk and young adult
Caucasian American men and women have the highest daily vitamin D intakes of 325 IU
86
and 293 IU, with 204IU and 124IU from fortified foods, respectively (252).
Supplement use contributes 80-120 IU/day (252). British men and women consume 186
IU and 148 IU/day with limited fortification of some breakfast cereals and mandatory
fortification of margarines (252). There is not fortification in Japan and limited
fortification in Norway but their intakes are higher (252). Japanese women consume
284 IU/day and Norwegian men and women consume 272 IU and 236 IU/day,
respectively (252). Their higher intakes are attributed to high fish consumption (252).
Overall patterns of vitamin D intake vary with gender, age, ethnicity, supplementation
practices and national fortification programs (252).
1.12 d Intake Recommendations
Dietary vitamin D can be toxic if too much is ingested (256) unlike exposure to
the sun which is regulated to prevent intoxication (257). In 1997 The Recommended
Daily Allowance (RDA) Standing Committee of the National Research Council made
recommendations about daily vitamin D intake assuming no sun exposure (258). There
was not enough scientific information to form an RDA so an Adequate Intake (AI) level
was set (258). They recommended 200 IU/day for children and younger adults (< 50
years of age), 400 IU/day for older adults (50-70 years of age), and 600 IU/day for
elderly adults (> 70 years of age) (258).
There is new literature showing the intake recommendations made in 1997, in the
absence of sun exposure, are inadequate (259-262). Studies have shown that 200 IU/day
had no effect on bone health (263). Even a daily intake of 600 IU in women with low
87
sun exposure was not enough to prevent deficiency (264). Most experts are now
recommending a minimum of 1,000 IU of vitamin D3/day to maintain 250HD levels
above 70 nmol/L (259-261). It has been suggested that for some groups even 2,000 IU
(the upper tolerable intake) does not deliver enough vitamin D to be optimal (259).
Concerns about toxicity are not founded when a total body exposure on a sunny day
produces the equivalent of dietary intake of ~10,000-20,000 IU (209). Veith argues that
toxic side effects are not encountered until 40,000 IU/day and that daily doses of 2,400
IU (four times the recommended intake) can be consumed safely (265).
Veith suggests that serum 250HD concentrations > 220 nmol/L that are found in
certain environments, are not unusual in the absence of vitamin D supplementation, and
should be regarded as begin within the physiologic range even though not typically
found in clothed, sun-avoiding populations (265). He argues that there is no harm in
250HD levels associated with sun exposure and they are likely optimal for human health
(265).
In order to reduce the risk of the diseases associated with vitamin D deficiency,
higher vitamin D intakes are required beyond those to just prevent deficiency but are
required to promote optimal health. It has been estimated using dose-response
relationships for vitamin D and cancer risk reduction that it takes 1,500 IU/day of
vitamin D3 for a 29% reduction in male cancer mortality rates in the US (266) and 50%
reduction in colorectal cancer incidence (267) and 4,000 IU for a 50% reduction in
breast cancer incidence (268).
If 75-100 nmol/L were the target range for the revised RDAs, then the new intake
level should meet the requirements of 97% of the population. Using the dose-response
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calculations of Heaney et al. of 0.6-1.0 nmol/L per 1 ug, a daily intake of 2,000 IU may
shift the NHANES III distribution so that only -10-15% of the population was < 75
nmol/L (269).
In recognition of the higher need for vitamin D, the 2005 Dietary Guidelines for
Americans made recommendations aimed at older adults, people with dark skin and
people exposed to insufficient sunlight to consume extra vitamin D from foods or
supplements (270).
1.12 e Risk Assessment for Vitamin D Intake
Hathcock et al. reviewed well-designed human clinical trials of vitamin D and
applied the risk assessment method used by the Food and Nutrition Board (FNB) to
derive a safe Tolerable Upper Intake Level (UL) for vitamin D intake (271). Currently,
the UL set by the FNB is 2,000 IU/day but this level is not based on current research and
is too restrictive (271). They used three basic, standardized steps to determine the UL:
hazard identification, dose-response evaluation and derivation of the UL (UL = No-
observed-adverse-effect-level (NOAEL) / Uncertainty factor (UF)) (271). They found
that more recent human clinical trials support a significantly higher UL and collectively,
the absence of toxicity (hypercalcemia) using > 10,000 IU/day supports a revised UL of
10,000 IU/day (UL = 10,000 IU /1.0) (271).
1.12 f Measuring Intake and Validating Measurement Tools

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Intake can be measured in several ways. Food intake can be measured directly
and recorded into a food record, food intake can be recalled the next day using a 24 hour
recall or usual food intake can be estimated with a food frequency questionnaire (272).
Once intake is recorded then the foods are entered into a database and vitamin D intake
can be determined.
A food-frequency questionnaire (FFQ) specific to only vitamin D does not exist
and would need to be validated. To valid a vitamin D specific FFQ, convergent
evidence would need to be determined by obtaining correlations between the FFQ and
other measures that theory suggest would be related to it (273). Convergent validity is a
measure of agreement between two measures, such as an FFQ and food records. The
effect size of the correlation must also be determined using the correlation coefficient (r)
and an r of 0.5 would be considered a large effect, an r of 0.3 would be intermediate and
an r of 0.1 would be weak support (274). Potosky et al. looked at correlation coefficients
between diet records and a diet history questionnaire and found that the apparent validity
of the questionnaire increases when it is compared to a greater number of 4-day food
records (275). They suggest that greater effort should be given to obtaining more diet-
record days when validating other dietary assessment instruments (275).
1.13 Setting Recommended Daily Allowances (RDA's) for Vitamin D Intake
1.13 a Supplementation Studies and Dose-Response Finding Studies

Lehtonen-Vernmaa et al. supplemented young girls in Finland daily with 400IU
of vitamin D2 for the first winter and then 800 IU of vitamin D2 for the next two winters
(276). They found that 400 IU did not significantly increase serum 250HD from
baseline but 800 IU did significantly increase serum 250HD in the wintertime (276).
They also found that sunlight exposure in the summer was more effective than 800 IU of
daily dietary vitamin D in the winter to raise serum 250HD (276). Another study in
children (10-17 years of age) found that doses of vitamin D3 equivalent to 2,000 IU/day
for one year was safe and resulted in desirable serum 250HD levels (277).
Viljakainen et al. tried to determine how much vitamin D3 is needed in the
elderly, by supplementing women (65-85 years old) with either 0, 200, 400 or 800
IU/day for 12 weeks (278). Serum 250HD increased significantly in all groups (p <
0.001) but decreased in the placebo group (278). Equilibrium was reached in all groups
by week 6 at 57.7 nmol/L, 59.9 nmol/L and 70.9 nmol/L for 200 IU, 400 IU and 800 IU,
respectively (278). The dose-response to supplementation decreased with increasing
vitamin D status at baseline (r = -0.51, p = 0.002) with a clear dose-response noted for
the different doses (278). They concluded that 600 IU may be adequate to maintain
serum 250HD levels around 40-55 nmol/L during the winter but higher intakes would
be needed for higher serum levels (278).
Veith et al. looked at the safety and efficacy of prolonged vitamin D3 intakes of
1,000 and 4,000 IU/day for 2-5 months, starting between January and February (279).
Baseline serum 250HD was 40.7 ±15.4 nmol/L and from month 3 on, serum plateaued
at 68.7 ± 16.9 nmol/L for the 1,000 IU/day group and 96.4 ± 14.6 nmol/L for the 4,000
IU/day group (279). Serum calcium and urinary calcium excretion did not significantly
91
change in either group during the study and they concluded that 4,000 IU/day is a safe
intake (279). This level is above the current UL of 2,000 IU/day but it did not have any
adverse effects.
Aloia et al. supplemented 208 African American women with 50 ug/day (2,000
IU/day) and were not able to raise serum 250HD > 75-80 nmol/L in 40% of the women
(280). There is a concern that responses to vitamin D intake may differ across races. It
is imperative to find a dose-response to vitamin D intake that will allow making
individualized recommendations for intake.
Byrne et al. conducted a meta-analysis of 10 studies which all gave supplemental
vitamin D to the elderly within the current dietary recommendations and found a dose
response of 2.2 nmol/L per 1 ug (40IU) vitamin D (281). Other studies have found a
range of 0.56 nmol/L per 1 ug (279) to 2.1 nmol/L per 1 ug (282) but most of these
studies did not control well for the vitamin D dose, the dose wasn't beyond the current
recommendations and adjustments were not made for season.
In a well conducted study, Heaney et al. administered vitamin D3 daily in
controlled doses of either 0 ug, 25 ug (1,000 IU), 125 ug (5,000 IU) or 250 ug (10,000
IU) for -20 weeks over two consecutive winters (126). Serum 250HD changed in direct
proportion to the dose with a slope of-0.70 nmol/L for each additional 1 ug (40 IU)
vitamin D3 input (126). It was estimated that to maintain the mean baseline value of
70.3 nmol/L a daily intake of 12.5 ug (500 IU) would be needed, whereas, 96 ug (3,800
IU) would be needed from all sources (supplements, food, tissue stores) (126). When
the Food and Nutrition Board made their recommendations for vitamin D intake in 1997,
the data needed for dose-response calculations in regards to intake was not available
92
(126). However, the authors conclude that the current recommended intakes are too low
and need to be reevaluated in light of the new evidence (126).
A current study conducted by Aloia et al. aimed to determine the vitamin D3
intake needed to raise 250HD > 75 nmol/L over a 6 month period with dose adjustments
every 2 months (283). They looked at the response in African American men and
women and Caucasian men and women to find the appropriate intake for each race
(283). To start, participants were given 50 ug (2,000 IU) if their 250HD was 50-80
nmol/L and 100 ug (4,000 IU) if their 250HD was < 50 nmol/L (283). Adjustments
were made in the doses given every two months if participants were < 80 nmol/L or >
140 nmol/L (283). After supplementation, almost all of the participants had serum levels
over 75 nmol/L and no one was over 220 nmol/L (283). They found that the mean slope
(change from baseline) did not differ significantly between races, however, the dose
needed for African Americans was 50% higher because of their low baseline level (283).
African Americans received an average dose of 97.9 ug (3,916 IU), Caucasians received
76.0 ug (3,040 IU) and the mean daily dose given was 96 ug (3,440 IU) (283). In a
multiple regression model, age, BMI and percent body fat did not significantly influence
the response to vitamin D (283). The response of 250HD to 1 ug of vitamin D was
inversely dependent on the baseline concentration (283). There was high variability in
the slopes with the overall slope of 0.66 ± 0.35 (0.15-1.49) (283). They then used a
computer program to find a dose that would be optimal for the entire sample based on
maximizing the number of participants projected to be within the 75-220 nmol/L range
and found a single dose of 115 ug/d (4,600 IU/d) to leave the lowest amount (13%) of
participants outside the range (283).

93
Aloia et al. determined the vitamin D intake needed for the population in the
National Health and Nutrition Survey III (NHANES III) to have a median serum 250HD
level of 105 nmol/L (283). Using their dose-response curve they projected that the
NHANES III population would need a dose of 95 ug/d (3,800 IU/d) for those above 55
nmol/L and 125 ug/d (5,000 IU/d) for those below 55 nmol/L (283).
Hollis et al. examined at the relationship between vitamin D 3 and 250HD 3 in two
separate populations, first, in Hawaiians who received significant sun exposure and
second, in women (lactation study) who received up to 6,400 IU/day of vitamin D3 for 6
months (284). They found that the relationship was saturable and controlled and that
optimal vitamin D status, 100 nmol/L, was achieved at the VmaxOf the 25-hydroxylase
enzyme (284). They argue that humans today operate well below the 25-hydroxylase
Vmax because of substrate (vitamin D3) deficiency (284). They also stated that when
humans are sun/dietary replete then the vitamin D endocrine system would not be
limited by substrate and that the relationship between vitamin D3 and 25OHD3
represents what normal vitamin D status should be (284).
Heaney et al. recently examined the hydroxylation of vitamin D3 to 250HD3
under various input conditions from 6 different studies that measured both vitamin D3
and 25OHD3 (285). Four of the studies were from oral supplementation, one was from
solar radiation in athletes and the last one from simulated radiation (285). From the
study with a single dose of 100,000 IU (286), they found that vitamin D peaked at day
one and 250HD peaked at day seven, at 103 nmol/L, while vitamin D reached baseline
by day seven and 250HD by day 112 (285). They suggest that an average rate of-1,000
IU/day from the complete conversion of the dose by day 112 (285). From the daily
94
dosing studies, they found that vitamin D concentration plateaued after - 3 weeks and
that 250HD rises rapidly at low vitamin D concentrations and then slowly with no
apparent tapering at higher vitamin D concentrations (285). The slow phase began at
-15 nmol/L for vitamin D, or at an intake of -2,000 IU/d, and -80-100 nmol/L for
250HD (285). For the linear portion of the curve, they found that - 4 3 % of the
supplemental vitamin D input was converted to 250HD while the rest must build up/be
stored (285). This was the first study to show that the input of vitamin D to 250HD is
biphasic (285). They authors also suggest that the point at which hepatic 250HD
production goes from a first order to zero order reaction could define the low end of
normal status (-88 nmol/L) (285). Thus, current dietary intake recommendations are too
low and must be revised to support optimal health based on new evidence.
1.14 Vitamin D2 versus Vitamin D3
Officially vitamin D2 and vitamin D2 are regarded as equivalent and
interchangeable but there has been much debate about the effectiveness of vitamin D2
versus vitamin D3. Houghton and Vieth argue that vitamin D2 should no longer be
considered equivalent to vitamin D3 because of differences in their efficacy in raising
250HD, diminished binding of vitamin D2 to the VDP, different metabolic fates and a
shorter shelf-life of vitamin D2 (287). Vitamin D2 may also have a higher risk of toxicity
because of a higher proportion of free metabolites (279). Armas et al. found that vitamin
D2 was only -30-50% as effective as vitamin D3 in maintaining serum 250HD
concentrations (288). Holick argues that this does not mean that vitamin D2 is less active
95
once it is metabolized to l,25(OH)2D2, however, higher doses would have to be given to
raise serum 250HD to above 75 nmol/L (289). However, Holick recently reported that
vitamin D2 was as effective as vitamin D3 in raising serum 250HD concentrations (89).
1.15 Determining Vitamin D Status using Serum 25-Hydroxyvitamin D (250HD)
1.15 a Overview
As already stated, liver production of 250HD is not tightly regulated and is
primarily dependent on substrate concentration (vitamin D) (1). This lack of
physiological regulation makes 250HD an excellent measure of vitamin D nutritional
status and is the accepted measurement to use when determining status (290). Also, the
half life of 250HD is 3 weeks while the half life for vitamin D is -24 hours and for
l,25(OH)2D is only ~4 hours (290).
The typical range of 250HD is 25-162.5 nmol/L (10-65 ng/ml) by most
commercial assays (291). Unfortunately, commercially available assays yield differing
results and this can confound the diagnosis of deficiency (292). There is a call for
standardization in 250HD assays throughout the literature to provide true values of
vitamin D status (292). A standardized assay could also provide a better understanding
of what levels to supplement individuals at in order to yield adequate vitamin D status.
1.15 b Cutoffs
Some researchers support changing the cutoff for 250HD deficiency from 27.5
nmol/L to 50 nmol/L (to minimize PTH levels) but this would dramatically change the
number of people that fall into the range of deficiency (165). This would increase the
number of African American women (15-49 years old) classified at deficient in the US
from 12.2% to 42.4% (84). When defining vitamin D deficiency and/or sufficiency it is
important to show the cutoff being used because it can make a huge difference.
Currently, there is not a consensus on what the optimal level of 250HD should
be and where the cut-off for insufficiency should be drawn. Deficiency typically
corresponds to 250HD levels indicative of a clinically evident disease state, such as
osteomalacia and rickets, while sufficiency refers to 250HD levels corresponding to the
absence of abnormalities in calcium homeostasis and/or maintaining PTH in the normal
range (165). In general, 250HD < 20-25 nmol/L is associated with clinical deficiency
(165). Defining the range of insufficiency is more difficult, however, studies, mostly in
older adults, suggest that 50-80 nmol/L (259) may be required to prevent increased bone
loss (293), fractures (294), secondary hyperparathyroidism (86, 282, 295), and to
maximize fractional calcium absorption (296). Heaney et al. found that calcium
absorption was 65% higher when 250HD was raised from 50 to 80 nmol/L (297). A
recent study looking at the associations with low 250HD levels to all-cause mortality
and cardiovascular mortality found that a serum 250HD level of > 50 nmol/L may be
best for maintaining general health (59). Vitamin D intoxication, which includes
hypercalcemia, is not typically observed until 250HD is at least 375 nmol/L (298).
A recent review conducted by Bischoff-Ferrari et al. estimated the optimal serum
concentrations of 250HD for multiple health outcomes (269). They examined studies
97
which evaluated the threshold for 250HD in relation to bone mineral density, lower
extremity function, dental health, risk of falls, fractures, and colorectal cancer (269).
They found that for all endpoints, the most advantageous 250HD level began at 75
nmol/L and the best was between 90-100 nmol/L (269). Reaching the optimal range for
bone health is expected to provide additional benefits to lower-extremity function, oral
health and colon cancer prevention (269). They concluded that for most people these
concentrations could not be reached with typical intakes and current recommendations
and that > 1,000 IU/day would be needed to bring 250HD concentrations in no less than
50% of the population to 75 nmol/L (269).
1.15 c Measuring 250HD using a Radioimmunoassay (RIA)
The first valid radioimmunoassay (RIA) procedure for 250HD was developed in
1985 (299). Hollis et al. were the first to use 125I-250HD as the tracer and they found
that an average of 97.3% of the added 250HD3 was accounted for (300). Their assay
precision (CV) for between-assay variation was ~15% and the within-assay was ~6%
(300). This is a simple assay with a strong correlation to High Performance Liquid
Chromatography (HPLC) analyses (300). This assay has been approved by the Food and
Drug Administration (FDA) for clinical use in the United States (290). The antibody
used in the RIA recognizes both 250HD2 and 25OHD3 as well as other hydroxylated
metabolites (< 6% of total) (290).

1.15 d Measuring 250HD using a Liquid Chromatography-Tandem Mass
Spectrometry Analysis (LC-MS)
LC-MS and MS is considered to be the most accurate of all methods and is the
gold standard (290). This method can separate and accurately quantify both 250HD2
and 250HD3 (290). This method has been shown to correlate well with the DiaSorin
RIA (r = 0.74 to 0.96) (301, 302).
1.16 Conclusion
It is estimated that one billion people worldwide are at risk for vitamin D
deficiency (83). Finally, vitamin D deficiency is being recognized as an epidemic
throughout the world and with 90-95% of our vitamin D requirement coming from the
sun, there is a lack of understanding to the benefits of moderate sun exposure (209).
Experts agree that in the absence of sun exposure, 1,000 IU is needed to satisfy the
body's requirement for vitamin D (126). Although chronic excessive exposure to
sunlight increases the risk of certain skin cancers but there is little evidence that sensible
limited sun exposure to satisfy the bodies need for vitamin D will increase the risk
substantially for skin cancer (209). Exposure of a healthy adult in a bathing suit to one
MED of sunlight or simulated light is equivalent to taking 10,000-20,000 IU of vitamin
D (209). However, elderly people and people with darker skin pigment are at an
increased risk of deficiency (145,158). African Americans require 5-50 times the sun
exposure (depending on skin pigmentation) than a Caucasian person to satisfy their

99
requirement for vitamin D because melanin acts as a sunscreen (145). For most people
sensible sun exposure of 5-10 minutes a day between 10am to 3pm during Summer and
Fall will satisfy their requirement for vitamin D (62). If a multivitamin provides 40%
(400IU) of the body's requirement then additional supplementation, foods with vitamin
D or moderate sun exposure will be needed to obtain 1,000 IU/day. This is the daily
intake level that experts believe is necessary to raise serum 250HD to a healthy level
above 75 nmol/L (30 ng/ml) (126).
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294. Trivedi DP, Doll R, Khaw KT. Effect of four monthly oral vitamin D3
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Chapter 2:
Vitamin D Intake, Skin Reflectance, and Sun Exposure, using
Polysulphone (PS) Dosimeter Badges, to Predict Serum 250HD
v
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2.1 Abstract
The contribution of sun exposure to vitamin D status is rarely assessed in
community studies using validated methods. The objective of our study was to measure
the contribution of sun exposure, dietary vitamin D intake and level of skin pigmentation
(reflectance) to vitamin D status as measured by serum 250H-vitamin D (250HD). Our
study was conducted with 4 cohorts of healthy young adults each followed for 7-8 weeks
in the Fall, Winter, Spring and Summer (2006-07) in Davis, CA (38.5 N latitude).
Subjects had a wide range of sun-exposure behavior and skin reflectance. A total of 72
subjects (-18 per season) enrolled and completed the study. Skin reflectance was
measured using a reflectance spectrophotometer. Dietary vitamin D intake was assessed
by food records. Sun exposure was assessed using polysulphone (PS) dosimeter badges
sensitive to ultraviolet (UV)-B light. Serum 250HD was measured by ultra-
performance liquid-chromatography-tandem mass spectrometry (LC-MS) and by
radioimmunoassay (RIA). Multiple linear regression analysis was used to predict serum
250HD using continuous variables for sun exposure, skin reflectance and vitamin D
intake, and dummy variables for cohort/season. Sun exposure, skin reflectance and
vitamin D intake were all significant independent predictors of serum 250HD (p < 0.05).
These results show that the contribution of these variables to vitamin D status can be
assessed using simple methods in free-living adults. These methods may help
nutritionists make more accurate individualized recommendations for vitamin D intake
based on skin reflectance and sun behavior.

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2.2 Introduction
"Vitamin D" refers to both vitamin D2 and vitamin D3. Vitamin D2
(ergocalciferol) is the naturally occurring form in plants and vitamin D3 (cholecalciferol)
is the form synthesized by vertebrates (1). Vitamin D is technically a prosteriod
hormone rather than a vitamin because it is made in the skin and converted to 25-
hydroxyvitamin D (250HD) in the liver and then to the active form, 1,25-
dihyroxyvitamin D (l,25(OH)2D), in the kidneys and other tissues (1).
When solar ultraviolet-B (UV-B) photons with energies between 290 and 315 nm
from sunlight strike the skin, photolysis of 7-dehydrocholesterol (7-DHC or provitamin
D3) to previtamin D3 occurs in the plasma membrane of skin cells (2). The amount of
UV-B available depends on the solar zenith angle (SZA) which is the difference in
degrees between the actual position of the sun and 90° vertical. During the winter the
SZA is larger than in the summer (at the same time of day) and with the sun lower in the
sky there is a longer atmospheric path for UV-B photons to travel before reaching the
ground. At higher latitudes and during the wintertime the amount of UV-B photons
reaching the earth may not be sufficient to stimulate significant vitamin D synthesis in
the skin (3). Not only is the SZA larger in the winter but people wear more clothes and
go outside less because of cold weather, further decreasing UV-B exposure. The SZA
also changes with time of day (3). The SZA is smallest at midday, resulting in minimal
UV-B scattering and atmospheric absorption and maximal UV-B radiation reaching the
earth. Thus, the SZA is affected by season, latitude and time of day and this in turn,
affects vitamin D production in the skin and consequently, the concentration of serum
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250HD. Other properties of the natural and human-made environment that affect
dermal vitamin D synthesis by affecting UV-B intensity are air pollution/aerosols, ozone
levels, clouds, reflective surfaces, and shade provided by trees and tall buildings in urban
environments.
There are also non-environmental factors that affect vitamin D production on the
individual level, including age, clothing, sunscreen use, behavior/time in the sun and
level of skin pigmentation. Melanin, the principal skin pigment, provides the body with
an effective sunscreen and reduces dermal synthesis of previtamin D3 (4). Holick et al.
first showed that skin with more melanin (darker skin) requires longer exposure or
higher intensity UV-B radiation to maximize previtamin D3 formation (5). Given
sufficient UV-B radiation skin pigmentation (melanin) does not prevent previtamin D3
formation (5). Thus, African Americans need longer or more intense UV-B exposure to
produce the same amount of vitamin D3 as someone with lighter skin (e.g., Caucasians).
It is well-established that sunlight and diet are sources of vitamin D for humans
but the relative importance of each is less well-defined. Specifically, few studies have
been conducted using quantitative methods to assess the contribution of usual sun
exposure to vitamin D status in free-living subjects. Sun exposure can be assessed either
by direct measurement or through questionnaires. Personal sun exposure can be
measured directly using polysulphone (PS) dosimeter film badges. PS badges measure
the amount of UV-B, energy to which a subject is exposed, reflecting the amount of time
in the sun and the intensity of sun exposure received by an individual over a specified
period of time (typically one day).

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The goals of our study were to quantify the relative contributions of sun
exposure, skin reflectance and vitamin D intake to vitamin D status (serum 250HD) in a
community-based longitudinal study, and to develop a multiple linear regression model
using these three variables to predict vitamin D status in individuals with high or low sun
exposure and light or dark skin reflectance throughout the year. Using this model we
estimated dietary vitamin D intake needed to maintain a healthy vitamin D status
throughout the year for individuals with different levels of sun exposure and skin
reflectance. These methods will be useful in helping define the Recommended Daily
Allowance (RDA) of vitamin D.
2.3 Subjects and Methods
2.3 a Subjects
Subjects were either students or employees recruited from the UC Davis (UCD)
campus. Flyers were posted around campus seeking volunteers with high or low levels
of outdoor activity to participate in the study to obtain a range of typical sun exposure.
E-mails were sent to different student organizations/associations to recruit subjects of
different ethnicities to represent the range of skin pigmentation seen in the US
population. However, because our recruitment was limited to the UCD campus, our
study sample may not be a representative sample of the population. Subjects were
screened for eligibility and included if they had a BMI between 18.5-30, were 19-39
years of age and self-reported high- or low-level daytime outdoor activity. Subjects
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were excluded if they reported consistent sunscreen use on all or most exposed skin,
frequent use of tanning beds or use of high-dose vitamin D supplements (excluding
RDA-level multiple vitamin and mineral supplements) within the past 2 months. These
exclusion criteria were selected because of difficulty in assessing sunscreen
application/use and the significant contribution of tanning-bed use or high-dose vitamin
D supplements to vitamin D status. Subjects were also excluded if they reported
pregnancy, or any disease or underlying condition requiring treatment that may affect
vitamin D absorption/metabolism or use of medications that affect vitamin D
metabolism.
The study was approved by the Institutional Review Board (IRB) of The
University of California, Davis and written informed consent was obtained from all
subjects by the study coordinator.
2.3 b Study Design
The study was an observational, prospective study conducted at the USDA
Western Human Nutrition Research Center (WHNRC) on the UCD campus over 8 week
periods each academic quarter, except Fall quarter where, for logistic reasons, the period
was 7 weeks, for one year from October 2006 to September 2007 (Figure 2.1). Each
quarter a new group of subjects were recruited with both high and low levels of sun
exposure and different levels of skin reflectance. Subjects attended an educational
meeting the week prior to the study to learn on how to keep food records, how to fill out
the sun exposure questionnaires and how to wear and maintain the PS dosimeter badges
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used in the study. They were also informed of the observational nature of the study and
asked to maintain their typical routines. The subjects came to the WHNRC to get their
blood drawn and skin reflectance measured at weeks 0, 4 and 8 (7 for Fall). Subjects
met with the study coordinator weekly to exchange PS badges and records. The study
coordinator also e-mailed reminders for wearing the PS badge and keeping food records.
2.3 c Sun Exposure Assessment
Personal UV-B exposure was measured using polysulphone (PS) dosimeter
badges (Kimlin, Australian Sun and Health Research Lab, Queensland University of
Technology, Brisbane, Qld 4059 Australia). PS is a photosensitive polymer that is used
as an objective way to measure broadband UV-B exposure and is sensitive to the same
wavelengths that affect human skin (6). PS film has a response to UV radiation that
peaks at 300 nm and drops to 1% at 330 nm. UV radiation causes deterioration of the
film and increases the film's absorption. The change in optical density of the PS film is
analyzed by the measurement of transmission at 330 nm in a spectrophotometer at four
locations for each dosimeter badge. The change in absorbance at 330 nm is
proportional to the amount of UV-B exposure for each badge. The PS badges are
typically used to estimate the amount of erythemal UV-B exposure in terms of a minimal
erythema dose (MED) for typical Caucasian-skin which is equivalent to 250 J/m2 of
erythemal action spectrum (EAS)-weighted UV energy (e.g., 1 MED causes slight
pinkness to the skin). The error associated with the UV-B measurements using PS film
was previously estimated to be -10% (7). Each PS film was mounted on a 3cm x 3cm
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white plastic holder with a central aperture measuring one cm . The PS badge
measurements (J/m ) were adjusted for clothing (ie. body surface area exposed (m ))
resulting in individual exposure measurements expressed as joules (J).
The PS badges were worn once per week on the same day by all participants on
their right wrist by attaching it to a white sweat band. For each cohort the PS badge days
were chosen randomly with the restriction that each day of the week and weekend were
included to sample weekly variation in sun exposure. Subjects received each PS badge
wrapped in foil and sealed in an envelope with the date and time for wearing the badge
written on the outside of the envelope. On the day specified the subjects wore the PS
badge from 7am to 7pm, indoors and outdoors. After 7pm they were asked to wrap it in
foil and place it back into the envelope. The subjects then exchanged the envelope for a
new PS badge. They were given the e-mail and phone number of the study coordinator
if they had any problems with the PS badge.
Subjects kept daily sun exposure logs from 7am-7pm that included specific
information about time of day, location, outdoor activity, time in the direct sun or shade,
sunscreen use and clothing worn. Thus, the clothing documented on sun exposure log
was used to adjust the PS badge to joules on the same day that they wore the badge.
2.3 d USDA UV-B Monitoring Station at the UC Davis Climate Center
To obtain maximum ambient exposures, a PS badge was placed in a horizontal
position in the direct sunlight (7am- 7pm) on the same day that the subjects wore their
PS badges. The PS badge was placed at the UC Davis Climate Station (Davis, CA, 38.5
N) next to a UV-B Broadband Pyranometer (Yankee Environmental Systems, Inc.,
Turners Falls, MA) which measures global irradiance on a continual basis in the UV-B
spectral range of 280-330 nm (8). The UV-B radiation detected by the instrument is
converted to visible light and this signal is measured in W/m every 15 seconds and
means are recorded every 3 minutes. We downloaded the erythemally-weighted UV-B
measurements from the USDA UV-B Network website
(http://uvb.nrel.colostate.edu/uvb/home page.html) and converted the measurements to
J/m to make them comparable to the PS badge measurements.
2.3 e Dietary Assessment
Vitamin D intake, from foods and supplements, was determined using
consecutive 4-day food records which were kept every other week for a total of 16
records. Food records have previously been validated as an accurate tool to measure
intake through observational studies (9). The 4-day food records included a weekend
day to capture typical intake and days included rotated from Wednesday-Saturday to
Sunday-Wednesday. The subjects were trained on how to keep accurate food records by
a Registered Dietitian (RD). The food records were analyzed for vitamin D content
using the Nutrition Data System for Research (NDSR) Program (2006, University of
Minnesota, Minneapolis, MN) and a daily intake in IU was determined.
2.3 f Skin Reflectance (Pigment) Assessment

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Skin reflectance was measured using a Minolta 2500d Spectrophotometer
(Konica Minolta Sensing Inc, Ramsey, NJ) on the middle upper inner right arm, the
dorsum of the right hand between the thumb and index finger and the middle of the
forehead at week 0, 4 and 7 or 8 when the participants came in for their blood draw.
Each site was measured 3 times and the instrument was moved slightly over the region
of interest to obtain an average of the site. Each time the instrument was turned on it
was calibrated against to the open air (zero calibration) and a white calibration plate.
The measurements were performed every time by the same trained operator. The
measurements are expressed in terms of the Commission International d'Eclairage (CIE)
system, which includes L* (lightness), a* (the amount of red or green) or b* (the amount
of yellow or blue). The L* value measures reflectance of the skin in which a value of 0
indicates that there was no reflectance (pure black color) and a value of 100 indicates
100% reflectance (pure white color). The L* value is highly correlated to the Melanin
Index of the inner arm and forehead (10) and therefore, the L* value was used in the
multiple linear regression model.
2.3 g Covariates
Weight and height was measured at week 0, 4, and 7 or 8 without shoes using a
calibrated scale and a stadiometer. Body mass index (BMI) was calculated (kg/m2) and
averaged over the 3 time points. Ancestry was self-reported. Subjects self-identified
with high or low levels of sun exposure and whether or not they are an athlete. Age was
determined upon entry into the study.

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2.3 h Serum 25-Hydroxyvitamin D (250HD)
Two 10 ml red-top tubes without an additive were collected at the Western
Human Nutrition Research Center (WHNRC) per subject per blood draw (week 0,4, 7
or 8) after a 4 hour fast from dietary fat using standard venipuncture techniques
performed by trained phlebotomists. The tubes were then wrapped in foil and left to clot
for ~1 hour at room temperature. The tubes were centrifuged for 10 minutes at 2510
RPMs and aliquoted to 1 ml tubes. Serum was stored at -70°C until analysis.
Serum 250HD was analyzed by ultra-performance liquid-chromatography-
tandem mass spectrometry (LC-MS), as described (11), and by radioimmunoassay (RIA)
for comparison using a standard protocol (DiaSorin, Stillwater, MN, USA) according to
the manufacturer's instructions with the following modification: the centrifugation
following the precipitation step was performed at 3000 x g for 60 minutes at 10°C, rather
than 1800 x g for 20 minutes at 20-25°C. This facilitated aspiration of supernatant from
above the pellet containing the labeled 250HD.
For the RIA procedure the coefficient of variation (CV) for duplicate samples
measured on the same-day was 5.0% (range, 0.04-10.0%) and for duplicate samples
measured on different days was 11.6% (range, 7.2-16.0%). For the LC-MS procedure
the same-day CV was 3.2% (range, 1.6-4.8%), as previously reported (11). The
WHNRC participates in the Vitamin D External Quality Assessment Scheme (DEQAS)
(http://www.deqas.org/) and calibration standards from DEQAS analyzed during this
period were all within acceptable limits (12).

2.3 I Parathyroid Hormone (PTH)
Intact Parathyroid hormone (PTH) was measured using an Immulite procedure
(Instrument model #K4610, Siemens Medical Solutions Diagnostics, Malvern, PA) at
week 7 or 8.
2.4 Statistical Analysis
Data was graphed for visual inspection using SIGMAPLOT software (version
9.0, Systat Inc, Pt. Richmond, CA). Statistical Analysis Software (SAS version 9.1.3,
Stat Corp, College Station, TX) was used to perform all statistical analyses. Continuous
data was tested for normality using the Kolmogorov-Smirnov test and variables that
were not normally distributed (p < 0.05) were transformed using Box-Cox
transformations. Analysis of variance (ANOVA) was performed for comparisons
between seasons/cohorts and time-repeated ANOVAs were performed within
seasons/cohorts. Duncan tests were performed for pair-wise comparisons. If a variable
couldn't be transformed to a normal distribution then a nonparametric test was
performed. Variables are expressed as mean ± standard deviations (SD) or as medians
(range) depending on their distribution. Spearman's correlation was used to examine
associations between variables.
To predict serum 250HD, multiple linear regression analysis was used with
continuous variables for skin reflectance, sun exposure, vitamin D intake because they
129
are known to influence status and dummy variables for cohort because of independent
groups each season. Covariates, such as age, gender, BMI, oral contraceptive use,
ethnicity, self-reported sun-level, and athlete were explored in the model along with
interactions, such as skin x sun, sun x diet, skin x diet, skin x sun x diet, and sun x each
season. Pitman's test was used to determine if a skin reflectance measurement from one
site in the model was a significantly better predictor than another site and to determine if
J/m2 and joules are significantly different predictors from each other. All p values <
0.05 were considered statistically significant.
2.5 Results
The baseline characteristics of each cohort are shown in Table 2.1. There were
more subjects of European ancestry than the other groups and more females than males.
Approximately 2/3rd (n= 45) of the subjects reported their usual sun exposure as "low"
and l/3 rd (n= 27) as "high" upon entering the study. The average age of the subjects was
24 ± 4 years and the average BMI was 23 ± 3.
2.5 a Sun Exposure
UV-B exposure (J/m2) was assessed one day per week for each subject using a
PS badge worn from 7am to 7pm. Individual doses (J) were determined by adjusting for
exposed skin. Individual daily doses for each subject in each cohort/season are shown in
Figures 2.2,2.3,2.4 and 2.5.

130
A dose-response curve for the personal PS badges relative to the UV-B
pyranometer readings was constructed using data from the same day that participants
wore the PS badges by exposing another PS badge in the direct sun near the
pyranometer. These results were significantly correlated (r = 0.91, p < 0.0001) (Figure
2.6) demonstrating the ability of the PS badge to accurately measure UV-B exposure.
Individual sun exposure varied by cohort and ambient sun exposure varied by
season. The daily UV-B exposure in joules was averaged for each participant. The
averages were then used to determine the median value for each cohort. The median
dose for Fall (3 J) was significantly lower than Winter (26 J) (Figure 2.7). Both Fall and
Winter were significantly lower than either Spring (169 J) or Summer (163 J), which did
not differ from one another (Figure 2.7). Data from the PS badges at the UV-B
monitoring station were averaged for each season and the median values followed the
same pattern with Fall (Oct-Dec, 643 J/m2) being significant lower than Winter (Jan-
March, 1830 J/m2) and Fall and Winter being significantly lower than either Spring
(April-June, 7230 J/m2) or Summer (July-Sept, 5818 J/m2) (Figure 2.8). Again, Spring
and Summer were not significantly different from one another (Figure 2.8). Therefore,
the maximum possible sun exposure varied by season which is in agreement with the
seasonal variation of UV-B intensity found in other studies (3, 13, 14).
Sun exposure could vary by ancestry due to cultural differences in behavior. The
median individual UV-B exposure for subjects of European (156 J), African (111 J), N.
Asian (76 J), and Hispanic (59 J) ancestry were not significantly different from each
other using a 2-way ANOVA with season (Figure 2.9). Subjects of Hispanic (59 J) and
S. Asian (16 J) ancestry were also not significantly different from each other but UV-B
131
exposure of S. Asian participants was significantly lower than N. Asian, African and
European subjects (Figure 2.9). The highest joules were from subjects of European
ancestry who were athletes and exercised outside. Furthermore, subjects who considered
themselves athletes did have higher sun exposure than those that weren't athletes
(adjusted for season, data not shown, p < 0.05). Thus, there was a difference in sun
exposure between subjects of different ancestries and sun exposure was higher in
athletes.
Sun exposure can be expressed as UV-B intensity (J/m2) or by the individual
UV-B dose (J) delivered to the body surface area (BSA) exposed to the sun. Using the
LC-MS data for serum 250HD, the average UV-B exposure in J/m2 was not
significantly correlated to the average serum 250HD level for each subject (Table 2.2)
but UV-B exposure dose adjusted for clothing (J), was significantly correlated to the
average serum 250HD level (r = 0.33, p = 0.0051, Table 2.2) as seen in Figure 2.10.
Therefore, sun exposure (J) adjusted for BSA exposed is a better predictor of serum
250HD then is environmental exposure (J/m2).
Participants, as they entered the study, were asked to characterize their typical
sun exposure as either "low" or "high". Exposure data were analyzed to determine if
their perception matched their actual sun exposure. As seen in Figure 2.11, subjects
who considered themselves "low" (77 ± 98 J) had significantly lower sun exposure than
those who considered themselves "high" (412 ± 527 J) for each season/cohort.
Regression analysis was used to predict sun exposure (J) from self-identified sun level
(low=0, high=l) and dummy variables for season/cohort. Level and season were
significant predictors of sun exposure (J) (R2= 0.72, p < 0.0001, Fall cohort used as the
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reference, data not shown). Therefore, self-identified level of sun exposure
corresponded closely with actual sun exposure.
2.5 b Dietary Vitamin D Intake
Vitamin D intake from food and supplements was measured 4 times for each
cohort. No differences were seen among their measurements within each cohort. The
median intakes of vitamin D for each cohort were 220IU for Fall, 171IU for Winter,
181IU for Spring and 206 IU for Summer (Figure 2.12). Vitamin D intake did not
differ by cohort/season (p > 0.05) (Figure 2.12). Therefore, dietary vitamin D intake did
not change over time within individuals and did not differ among cohorts.
Vitamin D intakes could differ between ancestry groups because of differences in
food habits or supplement use. Median vitamin D intakes of European (236 IU) and S.
Asian (223 IU) subjects were significantly higher than the intakes of Hispanic (117 IU)
subjects but not of N. Asian (184 IU) and African (148 IU) subjects (Figure 2.13).
Subjects who considered themselves an athlete were European and consequently,
athletes had higher vitamin D intakes than those that weren't athletes likely due to higher
caloric intakes (data not shown, p < 0.05). Therefore, dietary vitamin D intake differed
by ancestry groups and was higher in athletes.
Vitamin D from the diet contributes to circulating levels of 250HD. It was
therefore not surprising that vitamin D intake was significantly correlated to serum
250HD using LC-MS data (r = 0.31, p = 0.0082, Table 2.2, Figure 2.14). Thus,
vitamin D intake contributed significantly to the vitamin D status of these subjects.

2.5 c Skin Reflectance (Pigment)
Skin reflectance was assessed at three sites, one typically protected from the sun
(inner arm), and two typically exposed to the sun (hand and forehead). As seen in Table
2.2, reflectance (L*) measured at these sites significantly correlated with serum 250HD
using the LC-MS data, however, the forehead measurement had the highest correlation
coefficient (r = 0.43, p = 0.0002), followed by the back of the hand measurement (r =
0.41, p = 0.0003) and the inner arm measurement (r = 0.34, p = 0.0035). The difference
between the inner arm and forehead measurements were not significantly correlated to
250HD (data not shown). Correlations with the RIA data are also shown (Table 2.2).
The forehead skin reflectance had the highest correlation to serum 250HD likely
reflecting sun exposure to that site.
Differences in sun exposure can cause differences in skin pigmentation at
different sites or at the same site over time. There were no significant changes in
reflectance measured at the three sites over time within each cohort (adjusted for sun
level, data not shown, p > 0.05). The average inner arm reflectance measurement was,
however, significantly higher (lighter pigment) than the average hand and forehead
measurement after adjusting for ethnicity (Figure 2.15). The forehead reflectance
measurement was significantly higher (lighter pigment) in subjects who self-identified
with high sun exposure (adjusted for season, data not shown, p < 0.05), although the
inner arm and hand reflectance measurements were not different between subjects who
self-identified with low or high levels of sun exposure. In summary, the inner arm
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reflectance was higher than the other sites, but skin reflectance did not significantly
change over time in the three sites.
Level of skin pigmentation/reflectance varies by ancestry. As seen in Figure
2.16, for the forehead skin pigment measurement, African (L*= 44 ± 7) subjects had
significantly darker skin than the subjects of other ancestries. European (L*= 63 ± 4)
and N. Asian (L*= 59 ± 4) subjects were not significantly different from each other, as
were also N. Asian and Hispanic (L*= 58 ± 4) subjects, and Hispanic and S. Asian (L*=
54 ± 5) subjects (p > 0.05). The average inner arm and hand measurements also had the
same significant differences between the ancestries (data not shown). Subjects who self-
identified as an athlete had significantly higher reflectance (lighter skin) at the arm, hand
and forehead sites (adjusted for ethnicity, data not shown, p < 0.05). Thus, skin
reflectance varied by ancestry in the subjects, as expected, and was higher in athletes
because athletes were of European ancestry.
2.5 d Vitamin D Status
Serum 250HD levels as measured by RIA and LC-MS were significantly
correlated (r = 0.89, p < 0.0001) (Figure 2.17). The 250HD values from the LC-MS
procedure are used in the analyses because the LC-MS procedure is more accurate than
the RIA procedure.
Serum 250HD can vary seasonally due to differences in ambient UV-B intensity,
level and type of outdoor activity and type of clothing. The week 0 serum 250HD
measurements and week 7 or 8 measurements were highly correlated to each other (r -

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0.91, p < 0.0001, Table 2.2) using the LC-MS data. In the Fall cohort, serum 250HD at
week 0 (104 ± 39 nmol/L) was significantly higher than week 4 (97 ± 38 nmol/L) and
week 7 (92 ± 38 nmol/L) (Figure 2.18). For the Winter cohort, serum 250HD at week 0
(66 ± 30 nmol/L) was not significantly different than week 4 (69 ± 36 nmol/L) but was
significantly lower than week 8 (74 ±41 nmol/L) (Figure 2.19). Furthermore, week 4
was not significantly different from week 8 in the Winter Cohort (Figure 2.19). For the
Spring cohort, serum 250HD at week 0 (78 ±38 nmol/L) and week 4 (77 ± 40 nmol/L)
were significantly lower than week 8 (89 ±41 nmol/L) while week 0 and week 4 did not
differ (Figure 2.20). For the Summer cohort, serum 250HD at week 0 (86 ± 40 nmol/L)
was not different than week 4 (88 ± 37 nmol/L) or week 8 (82 ± 37 nmol/L), however,
week 4 was significantly higher than week 8 (Figure 2.21). Hence, the average serum
250HD in the Fall (~Oct-Dec) was trending down while the average serum 250HD in
the Winter (~Jan-March) was trending up. The average serum 250HD in the Spring
(-April-June) seemed to trend up while the average serum 250HD in the Summer
(~July-Sept) seemed to trend up and then back down. Thus, there were significant
changes in serum 250HD within the cohorts that corresponded to changes in the ambient
UVB intensity.
Average serum 250HD, using LC-MS data, in the Fall (97 ±38 nmol/L) was
significantly higher than the Winter (70 ± 35 nmol/L) while average serum 250HD in
the Spring (81 ± 39 nmol/L) and Summer (85 ± 38 nmol/L) were intermediate but not
significantly different from each other or Fall or Winter (Figure 2.22). Accordingly,
only serum 250HD in the Fall and Winter were significantly different from each other.
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Serum 250HD, using LC-MS data, was compared among the different ancestry
groups while adjusting for season. European (118 ± 37 nmol/L) subjects had
significantly higher serum 250HD levels than Hispanic (86 ± 20 nmol/L) subjects and
both European and Hispanic subjects had higher levels than African (63 ± 22 nmol/L), S.
Asian (63 ± 22 nmol/L) and N. Asian (59 ± 18 nmol/L) subjects (Figure 2.23).
Furthermore, athletes, all of whom were European, had significantly higher serum
250HD levels (adjusted for season, data not shown, p < 0.05). Consequently, serum
250HD differed between the different ancestry groups controlling for season while
Europeans had the highest levels.
2.5 e Parathyroid Hormone (PTH)
Parathyroid hormone (PTH) secretion is inhibited by the active form of vitamin
D and hyperparathyroidism is associated with vitamin D deficiency. PTH measured at
week 7 or 8 was negatively, but not significantly, correlated to week 7 or 8 serum
250HD using the LC-MS data (r = -0.20199, p = 0.0888, Figure 2.24). Looking at PTH
and serum 250HD values < 50 nmol/L did not make the correlation significant (data not
shown). However, typically when vitamin D is low, PTH is increased resulting in a
negative relationship.
2.5 f Model Predicting Vitamin D Status

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Multiple linear regression analysis was used to predict vitamin D status (250HD
by LC-MS) using sun exposure, vitamin D intake and skin reflectance as independent
variables. First, each continuous variable was used alone, along with dummy variables
for cohort/season, in the model to look at the independent effects of each.
Independently, average vitamin D intake explained 19% of the variation in 250HD (p =
0.0065), average forehead skin reflectance explained 28% of the variation in 250HD (p
< 0.0001), and the average UV-B dose (J) adjusted for clothing explained 38% of the
variation in 250HD (p < 0.0001). Together in the model they explained 53% of the
variation in 250HD (Table 2.3). Using J/m in the model as the sun exposure
measurement to predict serum 250HD was a significant predictor (R2 = 0.47, p =
0.0002, data not shown), however, Joules was a more significant predictor of status in
the model (p = 0.0186, data not shown). Sun exposure in joules had the largest impact
on 250HD, followed by skin reflectance and vitamin D intake (Table 2.3). Forehead
skin reflectance was a more significant predictor of status than arm reflectance (p =
0.0313, data not shown) but not significantly different than hand reflectance in the model
(p = 0.2822, data not shown). Hand reflectance was not a significantly better predictor
of status than arm reflectance in the model (p = 0.1178, data not shown) and therefore,
forehead skin reflectance was used in all the models. Independently, BMI, age, gender
and sunscreen use on PS badge days (No = 0, n = 49 or Yes - 1, n = 23) were not
significant in the full model. Several interactions were tested in the model and none
were significant (including sun x each season, sun x skin, sun x diet, skin x diet, or skin
x sun x diet). In summary, in the full model, sun exposure was the most significant
predictor of serum 250HD, followed by skin reflectance, and diet.

138
Several covariates were significant in the model. In Table 2.4, the full model
with skin reflectance, vitamin D intake and sun exposure, is shown in 4 models, along
with the significant covariates of ancestry/ethnicity, self-identified sun exposure level
(high = 1 or low = 0), athlete (yes = 1 or no = 0), and oral contraceptive (OC) users (yes
= 1 or no - 0). In the model with self-identified sun exposure level as a covariate, 56%
of the variance in 250HD was explained and with athlete as a covariate instead, 58% of
the variance was explained. Adding ancestry/ethnicity to the basic model, helped
explain 64% of the variance in 250HD, however, only N. Asian was a significant
negative predictor of 250HD. Within the model using data from females only (n = 53),
oral contraceptive use (n = 16) was a significant, positive predictor of 250HD,
explaining 62% of the variance in 250HD. Subjects that used oral contraceptives did
not have different sun exposure, vitamin D intake or skin pigment (data not shown).
However, oral contraceptive users had higher serum 250HD levels (106 ± 30 nmol/L)
than non-users (80 ± 43 nmol/L) (adjusted for season, p < 0.05), which is consistent with
the literature (12).
Table 2.5 shows the individual models for each season/cohort with sun exposure,
vitamin D intake and forehead reflectance to predict 250HD using the LC-MS data. In
the Fall model, sun exposure and forehead reflectance were both significant predictors of
250HD. In the Winter model, only vitamin D intake was a significant predictor of
250HD. In the Spring and Summer models, only sun exposure was a significant
predictor of 250HD. Several interactions were tested in the models and none were
significant (including sun x skin, sun x diet, skin x diet). The seasonal models
demonstrate how the variables change as significant predictors of 250HD depending on

the season. Table 2.6, shows the full model of sun exposure (J), vitamin D intake (IU)
and skin reflectance (forehead L*) using the RIA data for serum 250HD.
In the full model, sun exposure and skin reflectance are the two most importance
predictors of vitamin D status. To illustrate their association, we used the regression
model to predict serum 250HD (LC-MS data) in subjects with low and high levels of
skin reflectance and in subjects with low and high levels of sun exposure (4 groups).
Low and high sun exposure was defined as the 20 (low) and 80 (high) percentiles
from each cohort/season using the PS badge measurement in joules. Low and high sun
exposure, in that order, for each season were: 2 J vs. 56 J for Fall, 7 J vs. 158 J for
Winter, 24 J vs. 958 J for Spring, and 85 J vs. 576 J for Summer. On average, 20
minutes in the direct sun with -18% BSA (ie. face, neck, hands and arms (t-shirt))
exposed constituted low sun exposure while 90 minutes in the direct sun with -35%
BSA (ie. face, neck, hands, arms (tank top) and legs (long shorts)) exposed constituted
high sun exposure. Vitamin D intake was defined as the median value of 200 IU for all
seasons. Skin pigment was defined as the median reflectance value (L*) on the forehead
from African Americans (Low, L*= 42) or Europeans (High, L* = 63). In Figure 2.25,
the group with low skin reflectance and low sun exposure had the lowest levels of serum
250HD while the group with high skin reflectance and high sun exposure had the
highest levels of serum 250HD, as excepted, using the model prediction. Table 2.7
shows the predicted serum 250HD values, using the LC-MS data, each season for the 4
groups. The group with low reflectance (dark skin) and low sun exposure would be
below 75 nmol/L all year around while low reflectance (dark skin) and high sun
exposure group would be at risk of insufficiency in the Winter. The group with high
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reflectance (light skin) and low sun exposure would be below 75 nmol/L in the Winter
and Spring and only the high reflectance (light skin) and high sun exposure group would
be protected from insufficiency year around based on the model prediction. Thus, serum
250HD varies by season, as well as, level of sun exposure and level of skin pigment.
2.5 g Model Estimating Vitamin D Intake Needed To Maintain a Healthy
Vitamin D Status
In Table 2.8, vitamin D intake needed to maintain serum 250HD at -75 nmol/L
was determined using our model to estimate serum 250HD (LC-MS data) and the
response to supplementation reported by Heaney et al., a 0.7 nmol/L increase in serum
250HD for every additional 40 IU (1 ug) of vitamin D ingested (15). People with low
reflectance (dark skin) and low sun exposure need the highest intakes to reach 75
nmol/L. Higher intakes would be needed in the Winter and Spring for people with high
reflectance (light skin) and low sun exposure and in the Winter for people with low
reflectance (dark skin) and high sun exposure. Thus, the vitamin D intake needed to
maintain a healthy serum 250HD level (> 75 nmol/L) varies by season, as well as level
of sun exposure and level of skin reflectance/pigment.
2.6 Discussion
Vitamin D intake, sun exposure and level of skin reflectance all contribute to an
individual's vitamin D status. Therefore, assessing the quantity of UV-B radiation

141
received by an individual is essential when assessing their vitamin D status. PS
dosimeter badges worn by subjects are an objective way to quantify sun exposure.
Thieden et al. looked at the reliability of measuring UV radiation on the wrist with
electronic personal UV dosimeters and concluded that using the wrist for personal
dosimetry is practical and reliable (16). Our study used PS badges worn on the wrists of
participants once a week to assess their sun exposure and we adjusted the measurement
in J/m to Joules by multiplying by the amount of body surface area (BSA) in m
exposed to the sun to determine the UV-B dose received. Using the LC-MS data, there
was not a significant correlation between serum 250HD and the unadjusted J/m2 but
there was a significant correlation between serum 250HD and the adjusted joules (for
BSA exposed) (r = 0.33, p = 0.0051, Table 2.2, Figure 2.10). However, using J/m2 in
the model as the sun exposure measurement to predict serum 250HD was a significant
predictor (R2 = 0.47, p = 0.0002, data not shown) but the model with Joules was more
significant (R2 = 0.53, p < 0.0001, Table 2.3). Therefore, adjusting the PS badge
measurement to represent the actual dose received by an individual is a better was to
assess sun exposure as it correlates more closely with serum 250HD.
Herlihy et al. compared the measurements from PS badges to ambient UV-B
levels from a monitoring station and found good correlations (correlation of badge to
station was 1.08 ± 1.0), especially for activities in the open and direct sun and less for
partially shaded activities (17). For instance, the ratio of the badge/ambient in J/m was
0.87-1.00 for tennis to 0.26 for golfing (17). Our PS badge measurements correlated well
to ambient UV-B measurements from the UV-B monitoring station (r = 0.91, p <
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0.0001) (Figure 2.6) demonstrating the ability of the PS badge to accurately measure
UV-B exposure.
The PS badges captured the difference in individual and ambient sun exposure
which varied by cohort/season. The average individual and ambient measurements for
the Fall were significantly lower than Winter while Fall and Winter were significantly
lower than either Spring or Summer. Spring and Summer were not significantly
different from one another (Figure 2.7, Figure 2.8). Perhaps, at a location with a higher
latitude and consequently, a larger SZA, Spring and Summer would have been
significantly different from one another. However, the Spring and Summer cohorts had
significantly higher UV-B intensity, as expected, and this could stimulate greater dermal
vitamin D synthesis and thus, contribute more to an individual's vitamin D status.
Research in Australia has shown that the contribution of sun exposure in the
summer to vitamin D status is greater than sun exposure in the winter (16). In the
models predicting vitamin D status for each season, sun exposure was the most positive
important (p < 0.05) predictor in the Fall, Spring and Summer models (Table 2.5), while
it was not a significant predictor in the Winter Model (Table 2.5). The impact of sun
exposure to vitamin D status is significant when there is adequate available ambient UV-
B in the environment.
Many studies have shown a seasonal fluctuation with serum 250HD, with higher
levels in the Summer versus the Winter also indicating the relative importance of sun
exposure as a source of vitamin D (18-25). Dlugos et al. examined serum 250HD in
submariners and found that without sun exposure for two months their serum levels
declined by half, also emphasizing the importance of sun exposure in maintaining serum
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250HD levels (26). We found that serum 250HD in the Fall (97 ± 38 nmol/L) cohort
was significantly higher than the Winter (70 ± 35 nmol/L) cohort while Spring (81 ± 39
nmol/L) and Summer (85 ± 38 nmol/L) cohorts were not significantly different from
each other or from Fall or Winter (Figure 2.22). Surprisingly, Spring and Summer
levels were not significantly higher than Fall or Winter levels, however, Fall levels were
significantly higher than Winter levels demonstrating a seasonal difference in serum
250HD levels. However, our comparisons among cohorts cannot be attributed strictly
to seasonal differences because each cohort consisted of different groups of individuals.
Not only is sun exposure important when assessing vitamin D status, it is also
important to assess skin pigmentation. African Americans tend to have lower circulating
concentrations of 250HD and are more prone to developing vitamin D deficiency likely
because they have more melanin (darker skin pigment) which attenuates vitamin D
synthesis in the skin (27). Harris and Dawson-Hughes measured serum 250HD in
young African American and white women in Boston four times over one year (28).
They found that the African American women had significantly lower 250HD levels
throughout the year and a smaller seasonal change between Winter and Summer (28).
We found that African Americans had significantly lower skin reflectance (ie, darker
skin pigment) (Figure 2.16) than the other ancestry groups and lower serum 250HD
levels compared to Europeans (after adjusting for season, Figure 2.23). However,
African Americans did not have significantly different dietary vitamin D intakes (Figure
2.13) or sun exposure compared to Europeans (after adjusting for season, Figure 2.9).
This observation is consistent with darker skin pigment significantly reducing the dermal
production of vitamin D3 and consequently serum 250HD.

144
Armas et al. exposed UV-B light (200-800 J/m2) to participants on 90% of their
BSA three times a week for four weeks to subjects of various skin types, using the L*
(reflectance) value, and measured 250HD weekly (29). They found that 80% of the
variation in 250HD was explained by the UV-B dose and skin reflectance even though
they concluded that 4 weeks was not long enough to reach a steady state at the higher
doses (29). By using an average BSA of 1.7 m2 and with 90% BSA exposed, the doses
in joules given were 306-1224. In our study the median (and low-high) individual daily
sun exposure in joules were 3 (1-154), 26 (4-438), 169 (16-2138) and 163 (13-1085) for
Fall, Winter, Spring and Summer, respectively. Our median joules were lower than the
simulated sun exposure given by Armas et al. The values of Armas et al. are similar to
those received by subjects with high sun exposure in our study, particularly in the Spring
and Summer cohorts. Our full model with sun exposure, skin reflectance and vitamin D
intake explained 53% of the variation in serum 250HD. They found that UV-B dose
and skin reflectance explained more of the variation in serum 250HD (R2 = 0.79) than
we did, however, they induced sun exposure and increased 250HD while we monitored
sun exposure and 250HD in our free-living subjects. Thus, our results may reflect more
typical responses in serum 250HD to sun exposure.
Armas et al. also found an association between unexposed skin pigment and
250HD with lighter skin color being associated with higher 250HD levels (29).
However, Rockell et al. looked at skin pigment in sun-exposed and unexposed sites and
found that exposed skin color was more important than unexposed skin color in
determining vitamin D status (30). They concluded that the sun-exposed skin color
(adjusted for unexposed skin color) is an indicator of sun exposure because time spent
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outdoors was also associated with sun-exposed skin color (30). Malvy et al. also found
that lighter skinned phototypes had lower levels of 250HD in France (31). They
attributed this finding to the fact that participants with lighter skin are likely avoiding the
sun because of their tendency to burn (31). They also found that sun exposure, using an
assessment scale with four levels (none, low, moderate and high), had a significant
association with serum 250HD levels (p < 0.001) and with darker skin pigmentation
(31). In the present study, the inner arm measurement (unexposed skin) had
significantly higher reflectance (lighter) than the hand and forehead measurements
(exposed skin) (Figure 2.15). Skin reflectance at all three body sites correlated
significantly with serum 250HD, however, the forehead measurement was correlated the
highest (r = 0.43, p = 0.0002, Table 2.2), then the back of the hand measurement (r =
0.41, p = 0.0003, Table 2.2) and lastly the inner arm measurement (r = 0.34, p = 0.0035,
Table 2.2). The difference in reflectance between the inner arm and forehead
measurement was not significantly correlated with serum 250HD. The higher
correlation in exposed skin (hand and forehead) could reflect sun exposure received
which would contribute more to serum 250HD.
In our model (Table 2.3), skin reflectance, sun exposure, and vitamin D intake
were all significant predictors of vitamin D status, as determined by 250HD using LC-
MS data. Sun exposure was the most significant predictor (std. est = 0.65, p < 0.0001)
then skin pigment (std. est = 0.36, p = 0.0003) and lastly vitamin D intake (std. est =
0.19, p = 0.0314). Together they explained 53% of the variation in serum 250HD.
Ancestry/Ethnicity added to the model helped explain 64% of the variation in serum
250HD which could be attributed to genetic differences between ancestry groups.

146
Jacobs et al. looked at what correlated to 250HD in a sun-replete location, Arizona, and
constructed a model to determine which variables contributed to most circulating
250HD (23). In their predictive model, sun exposure and dietary vitamin D had a
larger effect on 250HD in whites than among African Americans and Hispanics (23)
perhaps due to the alteration of dermal vitamin D synthesis by melanin although skin
reflectance was not measured. Perhaps, ethnicity and not just skin reflectance is
important to assess when determining vitamin D status because of differences in genetics
and behavior.
Webb et al. found that the seasonal variation in serum 250HD was more
pronounced in individuals with low dietary vitamin D intakes (18). Other studies have
shown that dietary vitamin D correlates poorly with 250HD concentrations (25, 26) and
that 250HD remains around what is considered insufficient despite consumption of the
Al for vitamin D (26). We found a weak correlation between dietary vitamin D and
serum 250HD (r = 0.31, p = 0.0082, Figure 2.14). As already shown in the predictive
model (Table 2.3), sun exposure contributes more to vitamin D status than diet. In the
individual seasonal models, diet was a significant predictor of vitamin D status in the
Winter (Table 2.5). Thus, the relative (although not absolute) contribution of vitamin D
intake to status may be greater in those with lower sun exposure and in the wintertime.
Calvo et al. concluded that vitamin D intake is too low to sustain adequate
circulating concentrations of 250HD in countries without mandatory food fortification
(32). They also recognized that even in countries with fortification, vitamin D can be
low due to specific dietary patterns, such as loss of traditional high fish intakes, low milk
consumption, vegetarian diets, and limited use of supplements (32). They found that
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young adult Caucasian American men and women have the highest daily vitamin D
intakes of 325 IU and 293 IU, with 204IU and 124IU from fortified foods, respectively
(32). We also found that subjects of European ancestry (ie. Caucasian) had higher
median vitamin D intakes (236 IU) than Hispanic subjects (117 IU) but not significantly
different intakes from S. Asian (223 IU), N. Asian (184 IU), and African (148 IU)
subjects (Figure 2.13). Thus, vitamin D intake can vary by ethnicity likely due to
cultural differences in food habits.
In 1997 the Recommended Daily Allowance (RDA) Standing Committee of the
Food and Nutrition Board (FNB) made recommendations about daily vitamin D intake
assuming no sun exposure (33). There was not enough scientific information to warrant
an RDA so an Adequate Intake (AI), was set (33). They recommended 200 IU/day for
children and younger adults (< 50 years of age), 400 IU/day for older adults (50-70 years
of age), 600 IU/day for elderly adults (> 70 years of age) and they set an upper intake
level (UL) at 2,000 IU/day (33). However, recent findings indicate that the
recommendations made in 1997 are inadequate (29-31).
Hathcock et al. reviewed well-designed human clinical trials of vitamin D and
applied the risk assessment method used by the FNB to derive a UL for vitamin D intake
(34). They concluded that the UL set by the FNB at 2,000 IU/day is not based on
current research and is too restrictive (34). They determined that a UL of 10,000 IU of
vitamin D3 would be more appropriate (34).
Dawson-Hughes et al. showed that 200 IU/day was not sufficient to minimize
bone loss from the femoral neck (33). Glerup et al. concluded that a daily intake of 600
IU in women with low levels of sun exposure is not enough to prevent vitamin D
148
deficiency (34). Aloia et al. estimated the vitamin D intake needed for the NHANES III
population to have a median serum 250HD level of 105 nmol/L (35). Using their dose-
response curve they projected that the NHANES III population would need a dose of
3,800 IU/d for those above 55 nmol/L and 5,000 IU/d for those below 55 nmol/L (35).
We used 75 nmol/L as cut-off for insufficiency, however, there is currently not a
consensus on what the optimal level of 250HD should be and where the cut-off for
insufficiency should be drawn. In general, serum 250HD levels < 20-25 nmol/L are
associated with a significant risk of deficiency (36). Defining the range of insufficiency
is more difficult, however, studies, mostly in older adults, suggest that 50-80 nmol/L
(37) may be required to prevent increased bone loss (38), fractures (39), secondary
hyperparathyroidism (26, 39, 40), and to maximize fractional calcium absorption (40).
A recent review conducted by Bischoff-Ferrari et al. looked at estimating optimal serum
concentrations of 250HD for multiple health outcomes and found that for all endpoints,
the most advantageous 250HD level began at 75 nmol/L and the best was between 90-
100 nmol/L (41). They concluded that for most people these concentrations could not be
reached with typical intakes and current recommendations and that > 1,000 IU/day
would be needed to bring 250HD concentrations in no less than 50% of the population
to 75 nmol/L (41). Accordingly, most experts are now recommending a minimum of
1,000 IU of vitamin D3/day to maintain 250HD levels above 75 nmol/L (29-31). In
accordance with the wide use of this value, we chose 75 nmol/L as a reasonable cut-off
for vitamin D insufficiency in our study.
Webb and Engelsen using a UV-B computerized simulation tool (42) determined
that the current vitamin D intake recommendation of 400 IU/day is achievable through
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sun exposure, without risk of erythema (skin reddening), for all or part of the year (43).
For an intake recommendation of 1,000 IU/day, they determined that sun exposure of
less than one hour can still serve as a single vitamin D source at low latitudes and
middle-high latitudes with sufficient BSA exposed for all or part of the year (43). Of
course, increases in latitude and skin pigment make the recommendations harder to
achieve without supplementation. Using our predictive model with sun exposure, skin
reflectance and vitamin D intake, in Figure 2.25/Table 2.3, and applying the dose
response curve by Heaney et al. (15), we found that someone with high skin reflectance
(light skin) and high sun exposure would not need additional dietary vitamin D to
maintain their serum 250HD level above 75 nmol/L but someone with high skin
reflectance and low sun exposure would need additional vitamin D intake during the
Winter and Spring, 1,029 IU and 571IU, respectively (Table 2.8). Someone with high
skin reflectance (dark skin) and high sun exposure would need additional dietary vitamin
D during the Winter only (800 IU), however, someone with high skin reflectance and
low sun exposure would need additional dietary vitamin D year long to maintain their
serum 250HD at or above 75 nmol/L (1,086 IU for Fall, 2,114 IU for Winter, 1,771 IU
for Spring and 971 IU for Summer) (Table 2.8). Thus, sun exposure cannot maintain
serum 250HD above 75 nmol/L for all times of the year unless sun exposure is on the
high end (-90 mins/day) and skin reflectance is high (light skin). Supplementation of
vitamin D is needed for most individuals at higher levels than the current AL Thus, the
current AI for vitamin D intake is inadequate for most individuals and should be
adjusted in light of new evidence.

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The strengths of the present study include longitudinal data collected over each
season while participants went about their daily lives and their sun exposure was
measured. The study also included individuals with a wide range of skin reflectance and
sun exposure levels. Some of the limitations of the present study include a small sample
size (particularly for subjects with low skin reflectance and high sun exposure) although
adequate to show statistically significant differences, different groups of individuals each
season, a limited range of dietary intake and all self-identified athletes were European
and had the highest levels of sun exposure. In the full model, age and BMI were not
significant predictors of 250HD because we recruited subjects in a narrow age and BMI
range, however, studies have shown that older age (44) and/or having a higher BMI (45)
is associated with a lower vitamin D status. We also thought that there would be a
significant interaction between sun exposure and skin pigment in the model, however,
we may need a larger sample size to find significance.
Our study showed that there was a significant contribution of sun exposure, skin
reflectance and vitamin D intake to vitamin D status in our study population which was
assessed using simple methods in the free-living adults and these methods could be used
in future population-based studies. Hopefully, these methods will help nutritionists
make more accurate individualized recommendations for vitamin D intake based on skin
reflectance/pigmentation and sun exposure.
2.7 Acknowledgements
151
I would like to thank Dr. Stephensen for helping with all aspects of the study. I
would also like to thank the student volunteers who helped enter and check data. I
would like to thank Dr. Bonnel and the phlebotomists, Emma White and Evelyn
Holguin, for their help with scheduling and managing the participants. I would also like
to thank Dr. Woodhouse and Manuel Tengonciang for their help with running the RIA
and Dr. Aronov for running the LC-MS. I would like to thank Joseph Domek for
running the PTH samples. I would also like to thank Dr. Slusser for helping us with the
USDA Monitoring Station. I would like to thank Dr. Kimlin and his staff for donating
the PS badges and analyzing them. I would also like to thank Thuan Nguyen and Jan
Peerson for their advice on statistics. I am grateful for all the help and guidance I
received.
2.8 References

Science 1982;216:1001-3.
3. Webb AR, Kline L, Holick MF. Influence of season and latitude on the
cutaneous synthesis of vitamin D3: exposure to winter sunlight in Boston and
Edmonton will not promote vitamin D3 synthesis in human skin. J Clin
5. Holick MF, MacLaughlin JA, Doppelt SH. Regulation of cutaneous previtamin
D3 photosynthesis in man: skin pigment is not an essential regulator. Science
1981;211:590-3.
6. Kimlin MG, Parisi AV, Wong JC. Quantification of personal solar UV exposure
of outdoor workers, indoor workers and adolescents at two locations in Southeast
Queensland. Photodermatol Photoimmunol Photomed 1998;14:7-11.
7. Parisi A, Wong CF. A dosimeter technique for the measurement of ultraviolet
exposure to plants. Photochem Photobiol 1994;60:470-474.
152
9. Karvetti RL, Knuts LR. Validity of the estimated food diary: comparison of 2-
day recorded and observed food and nutrient intakes. J Am Diet Assoc
1992;92:580-4.
different biological ancestry. Am J Phys Anthropol 2000;112:17-27.
11. Aronov PA, Hall LM, Dettmer K, Stephensen CB, Hammock BD. Metabolic
profiling of major vitamin D metabolites using Diels-Alder derivatization and
ultra-performance liquid chromatography-tandem mass spectrometry. Anal
Bioanal Chem 2008;391:1917-30.
12. Carter GD, Carter CR, Gunter E, et al. Measurement of Vitamin D metabolites:
an international perspective on methodology and clinical interpretation. J Steroid
Biochem Mol Biol 2004;89-90:467-71.
13. Holick MF. Environmental factors that influence the cutaneous production of
vitamin D. Am J ClinNutr 1995;61:638S-645S.
14. Marts PJ. Solar ultraviolet radiation: definitions and terminology. Dermatol Clin
2006;24:1-8.
Am J ClinNutr 2003;77:204-10.
16. Thieden E, Agren MS, Wulf HC. The wrist is a reliable body site for personal
dosimetry of ultraviolet radiation. Photodermatol Photoimmunol Photomed
2000;16:57-61.
17. Herlihy E, Gies PH, Roy CR, Jones M. Personal dosimetry of solar UV radiation
for different outdoor activities. Photochem Photobiol 1994;60:288-94.
18. Webb AR, Pilbeam C, Hanafin N, Holick MF. An evaluation of the relative
contributions of exposure to sunlight and of diet to the circulating concentrations
of 25-hydroxyvitamin D in an elderly nursing home population in Boston. Am J
ClinNutr 1990;51:1075-81.
2000;51:439-51.
6.
21. Lamberg-Allardt CJ, Outila TA, Karkkainen MU, Rita HJ, Valsta LM. Vitamin
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in other parts of Europe? J Bone Miner Res 2001;16:2066-73.
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153

2007;85:860-8.
24. Levis S, Gomez A, Jimenez C, et al. Vitamin d deficiency and seasonal variation
in an adult South Florida population. J Clin Endocrinol Metab 2005;90:1557-62.
2002;87:4952-6.
26. Dlugos DJ, Perrotta PL, Horn WG. Effects of the submarine environment on
renal-stone risk factors and vitamin D metabolism. Undersea Hyperb Med
1995;22:145-52.
27. Bell NH, Greene A, Epstein S, Oexmann MJ, Shaw S, Shary J. Evidence for
alteration of the vitamin D-endocrine system in blacks. J Clin Invest
1985;76:470-3.
28. Harris SS, Dawson-Hughes B. Seasonal changes in plasma 25-hydroxyvitamin D
concentrations of young American black and white women. Am J Clin Nutr
1998;67:1232-6.
29. Armas LA, Dowell S, Akhter M, et al. Ultraviolet-B radiation increases serum
25-hydroxyvitamin D levels: the effect of UVB dose and skin color. J Am Acad
Dermatol 2007;57:588-93.
30. Rockell JE, Skeaff CM, Williams SM, Green TJ. Association between
quantitative measures of skin color and plasma 25-hydroxyvitamin D.
Osteoporos Int 2008.
31. Malvy DJ, Guinot C, Preziosi P, et al. Relationship between vitamin D status and
skin phototype in general adult population. Photochem Photobiol 2000;71:466-9.
32. Calvo MS, Whiting SJ, Barton CN. Vitamin D intake: a global perspective of
current status. J Nutr 2005;135:310-6.
34. Hathcock JN, Shao A, Vieth R, Heaney R. Risk assessment for vitamin D. Am J
Clin Nutr 2007;85:6-18.
35. Aloia JF, Patel M, Dimaano R, et al. Vitamin D intake to attain a desired serum
25-hydroxyvitamin D concentration. Am J Clin Nutr 2008;87:1952-8.
38. Scharla SH, Scheidt-Nave C, Leidig G, et al. Lower serum 25-hydroxyvitamin D
is associated with increased bone resorption markers and lower bone density at
the proximal femur in normal females: a population-based study. Exp Clin
Endocrinol Diabetes 1996;104:289-92.
39. Trivedi DP, Doll R, Khaw KT. Effect of four monthly oral vitamin D3
(cholecalciferol) supplementation on fractures and mortality in men and women
living in the community: randomised double blind controlled trial. Bmj
2003;326:469.
154
40. Heaney RP. Vitamin D depletion and effective calcium absorption. J Bone Miner
Res 2003;18:1342; author reply 1343.
41. Bischoff-Ferrari HA, Giovannucci E, Willett WC, Dietrich T, Dawson-Hughes
B. Estimation of optimal serum concentrations of 25-hydroxyvitamin D for
multiple health outcomes. Am J Clin Nutr 2006;84:18-28.
42. Engelsen O, Kylling A. Fast simulation tool for ultraviolet radiation at the earth's
surface. Opt Eng 2005;44:1-7.
43. Webb AR, Engelsen O. Ultraviolet exposure scenarios: risks of erythema from
recommendations on cutaneous vitamin D synthesis. Adv Exp Med Biol
2008;624:72-85.
44. MacLaughlin J, Holick MF. Aging decreases the capacity of human skin to
produce vitamin D3. J Clin Invest 1985;76:1536-8.
45. Wortsman J, Matsuoka LY, Chen TC, Lu Z, Holick MF. Decreased
bioavailability of vitamin D in obesity. Am J Clin Nutr 2000;72:690-3.
155
2.9 Figures
Figure 2.1: Study Design
Cohort 1 Cohort 2 Cohort 3 Cohort 4
Fall '06 Winter '07 Spring '07 Summer '07

(Oct-Dec) (Jan-March) (April-June) (July-Sept)
->n=72
7 wks 8 wks 8 wks 8 wks
n-17 n=17 n=20 n=18
Each Cohort:
WeekO Week 4 Week 7 or 8
Blood draw Blood draw Blood draw

Skin pigment Skin pigment Skin pigment
Diet & sun recall Diet & sun recall Diet & sun recall
Weekly PS dosimeter badges and daily sun exposure logs

2.0
1.5
o 1.0 H
<
95
pa 0.5
0.0 A
— 1 1 — • —
Wkl Wk2 Wk3 Wk4 Wk5 Wk 6 Wk 7

Dates worn: 11/2 11/8 11/17 11/19 11/28 12/9 12/11/06
Figure 2.2: Individual daily sun exposure for the 17 subjects in the Fall cohort. PS
badges were worn from 7am to 7pm once a week to assess UV-B exposure (J/m2).
Individual doses (J) were determined by multiplying the area of skin exposed on the
same day when outdoors. Measurements are shown transformed (JA0.1). Each symbol
represents an individual subject.
z.t> -
2.0 -
V v
1.5 -
•v.
- X X<*
<
*^zrry^ ^^L\KI^^TJv—•-^8
M
X)
1.0 -
pr ^-^/ \ r
OS
to
1 \ \ /
0.5- 1 \ \ /
1 \ •••'' \ /
1 \ / \ /
0.0 -
v V
\ 1 1 i I- • ' i i • i
Wkl Wk2 Wk3 Wk4 Wk5 Wk6 Wk7 Wk8

Dates worn: 1/26 1/31 2/6 2/15 2/24 2/28 3/5 3/11/07
Figure 2.3: Individual daily sun exposure for the 17 subjects in the Winter cohort. PS
badges were wornfrom7am to 7pm once a week to assess UV-B exposure (J/m2).
158
1.5 H
— i 1 1 1 1 1 1 1

Dates worn: 4/18 4/24 4/29 5/7 5/17 5/25 6/2 6/4/07
Figure 2.4: Individual daily sun exposure for the 20 subjects in the Spring cohort. PS
2.5 -,
2.o ^
1.5 H
<
1-9 1.0 H
wo
•9
pa
0.5
0.0
1 1 1 1 1 1 1 1

Dates worn: 7/28 7/31 8/9 8/13 8/22 8/31 9/5 9/9/07
Figure 2.5: Individual daily sun exposure for the 18 subjects in the Summer cohort. PS
10000 i
C 8000 A
0 4-/ , , , , ,
0 2000 4000 6000 8000 10000
PS Badge at Monitoring Station (UV-B 31m)
Figure 2.6: Correlation of PS Badge UV-B at Monitoring Station and UV-B

Measurement from Monitoring Station Pyranometer
*r = Spearman Correlation Coefficient

Significant p-value < 0.05
Dashed lines = 95% Confidence Interval
Regression Equation: Station UV-BA0.3 = 3.11913 + 0.27624*Badge UV-BA0.4 (R2 =
0.96, p < 0.0001, not normally distributed)
2500
c
o
2000
1500
c
s
\m*>
o
*L 1000
Ml o
-e o
«
03 b
X/l o
0< 500 o
a 8
o 8
8 Q
Fall Winter Spring Summer

3 26 169 163 Median J
(1-154) (4-438) (16-2138) (13-1085) (Range)
0.47% 2.0% 2.3% 2.8% % of Fig. 2.8
Figure 2.7: Individual Average PS Film Badges for each Cohort/Season
*Average J over 7-8 weeks, adjusted for clothing/body surface area exposed to the sun
Different letters represent a significant difference (p < 0.05) between cohorts/seasons
using an ANOVA for Box-Cox transformed values.
10000
c c
o
o o
8000
oo
6000
a o 8
b o
Si 4000-
T3
a 0
SO a ©
04
2000 o o o
§
o
0-
1 i 1

643 1830 7230 5818 Median J/m2
(213-2100) (1124-3431) (5282-8454) (1995-8309) (Range)
8.9% 25% 100% 80% % of Spring
Figure 2.8: Monitoring Station PS Badges of Total Available UV-B for each
Cohort/Season

using an ANOVA for Box-Cox transformed values.
163
2500 n
b
o
2000
1500
3
O
>-»
s^
ft. 1000
W>
"0
«S
CO
CZ!
500
a,b
a. o
a o o
o
S Asian Hispanic N Asian African European

16 50 76 111 156 Median J
(2-227) (5-369) (1-305) (13-486) (1-2138) (Range)
Figure 2.9: Ancestry/Ethnicity and Average Individual PS Badges
* Average J over 7-8 weeks, adjusted for clothing/body surface area exposed to the sun
Different letters represent a significant difference (p < 0.05) between ancestries with a 2-
way ANOVA for season using Box-Cox transformed values.
164
250 -i
0 A 1 1 1 1 1 1 1 1
0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4
PS Badge (JA0.1**)
Figure 2.10: Correlation of Ave. Serum 250HD (LC-MS data) and Ave. PS badges
transformed (JA0.1)

Regression equation: 25OHDA0.1 = 1.34109 + 0.0.19137*JA0.1 - 0.10223*winter-
0.12492*spring - 0.11483*summer (R2 = 0.38, p < 0.0001, fall as ref.) **Average J over
7-8 weeks, adjusted for BSA exposed to the sun.
165
2.4 -i * *
2.2 -
1
o
1
* o 3/4
2.0- 9/12 o 0
* o
* o o
o o
9
*
o*
1.8 -
I 3/4 0
o 0
9 o
<
^
T3
1.6-
5/7
o
0
o
§
0
8 1
8 o
« 1.4- Q o
o
0-
o
0
8 o
o o
CODOO
1.2 -
e
o
1.0 - O o
0.8- I • I '"
._,.._ 'I -I .,, __. 1
A\V 0 ^ w$& <V*" +<$& aV^ - ^ <V0^ *<$&
2 18 24 197 107 352 136 706 Median J

(1-7) (1-154) (4-51) (21-438) (16-189) (24-2138) (13-486) (263-1085) (Range)
Figure 2.11: Self-Identified Sun Exposure Level (High or Low) each Cohort/Season
and Actual Sun Exposure (PS Badges)
** Average J over 7-8 weeks, adjusted for clothing/body surface area exposed to the sun.
* Significant difference (p < 0.05) between high and low sun level and PS badge joules
with a 2-way ANOVA for season using Box-Cox transformed values. Subjects who self-
reported high sun exposure and who had higher sun exposure than those who reported
low sun exposure were 5/7 for Fall, 3/4 for Winter, 9/12 for Spring and 3/4 for Summer.
a
a
o
o a
a
o
8
o o o
o
o o
o o
o
o
o
o
s
8
o
8
o

220 171 181 206 Median IU
(66-692) (36-621) (55-599) (50-719) (Range)
Figure 2.12: Average Dietary Vitamin D Intake including Supplements between

Cohorts/Seasons

using an ANOVA with Box-Cox transformed values.
BUU -
a,b b
o
700 - o
b
a,b o
600 - o o
8
$ 500 -
o o
g 400 - 0
'a o
o
£ 300-
o 8
o
200 - a o
o
9 8
100 - o o
o o o
0- I o
Hispanic African N Asian S Asian European
117 148 184 223 236 Median IU
(66-150) (50-581) (72-719) (36-599) (71-692) (Range)
Figure 2.13: Ancestry/Ethnicity and Average Dietary Vitamin D Intake Including

Supplements
Different letters represent a significant difference (p < 0.05) between groups using an
ANOVA and Box-Cox transformed values.
250
200 r = 0.31*
p - 0.0082
1 1!
"S 150
a
s
o 100
IT)
50 o <? u%> o<f?°

o o
> o
100 200 300 400 500 600 700 800
Dietary Vitamin D (IU)
Figure 2.14: Correlation Between Average Serum 250HD (LC-MS data) and Average
Dietary Vitamin D including Supplements (IU)

Regression equation: 25OHDA0.1 = 1.35835 + 0.03498*nl Diet (R2 = 0.11, p = 0.0054)
169
80 -i
70
^ 60
I O o
8" 8 o
8 8
40
o
30
Forehead Hand Inner Arm

56 ±8 57 ±9 62 ±8 Mean L* ± SD
Figure 2.15: Skin Pigmentation1 Measured at 3 Different Body Sites
'Skin Pigmentation assessed using L*= 0 (pure black) to 100 (pure white).
Different letters represent a significant difference (p < 0.05) between sites using a 2-way
ANOVA with ethnicity and Box-Cox transformed values.
170
80 i
d
c,d 0
b,c
a
o 0 o
o 0
8
8
I- o
o o
1 40
30
African S Asian Hispanic N Asian European MeanL*
44 ±7 54 ±5 58 ±4 59 ±4 63 ± 4 ± SD
Figure 2.16: Ancestry/Ethnicity and Forehead Skin Pigmentation1
'Skin Pigmentation assessed using L*= 0 (pure black) to 100 (pure white).
Different letters represent a significant difference (p < 0.05) between groups using an
ANOVA with Box-Cox transformed values. Skin pigment measured on the inner arm
and hand also followed the same pattern of significance using an ANOVA with Box-Cox
transformed values.
171
100 150 200 250

RIA250HD(nmoI/L)
Figure 2.17: Correlation of LC-MS1 & RIA2 250HD Data

Regression Equation: LC-MS 25OHDA0.1 = 1.02873 + 0.13195*RIA nl250HD (R2 =
0.8351, p < 0.0001)
!
LC-MS = Ultra-performance liquid-chromatography-tandem mass spectrometry (11).
2
RIA = Radioimmunoassay using a standard protocol according to the manufacturer's
instructions with the following modification: the centrifugation following the
precipitation step was performed at 3000 x g for 60 minutes at 10°C, rather than 1800 x g
for 20 minutes at 20-25°C to permit aspiration of the pellet.
180
160
140
^ 120
o
S
^ 100-1
$ 80 H
60
40
20
WeekO Week 4 Week 7 Mean nmol/L
104 ±39 97 ±38 92 ± 38 ± SD
Figure 2.18: Fall* 250HD Status using LC-MS Data
*Week 0 = ~ Oct. 26, 2006, Week 4 = ~ Nov. 21,2006, Week 7 = ~ Dec. 14,2006
Different letters represent a significant difference (p < 0.05) between weeks using an
ANOVA with Box-Cox transformed values. Each symbol represents an individual
subject.
b
180-1 a,b
' ...A
160 - a
...A"""'
140-
A
'""".'.''.••• .^^4
-^ 120- A"" __-V
OHD (nmol/I
100-
-•
80 -
in
60 - S-^-^-.-- -£--~~ ~~~—-•
40-
20 -
1 1 1
0-
66 ±30 69 ±36 74 ±41 ±SD
Figure 2.19: Winter* 250HD Status using LC-MS Data
*Week 0 = ~ Jan. 18,2007, Week 4 = ~ Feb. 15,2007, Week 8 = ~ March 15, 2007
ANOVA with Box-Cox transformed variables. Each symbol represents an individual
subject.
WeekO Week 4 Week 8 Meannmol/L
78 ±38 77 ±40 89 ±41 ± SD
Figure 2.20: Spring* 250HD Status using LC-MS Data
*Week 0 = ~ April 12,2007, Week 4 = ~ May 10,2007, Week 8 = ~ June 7,2007

subject.
a,b
220
V— .___
200
180 --v
160
o 140
S
s©120 ^
m
o 100
in
80 H
60
40
20
86 ±40 88 ±37 82 ±37
±SD
Figure 2.21: Summer* 250HD Status using LC-MS Data
*Week 0 = ~ July 19,2007, Week 4 = ~ Aug. 16,2007, Week 8 = ~ Sept. 13,2007

subject.
250
a,b
a,b
o
200 A o
o b
o
o 150 o
S 8
a o o
*
o
o
= 100 4
O
50
0 o
e
Fall Winter Spring Summer Mean±SD
97 ±38 70 ±35 81 ±39 85 ± 38 Median nmol/L
96(45-165) 66(23-145) 73(44-212) 77 (40-197) (Range)
Figure 2.22: Average Serum 250HD between Cohorts/Seasons
*LC-MS data
using an ANOVA with Box-Cox transformed values.
177
250 i
C
o
200 o
o 150
S
& a
* a b
ft a o
100 H o o 8
O
IT)
o
8
8
50
N Asian S Asian African Hispanic European Mean ± SD

59 ±18 63 ±22 63 ± 22 86 ±20 118 ±37 Median nmol/L
57(35-105) 65(23-96) 61(34-114) 87(66-106) 117(58-212) (Range)
Figure 2.23: Ethnicity/Ancestry and Average Serum 250HD
*LC-MS data
Different letters represent a significant difference (p < 0.05) between ancestries with a 2-
way ANOVA for Season and Box-Cox transformed values.
178
140
120 4 r = - 0.20* o
p = 0.0888
100
B 60
°<F*g<5> (CO °"~

20
o o o ©
o ~ 6> o °o
50 100 150 200 250
250HD (nmoI/L)
Figure 2.24: Correlation between Week 7 or 8 Serum 2 5 0 H D (LC-MS data) and Week
7 or 8 PTH

Significant p-value < 0.05. Dashed line = 9 5 % Confidence Interval
Regression Equation: nl 250HD = 4.99281 - 0.20845*PTH A 0.3 (R2 = 0.0437, p
0.0779)
179
Dark skin, High sun

o Dark skin, Low sun
140 -i T Light skin, High sun
_.._A Light skin, Low sun
*
120 -
\
\ / — - - - ,
^ - S
\ /
d \ /
\ /
|OlU
100 -
\ /
\ /
£ \ /
a m . A
X
o
to
80 -
• '""^X / /
w \ \ / /
ve.
60 -
o
vV •*•- ~"~
^ - • • u
' '
< .--O
' • - .
o •''
40 - 'O-
Figure 2.25: Model Prediction* for Serum 250HD in 4 Groups
^Predicted 250HD 1 using LC-MS data with the 20th (low) & 80th (high) percentiles for
sun exposure each season (J), median for diet (200IU) & median L*for forehead skin
reflectance (African & European).
'Equation:
25OHDA0.1=(1.19778+(0.15948*badgeA0.1)+(0.00000006049589*skinA3.4)
+(0.02054*nldiet)-(0.08238*winter)-(0.08602*spring)-(0.07535*summer))
180
2.10 Tables
Table 2.1:
Baseline Characteristics
Season/Cohort Fall Winter Spring Summer Tota

No. of Subjects 17 17 20 18 72
1
Ethnicity/Ancestry
European 10 5 6 5 26
Hispanic 1 2 1 0 4
N. Asian 1 3 2 6 12
S. Asian 5 3 6 1 15
African 0 4 5 6 15
Gender
Female 14 11 16 12 53
Male 3 6 4 6 19
Reported Sun Exposure2
Low 10 13 8 14 45
High 7 4 12 4 27
3
Age (x±SD) 23 ± 3 27 ± 5 23 ± 4 22 ± 3 24 ±
BMI 4 (x±SD) 23 ± 2 24 ± 2 23 ± 2 22 ± 3 23 ±
1
Self-reported ethnicity/ancestry.
Nationalities for European included Russian/Kazakhstan and American, Hispanic
included South American and Mexican, N. Asian included Chinese and Korean, S. Asian
included Indian, Vietnamese, Sri Lankan, Thai, Cambodian/Chinese/Malaysian and
Philipino and African included American, African/Peurto Rican, African/European
/Italian and African/Mexican.
2
Self-reported level of sun exposure upon entry to study.
3
Age upon entry to study.
4
Body mass index (kg/m2) averaged over wk 0, 4 and 7 or 8.
181
Table 2.2:
Correlations of Average 250HD with Average Sun Exposure,
Diet and Skin Reflectance
LC-MS1 RIA2
Spearman Correlations r p-value R p-value
Wk 0 & Wk 7 or 8 Serum 250HD 0.91 O.0001* 0.84 O.0001*
250HD & PS Badge (J**) 0.33 0.0051* 0.36 0.0017*
250HD & PS Badge (J/m2) 0.19 0.1106 0.23 0.0474*
250HD & Vit D Intake (IU) 0.31 0.0082* 0.27 0.0195*
250HD & Skin Reflectance (Arm) 0.34 0.0035* 0.29 0.0136*
250HD & Skin Reflectance (Hand) 0.41 0.0003* 0.38 0.0011*
25QHD & Skin Reflectance (Forehead) 0.43 0.0002* 0.39 0.0008*
r = correlation coefficient
*Significant p-value < 0.05 for Spearman Correlation between values.
Average = mean of all measurements for each subject.
^C-MS = Ultra-performance liquid-chromatography-tandem mass spectrometry
rocedure to determine serum 250HD.
IRIA = Radioimmunoassay to determine serum 250HD.
** Joules (UV-B exposure) adjusted for clothing worn (BSA exposed). Skin reflectance
= L* value, measured either on the upper inner arm, the back of the hand or the
forehead.
Table 2.3:
Multiple Linear Regression Model to Predict Serum 25QHD1 ^^^
Parameter Standardized
Model Est SE Est p-value R2
Full Model (n=72) 0.5324
PS Badge (J) 0.15948 0.0311 0.65480 O.0001*
Skin (Forehead) 6.05E-08 1.6E-08 0.35544 0.0003*
Vit D Intake (IU) 0.02054 0.00933 0.19075 0.0314*
Fall Ref
Winter -0.08238 0.0188 -0.50912 O.0001*
Spring -0.08602 0.02394 -0.56070 0.0006*
Summer -0.07535 0.02411 -0.47478 0.0027*
Equation using LC-MS data:

25OHDA0.1 = 1.19778+(0.15948*badgeA0.1)+(0.00000006049589*skinA3.4)
+(0.02054*nldiet)-(0.08238*winter)-(0.08602*spring)-(0.07535*summer)
*Significant p-value < 0.05
Ref- Reference dummy variable in the model.
J= mean joules per subject.
Forehead = mean forehead L* skin reflectance per subject.
IU= mean dietary vitamin D including supplements per subject.
183
Table 2.4:
Significant Covariates in Multiple Linear Regression Model to Predict Serum 25QHD**
Model Est. SE Est. p-value Rl
Full Model with
Ethnicity1 72 0.6406
PS Badge (J) 0.0964 0.03882 0.39581 0.0158*
Skin (forehead) 5.97E-08 2.66E-08 0.35057 0.0283*
Vit D Intake (IU) 0.02333 0.00885 0.21669 0.0106*
Fall Ref
Winter -0.06102 0.01943 -0.37710 0.0026*
Spring -0.04710 0.02586 -0.30701 0.0735
Summer -0.02726 0.02631 -0.1718 0.3042
Hispanic Ref
European -0.01723 0.02781 -0.12045 0.5379
African -0.03401 0.03014 -0.20100 0.2636
S Asian -0.05320 0.02812 -0.31443 0.0632
N Asian -0.08739 0.02786 -0.47393 0.0026*
Full Model with Level2 72 0.5602
PS Badge (J) 0.11391 0.03791 0.46770 0.0038*
Skin (forehead) 5.27E-08 1.61E-08 0.30990 0.0017*
Vit D Intake (IU) 0.01798 0.00921 0.16702 0.0553
Fall Ref
Winter -0.06777 0.01976 -0.41887 0.0011*
Spring -0.07240 0.02436 -0.47194 0.0042*
Summer -0.04968 0.02679 -0.31304 0.0683
Sun Level 0.03342 0.01662 0.23548 0.0485*
Full Model with
Athlete3 72 0.5747
PS Badge (J) 0.07928 0.04363 0.32551 0.0739
Skin (forehead) 5.54E-08 1.55E-08 0.32569 0.0007*
Vit D Intake (IU) 0.01474 0.00926 0.13689 0.1164
Fall Ref
Winter -0.06568 0.01924 -0.40591 0.0011*
Spring -0.04936 0.02721 -0.32177 0.0744
Summer -0.03427 0.02832 -0.21592 0.2307
Athlete 0.05931 0.02351 0.32196 0.0141*
Full Model with OC
Users4 53 0.6201
PS Badge (J) 0.17377 0.03318 0.72583 <0.0001*
Skin (forehead) 5.25E-08 1.85E-08 0.29094 0.0069*
Vit D Intake (IU) 0.01954 0.01105 0.16768 0.0838
Fall Ref
Winter -0.08225 0.02098 -0.46389 0.0003*
Spring -0.09286 0.02621 -0.59287 0.0009*
Summer -0.08607 0.02761 -0.50094 0.0032*
OCUse 0.03926 0.01528 0.25067 0.0135*
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Table 2.4 continued

**LC-MS data.
*Significant p-value < 0.05.
Ref = Reference dummy variable in the model.
Ancestry/Ethnicity self-categorized without an 'other' category (dummy variables).
2
Self-Identified sun exposure level (Low=0, n=45 or High=l, n=27).
3
Self-Identified athlete (No=0, n=60 or Yes=l, n=12).
4
OC=oral contraceptive users (No=0, n=37 or Yes=l, n=16), model with females only.
Forehead= mean forehead skin reflectance L* per subject.
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Table 2.5:
Seasonal Multiple Linear Regression Models to Predict Serum 250HD**
Model n Est. SE Est. p-value R2
Fall 17 0.6372
PS Badge (J) 0.54396 0.18023 0.56257 0.0099*
Skin (forehead) 2.94944 0.86286 0.59357 0.0046*
Vit D Intake (IU) -0.01736 0.04329 -0.07735 0.6949
Winter 17 0.7064
PS Badge (J) 0.06719 0.06791 0.21295 0.3406
Skin (forehead) 1.09356 0.69329 0.25362 0.1387
Vit D Intake (IU) 0.12291 0.04669 0.55627 0.0207*
Spring 20 0.3570
PS Badge (J) 0.00033 0.00014 0.47231 0.0343*
Skin (forehead) 0.01304 0.00934 0.28810 0.1818
Vit D Intake (IU) 0.00022 0.00047 0.09766 0.6389
Summer 18 0.4972
PS Badge (J) 0.08321 0.02661 0.6415 0.0074*
Skin (forehead) 0.70012 1.00074 0.14804 0.4956
Vit D Intake (IU) -0.01149 0.05087 -0.04451 0.8246
**LC-MS data for 250HD and non-transformed dependent variables.

*Significant p-value < 0.05
Table 2.6:
Multiple Linear Regression Model using RIA Data to Predict Serum 25QHD
RIA Model Est. SE Est. p-value Rl
Full Model 0.5482
PS Badge (J) 1.12760 0.20888 0.67776 O.0001*
Skin (forehead) 3.75E-07 1.08E-07 0.32280 0.0009*
VitD Intake (IU) 0.12436 0.06269 0.16904 0.0515
Fall Ref
Winter -0.57209 0.12627 -0.5175 O.0001*
Spring -0.60856 0.16080 -0.5805 0.0003*
Summer -0.37243 0.16190 -0.3435 0.0246*
* Significant p-value < 0.05

187
Table 2.7: Model Prediction of Serum 250HD* in 4 Groups

Low Low High High
reflectance reflectance reflectance reflectance
(dark skin), (dark skin), (light skin), (light skin),
Low sun High sun Low sun High sun
Fall: 56 88 82 128 nmol/L
Winter: 38 61 57 90 nmol/L
Spring: 44 84 65 122 nmol/L
Summer: 58 81 85 118 nmol/L
*Predicted 250HD 1 using LC-MS data with the 20th (low) & 80th (high)
percentiles for sun exposure each season (J), median for diet (200IU) & median
L*for forehead skin reflectance (African & European).
'Equation:
25OHDA0.1 = 1.19778+(0.15948*badgeA0.1)+(0.00000006049589*skinA3.4)
+(0.02054*nldiet)-(0.08238*winter)-(0.08602*spring)-(0.07535*summer)
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Table 2.8: Estimation of Dietary Vitamin D (IU) Intake Needed to Maintain Serum
250HD -75 nmol/L*
Low Low High High
Fall 1,086 0 0 0 IU
Winter 2,114 800 1,029 0 IU
Spring 1,771 0 571 0 IU
Summer 971 0 0 0 IU
*Dietary vitamin D intake predicted using model with LC-MS data (table 2.7) and by
dose response expected based on a supplementation study by Heaney et al. in which they
found a 0.7 nmol/L increase in serum 250HD for every additional 40 IU (1 ug) of
vitamin D ingested (15).
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Chapter 3:
Vitamin D Intake, Skin Reflectance, and Sun Exposure,
using Daily Sun Exposure Logs, to Predict Serum 250HD

190
3.1 Abstract
Individual sun exposure is difficult to quantify and is typically estimated using
non-validated methods. The goals of our study were to validate our daily log as a useful
tool for measuring sun exposure by an objective measure (polysulphone (PS) badge) and
to measure the contribution of sun exposure to vitamin D status as measured by serum
250H-vitamin D (250HD) compared to other sun exposure assessment tools. Our study
was conducted with 4 cohorts of healthy young adults each followed for 7-8 weeks in the
Fall, Winter, Spring and Summer (2006-07) in Davis, CA (38.5 N latitude). Subjects
had a wide range of sun-exposure behavior and skin reflectance. A total of 72 subjects
(-18 per season) enrolled and completed the study. Skin reflectance was measured using
a reflectance spectrophotometer. Dietary vitamin D intake was assessed by food
records. Sun exposure was assessed using daily sun exposure logs and weekly PS
dosimeter badges. Serum 250HD was measured by ultra-performance liquid-
chromatography-tandem mass spectrometry (LC-MS) and by radioimmunoassay (RIA).
Spearman's correlation coefficients were used to look at the association between serum
250HD and different measures of sun exposure including time outside, a sun exposure
index and joules. Regression analysis was used to validate the sun exposure logs against
the PS badges. Multiple linear regression analysis was used to predict serum 250HD
using dummy variables for cohort/season and continuous variables for skin reflectance,
vitamin D intake, and sun exposure using the various measures of sun exposure. The
different measurements of sun exposure were all significant independent predictors of
serum 250HD (p < 0.05). However, joules of sun exposure and the sun exposure index
191
were significantly better predictors of status in the model than time spent outside. Joules
averaged from 5-6 weeks before a blood draw were significantly better predictors in the
model than joules measured less than 5 weeks out. These results show that the
contribution of sun exposure to vitamin D status can be assessed using simple methods.
3.2 Introduction
Vitamin D was originally considered a vitamin because of its' antirachic activity
in cod liver oil and has since kept the name. However, contemporary views actually
classify vitamin D as a prosteroid hormone rather than a vitamin because in mammals
vitamin D3 is made in the skin (1). When sunlight, namely ultraviolet (UV)-B photons,
with wavelengths between 290-315nm, strike 7-DHC in the skin, this results in the
cleavage of the B-ring of the steroid structure forming previtamin D3 (1). This structure
is thermodynamically unstable and undergoes an internal transformation of its' double
bonds which is stimulated by the body's temperature to form vitamin D3 over a few
hours (1). This conversion facilitates it's translocation from the skin into the blood
stream. Vitamin D3 produced in the body and dietary vitamin D (D2 or D3) both
circulate to the liver attached to the vitamin D-binding protein (DBP), ai -globulin, where
they are hydroxylated to 250HD (1). Serum 250HD is measured to determine an
individuals' vitamin D status because the 25-hydroxylase enzyme is not tightly regulated
and therefore, any increase in vitamin D intake or production leads to an increase in
250HD. The unbound form of 250HD enters the kidney tubular cells, where it is
hydroxylated to form l,25(OH)2D3, the active form of vitamin D which is responsible

192
for carrying out most of its' functions, such as, regulating calcium absorption and bone
mineral mobilization in order to maintain calcium homeostasis (1). Extrarenal sites of
production have also been identified in the liver, bone, placenta, macrophages and skin
(2).
Sun exposure is required for vitamin D3 production, however, previtamin D3 and
vitamin D3 can be broken down with further UV-B radiation to inert photoproducts (3,
4). For instance, previtamin D3 can be converted to vitamin D3 or into biologically inert
photoisomers, lumisterol and tachysterol upon further UV-B exposure (3). Once vitamin
D3 is formed it can either enter circulation or isomerize to at least 3 photoproducts,
suprasterol I, suprasterol II, and/or 5,6-trans-vitamin D3 (4). Therefore, sunlight can act
as a regulator of vitamin D3 production and prevent against intoxication.
Sun exposure can be estimated using environmental estimates and/or personal
estimates. However, sun exposure is difficult to quantify because there are many factors
that affect ambient levels on any given day, these include latitude, altitude, day of the
year (season) and time of day, which vary as the solar zenith angle (SZA) from the sun
to the earth changes. On top of a somewhat predictable seasonal cycle, there are other
components in the environment, such as clouds, aerosols, pollution, ozone and albedo
(ie. the fraction of radiation striking a surface that is reflected by that surface), which can
change unpredictably and also affect ambient radiation. Engelsen et al. used a computer
simulation tool and found that a thick cloud cover at the equator could halt dermal
vitamin D synthesis (5). Thick clouds scatter UV-B radiation back to space while thin
clouds actually scatter UV-B radiation toward the earth (6). Therefore, depending on the
type of clouds overhead UV-B radiation can change instantaneously. Thus, sun
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exposure received by individuals depends first on the total available UV-B radiation in
the environment.
On top of environmental factors, personal factors also affect sun exposure, such
as skin pigment, clothing worn and sunscreen use (protective behaviors). Melanin, or
skin pigment, reduces the efficiency of dermal synthesis of previtamin D3 in the skin (7).
In people with lighter skin, 20-30% of the UV-B radiation received is transmitted
through the epidermis and absorbed in the stratum spinosum and basale layers of the
skin (8). However, in darker skin the melanin in the epidermis absorbs UV-B radiation
and less than 5% of the UV-B radiation is transmitted through (8). Consequently,
production in darker skin is predicted to be 40% of Caucasian skin (9). Sunscreens are
used because they prevent against sunburn, skin cancer and skin damage that is
associated with sunlight exposure. However, the UV-B radiation which is responsible
for causing damage to the skin is also responsible for vitamin D synthesis. Sunscreens
absorb the solar UV-B radiation on the surface of the skin before they can reach the
deeper layers of the skin diminishing vitamin D3 formation (10). Clothing also absorbs
UV-B radiation and prevents the dermal synthesis of vitamin D3 in the covered areas
(11). Sunscreen use and clothing even vary seasonally with the weather. For example,
individuals are going to cover-up when the weather is cold. All of these personal factors
must be considered when trying to assess sun exposure on the individual level.
Together, with environmental UV-B information, the contribution of sun exposure to
vitamin D status can be determined.
A recent review of the peer-reviewed literature in relation to sunlight exposure
assessment and the validity of using sunlight exposure questionnaires to quantify vitamin
D status concluded that questionnaires currently provide imprecise estimates of vitamin
D status (12). McCarty emphasized that individual-level sun exposure data is needed
because personal sun protection and behavior has a greater influence on personal UV-B
exposure than do ambient UV-B levels (12). Consequently, results have differed from
studies that included personal-level data with those that included only environmental-
level data (12). Personal sun exposures have been calculated by knowing the amount of
time spent outdoors with respect to specific time of the year and day, use of sun
protection such as sunscreen and clothes, position to the sun (anatomical distribution of
UV-B exposure) and the available ambient UV-B in the environment (12). Again, both
environmental and personal influences need to be considered when assessing sun
exposure. However, studies that assess sun exposure in detail are limited.
Studies have categorized (13, 14) or ranked (15-17) individuals based on their
reported sun exposure using a variation of recalled time, clothing and sunscreen use.
Some studies have only used time (minutes) in the sun to estimate individual sun
exposure without taking into account skin pigment, clothing, sunscreen use or even
ambient UV-B levels. Other studies have set up a sun exposure index (SEI) which
includes time in the sun, clothing and sunscreen (18-20). Average time outdoors per
week is multiplied by usual body surface area (BSA) exposed to the sun, using an
adapted rule of nines for BSA (13). Sunscreen is also taken into account as BSA
covered from the sun. However, most studies use non-validated questionnaires to assess
sun exposure relying on the subjects' memory and most questionnaires only recall sun
exposure over the past week. Most studies either lack vital personal information or do
not take into account ambient UV-B levels. Thus, sun exposure is not accurately
quantified and low correlations to serum 250HD are found.
The objective of our study was to quantify sun exposure over each season using
daily sun exposure logs and to validate our sun exposure log as a useful tool for
measuring sun exposure. The sun exposure log entries were used to calculate joules per
day of UV-B exposure with both the erythemally-weighted spectrum and the vitamin D-
weighted spectrum. Other measures of sun exposure were also considered, such as time
outdoors and the sun exposure index (SEI) which takes into account time outdoors and
body surface area (BSA) exposed. Using multiple linear regression analysis, the
contribution of sun exposure, using the different measurements, to vitamin D status was
assessed, along with vitamin D intake and skin reflectance.
3.3 a Subjects
Subjects were either students or employees recruited from the UC Davis (UCD)
campus. Flyers were posted around campus seeking volunteers with high or low levels
of outdoor activity to participate in the study to obtain a range of typical sun exposure.
E-mails were sent to different student organizations/associations to recruit subjects of
different ethnicities to represent the range of skin pigmentation seen in the US
population. However, because our recruitment was limited to the UCD campus, our
study sample may not be a representative sample of the population. Subjects were
screened for eligibility and included if they had a BMI between 18.5-30, were 19-39
years of age and self-reported high- or low-level daytime outdoor activity. Subjects
were excluded if they reported consistent sunscreen use on all or most exposed skin,
frequent use of tanning beds or use of high-dose vitamin D supplements (excluding
RDA-level multiple vitamin and mineral supplements) within the past 2 months. These
exclusion criteria were selected because of difficulty in assessing sunscreen
application/use and the significant contribution of tanning-bed use or high-dose vitamin
D supplements to vitamin D status. Subjects were also excluded if they reported
pregnancy, or any disease or underlying condition requiring treatment that may affect
vitamin D absorption/metabolism or use of medications that affect vitamin D
metabolism.
3.3 b Study Design

197
asked to maintain their typical routines. The subjects came to the WHNRC to get their
blood drawn and skin reflectance measured at weeks 0,4 and 8 (7 for Fall). Subjects
met with the study coordinator weekly to exchange PS badges and records. The study
coordinator also e-mailed reminders for wearing the PS badge and keeping food records.
3.3 c USDA UV-B Monitoring and Research Program (UVMRP)
Information from the USDA UV-B Monitoring Station at the UC Davis Climate
Center (Davis, CA, 38.5 N) was used to determine ambient UV-B levels in the
environment. The station uses a UV-B Broadband Pyranometer (Yankee Environmental
Systems, Inc., Turners Falls, MA) which measures global irradiance on a continual basis
in the UV-B spectral range of 280-330 nm (14). There are 34 climatologic sites around
the United States including one climate station at the UCD campus. The UV-B radiation
detected by the instrument is converted to visible light and this signal is measured in
W/m2 every 15 seconds and repeated in 3 minute averages. We downloaded the
erythemally-weighted (ie. erythemal-producing UV-B spectrum) measurements from the
USDA UV-B Network website (http://uvb.nrel.colostate.edu/uvb/homejpage.html) and
averaged the values for each hour from 7am-7pm (14). We then converted the hourly
averages to J/m2 to make them comparable to the PS badge measurements and to
determine joules from the daily sun exposure logs.

198
We also obtained vitamin D-weighted (ie. previtamin D3-producing UV-B
spectrum) measurements from the UV-B Monitoring Stations and converted the hourly
averages to J/m2. The vitamin D index was derived from measurements made using a
Yankee Environmental Systems (YES) Inc. UV Multiple Filter Rotating Shadowband
Radiometer (UVMFRSR) (Yankee Environmental Systems, Inc., Turners Falls, MA).
Measurements of the total horizontal irradiance, which are made in seven narrow
bandwidth channels, were used to derive the amount of solar irradiance arriving at the
ground at each nanometer in the 295 to 400 nm range. The surface solar UV irradiance
is integrated over wavelength, weighted by the vitamin D action spectrum resulting in a
single number for each hour and thus, the vitamin D spectrum measurements used in this
study.
3.3 d Daily Sun Exposure Assessment
Subjects kept daily sun exposure logs from 7am-7pm everyday which totaled an
average of 53 days per subject. The daily sun exposure log collected information about
time of day when subjects were outside and time of day was listed hourly as 7-8am, 8-
9am, 9-10am, 10-1 lam, llam-12pm, 12-lpm, 12-lpm, l-2pm, 2-3pm, 3-4pm, 4-5pm, 5-
6pm, or 6-7pm. Next, they were asked to record their location and how many minutes
that they were in the direct sun or shade which was greater than 5 minutes. They also
recorded their outdoor activity for those minutes. Then they had to record what they
were wearing using a key provided to estimate body surface area (BSA) exposed to the
sun during each time period. The key divided the body into 4 sections, (A) head, (B)
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torso, (C) legs, and (D) feet and is shown in Table 3.1. Then within each section, A-D,
the body is progressively covered up and described using numbers. For instance in (B)
torso, Bl equates to nothing worn over the torso, B2 equates to a sports bra, B3 equates
to a tank top, B4 equates to a tee-shirt, B5 equates to a quarter-length sleeved shirt and
finally, B6 equates to a jacket coving all of the torso. In Table 3.1 the corresponding
percent BSA exposed to the sun in the key is shown. The percent BSA exposed was
determined using the "rule of nines" commonly used for burn victims (15) with
confirmation from estimating the BSA exposed for different clothing using a male and
female volunteer and a measuring tape. The subjects also had to record if they were
using sunscreen, what SPF was used and where the sunscreen was applied using the
same key for BSA exposed. They were trained on how to fill out the sun logs and asked
to keep track of their sun exposure as they go and not to change their normal behavior or
use their memory later. They were also asked to time themselves using a watch or cell
phone for accuracy. Subjects periodically turned in their completed sun exposure logs to
check for compliance and to clarify any questions or concerns.
The daily sun exposure logs were used to calculate a daily value in joules for
each subject. This was accomplished by including the average UV-B number (from the
erythemal-producing spectrum and the previtamin D3-producing spectrum) in J/m2 for
each hour that the person was outside using the ambient measurements from the UV-B
Monitoring Station and adjusting this value for minutes outside and BSA exposed (m ).
This gave a UV-B value in joules for each time they reported being outside and the
joules were then totaled for every time they were outside to obtain their daily joules.
Depending on where the subject was located the closest UV-B Monitoring
200
Station measurements were used to determine their ambient UV-B exposure. This is
important because UV radiation varies by location/latitude (134). However, studies have
shown that the UV-B measurements from the Monitoring Stations have provided the
ability to accurately interpolate solar radiation over distances less than 100 km (16, 17).
Grant and Slusser found a 0.7-0.8 linear correlation between locations for 100 km
spacing distance (18). Ambient UV-B measurements were adjusted appropriately for
altitude because UV-B radiation increases with the decreasing amount of UV scattering
and absorbing that occurs in higher altitudes (19). The resulting increase in UV
radiation is described as the altitude effect (AE) (19). Pfeifer et al. analyzed the AE due
to variations in solar elevation, albedo and aerosol properties on UV radiation (19).
Their model showed that depending on the solar elevation and albedo the AE ranges
between 3-7% per 1,000 m (19). Therefore, we used a 5% increase in UV-B radiation
per every 1,000 m increase in altitude. The UV-B measurements were also adjusted for
shade versus direct sun. Turnbull et al. found that tree shade was 55% UV exposure
compared to the full sun, umbrella shade was 52%, a northern facing veranda was 11%
and a car with closed windows was 0% (20). Accordingly, shade UV-B was estimated
to be 50% of direct sun UV-B. Consequently, the personal UV-B measurements
reflected location (latitude and altitude), time of day, minutes in the direct sun or shade
and %BSA exposed to the sun.
Subjects wore polysulphone (PS) dosimeter badges (Kimlin, Australian Sun and
Health Research Lab, Queensland University of Technology, Brisbane, Qld 4059
Australia) once a week as an objective measure of sun exposure in joules and they were
used to validate the sun exposure logs in joules, both adjusted for clothing.
3.3 e Dietary Assessment
consecutive 4-day food records which were kept every other week for a total of 16
records. Food records have previously been validated as an accurate tool to measure
intake through observational studies (21). The 4-day food records included a weekend
day to capture typical intake and days included rotated from Wednesday-Saturday to
Sunday-Wednesday. The subjects were trained on how to keep accurate food records by
a Registered Dietitian (RD). The food records were analyzed for vitamin D content
using the Nutrition Data System for Research (NDSR) Program (2006, University of
Minnesota, Minneapolis, MN) and a daily intake in IU was determined.
3.3 f Skin Reflectance (Pigment) Assessment
202
3.3 g Covariates
Weight and height was measured at week 0,4, and 7 or 8 without shoes using a
3.3 h Serum 25-Hydroxyvitamin D (250HD)
Human Nutrition Research Center (WHNRC) per subject per blood draw (week 0, 4, 7
203
measured on the same day was 5.0% (range, 0.04-10.0%) and for duplicate samples
WF1NRC participates in the Vitamin D External Quality Assessment Scheme (DEQAS)
9.0, Systat Inc, Pt. Richmond, CA). Statistical Analysis Software (SAS version 9.1.3,
Stat Corp, College Station, TX) was used to perform all statistical analyses. Continuous
data was tested for normality using the Kolmogorov-Smirnov test and variables that
204
were not normally distributed (p < 0.05) were transformed using Box-Cox
transformations. Analysis of variance (ANOVA) was performed for comparisons
between seasons/cohorts. Duncan tests were performed for pair-wise comparisons. If a
variable couldn't be transformed to a normal distribution then a nonparametric test was
associations between variables.
groups each season. Covariates, such as age, gender, BMI, oral contraceptive use,
ethnicity, self-reported sun-level, and athlete were explored in the model along with
interactions, such as skin x sun, sun x diet, skin x diet, skin x sun x diet, and sun x each
season. Pitman's test was used to determine if a sun exposure measurement in the model
was a significantly better predictor than another sun exposure measurement. All p
values < 0.05 were considered statistically significant.
3.5 Results
Approximately 2/3rd (n= 45) of the subjects reported their usual sun exposure as "low"
205
and l/3 r (n= 27) as "high" upon entering the study. The average age of the subjects was
24 ± 4 years and the average BMI was 23 ± 3.
3.5 a Daily Sun Exposure Logs
Daily UV-B exposure was assessed for each subject using sun exposure logs
from 7am to 7pm. Individual doses (J) were determined by adjusting ambient
measurements (J/m2) for time and exposed skin. UV-B exposure was also assessed
objectively by PS badges worn by subjects once a week from 7am to 7pm. The average
sun exposure logs kept on the same day were significantly correlated to the average PS
badge measurements in joules (r = 0.94, p < 0.0001) (Figure 3.2) demonstrating the
ability of the sun exposure log to accurately measure personal UV-B exposure.
Environmental UV exposure varies seasonally as the earth rotates around the sun
and the solar zenith angle (SZA) changes. Solar wavelengths also strike the earth at
different angles depending on the SZA. For instance, at larger SZAs (eg. wintertime,
early morning and late afternoon) there is greater attenuation of UV-B than UV-A
wavelengths due to ozone filtering (25). Accordingly, the vitamin D-producing
spectrum is confined to the UV-B range while the erythemal producing spectrum
extends into the UV-A spectrum and therefore, levels of vitamin D UV reduce faster
than levels of erythemal UV with increasing SZA (25). Therefore, vitamin D production
is greatest, relative to erythemal, with smaller SZAs, such as during the summer and
around noontime. Ambient UV-B exposure was collected from the UV-B Monitoring
station. The average daily ambient irradiance in J/m2 for the vitamin D-weighted and
206
erythemally-weighted spectrum are shown in Figure 3.3 for each study week. Ambient
levels in the Fall were significantly lower than the levels in the Winter and both the Fall
and Winter were significantly lower from both the Spring and Summer levels which did
not differ from one another (Figure 3.3). This pattern was true for both the erythemal
and vitamin D measurements. The UV-B available for vitamin D production is higher
during the Spring and Summer relative to the Fall and Winter. In Figure 3.4, the UV
intensity for one day during each cohort is shown from 7am to 7pm using averages for
each hour increment. There is more UV-B available for vitamin D production during the
middle of the day in all seasons and the ratio of vitamin D UV to erythemal UV was 1.7
times in the Winter and almost 2 times higher in the Summer. Consequently, in the Fall
and Winter because there is less available UV-B, longer exposure would be necessary.
However, the actual dose received by the individual depends on how much skin they
have exposed.
The individual daily doses in erythemally-weighted joules as determined from
the daily sun exposure logs which reflect BSA exposed for each subject are shown in
Figures 3.5a, 3.6a, 3.7a and 3.8a and the transformed joules are shown in Figures 3.5b,
3.6b, 3.7b and 3.8b. The vitamin D-weighted joules and transformed joules are shown
in Figures 3.9ab, 3.10ab, 3.11ab and 3.12ab. The daily joules were averaged for each
subject and the averages were then used to determine the median value for each cohort.
For the erythemal spectrum, the median dose for Fall (11 J) was not different than the
median dose for Winter (11 J) (Figure 3.13). Both Fall and Winter were significantly
lower than either Spring (167 J) or Summer (161 J), which did not differ from one
another (Figure 3.13). The vitamin D spectrum followed the same pattern as the
207
erythemal spectrum with the median dose for Fall (13 J) and Winter (16 J) not differing
from one another, however, both Fall and Winter were significantly lower than either
Spring (289 J) or Summer (295 J), which did not differ from one another (Figure 3.14).
Therefore, individual sun exposure varied by cohort which reflected ambient UV-B
levels, time of day and clothing. The ambient UV-B levels were lower in the Fall and
Winter and subjects were more likely to wear warmer clothing and have less BSA
exposed than in the Spring and Summer. Thus, the joules reflect both environmental and
personal information.
Dermal vitamin D synthesis via sun exposure contributes to circulating levels of
serum 250HD. The average erythemally-weighted joules were positively associated
with the average serum 250HD from the LC-MS procedure (r = 0.36, p =0.0018, Figure
3.15, Table 3.3). The average vitamin D-weighted joules were also positively
associated with serum 250HD (r = 0.34, p =0.0032, Figure 3.16, Table 3.3). Thus, sun
exposure is a positive contributor to vitamin D status.
To evaluate the daily sun exposure logs and the association with serum 250HD
for each season/cohort, a separate correlation was performed for each using an average
erythemally-weighted value in joules and an average serum 250HD value. As seen in
Figure 3.17, the Fall and Winter joules were not significantly associated with serum
250HD but there was a significant correlation in the Spring (r = 0.52, p = 0.0195) and
Summer (r = 0.73, p = 0.0005). Separate correlations were also performed for the
average vitamin D-weighted joules and the average serum 250HD. Joules in the fall
were not significantly associated with serum 250HD but there was a significant
correlation in the Winter (r = 0.48, p = 0.0496), Spring (r = 0.49, p = 0.0293) and

Summer (r = 0.77, p = 0.0002) (Figure 3.18). Thus, sun exposure was more important
in the Winter, Spring and Summer cohorts when there is more available ambient UV-B
irradiance.
Individual UV-B doses can be defined as the percent received from the available
UV-B in the environment. The daily sun exposure logs were used to calculate joules per
day (vitamin D-weighted spectrum) for the subjects and the UV-B Monitoring Station
measured the total J/m2 in the environment each day. To evaluate the association of the
average percent of individual UV-B exposure from the total ambient UV-B to average
serum 250HD, a correlation was performed. As seen in Figure 3.19, the percent of
ambient UV-B was significantly correlated to serum 250HD (r = 0.48, p < 0.0001,
Table 3.3). In Figure 3.20, the percent of ambient UV-B was significantly correlated to
serum 250HD in the Winter (r = 0.55, p = 0.0225), Spring (r = 0.51, p = 0.0231) and
Summer (r = 0.73, p = 0.0005) cohorts but not in the Fall cohort. Thus, the percent of
ambient UV-B exposure was associated with serum 250HD, especially in the cohorts
with higher available ambient UV-B. The average percent of ambient was 4 ± 5% and
this is consistent was other studies that have found that adults that work indoors receive
- 3 % (2-4%) of the annual terrestrial UV radiation (26).
To determine the strongest relationship between the time in which sun exposure
(erythemal spectrum) is measured and serum 250HD, sun exposure was averaged for
each week of the study working backwards from the week 7 (Fall cohort) or 8 (Winter,
Spring, Summer cohorts) serum 250HD measurement (Table 3.4). The average
erythemally-weighted joules were used in these correlations because overall the values
had a slightly higher correlation to serum 250HD than the vitamin D-weighted joules.
The average joules in week -1 from the final blood draw were not significantly
associated with serum 250HD, using the LC-MS data (p > 0.05, Table 3.4). Estimating
only one week of sun exposure before a blood draw may not be enough to find a
relationship to serum 250HD because physiologically the half life of 250HD is ~3
weeks (27). Average sun exposure for weeks -2 through -7 alone were significantly
associated with the final serum 250HD (Table 3.4). Sun exposure for the average of
weeks -1 and -2 were significantly correlated to serum 250HD, along with weeks -1
through -3, weeks -1 through -4 (month average), weeks -1 though -5, weeks -1 through
-6, and the average for the whole study (7 weeks for the Fall cohort and 8 weeks for
Winter, Spring and Summer cohorts) (Table 3.4). Thus, when solely evaluating the
contribution of sun exposure to vitamin D status it would be better to measure joules at
least 2 weeks prior to the blood draw.
3.5 b Other Measures of Sun Exposure
As subjects entered the study, they were asked to characterize their typical sun
exposure as either "low" or "high". The sun exposure logs were analyzed to determine
if their perception matched their actual sun exposure in joules. Subjects who considered
themselves "low" (111±137J) had significantly lower sun exposure than those who
considered themselves "high" (529 ±591 J) for each cohort. Regression analysis was
used to predict vitamin D-weighted UV-B exposure from self-identified sun level
(low=0, high=l) and dummy variables for cohort. Level and cohort (Spring and
Summer) were significant predictors of sun exposure (R2= 0.76, p < 0.0001, Fall cohort
used as the reference, data not shown). Therefore, self-identified level of sun exposure
corresponded closely with actual sun exposure from the daily sun exposure logs.
Time outside has been used as a marker for sun exposure. Therefore, we
determined the minutes spent outside in the direct sun (7am-7pm) from the daily sun
exposure logs to see how this measure of sun exposure compares to the other measures.
There was not a significant difference in minutes outside between the cohorts (p > 0.05,
Figure 3.21). This would be expected because time does not reflect ambient UV-B
levels available for vitamin D production or BSA exposed to the sun. However, there
was a significant correlation of average time outside to average serum 250HD (r = 0.38,
p = 0.0009, Figure 3.22, Table 3.3). Time spent outside in minutes was narrowed from
7am-7pm to 9am-3pm and 10am-2pm to see if there was a higher correlation to serum
250HD when the SZA is smaller. Although time from 9am-3pm and 10am-2pm was
significantly correlated to serum 250HD, the correlation coefficient for 9am-3pm (r =
0.39, p = 0.0008, Table 3.3) closely matched the 7am-7pm coefficient and the
correlation coefficient from 10am-2pm was the same as 7am-7pm (r = 0.38, p = 0.0009,
Table 3.3). Thus, time outside was not different between the cohorts but there was an
association with serum 250HD, likely reflecting sun exposure.
To evaluate the association of time outside (7am-7pm) with serum 250HD for
each season/cohort, a separate correlation was performed for each cohort using an
average time in minutes and an average serum 250HD value. As seen in Figure 3.23,
time outside in the Fall, Winter or Summer cohorts was not significantly associated with
serum 250HD but there was a significant correlation in the Spring cohort (r = 0.51, p =
0.0228). Thus, time outside was only associated with serum 250HD in the Spring
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cohort and does not reflect how much personal sun exposure is actually received because
ambient UV-B levels and BSA exposed to the sun is not taken into account. Therefore,
a significant association with serum 250HD was not found for three of the four cohorts.
Body surface area (BSA) exposed to the sun is important because the more skin
which is exposed the more dermal synthesis that will occur upon exposure to the sun.
The subjects were given a key to record what they wore when they went outside which
corresponded to percent BSA exposed (Table 3.1). The average percent BSA exposed
was determined every day that a subject went outside and the average did not take into
account days in which the subjects did not go outside. The percent BSA exposed also
does not take into account actual BSA. In the Fall and Winter cohorts, the median
percent BSA exposed (10%) corresponded to the face, neck and hands being exposed to
the sun (Figure 3.24). In the Spring cohort, the median percent BSA exposed (24%)
corresponded to the face, neck, arms and hands (~t-shirt) being exposed to the sun
(Figure 3.24). In the Summer cohort, the median percent BSA exposed (28%)
corresponded to the face, neck, arms, hands and legs (~t-shirt and shorts covering the
thighs) being exposed to the sun (Figure 3.24). The percent BSA exposed was not
significantly different between the Fall and Winter cohorts, however, in both the Fall and
Winter cohorts there was less BSA exposed than in the Spring and Summer cohorts
(Figure 3.24). However, in the Summer cohort there was more BSA exposed than in the
Spring cohort (Figure 3.24). This was expected because when the weather is warm,
people wear less clothing.
Studies have used a sun exposure index (SEI) as a marker for sun exposure. The
SEI is calculated by multiplying time outside and BSA exposed. Using the daily sun
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exposure logs, we calculated an average SEI for each subject. There wasn't a difference
in the SEI for the Fall and Winter cohorts but they were significantly lower than the
Spring and Summer cohorts (Figure 3.25). However, the Spring and Summer SEI's
were not different from one another (Figure 3.25). Accordingly, the SEI for Fall and
Winter was significantly lower than the Spring and Summer as expected because the
percent BSA exposed was also significantly lower in the Fall and Winter. In Figure
3.26, the average SEI was more significantly correlated to the average serum 250HD (r
= 0.47, p < 0.001, Table 3.3) than time outside. Thus, the SEI is a better marker of sun
exposure than time alone because it takes into account BSA exposed and likely reflects
the dose of sun exposure received.
Sunscreens block the dermal synthesis of previtamin D3 (10). Subjects kept track
of sunscreen use using the same key as clothing worn (Table 3.1) to estimate BSA
covered. Depending on where the subject applied sunscreen we assumed that BSA
exposed to be zero. Then assuming that sunscreen loses efficiency over time, we
estimated that sunscreen would wear off after 4 hours and then BSA would be exposed
again, accordingly. Using the SEI, with sunscreen taken into consideration, we
examined the relationship to serum 250HD. We found that the average SEI with
sunscreen was also significantly associated to the average serum 250HD (r = 0.45, p <
0.0001, Table 3.3) although slightly less than SEI alone. Typically sunscreens aren't
applied properly (28) and do not fully prevent previtamin D3 production. Subjects that
applied sunscreen likely did so because they knew they were going to be outside longer
than normal and, therefore, sunscreen use could reflect more sun exposure. Since there
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was not a large difference when sunscreen was taken into account, we used the sun
exposure measurements without considering sunscreen.
To evaluate the association of the SEI with serum 250HD for each
season/cohort, a separate correlation was performed for each cohort using an average
SEI and an average serum 250HD value. As seen in Figure 3.27, the SEI in the Fall
and Winter cohorts were not significantly associated with serum 250HD but there was a
significant correlation in the Spring (r = 0.57, p = 0.0085) and Summer (r = 0.69, p =
0.0016) cohorts. Thus, the SEI was significantly associated with serum 250HD in the
Spring and Summer cohorts when individuals spend more time outside and exposure
more BSA to the sun.
Average time outside and the average SEI were significantly correlated (r = 0.86,
p < 0.0001, data not shown). The average SEI and the average erythemally-weighted
joules were also significantly correlated (r = 0.91, p < 0.0001, data not shown). The
average time outside and the average erythemally-weighted joules were correlated but to
a lesser extent (r = 0.96, p < 0.0001, data not shown). All 3 sun exposure measures are
correlated likely because they build on each other. First, time outside is the most simple
measure, then the SEI multiples time by BSA exposed and finally, the measurement in
joules accounts for time, BSA exposed and the ambient UV-B levels. Thus, the most
accurate measure of sun exposure is to use joules per subject.
3.5 c Other Information Collected From the Daily Logs

The sun exposure logs also gathered information about daily outdoor activities
and daily multivitamin use. As shown in Figure 3.28, the majority of time outside was
spent walking (32%) and then biking (26%). Ten percent of the time subjects were
standing, 8% of the time they were sitting and 7% of the time they were swimming. As
for multivitamin use, 41% of the subjects in the Fall Cohort, 29% in the Winter Cohort,
40% in the Spring Cohort and 22% in the Summer Cohort took at least one multivitamin
during the course of the study (Figure 3.29).
3.5 d Dietary Vitamin D Intake
Vitamin D intake from food and supplements was measured 4 times for each
cohort. No differences were seen among their measurements within each cohort. The
median intakes of vitamin D for each cohort were 220IU for Fall, 171IU for Winter,
181IU for Spring and 206 IU for Summer which did not differ (p > 0.05) (Data not
shown). Therefore, dietary vitamin D intake did not change over time within individuals
and did not differ among cohorts.
Vitamin D from the diet contributes to circulating levels of 250HD. It was
therefore not surprising that vitamin D intake was significantly correlated to serum
250HD (r = 0.31, p = 0.0082, data not shown). To examine the association with diet
each season/cohort, a correlation was performed between average vitamin D intake and
average serum 250HD for each cohort. As seen in Figure 3.30, vitamin D intake was
only significantly associated with serum 250HD in the Winter cohort (r = 0.51, p =
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0.0376). Thus, vitamin D intake may be a more significant contributor to vitamin D
status when sun exposure is lower, such as in the wintertime.
3.5 e Skin Reflectance (Pigment)
Skin reflectance was assessed at three sites, one typically protected from the sun
(inner arm), and two typically exposed to the sun (hand and forehead). Differences in
sun exposure can cause differences in skin pigmentation at different sites or at the same
site over time. There were no significant changes in reflectance measured at the three
sites over time within each cohort (adjusted for sun level, data not shown, p > 0.05).
However, the average inner arm reflectance measurement was significantly higher
(lighter pigment) than the average hand and forehead measurement after adjusting for
ethnicity (data not shown). In summary, the inner arm reflectance was higher than the
other sites, but skin reflectance did not significantly change over time in the three sites.
3.5 f Vitamin D Status
Serum 250HD levels as measured by RIA and LC-MS were significantly
correlated (r = 0.89, p < 0.0001, data not shown), however, the 250HD values from the
LC-MS procedure are used in the analyses because the LC-MS procedure is more
accurate than the RIA procedure.
Average serum 250HD, using LC-MS data, in the Fall (97 ± 38 nmol/L) was
significantly higher than the Winter (70 ± 35 nmol/L) while average serum 250HD in
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the Spring (81 ± 39 nmol/L) and Summer (85 ± 38 nmol/L) were intermediate but not
significantly different from each other or Fall or Winter (data not shown). Accordingly,
only serum 250HD in the Fall and Winter cohorts were significantly different from each
other, likely corresponding to changes in the ambient UV-B intensity available for
vitamin D production. We would have expected serum 250HD in the Spring and
Summer to be significantly higher than Fall or Winter, however, separate individuals
were in each cohort.
3.5 g Models Predicting Vitamin D Status
The different measures of sun exposure were used independently in a model to
predict vitamin D status (LC-MS data), along with continuous variables for vitamin D
intake, skin reflectance and dummy variables for cohort (Table 3.5). Time in the direct
sun was the most significant predictor of vitamin D status when used for sun exposure in
the model. After time, skin reflectance was the highest predictor and then vitamin D
intake. Time along, with skin reflectance, vitamin D intake and cohort, 46% of the
variation in serum 250HD was explained (Table 3.5). When the SEI was used in the
model, it was also the most significant predictor of status and 53% of the variation was
explained along with skin reflectance, vitamin D intake and cohort (Table 3.5). Using
self-identified level of sun exposure (High = 1, Low = 0) explained 50% of the variation
in serum 250HD (Table 3.5). When the vitamin D-weighted joules and the
erythemally-weighted joules were used 52% and 53%, respectively, of the variation in
serum 250HD was explained (Table 3.5). Table 3.6 shows the model with the vitamin
D-specific joules to predict status using the RIA data and 53% of the variation in serum
250HD was explained. All of the different measures of sun exposure were significant
predictors of serum 250HD and had the highest standardized coefficients followed by
skin reflectance and then vitamin D intake. The different sun exposure measurements
are all useful tools for measuring sun exposure and they all contributed to vitamin D
status. However, to determine which sun exposure measurement in the full model (with
skin reflectance, vitamin D intake and cohort) was the most significant predictor of
vitamin D status, the different models were compared. As seen in Figure 3.31, the
models which used the PS badge joules, level, erythemally-weighted joules, vitamin D-
weighted joules, percent ambient and the SEI as measures of sun exposure were not
significantly better predictors of vitamin D status in the full model than one another.
The SEI was a significantly better predictor in the model than using the SEI with
sunscreen (Figure 2.31). Using time from 10am-2pm in the model was not a
significantly better predictor than time from 7am-7pm, time from 9am-3pm and level.
Consequently, the most difficult tool to use is the daily sun exposure log which takes
into account the ambient UV-B levels every day. Since the joules (vitamin D-weighted
and erythemally-weighted) were not significantly better predictors than the SEI in the
model, the SEI would be an accurate measurement to use and is an easier tool to use.
However, joules would be a comparable measure of sun exposure between different
latitudes.
Covariates and interactions were tested in the full model, with vitamin D-
weighted joules as the measure of sun exposure, forehead skin reflectance, vitamin D
intake and cohort. Independently, BMI, age and gender were not significant in the full
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model. Several interactions were tested in the model and none were significant
(including sun x each season, sun x skin, sun x diet, skin x diet, or skin x sun x diet).
However, several covariates were independently significant in the full model including,
sunscreen use greater than 5 times (No = 0, n = 46 or Yes = 1, n = 26), athlete (No = 0, n
= 60 or Yes = 1, n = 12), oral contraceptive (OC) use (No = 0, n = 56 or Yes = 1, n =
16), and ethnicity/ancestry (SE Asian and N Asian were significant) (Table 3.7). In the
model with sunscreen use (greater than 5 times) as a covariate, 56% of the variance in
250HD was explained. Sunscreen use actually predicts higher serum 250HD levels,
most likely from use with anticipated outdoor activity and improper application and
therefore, higher sun exposure received. With athlete as a covariate instead, 57% of the
variance was explained. Being an athlete predicts higher serum 250HD likely because
most athletes train and compete outside in the sun. In the model with OC use, 59% of
the variance in 250HD was explained. OC users had significantly higher serum 250HD
levels than non-users (p < 0.05, data not shown). In the model with ancestry/ethnicity,
63% of the variance in 250HD was explained, however, only SE Asian and N Asian
were significant negative predictors of 250HD. Sun exposure had the highest
standardized coefficient in the all the models except for the model with athlete where
athlete had the highest coefficient. Accordingly, athletes had higher levels of sun
exposure than non-athletes (p < 0.05, data not shown). Thus, there were other variables
that contributed to vitamin D status in the full model.
To see if any of the weekly sun exposure measurements (erythemally-weighted)
from the daily log were significantly better predictors of serum 250HD, each
measurement was used in the full model, along with vitamin D intake, skin reflectance
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and cohort. The averages of the individual weeks were not significantly better predictors
of serum 250HD from one another (data not shown). However in Figure 3.32, the
average of weeks -1 through -5 and weeks -1 through -6 were significantly better
predictors than weeks -1 through -2, -3 and -4. They were not significantly better
predictors than the study average (weeks -1 through -7 or -8) which was not significantly
better than weeks -1 through -3 or -4. Thus, when evaluating the contribution of sun
exposure to vitamin D status it would be better to measure joules at least 5 weeks prior
to the blood draw and it may not be necessary to measure more than 6 weeks out.
We used the regression model with the vitamin D-weighted joules as the measure
of sun exposure to predict serum 250HD (LC-MS data) in subjects with low and high
levels of skin reflectance and in subjects with low and high levels of sun exposure (4
groups). Low and high sun exposure was defined as the 20th (low) and 80th (high)
percentiles from each cohort/season using vitamin D-weighted joules. In Table 3.8, the
20th (low) and 80th (high) percentiles for sun exposure are shown for each cohort along
with the relative time and BSA exposed. On average, 24 minutes in the direct sun with
~15% BSA (ie. face, hands and arms (t-shirt)) exposed constituted low sun exposure
while 113 minutes in the direct sun with -26% BSA (ie. face, hands, arms (tank top))
exposed constituted high sun exposure. Vitamin D intake was defined as the median
value of 200 IU for all seasons. Skin pigment was defined as the median reflectance
value (L*) on the forehead from African Americans (Low, L*= 42) or Europeans (High,
L* = 63). Using these variables, serum 250HD (LC-MS data) was predicted for each of
the 4 groups in each season as shown in Table 3.9. Using 75 nmol/L as the cut-off for
vitamin D insufficiency, the group with low reflectance (dark skin) and low sun
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exposure would be insufficient all year around while low reflectance (dark skin) and
high sun exposure group would be at risk of insufficiency in the Winter. The group with
high reflectance (light skin) and low sun exposure would be insufficient in the Winter
and Spring and only the high reflectance (light skin) and high sun exposure group would
be protected from insufficiency year around based on the model prediction. Thus, three
of the four groups would need to either increase their vitamin D intake or their sun
exposure to decrease their risk for insufficiency.
3.5 h Model Estimating Vitamin D Intake Needed To Maintain a Healthy
Vitamin D Status
In Table 3.10, vitamin D intake needed to maintain serum 250HD at -75 nmol/L
was determined using our model which estimated serum 250HD (LC-MS data) in 4
different groups throughout the year and by using the response to supplementation
reported by Heaney et al., a 0.7 nmol/L increase in serum 250HD for every additional
40IU (1 ug) of vitamin D ingested (29). Individuals with low reflectance (dark skin)
and low sun exposure need the highest intakes to reach the level of sufficiency, 75
nmol/L, year around. They would need anywhere from 857 IU to 1,829 IU of vitamin
D/day. Individuals with high reflectance (light skin) and low sun exposure would need
800 IU in the Winter and 114 IU in the Spring and for individuals with low reflectance
(dark skin) and high sun exposure 171 IU would be needed in the Winter. Thus, the
vitamin D intake needed to maintain a healthy serum 250HD level (> 75 nmol/L) varies
by season, as well as level of sun exposure and level of skin reflectance/pigment with
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those having low skin reflectance (dark skin) and low sun exposure needing the highest
vitamin D intakes.
3.5 i Model Estimating Joules of Sun Exposure Needed To Maintain a
Healthy Vitamin D Status
In Table 3.11, our model was used to estimate vitamin-D weighted joules needed
to maintain serum 250HD levels at ~75 nmol/L in individuals with low and high levels
of skin reflectance (2 groups) for each season. In the model vitamin D intake was
defined as the median value of 200 IU for all seasons. Skin pigment was defined as the
median reflectance value (L*) on the forehead from African Americans (Low, L*= 42)
or Europeans (High, L* = 63). Serum 250HD was set at 75 nmol/L, the level of
sufficiency. Individuals with low reflectance (dark skin) need more joules per day in
every season than those with high reflectance (light skin) because melanin decreases the
dermal synthesis of vitamin D. In Table 3.12, the joules from the model prediction were
used to determine time needed in the direct sun to maintain serum 250HD at -75
nmol/L. Time was estimated from the joules using average ambient J/m2 from 12pm-
lpm and an average BSA of 1.75 m2 with 20% BSA exposed each season. Twenty
percent BSA exposed corresponds to the face, neck, hands and arms (t-shirt) exposed to
the sun and was used across all seasons although in the Fall and Winter less BSA may be
exposed due to colder weather. It was determined that individuals with low reflectance
(dark skin) need 6-9 times more time in the sun around 12pm-lpm each day compared to
those with high reflectance (light skin) because of the melanin in their skin. Some of the
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exposure times or the percent BSA exposed may not be realistic or reasonable to
achieve; therefore, dietary or supplementary vitamin D will be needed to maintain
individuals at a healthy status instead of sun exposure.
3.6 Discussion
Daily sun exposure logs were collected for 7-8 weeks each cohort/season
including location, time of day, time in the direct sun or shade, clothing worn and
sunscreen use. Ambient J/m2, either erythemally-weighted or vitamin D-weighted, was
collected daily from a UV-B Monitoring Station for each hour from 7am-7pm. Every
time a subject reported being outside, the hourly average of the ambient J/m was
adjusted for the percent of time in the sun for that hour and adjusted for their body
surface area (BSA) exposed to the sun using a similar estimation as the rule of nines.
This converted the ambient UV-B level (J/m2 x m2 = J) into a personal UV-B dose of
joules per day. To validate the sun exposure logs we compared the measurement to an
object measure of sun exposure (ie. PS badge). The sun exposure logs were significantly
correlated to the PS badge measurements in joules worn on the same day (r = 0.94, p <
0.0001) (Figure 3.2) reinforcing the ability of the sun exposure log to accurately
measure personal UV-B exposure. This method of assessing sun exposure is novel
because it encompasses ambient UV-B levels which reflect latitude, altitude, season and
time of day and personal information on time in the direct sun or shade and
clothing/sunscreen to obtain a single daily value in joules. Consequently, joules of sun
exposure would be comparable across studies.

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Other studies have compared their sun exposure questionnaires to objective
measures of sun exposure. Chodick et al. assessed how well a 7-day record of time
spent outdoors compared to PS badges worn each of the seven days (30). In a linear
regression model, time spent outdoors was associated with an increase of 8.2% in the
badge exposure with every hour spent outdoors (30). They found a significant
correlation between records and personal UV-B measurements (37). Sullivan et al. had
young girls keep track of their outdoor activities for one day and wear a PS badge on the
same day (31). Their questionnaire assessment took into account the changing strength
of UV-B radiation throughout the day by using average UV-B each hour (7am-5pm)
from the USDA UV-B Monitoring Program in Presque Isle, Maine (31). They showed a
correlation between PS badges and self-reported minutes outdoors adjusted for the time
of day (r = 0.64) in adolescents (31). Dwyer et al. compared estimates of erythemally
effective doses (EED) obtained from PS badges worn on 4 consecutive weekend days in
late spring to several questions on habitual sun exposure in 14-15 year olds (32). The
Pearson correlation coefficients for the association with EED were 0.36 for girls and
0.23 for boys for the question "During weekends and school holidays, how much time
do you spend in the sun each day" (32). They conclude that habitual sun exposure in
teens was reported with an acceptable degree of reliability and validity (32). In a study
in Australia, O'Riordan et al. compared UV exposures from diaries to dosimeter
readings (33). They found statistically significant yet modest correlations for mothers (r
= 0.32) and children (r = 0.34) (33). In comparison, we found a higher correlation
between the PS badges and the sun exposure logs (r = 0.79). We adjusted both
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measurements for doses received in joules and we also had more comparisons per
subject and across all seasons.
As subjects entered the study, they categorized themselves as "high" or "low"
sun exposure. Their self-categorization matched their actual sun exposure from the daily
logs. For instance, subjects who considered themselves "high" sun exposure (529 ±591
J) had higher levels of sun exposure than those who considered themselves "low" (111 ±
137 J). This self-identified sun level was used in the full model as the sun exposure
measurement to predict vitamin D status and it was a significant predictor of serum
250HD (R2 = 0.50, p < 0.0001, Table 3.5), demonstrating the ability of a simple tool to
quantify sun exposure. Lips et al. developed a questionnaire using two categories of
time outdoors (little or frequent) and two categories of reported sun exposure level (low
or high) to get a sunshine score of low, intermediate or high to categorize people (34).
Salamone et al. used this categorization with another questionnaire that asked about time
spent outside and sunscreen use over the past week (35). They used this to categorize
the sun exposure levels of healthy elderly women from one to nine and found a
significant difference between summer and winter rankings (35). Lawson et al. used a
14-point scale assigned to the number of times a week that the subject went shopping,
visiting, gardening or on holiday (36). The scores were used to assess the relative
variation in potential UV exposure not an actual amount of time spent outdoors (36).
They found that the outdoor score and 250HD was significantly correlated in August-
September (r = 0.62, p < 0.01) (36). Brot et al. used never, occasionally and regularly
for categorizing sun exposure and in a multiple linear regression model they found that
sun exposure (never = 0, occasionally/regularly =1) was a significant predictor of

225
250HD (37). In the whole population sun exposure (occasionally/regularly) was
associated with 27.6% higher serum 250HD (37). Thus, ranking or categorizing
individual sun exposure can significantly capture differences in sun exposure.
Using the daily sun exposure logs, we examined other measures of sun exposure,
such as time outside and the sun exposure index (SEI) which encompasses time and
BSA exposed. Time outside significantly correlated to serum 250HD (r = 0.38, p =
0.0009, Figure 3.22). Villareal et al. also found a significant correlation between time
spent outdoors and serum 250HD (r = 0.23) with the time between 12pm-2pm showing
the highest correlation to serum 250HD (r = 0.33) (38). A study in Korean women also
found that serum 250HD correlated with sun exposure between 12pm-2pm as assessed
by time spent outdoors (r = 0.33, p < 0.0001) (39). We narrowed the time to 9am-3pm
and 10am-2pm but this did not seem to improve the correlation coefficient (Table 3.3).
Tangpricha et al. also found a weak but positive relationship between time spent
outdoors during the summer and 250HD levels (r = 0.20, p = 0.001) (40). In the
Netherlands (52°N), van der Meer et al. found significant associations between season,
area of uncovered skin and preference for the sun but not for time spent outdoors (41).
They reasoned that the effect of clothing and skin pigmentation might overrule the effect
of time spent outdoors (41). HyppOnen and Power found that time spent outdoors was
strongly associated with 250HD during the Summer and Fall but there was no apparent
association during the winter months (p < 0.0001) (42). We found that time outside was
not significantly correlated to serum 250HD in the Fall, Winter and Summer and was
only significant in the Spring, limiting the usefulness of time outside as a measure of sun
226
exposure (Figure 2.23). Time alone does not take into account the ambient UV-B levels
or BSA exposed to the sun.
Using the daily sun exposure logs to calculate a SEI (mins x BSA exposed), we
found a significant correlation of the average SEI to the average serum 250HD (r = 0.47,
p < 0.001, Figure 3.26). For the individual cohorts, the SEI was significantly associated
with serum 250HD in the Spring (r = 0.57, p = 0.0085, Figure 3.27) and Summer (r =
0.69, p = 0.0016, Figure 3.27) when individuals spend more time outside and expose
more BSA to the sun. Thus, the SEI is a better marker of sun exposure than time alone
because it takes into account BSA exposed and likely reflects the dose of sun exposure
received. Sowers et al. used a SEI to rank women based on self-reported time outside
adjusted for percent BSA exposed considering sunscreen but not time of day and found
that it was correlated to 250HD (r = 0.26) (43). Barger-Lux and Heaney looked serum
250HD in outdoor workers (n = 26) who had just completed a summer season of
extended outdoor activity and -175 days later after the winter sun deprivation in Omaha,
Nebraska (13). They examined the relationship between three measures of sun exposure
and the summer increment in 250HD and found a significant correlation for hours of
sun exposure per week (r = 0.39, p < 0.05), for fraction of BSA exposed (r = 0.66, p <
0.001) and for the SEI (r = 0.67, p < 0.001) (13). Limitations include recalled sun
exposure data, no consideration of sunny versus overcast days, time of day or time per
exposure (13). On the other hand, Binkley et al. used this SEI and didn't find a
correlation with 250HD, even though the lowest quartile of 250HD had a lower index
than the rest of the cohort (p < 0.05) (44). Atli et al. used a questionnaire similar to the
SEI to collect information about clothing and level of sunlight exposure times called the
227
"benefiting from ultraviolet index (BFUI)" (45). The BFUI represented the degree of
sunlight exposure calculated as the ratio of sunlight exposure to clothing using points
(45). For instance for sun exposure, 1 point was given for not being exposed directly to
the sun and 4 points was given for being exposed to the sun all day long (45). For
clothing, 1 point was given for the least amount of clothing and 4 points for being
covered up (45). The BFUI = points for sun exposure / points for sunshine prevention
capacity of the outfit (45). They found a positive correlation between 250HD and BFUI
(r = 0.34, p < 0.001) (45). Thus, taking into account more than just time is helpful when
assessing sun exposure because they are other factors to consider, such as BSA exposed.
Sun exposure and time spent outdoors are better predictors of 250HD than is
dietary vitamin D intake (47). Sowers et al. found that 250HD levels were more closely
correlated with estimated sun exposure (r = 0.26) than estimated dietary intake (119 ±
148 IU) (r = 0.11) and supplement use (319 ± 463 IU) (r = 0.21) in women aged 20 to 80
(43). However, dietary intake was assessed from one 24-hour recall (43). Salamone et
al. evaluated the contribution of vitamin D intake and sunlight exposure to plasma
250HD in free-living elderly women during both the Summer and the Winter (35).
Subjects were categorized into high and low vitamin D intakes and then within each
intake group they were categorized into high and low sunlight exposure groups based on
summer exposure (35). In subjects with low sun exposure there was a strong correlation
between vitamin D intake and 250HD in the Summer (r = 0.71, p < 0.01) and the Winter
(r = 0.80, p < 0.01) (35). However, in the high sun exposure group the correlations
weren't as strong (35). A study in Korean women found a significant correlation
between dietary intake and 250HD (r = 0.35, p < 0.0001) (39). Lawson et al. estimated
228
dietary vitamin D from daily food records and found that there was not a seasonal
variation in vitamin D intake, however, average vitamin D intake was significantly
related to 250HD in the winter (r = 0.55, p < 0.02) but not the summer (r - 0.17, NS)
(36). We also found that vitamin D intake was significantly correlated to serum 250HD
(r - 0.31, p = 0.0082) but the correlation of serum 250HD with the different sun
exposure measurements were stronger (ie. SEI, r = 0.47, p < 0.0001, Table 3.3).
Individually, vitamin D intake in the Winter was the only cohort significantly associated
with serum 250HD (r = 0.51, p = 0.0376, Figure 3.30) when ambient UV-B levels are
lower. Similarly, sun exposure was the strongest predictor of serum 250HD in all of the
models (Table 3.5). Thus, sun exposure is the most significant contributor to vitamin D
status.
Using a linear regression model we examined which variables are significantly
associated with serum 250HD and then used the model to predict vitamin D status.
Other studies have set up a similar model to predict vitamin D status. Kim and Moon
found in their regression model, that dietary intake explained 33.6% of the variation in
250HD and time spent outdoors explained 19.7% of the variation (39). Age also had a
significant effect on 250HD (39). However, consumption of vitamin D was not
calculated exactly and had to be assumed (39). This explained more of the variation in
250HD, however, average time spent outdoors (76.5 ±51.5 mins.) may not represent
actual sun exposure since clothing and sunscreen use was not taken into account.
However, we found time outside to be a significant predictor of vitamin D status (Table
3.3). A cross-sectional study looked at predictors of vitamin D status in Turkish
immigrants living in Germany, Germans and Turkish residents of Turkey (48). Using a
229
multivariate regression analysis it was found that women wearing a scarf had a five-fold
higher risk of being insufficient (< 50 nmol/L) and women with multiple children had a
two-fold higher risk (48). An unconditional logistic regression identified the most
important predictors for low 250HD as sex, BMI, lack of sun exposure and living at a
higher latitude (48). Brot et al. used multiple linear regression analysis to predict
250HD with a continuous variable for dietary vitamin D and categorical variables for
tanning bed use (no = 0 or yes =1), supplement use (no = 0 or yes =1), and whole body
sun exposure (never = 0 and occasionally/regularly =1) (37). Dietary vitamin D intake,
vitamin D supplementation (200IU), sun exposure, and tanning bed use were all
significantly related to serum 250HD (37). In the whole population sun exposure
(occasionally/regularly) was associated with 27.6% higher serum 250HD (37). Those
reporting regular sun exposure had the highest 250HD year round, then those reporting
occasional sun exposure and lastly those reporting no sun exposure had the lowest
250HD (37). In the Norwegian arctic, Brustad et al. looked at the vitamin D status of a
cross-sectional sub-set of middle aged women from a larger cohort from November 2001
to June 2002 (46). They used a multiple linear regression model to predict plasma
250HD with diet, season and a variable named 'UV-hours' representing hours per day
of sun exposure above the threshold for vitamin D3 production (BED > 0.472) (46).
Significant predictors of 250HD included vitamin D intake (p = 0.0003), time of year of
blood draw (p = 0.005), sun holiday (Y/N) (p = 0.02), tanning bed use (Y/N) (p <
0.0001), and 'UV-hours' (p = 0.0002) while residing in Norway during the summer prior
to the blood draw was negatively associated (p = 0.001) (46). Their model with vitamin
D intake, age, BMI, use of dietary supplements, tanning bed use, sun holiday, 'UV-
230
hours' and summer residency in Norway, explained 25% (R2adj) of the variance in
plasma 250HD (p < 0.0001) (46). They found that 'UV-hours' was a better
measurement of UV exposure than 'hours in daylight last week prior to blood sampling',
'time of year when the blood sample was collected' and 'latitude' (46). In comparison,
our model with vitamin D-weighted joules, skin reflectance, vitamin D intake and
dummy variables for cohort explained 52% of the variation in serum 250HD (Table
3.5). All variables were significant predictors of vitamin D status. Different sun
exposure measurements were also examined in the model. The sun exposure
measurements including vitamin D-weighted joules, percent ambient (vitamin D-
weighted), erythemally-weighted joules, the SEI, the PS badge joules, and self-identified
sun-level were all the most significant predictors of serum 250HD in the model and they
were not significantly better predictors than each other in the models (Figure 3.31).
They were all useful measures of sun exposure.
Using the full model, with sun exposure, skin reflectance, vitamin D intake and
cohort, we predicted serum 250HD for individuals with high or low skin reflectance and
high or low sun exposure each season (Table 3.9). Individuals with low reflectance
(dark skin) and low sun exposure would be insufficient (< 75 nmol/L) year around and
would need higher vitamin D intakes to maintain their serum levels at ~75 nmol/L year
around (Table 3.10). The common advice that sun exposure of 5-10 minutes a day
between 10am-3pm in the Summer and Fall will satisfy most individuals vitamin D
requirement (47) may only be true if they have high reflectance (light skin) and at least
their face, hands and arms exposed (Table 3.12). We estimated that individuals with
low reflectance (dark skin) would need 6-9 times the amount of time in the sun as an
231
individual with high reflectance (light skin) and that this may not be realistic given cold
weather or cultural beliefs about clothing or tanning. Thus, supplementary or dietary
vitamin D would be needed to maintain serum levels in a healthy range year around.
The strengths of the present study include longitudinal data collected over each
season while participants went about their daily lives and we measured their sun
exposure. By collecting daily sun exposure logs we were able to compare our method of
calculating doses received in joules to other assessment methods that have used only
time or a SEI. The study also included individuals with a wide range of skin reflectance
and sun exposure levels. Some of the limitations of the present study include a small
sample size (particularly for subjects with low skin reflectance and high sun exposure)
although adequate to show statistically significant differences, different groups of
individuals each season and a limited range of dietary intake. However, our study has
shown that there is a significant contribution of sun exposure, skin reflectance and
vitamin D intake to vitamin D status in our study population which was assessed using
simple methods in the free-living adults and these methods could be used in future
population-based studies.
232
and Dr. Aronov for running the LC-MS. I would also like to thank Dr. Slusser for
helping us with the USDA UV-B Monitoring Station, the UCD Department of Land Air
Water Resources (LAWR) (Dr. Mata) for giving us access to the station and Dr. Davis
for providing the vitamin D-weighted UV-B values. I would like to thank Dr. Kimlin
and his staff for donating the PS badges and analyzing them. I would also like to thank
Thuan Nguyen and Jan Peerson for their advice on statistics. I am grateful for all the
help and guidance I received.
3.8 References

2. Armbrecht H, Okuda, K, Wongsurawat, N, Nemani, RK, Chen, ML, Boltz, MA.
Characterization and Regulation of the Vitamin D Hydroxylases. J Steroid
Biochem 1992;43:1073-1081.
3. Holick MF, MacLaughlin, J A, Doppelt, SH. Factors that Influence the Cutaneous
Photosynthesis of Previtamin D3. Science 1981;211:590-593.
4. Webb AR, DeCosta BR, Holick MF. Sunlight regulates the cutaneous production
of vitamin D3 by causing its photodegradation. J Clin Endocrinol Metab
1989;68:882-7.
5. Engelsen O, Brustad M, Aksnes L, Lund E. Daily duration of vitamin D
synthesis in human skin with relation to latitude, total ozone, altitude, ground
cover, aerosols and cloud thickness. Photochem Photobiol 2005;81:1287-90.
6. Gibson J. UVB Radiation: Definition and Characteristics.
http://uvb.nrel.colostate.edu/UVB/publications/uvb_primer.pdf.
8. Holick MF. Photosynthesis of vitamin D in the skin: effect of environmental and
life-style variables. Fed Proc 1987;46:1876-82.
9. Beadle PC. The epidermal biosynthesis of cholecalciferol (vitamin D3).
233
10. Matsuoka LY, Ide L, Wortsman J, MacLaughlin JA, Holick MF. Sunscreens
suppress cutaneous vitamin D3 synthesis. J Clin Endocrinol Metab
1987;64:1165-8.
11. Matsuoka LY, Wortsman J, Dannenberg MJ, Hollis BW, Lu Z, Holick MF.
Clothing prevents ultraviolet-B radiation-dependent photosynthesis of vitamin
D3. J Clin Endocrinol Metab 1992;75:1099-103.
12. McCarty CA. Sunlight exposure assessment: can we accurately assess vitamin D
exposure from sunlight questionnaires? Am J Clin Nutr 2008;87:1097S-101S.
2002;87:4952-6.
Obstretrics 1944;79:352-358.
16. Long C, Akerman T. Surface measurements of solar irradiance: a study of the
spatial correlation between simultaneous measurements at seperated sites. J Appl
Meterol 1995;34:1039-46.
17. Hay J. An assessment of the mesoscale variability of solar radiation at the earth's
surface. Solar Energy 1984;32:425-434.
18. Grant RH, Slusser JR. Spatial Variability in UV Radiation During the Growing
Season Across the Continental USA. Theoretical and Applied Climatology
2003;74:167-177.
Geophys Res 2006;111:1-11.
20. Turnbull DJ, Parisi AV, Kimlin MG. Vitamin D effective ultraviolet wavelengths
due to scattering in shade. J Steroid Biochem Mol Biol 2005;96:431-6.
1992;92:580-4.
Bioanal Chem 2008;391:1917-30.
25. Olds WJ, Kimlin MG. Availability of Vitamin D UV and a "Vitamin D Index"
for Public UV Exposure Advice.
26. Godar DE. UV doses worldwide. Photochem Photobiol 2005;81:736-49.
27. Zerwekh JE. Blood biomarkers of vitamin D status. Am J Clin Nutr
2008;87:1087S-91S.
234
28. Bech-Thomsen N, Wulf HC. Sunbathers' application of sunscreen is probably

inadequate to obtain the sun protection factor assigned to the preparation.
Am J Clin Nutr 2003;77:204-10.
30. Chodick G, Kleinerman RA, Linet MS, et al. Agreement between diary records
of time spent outdoors and personal ultraviolet radiation dose measurements.
31. Sullivan S, Cobb JL, Rosen CJ, Holick MF, Chen TC, Kimlin MG, Parisi AV.
Assessment of sun exposure in adolescent girls using activity diaries. Nutrition
Research 2003;23:631-644.
32. Dwyer T, Blizzard L, Gies PH, Ashbolt R, Roy C. Assessment of habitual sun
exposure in adolescents via questionnaire—a comparison with objective
measurement using polysulphone badges. Melanoma Res 1996;6:231-9.
33. O'Riordan DL, Stanton WR, Eyeson-Annan M, Gies P, Roy C. Correlations
between reported and measured ultraviolet radiation exposure of mothers and
young children. Photochem Photobiol 2000;71:60-4.
control subjects. Am J Clin Nutr 1987;46:1005-10.
6.
1979;2:303-5.
J Nutr 2001;86 Suppl 1:S97-103.
38. Villareal DT, Civitelli R, Chines A, Avioli LV. Subclinical vitamin D deficiency
in postmenopausal women with low vertebral bone mass. J Clin Endocrinol
Metab 1991;72:628-34.
2000;51:439-51.
41. van der Meer IM, Boeke AJ, Lips P, et al. Fatty fish and supplements are the
greatest modifiable contributors to the serum 25-hydroxyvitamin D concentration
in a multiethnic population. Clin Endocrinol (Oxf) 2008;68:466-72.
2007;85:860-8.
235
45. Atli T, Gullu S, Uysal AR, Erdogan G. The prevalence of Vitamin D deficiency
and effects of ultraviolet light on Vitamin D levels in elderly Turkish population.
Arch Gerontol Geriatr 2005;40:53-60.
46. Brustad M, Alsaker E, Engelsen O, Aksnes L, Lund E. Vitamin D status of
middle-aged women at 65-71 degrees N in relation to dietary intake and exposure
to ultraviolet radiation. Public Health Nutr 2004;7:327-35.
2002;9:87-98.
3.9 Figures
lire 3.1: Study Design

^n=72
n=17 n=17 n-20 n=18
ich Cohort:


237
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Sun Exposure Log (JA0.1)
Figure 3.2: Correlation of the Average Joules1 from Sun Exposure Log and the Average
Joules from PS Badge2 on the same day

Regression Equation: PS Badge JA0.1 = 0.07865 + 0.99550*Sun Log JA0.1 (R2 = 0.87, p
< 0.001)
^rythemally-weighted j oules
2
PS Badges were worn by subjects on the wrist once a week as an objective measure of
sun exposure and adjusted for clothing to calculate the dose received in joules
1000 -i
Vit D Spectrum1
800 I I Erythemal Spectrum
600
a
&
2 400
3
200 ^
Fall
1
Winter Spring Summer
10/28/2006- 1/17/2007- 4/11/2007- 7/18/2007
12/15/2006 3/16/2007 6/8/2007 9/17/2007
Figure 3.3: Average Ambient Daily Sun Exposure per Weekfromthe USDA UV-B
Monitoring Station Pyranometer for 7 Weeks in the Fall Cohort and 8 Weeks in the
Winter, Spring and Summer Cohorts
1
Vit D-weighted spectrum for dermal previtamin D3 synthesis
2
Erythemally-weighted spectrum for producing erythema (ie. skin reddening)
Different letters represent a significant difference (p < 0.05) between seasons (for both
the erythemal and vitamin D spectrums)
1800 - 1800 -|
1600 - 1600 -
1400 1400
1200 1200 -
1000 1000
-S 900 - -S 800-
600 eoo
400 - 400 -
200 200
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November 15,2006 February 14,2007
PALL WINTER
1600 i
1800
1600 1600
1400 1400 -
1200 1200
1000- 1000
800 ^ 800
600 600
400 - 400
200 - 200
0 0
4/r . , « • ' .<)»•>- <**• ««#>•,

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May 9,2007
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August 15, 2007
V
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SPRING SUMMER
Figure 3.4: Average Hourly Ambient Sun Exposure from USDA UV Monitoring
Station Pyranometer from 7am-7pm for One Day (Middle of Week 4) Each Cohort
Black Circles = Vit D-weighted spectrum for dermal previtamin D3 synthesis

White Circles = Erythemally-weighted spectrum for producing erythema (ie. skin
reddening)
1000 H
800
g. 600
05
J? 400 H
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Figure 3.5a: Daily Sun Exposure for the Fall Cohort (n=17) using Sun Exposure Logs
and Erythemal Spectrum Ambient UV-B Information to Calculate Joules per Subject.
Different symbols represent different subjects.
-=• 1.5
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Figure 3.5b: Transformed Daily Sun Exposure for the Fall Cohort (n=17) in JA0.1
A
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Figure 3.6a: Daily Sun Exposure for the Winter Cohort (n=17) using Sun Exposure
Logs and Erythemal Spectrum Ambient UV-B Information to Calculate Joules per
Subject. Different symbols represent different subjects.
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Figure 3.6b: Transformed Daily Sun Exposure for the Winter Cohort (n=17) in JA0.1
242
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s 2000
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Figure 3.7a: Daily Sun Exposure for the Spring Cohort (n=20) using Sun Exposure
2.0
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Figure 3.7b: Transformed Daily Sun Exposure for the Spring Cohort (n=20) in JA0.1
5000 i
4000
<«" 3000
« 2000
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i - i — r - r - I " i i i t i i i i i i i i i i i i i ""I 'I" '! |""T"-r T " I I I I I I I I I I I T - I I I I I

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Figure 3.8a: Daily Sun Exposure for the Summer Cohort (n=18) using Sun Exposure
2.0
1.5
a.
1.0
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o
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0.5 A
0.0 A
i t i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i t t i t i i i i i i i i i i i i i i i i i i i i i i i i—
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Figure 3.8b: Transformed Daily Sun Exposure for the Summer Cohort (n=18) in JA0.1
1400 A
1200
1000
I 800
a
I 600
I 400 A
200
T- nr-i—i—i—i—i—r- -i—i—r- -i—i—I—f—i—i—i—i—i—I—r- -i—i—i—i—i—i—i—i—i—i—i—r

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g OS O O O O O O O Q O O Q Q O O O O O O O O O O O O O O O Q O O O Q O O O O O Q O O O O O O O O
o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o
O O O H H ^ ^ - H H H H H H H H W - H H M H H H « H e . H « H H p H H H H M H H H H H H H H M M ( S M M M
Figure 3.9a: Daily Sun Exposure for the Fall Cohort (n=17) using Sun Exposure Logs
and Vitamin D Spectrum Ambient UV-B Information to Calculate Joules per Subject.
Different symbols represent different subjects.
2.0
~ 1-5
i
c
a to
o
-s 0.5
0.0
—i—|—r—|—|—|—|—|— i—i—r- -\—i—r~
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o o o rsjNMC^f^fôirsfsriEr* (Sts tsr^M N M rqts• CS ( N CS <N (N CS
Figure 3.9b: Transformed Daily Sun Exposure for the Fall Cohort (n=17) in JA0.1
4000 -i
3500 H
3000
S
£ 2500
a.
© 2000
ii
S 1500 H
£
1000 A
500
4¥ X /I :~:Y /\/¥.4. \/v-
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oooSooooooSooooooooSoooooooooooooooo
J(N<N<M<N(Nc4<NC-t
Figure 3.10a: Daily Sun Exposure for the Winter Cohort (n=17) using Sun Exposure
Logs and Vitamin D Spectrum Ambient UV-B Information to Calculate Joules per
2.0
1.5
CO
Q
1.0 A
o
••9 0.5 A
0.0
i i i i i r i i i i i i i i i i i i i i i i i i i i i i i i i i i
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n n f o m m n f i m f i SSSSSSiS
sssssssssssssssssss
Figure 3.10b: Transformed Daily Sun Exposure for the Winter Cohort (n=17) in JA0.1
12000
10000
8000
ft,
05
® 6000 -
E
J 4000
2000
IIIIT" I I I I I III' I I I I I I I I I I I I I I 1 1 t I I I I 1 I I 1 I
r- r--1-» r-r->£• C: C- P"- f**t~* P** *-* P"- » r ^ t ^ P-*P- » P - o P"-P- P"- P"- t~- r~- c- r~- r-» [^c^r^r^r^^r^r^r^r^r^r^r^c^r^r^r^r^r^r^r^r^r^r^r^r-r^r--
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Figure 3.11a: Daily Sun Exposure for the Spring Cohort (n=20) using Sun Exposure
2.5 H
2.0 H
a
s
fa
'S
a 1-5 H
05
Q
1.0
I 0.5
0.0
"T F T " "T—I—I—T" rM"i r~ r n " T - i ' " - j - -r-r-i" "rn-t"T"T~
t--t-» r-r-r-r-- i> r-r*-1-« r-.
oo oo o o o o o o o o o o o o o o o o OOOOOOOOOOOOOQOOOOOOOOOOOOOOOOOOOOOOOOO
<NCN
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JNJNJNJNJN
m</")M"iv"iv"}<n»/'>v"ii
^sssssssssssssssssssss- ÔÔVO^\0>0'O^D
Figure 3.11b: Transformed Daily Sun Exposure for the Spring Cohort (n=20) in JA0.1
247
10000
8000 H
g
a 6000
t
t
so
Q
4000
o
"a
2000
OH
I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I 1 I I M I I I I I I I I I I I I I I I I I I I I I
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r- r- r- c- r-r-r- r-r- r- 55saa55saSo5eaas5S5Sc&5sSc&a56SiS5: O N CTV ON ON < ^ CPs O N
Figure 3.12a: Daily Sun Exposure for the Summer Cohort (n=18) using Sun Exposure
2.5i
2.0 A
1.5
1.0 H
0.5
0.0
" i " i i i i"" c ' i " i i ' V" 1 r r" i i "i i i'""i"i 'i"i'"i' j i "i T T T "i-'i 1 r1 r- i i""i i i""i T r r i r " r
t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t ! : - o- t--t*- r-1--c~-t-*- t-»r-- r- t> r-r- r- r- r- f- r- r- r- r- r- t-»
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t~-r"-r~-t--r-r-t--r--r--t--t--r--t--t~-- oooooooooooooooooooooooooooooooooooooooooooo 0\0,ooa\a\0
Figure 3.12b: Transformed Daily Sun Exposure for the Summer Cohort (n=18) in J A0.1
248
1600 -i
1400 -
1"
9
1200 -
t.
•11
** 1000 -
a
cc
800 -
s4>
ryth
600 -
W o o
w«e o
400 - o
V
•-i
g 200 -
£
0 -
1 1 1 1

11 11 167 161 Median J
(1-172) (2-188) (20-836) (21-1398) (Range)
Figure 3.13: Average Erythemally-Weighted Joules from the Daily Sun Exposure Logs
for each Season/Cohort
Different letters represent a significant difference (p < 0.05) between cohorts using Box-
Cox transformed variables.
249
3000 b
2500
S 2000 A
o
o. 1500
«5
b 1000
o
a o
£ 500

13 16 289 295 Median J
(2-217) (2-296) (32-1348) (38-2534) (Range)
Figure 3.14: Average Vitamin D-Weighted Joulesfromthe Daily Sun Exposure Logs
250 -i
200
r = 0.36*
p = 0.0018
o 150
aa
• * — '
O
= 100
o
m
50 H
0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2

1J 1024 J
Daily Sun Log (Erythemal Spectrum JA0.1)
Figure 3.15: Correlation between Serum 250HD** and the Erythemally-Weighted

Spectrum JoulesA0.1 from the Daily Sun Exposure Logs

**LC-MS Data
Regression Equation: 25OHDA0.1 = 1.27 + 0.2266*Eryth JA0.1 - 0.06483 * Winter-
0.10987*Spring - 0.10346*Summer (R2 = 0.4015, p < 0.0001, Fall as Reference)
250
r = 0.34* o
200 - p = 0.0032 o
o
o ___^
r° o
o
o
o
-^
., -~"
o 100 o _ ——
o?_
_- — — -~~°~~ o
""o noo o °
50 ^_-"- o <s Oo O O <->
o Q o o
o o
" 1" ' T ""1 1 "• 1 1 1
1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4

6.2 J 2656 J
Daily Sun Log (Vit D Spectrum JA0.1)
Figure 3.16: Correlation between Serum 250HD** and the Vitamin D-Weighted
Spectrum JoulesA0.1 from the Daily Sun Exposure Logs

**LC-MS Data
Regression Equation: 25OHDA0.1 = 1.28 + 0.2234*VitD JA0.1 - 0.06773* Winter
160 -| y
200 -\
140 •
o //
180
r = 0.47*
p = 0.0596
160 120 -
-f 140 ^ o / ^^
100 -
a 120
& 80 -
I5 10° 0
^,o-—*""*
V)
! 60 -
« 80 **» ' o
40-
^ CO
o / o
20-
0 -
1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.G 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 Li
Erythemal Exposure (JA0.1) Erythema! Exposure (JA0.1)
FALL WINTER
a
s 10
8 °
1.5 1.6 1.7 1.5 1.6 1.7 1.8 1.9

Erythemal Exposure (JA0.1) Erythemal Exposure (JA0.1)
SPRING SUMMER
Figure 3.17: Correlation Between the Average Erythemally-Weighted Joules and the
Average Serum 250HD** for each Season/Cohort

**LC-MS Data
1.0 1.1 1.2 1.3 1.4 1.5
Vitamin D Spectrum (JA0.1) Vitamin D Spectrum (JA0.1)
FALL WINTER
Vitamin D Spectrum (JA0.1) Vitamin D Spectrum (JA0.1)

SPRING SUMMER
Figure 3.18: Correlation Between the Average Vitamin D-Weighted Joules and the

**LC-MS Data
254
250 n
0.5 0.6 0.7 0.8 0.9

A
Percent of Ambient UV-B (Vitamin D Spectrum J 0.1)
Figure 3.19: Correlation between Serum 250HD** and the Percent of Ambient UV-B
Vitamin D-Weighted Spectrum JoulesA0.1 from the Daily Sun Exposure Logs

**LC-MS Data
Regression Equation: 25OHDA0.1 = 1.24 + 0.49814*%Ambient JA0.1 - 0.05569*Winter
- 0.06104*Spring - 0.05330*Summer (R2 = 0.3939, p < 0.0001, Fall as Reference)
200 T
160 i
180
/
140- o y /
160 - y ^y
y o
120- r = 0.55* , ^ ^
140 p = 0.0225 y y^
120 - £2 100- y
y
^ y
y ^
^ ^
80 - s>^^ y ^ _,--"•"""""
100
1 ° , - —"" ° y ^ y~~
1
60-
--"""" y^y'*
40- ^y yo o
y ^ o /
20- y ^ /
^ y y
•^ y
0- y
y
y
0.45 0.60 0.55 0.60 0.65 0.70 0.75 0.80 0.85 0.4 0.5 0.6 0.7 0.8 0.
Percent Ambient UV-B (Vitamin D Spectrum J A 0.1) Percent Ambient UV-B (Vitamin D Spectrum J A 0.1)
FALL WINTER
0.65 0.70 0.75 0.80 0.85 0.90 0.55 0.60 0.65 0.70 0.75 0.80 0.85 0.90
Percent Ambient UV-B (Vitamin 0 Spectrum JA0.1) Percent Ambient UV-B (Vitamin D Spectrum .I'O.l)
SPRING SUMMER
Figure 3.20: Correlation Between the Percent of Ambient UV-B Vitamin D-Weighted
Spectrum JoulesA0.1 and the Average Serum 250HD** for each Season/Cohort

**LC-MS Data
256
a
o
o
o
o 8 o
8 o
o
o o
o 8
8
Fall Winter Spring Summer Meanmins

47 ±32 47 ±41 67 ±35 50 ±49 ± SD
Figure 3.21: Average Time Spent Outside in the Direct Sun for Each Season/Cohort
250-.
0 50 100 150 200 250

Time Outside (mins)
Figure 3.22: Correlation of Average Time Spent Outside in the Direct Sun and Serum
250HD**

**LC-MS Data
Regression Equation: 25OHDA0.1 = 1.39 + 0.04907*nl time - 0.05522*Winter -
200-
/
180-
r = 0.41* /
160 • p = 0.1051
140 -
/ ^^
/ ^^
250HD (nmol/L
120-
100-
o ^ ' '^^
' ^ " ^~~-^"~~
80-
s*^^ -D""
60 - jS*"^ s*
—- 0
40-
0 o
20 -
// /
40 60 80 100 0 20 40 60 80 100 120 140 160 180
Time Outside (mins) Time Outside (mins)
FALL WINTER
80 100 120 140 160 100 150

Time Outside (mins) Time Outside (mins)
SPRING SUMMER
Figure 3.23: Correlation Between the Average Time Outside in Minutes and the

**LC-MS Data
60 n
50 H
o
o
x, 40
©
a
W 30
03
20 ^
o
o o
o o
o
0 o
10
o

10 10 24 28 Median %BSA
(5-33) (5-21) (8-47) (13-55) (Range)
Figure 3.24: Percent Average Body Surface Area (BSA) Exposed for Each Cohort
Different letters represent a significant difference (p < 0.05) between cohorts using a
Friedman test for non-parametric variables.
250
b
o
200
o
150
o
pa
o
09
100 o
o
o
50 o o
o
OH

6 5 23 21 Median SEI
(1-104) (1-62) (3-158) (3-211) (Range)
Figure 3.25: Average Sun Exposure Index (SEI) for each Cohort
SEI= mins x BSA
250 -i
r = 0.47*
p < 0.0001
SEI (nl (mins x BSA))
Figure 3.26: Correlation of Average Sun Exposure Index (SEI) and Serum 250HD*

**LC-MS Data
SEI= mins x BSA
Regression Equation: 25OHDA0.1 = 1.497 + 0.03393*nl SEI - 0.05250* Winter -
r
262
2Q0
180
160
T 140
a 120
§ 100
o
SEI (nl (mini x BSA|) SEI (nl (mim xBSA))

FALL WINTER
SEI(nl(minsxBSA)) SEI (nl (mins x BSA)

SPWNG SUMMER
Figure 3.27: Correlation of Average Sun Exposure Index (SEI) and Serum 250HD**
*r - Spearman Correlation Coefficient

**LC-MS Data
25 A
I 20 i
m m m i;;l ^ H
ê^^^^^vv^ t ^ A ^ V * <^*&
^ *
iP
c& '>°
Outdoor Activities
Figure 3.28: Percent of Time Spent Performing the Different Outdoor Activities as
Documented on Daily Logs for All Four Cohorts
264
% 1-5 days
] % 6-20 days
% 21-40 days
% > 40 days
a
Figure 3.29: Percent of Subjects Who Took a Multivitamin Supplement Each

Cohort/Season as Documented on the Daily Logs and Categorized By How Many Times
(Days) They Took a Multivitamin Out of the Total* Number of Days in Each Cohort
*Fall = average of 47 daily logs

* Winter = average of 57 daily logs
* Spring = average of 57 daily logs
* Summer = average of 57 daily logs
Vitamin D Intake (IU) Vitamin D Intake (IU)
FALL WINTER
240
220-
200-
180 r" 0.003*

p = 0.9900
ftf 160
| 140
a 120-
~o
<t
~*5~~ ' ~o~
60-
40-
20
100 200 300 400 500 600 100 200 300 400 500 600 700 600
Vitamin D Intake (IU) Vitamin D Intake (ID)

SPRING SUMMER
Figure 3.30: Correlation between Average Vitamin D intake and Average Serum
250HD** for each Cohort/Season

**LC-MS Data
266
0.55 -, d,e b,c,d

d,e b,d,e
b,e a,d,e
0.50
a,b
0.45 H
a,c
£ 0.40 -\
0.35
0.30
&<*** ?^J&<>*' *£*>*

o/o ^
Different Measures of Sun Exposure
Figure 3.31: R2 for the Different Measures of Sun Exposure in the Full Model*
Different letters represent a significant difference (p < 0.05) between the measures of
sun exposure in the full model* with skin reflectance, vitamin D intake and dummy
variables for cohort to predict week 7 or week 8 serum 250HD using the LC-MS data.
Time = mean minutes outdoors in the direct sun per subject. Time (10am-2pm) = mean
minutes outdoors in the direct sun limited to 10am to 2pm per subject. Time (9am-
3pm) = mean minutes outdoors in the direct sun limited to 9am to 3pm per subject. SEI
= mean sun exposure index includes minutes in direct sun x BSA exposed per subject.
SEI with sunscreen = mean sun exposure index includes minutes in direct sun x BSA
exposed without sunscreen (4 hours per application of sunscreen) per subject. J (Vit D
Spectrum) = mean joules includes ambient vitamin D-weighted UV-B, time & BSA
exposed per subject. % Ambient (Vit D Spectrum) = mean joules of vitamin D-weighted
UV-B divided by ambient vitamin D-weighted UV-B per subject. J (Erythemal D
Spectrum) = mean joules includes ambient erythemally-weighted UV-B, time & BSA
exposed per subject. Level = Self-identified sun exposure level (High=l, Low=0) per
subject. J (PS Badge) = mean joules adjusted for BSA exposed from PS badges (worn
once a week) per subject.
267
u.oo -
c
C b,c
0.50 -
a,b
* a,b
o 0.45 -
a
"3
ta
J| 0.40 -
P*
0.35 -
ot
^ x&> ***** vvv^ v «^ A:i
Sun Exposure (Joules)
Figure 3.32: R for the Different Weeks of the Erythemally-Weighted Joules from the
Daily Sun Exposure Logs in the Full Model*
Different letters represent a significant difference (p < 0.05) between the different
weekly averages of sun exposure in the full model* with skin reflectance, vitamin D
intake and dummy variables for cohort to predict week 7 or week 8 serum 250HD using
the LC-MS data.
Wk -1 thru -2 = the average joules/person for the first and second week before the final
blood draw.
Wk -1 thru -3 = the average joules/person for the first, second and third week before the
final blood draw.
Wk -1 thru -4 = the average joules/person for the month before the final blood draw.
Wk -1 thru -5 = the average joules/person for the month and fifth week before the final
blood draw.
Wk -1 thru -6 = the average joules/person for the month, fifth and sixth week before the
final blood draw.
Wk -1 thru -7 or -8 = the average joules/person (7 weeks for Fall and 8 weeks for
Winter, Spring, Summer) before the final blood draw.
3.10 Tables
Table 3.1: Percent Body Surface Area (BSA) Exposed*

Baseball Cowboy
A Nothing Beanie/helmet cap hat Neck Hands
Head 4% 3% 2% 1% 2% 4%
Quarter
Bikini top/ length Long sleeves/
B Nothing Sports bra Tank top Tee shirt shirt Coat
Torso 47% 42% 18% 10% 3% 0%
Bikini Short shorts/ Shorts to
C bottom Skirt the knee Pants
Legs 38% 24% 8% 0%
Tennis
D Nothing Sandals shoes
Feet 2% 1.5% 0%
* Clothing key for the daily sun exposure logs, BSA based on "rule of nines" for burn
patients (15).
269
Table 3.2:

1
Ethnicity/Ancestry
Hispanic 1 2 1 0 4
N. Asian 1 3 2 6 12
S. Asian 5 3 6 1 15
African 0 4 5 6 15
Gender
Female 14 11 16 12 53
Male 3 6 4 6 19
Reported Sun Exposure
Low 10 13 8 14 45
High 7 4 12 4 27
3
Age (x ± SD) 23 ± 3 27 ± 5 23 ± 4 22 ± 3 24 ±
BMI 4 (x±SD) 23 ± 2 24 ± 2 23 ± 2 22 ± 3 23 ±
1
Self-reported ethnicity/ancestry.
2
3
4
Body mass index (kg/m2) averaged over wk 0, 4 and 7 or 8.
Table 3.3:
Correlation of Average 25QHD with Different Sun Exposure Measurements
LC-MS1 RIA2
Spearman Correlations r p-value r p-value
250HD & Time 0.38 0.0009* 0.37 0.0014*
250HD & Time (10am-2pm only) 0.38 0.0009* 0.35 0.0028*
250HD & Time (9am-3pm only) 0.39 0.0008* 0.36 0.0021*
250HD & SEI 0.47 O.0001* 0.49 0.0001*
250HD & SEI with sunscreen 0.45 O.0001* 0.45 0.0001*
250HD & J (Vit D Spectrum) 0.34 0.0032* 0.40 0.0006*
250HD & % Ambient (Vit D Spectrum) 0.48 O.0001* 0.50 0.0001*
250HD & J (Erythemal Spectrum) 0.36 0.0018* 0.41 0.0003*
25QHD & J (PS Badge) 0.33 0.0051* 0.36 0.0017*
r = Spearman correlation coefficient

* Significant p-value < 0.05 for Spearman Correlation between values.
Average = mean of all measurements for each subject.
1
LC-MS = Ultra-performance liquid-chromatography-tandem mass spectrometry
procedure to determine serum 250HD.
RIA = Radioimmunoassay to determine serum 250HD.
Table 3.4:
Correlation of Week 7 or 8 Serum 250HD and Sun Exposure
LC-MS1 R1A2
Spearman Correlations r p-value r p-value
250HD & Wk -1 Joules 0.21 0.0729 0.28 0.0173*
250HD & Wk -2 Joules 0.30 0.0097* 0.37 0.0014*
250HD & Wk -3 Joules 0.33 0.0041* 0.39 0.0007*
250HD & Wk -4 Joules 0.39 0.0008* 0.47 O.0001*
250HD & Wk -5 Joules 0.33 0.0044* 0.43 0.0002*
250HD & Wk -6 Joules 0.42 0.0002* 0.45 O.0001*
250HD & Wk -7 Joules 0.30 0.0102* 0.37 0.0015*
250HD & Wk -1 to -2 Joules 0.28 0.0168* 0.35 0.0026*
250HD & Wk -1 to -3 Joules 0.34 0.0040* 0.39 0.0006*
250HD & Wk -1 to -4 Joules 0.35 0.0028* 0.41 0.0003*
250HD & Wk -1 to -5 Joules 0.38 0.0009* 0.46 O.0001*
250HD & Wk -1 to -6 Joules 0.43 0.0001* 0.50 <0.0001*
250HD & Ave. Joules 0.41 0.0004* 0.49 O.0001*
r = Spearman correlation coefficient

* Significant p-value < 0.05 for Spearman Correlation between values.
Average = mean joules of all the sun exposure logs.
^C-MS = Ultra-performance liquid-chromatography-tandem mass spectrometry
procedure to determine serum 250HD.
RIA = Radioimmunoassay to determine serum 250HD.
272
Table 3.5: Multiple Linear Regression Models to Predict Serum 250HD** using Different Sun
Exposure Measurements
Model Est. SE Est. p-value Rz
Full Model with Time 0.46
Time Outside (mins) 0.03671 0.00981 0.37596 0.0004*
Skin Reflectance (forehead) 5.54E-08 1.77E-08 0.32551 0.0026*
Vit D Intake (IU) 0.02241 0.01002 0.20812 0.0287*
Fall Ref
Winter -0.04422 0.01848 -0.27332 0.0196*
Spring -0.02344 0.01950 -0.15276 0.2339
Summer 0.00178 0.01915 0.01123 0.9262
Full Model with SEI 0.53
Sun Exposure Index (SEI) 0.02757 0.00546 0.49568 <0.0001*
Vit D Intake (IU) 0.01933 0.00941 0.17955 0.0440*
Fall Ref
Winter -0.04174 0.01727 -0.25797 0.0185*
Spring -0.03593 0.01867 -0.23422 0.0587
Summer -0.02254 0.01884 -0.14206 0.2358
Full Model with Joules (Vit D Spectrum) 0.52
Joules (Daily log) 0.17149 0.03448 0.66705 <0.0001*
Vit D Intake (IU) 0.02086 0.00941 0.19378 0.0301*
Fall Ref
Winter -0.05457 0.01748 -0.33728 0.0027*
Spring -0.07847 0.02337 -0.51145 0.0013*
Summer -0.07341 0.0243 -0.46257 0.0036*
Full Model with Joules (Erythemal Spectrum) 0.53
Joules (Daily log) 0.18255 0.03619 0.64429 O.0001*
Vit D Intake (IU) 0.02067 0.00938 0.19198 0.0311*
Fall Ref
Winter -0.0523 0.01736 -0.32323 0.0037*
Spring -0.0738 0.02257 -0.48101 0.0017*
Summer -0.06626 0.02316 -0.41755 0.0057*
Full Model with Self-Identified Sun Level 0.50
Sun Level (High or Low) 0.06327 0.01412 0.44574 <0.0001*
Vit D Intake (IU) 0.01847 0.00976 0.17153 0.0630
Fall Ref
Winter -0.03681 0.01787 -0.22748 0.0434*
Spring -0.02117 0.01844 -0.13802 0.2551
Summer 0.01166 0.01839 0.07349 0.5283
*Significant p-value < 0.05 Ref = Reference dummy variable in the model. Time Outside = mean
minutes in the direct sun per subject. SEI = mean sun exposure index includes minutes in direct sun x
BSA exposed per subject. Joules = mean joules includes ambient UV-B, time & BSA exposed per subject.
Level = Self-identified sun exposure level (High=l, Low=0). Forehead = mean forehead L* skin
reflectance per subject. IU = mean vitamin D intake including supplements per subject. **LC-MS data.
Table 3.6:
Multiple Linear Regression Model using RIA Data to Predict Serum 25QHD
RIA Model Est SE Est. p-value R2
Full Model with Joules
(Vit D Spectrum) 0.53
Joules (Daily log) 1.18090 0.23426 0.67223 <0.0001*
Skin (forehead) 3.29E-07 1.12E-07 0.28296 0.0045*
Vit D Intake (IU) 0.12771 0.06394 0.17360 0.0500*
Fall Ref
Winter -0.37344 0.11880 -0.33778 0.0025*
Spring -0.54085 0.15877 -0.51594 0.0011*
Summer -0.34365 0.16508 -0.31693 0.0413*

Joules = mean joules (Vitamin D-Spectrum) per subject.
Table 3.7: Multiple Linear Regression Model using Vitamin D-Weighted Joules as Sun Exposure
Measurement to Predict Serum 25QHD** with Significant Covariates
Model Est. SE Est. p-value R2
1
Full Model with Sunscreen 0.56
Joules (Vit D Spectrum) 0.16686 0.03366 0.64902 O.0001*
Vit D Intake (IU) 0.02116 0.00917 0.19655 0.0242*
Fall Ref
Winter -0.05429 0.01703 -0.33552 0.0022*
Spring -0.08440 0.02294 -0.55016 0.0005*
Summer -0.08065 0.02391 -0.50819 0.0013*
Sunscreen Use 0.02796 0.01320 0.18970 0.0381*
Full Model with Athlete2 0.57
Joules (Vit D Spectrum) 0.07652 0.0496 0.29765 0.1278
Vit D Intake (IU) 0.01472 0.00934 0.13672 0.1200
Fall Ref
Winter -0.05144 0.01682 -0.31792 0.0032*
Spring -0.04199 0.02654 -0.27369 0.1185
Summer -0.02919 0.02897 -0.18397 0.3174
Athlete 0.06248 0.02431 0.33886 0.0125*
Full Model with OC Use3 0.59
Joules (Vit D Spectrum) 0.17138 0.03213 0.66660 O.0001*
Vit D Intake (IU) 0.02415 0.00883 0.22429 0.0080*
Fall Ref
Winter -0.04509 0.01655 -0.27865 0.0083*
Spring -0.06733 0.02204 -0.43884 0.0033*
Summer -0.06810 0.0227 -0.42916 0.0038*
OC 0.04614 0.01402 0.27914 0.0016*
Full Model with Ancestry4 0.63
Joules (Vit D Spectrum) 0.08712 0.03973 0.33888 0.0321*
Skin Reflectance (forehead) 4.21E-08 2.49E-08 0.24761 0.0954
Vit D Intake (IU) 0.02344 0.00894 0.21774 0.0110*
Fall Ref
Winter -0.04253 0.01673 -0.26285 0.0136*
Spring -0.03583 0.02409 -0.23353 0.1421
Summer -0.02035 0.02601 -0.12820 0.4372
Hispanic Ref
African -0.04852 0.02906 -0.28674 0.1002
Caucasian -0.01361 0.02791 -0.09516 0.6275
SE Asian -0.06325 0.02744 -0.37383 0.0246*
N Asian -0.08769 0.02820 -0.47556 0.0028*
*Significant p-value < 0.05 Ref = Reference dummy variable in the model. **LC-MS data.
'Sunscreen use greater than 5 times (No=0, n=46, or Yes=l, n=26) using the daily sun exposure logs.
2
Self-Identified Athlete (No=0, n=60, Yes=l, n=12). 3Oral contraceptive users (No=0, n=56, Yes=l,
n=16). "Ancestry self-categorized without an 'other' category (dummy variables). Joules = mean vitamin
D-weighted joules from daily sun exposure logs per subject. Forehead = mean forehead skin reflectance
L* per subject. IU = mean dietary vitamin D including supplements per subject.
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Table 3.8: Average Time* and Percent BSA* Exposed of Subjects

with Low and High Sun Exposure** each Season/Cohort
Low Sun Exposure High Sun Exposure
(2(T Percentile) (80tn Percentile)
Joules Time BSA Joules Time BSA
(J) (mins) (%) (J) (mins) (%)
Fall 9 34 71 83 98 172
Winter 12 24 71 229 102 21 2
Spring 89 15 25 3 915 130 364
Summer 115 23 222 904 121 283
Average 24 152 113 263
* Average time in minutes and average percent of body surface area (BSA) exposed
**20th and 80th percentiles for sun exposure (vitamin-D weighted joules) in each cohort
which are used in the model prediction (Table 3.7) to represent low (20* ) and high (80th)
sun exposure
1
Comparable to face and hands exposed
2
Comparable to face, hands and arms (t-shirt) exposed
3
Comparable to face, hands and arms (tank top) exposed
4
Comparable to face, hands, arms (tank top) and legs (shorts to the knee) exposed
Table 3.9: Model Prediction of Serum 250HD* in 4 Groups
Low Low High High
Fall: 60 85 84 118 nmol/L
Winter: 43 72 61 100 nmol/L
Spring: 51 81 73 113 nmol/L
Summer: 55 84 78 117 nmol/L
* Predicted 250HD 1 using LC-MS data with the 20th (low) & 80th (high) percentiles
for sun exposure each season (vitamin D-weighted joules, Table 3.6), median for
diet (200IU) & median L*for forehead skin reflectance (African & European).
Equation:
25OHDA0.1=(1.16367+(0.17149*joulesA0.1)+(0.0000000534072*skinA3.4)
+(0.02086*nldiet)-(0.05457*winter)-(0.07847*spring)-(0.07341*summer))
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Table 3.10: Estimation of Dietary Vitamin D (IU) Intake Needed to Maintain

Serum 250HD ~75 nmol/L* each Season
Low Low High High
Fall 857 0 0 0 IU
Winter 1,829 171 800 0 IU
Spring 1,371 0 114 0 IU
Summer 1,143 0 0 0 IU
*Dietary vitamin D intake predicted using model with LC-MS data (table 3.6) and by
dose response expected based on a supplementation study by Heaney et al. in which they
found a 0.7 nmol/L increase in serum 250HD for every additional 40 IU (1 ug) of
vitamin D ingested (29).
Table 3.11: Estimation of Daily Sun Exposure in Joules
(Vitamin D-Weighted) Needed to Maintain Serum 250HD
~75nmol/L* each Season
Low reflectance High reflectance
(dark skin) (light skin)
Fall 40 4 J
Winter 293 44 J
Spring 627 109 J
Summer 537 91 J
*Predicted Joules (Vitamin D-weighted) for low and high skin reflectance each season
using model with median for diet (200IU), median L* forehead skin reflectance
(African & European) and serum 250HD set at 75 nmol/L.
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Table 3.12: Estimation of Time* Needed Each Season to Maintain Serum

250HD ~75nmol/L
Low reflectance (dark skin)
Season Fall Winter Spring Summer
1
Joules needed 40 293 627 537
Ave. ambient J/m2 (12-lpm) 2 245 448 1237 1431
BSA3 1.75 1.75 1.75 1.75
% BSA exposed4 20 20 20 20
5
Estimated time 28 112 87 64
High reflectance (light skin)

Season Fall Winter Spring Summer
Joules needed1 4 44 109 91
Ave. ambient J/m2 (12-lpm) 2 245 448 1237 1431
BSA3 1.75 1.75 1.75 1.75
% BSA exposed4 20 20 20 20
Estimated time5 3 17 15 11
Model estimation (table 3.8) of joules needed to maintain serum at 75nmol/L in

individuals with low and high skin reflectance each season
2
Average ambient J/m2 using UV-B Monitoring Station UV-B data (Vitamin D-
weighted) from 12-lpm each cohort/season
3
Body surface area (BSA) in m2
4
20% BSA represents face, neck, hands and arms (t-shirt) exposed
5
Time needed in minutes around noon each day to obtain estimated joules for individuals
with low and high skin reflectance each season
*Equation: Time= (Joules/(J/m2)(BSA*%BSA))*60

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Chapter 4:
Validation of Sun Exposure and Vitamin D Intake
Recall Questionnaires for Estimating Vitamin D Status

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4.1 Abstract
Individual sun exposure and dietary intake both contribute to vitamin D status
however, simple, validated recall methods to assess these exposures are lacking. The
objectives of our study were to validate both a sun exposure recall questionnaire, relative
to a validated daily sun exposure logs, and a vitamin D-specific food frequency
questionnaire (FFQ), relative to food records. We then assessed the ability of these
methods, in conjunction with skin reflectance, to predict serum 25-hydroxyvitamin D
(250HD). Our study was conducted with 4 cohorts of healthy young adults each
followed for 7-8 weeks in the Fall, Winter, Spring and Summer (2006-07) in Davis, CA
(38.5 N latitude). Subjects had a wide range of sun-exposure behavior and skin
reflectance. A total of 72 subjects (~18 per season) enrolled and completed the study.
Skin reflectance was measured using a reflectance spectrophotometer. Vitamin D intake
was assessed by food records (reference method) and a vitamin D-specific FFQ. Sun
exposure was assessed using daily sun exposure logs (reference method), weekly
polysulphone (PS) dosimeter badges (reference method) and sun exposure recall
questionnaires. Serum 250HD was measured by ultra-performance liquid-
chromatography-tandem mass spectrometry (LC-MS) and by radioimmunoassay (RIA).
Spearman's correlation coefficients were used to correlate the recalled sun exposure
measurements to the daily sun exposure logs and to correlate the vitamin D-specific FFQ
measurement to the food records. Spearman's correlation coefficients were also used to
assess the association between the recalled measurements and serum 250HD. The
method of triads was used to calculate a validity coefficient for the recall questionnaires
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and regression analysis was used to compare our recall methods to the reference
methods. Multiple linear regression analysis was used to predict serum 250HD using
dummy variables for cohort/season and continuous variables for skin reflectance,
vitamin D intake, and sun exposure using the reference methods and the recall methods.
The recall methods were highly correlated with the reference methods (sun: r = 0.83-
0.95, diet: r = 0.69-0.77, p < 0.0001), moderately correlated to serum 250HD (sun: r =
0.40-0.51, diet: r = 0.36-0.37, p < 0.005) and they had high validity coefficients (sun:
0.84-0.94, diet: 0.90-0.91), validating the recall methods as useful tools for assessing
vitamin D. The recall measurements in the model were all significant independent
predictors of serum 250HD (p < 0.05) and these models predicted vitamin D status as
well as the reference model (p > 0.05). These results show that the contribution of sun
exposure and vitamin D intake to vitamin D status can be assessed using simple recall
methods in free-living adults.
4.2 Introduction
Vitamin D is unique among nutrients in that it can be made in the skin from sun
exposure and/or obtained in the diet. However, very few foods naturally contain vitamin
D; some sources include oily fish (ie. salmon, mackerel, herring), cod liver oil, and sun
dried mushrooms (1). Vitamin D is also made in the skin when solar ultraviolet (UV)-B
photons with energies between 290 and 315 nm strike the skin initiating photolysis of 7-
dehydrocholesterol (provitamin D3) to previtamin D3 (2). Previtamin D3 in its' cis,cis
isomer is thermodynamically unstable and is rapidly transformed to vitamin D3 by

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rearrangement of its' double bonds (3). Circulating vitamin D from sunlight exposure
and from the diet is converted to 25-hydroxyvitamin D (250HD) in the liver and then to
1,25-dihyroxyvitamin D (l,25(OH)2D) in the kidneys (4).
Dietary vitamin D can be toxic if too much is ingested (5) unlike vitamin D
produced by exposure to the sun, which is regulated to prevent intoxication (6). In 1997
The Recommended Daily Allowance (RDA) Standing Committee of the National
Research Council made recommendations about daily vitamin D intake assuming no sun
exposure (7). There was not enough scientific information to form an RDA so an
Adequate Intake (AI) level was set (7). They recommended 200 IU/day for children and
younger adults (< 50 years of age), 400 IU/day for older adults (50-70 years of age), and
600 IU/day for elderly adults (> 70 years of age) (7). However, Moore et al. estimated
vitamin D intake in the US using NHANES III data and/or the Continuing Survey of
Food Intakes by Individuals (CSFII) 1994-1996,1998 data and concluded that the
intake of vitamin D from food sources and supplements in the US does not meet the
recommended levels (8).
Currently, there is not a consensus on what the optimal level of 250HD should
be and where the cut-off for insufficiency should be drawn. Deficiency typically
corresponds to 250HD levels, < 20-25 nmol/L, which are indicative of a clinically
evident disease state, such as osteomalacia and rickets (9). On the other hand,
sufficiency refers to 250HD levels, -50-80 nmol/L, which correspond to the absence of
abnormalities in calcium homeostasis and/or maintaining PTH in the normal range (9,
10).
Currently, it is estimated that one billion people worldwide are at risk for vitamin
D levels < 75 nmol/L (11). Not only are people not getting enough vitamin D in their
diets (8), people may be avoiding the sun and thus, not making enough vitamin D from
casual sun exposure to sustain an adequate vitamin D status. Nesby-O'Dell et al. found
that 42% of African American women (15-49 years old) had serum 250HD levels < 37.5
nmol/L at the end of winter (ie. after sun deprivation) throughout the United States (10).
Tangpricha et al. found the 36% of healthy young men and women (18-29 years old)
were < 50 nmol/L at the end of winter in Boston (42°N) (12). Holick found that 30%,
42% and 84% of free-living white, Hispanic and Black elderly, respectively, were < 50
nmol/L at the end of August in Boston (13). Also in Boston, 57% of medical inpatients
were vitamin D deficient (< 37.5 nmol/L) and 22% were severely deficient (< 20
nmol/L) (13). Sixty-six of the patients that didn't consume the recommended intake
level were < 37.5 nmol/L and 37% of those that met the Al were still < 37.5 nmol/L
(13). Even in high-sun areas, vitamin D deficiency is a problem. For instance, in
Geelong, Australia (38°S), Pasco et al. found that over the winter months 43% of
females were < 50 nmol/L and 8% < 25 nmol/L (14). In Honolulu, Hawaii (21°N),
Binkley et al. found that 51% of their study population was < 75 nmol/L in late March
(15). In Arizona (32-3 3°N), 25.4% of the study population had serum 250HD levels <
50 nmol/L and 2% were < 25 nmol/L (16). In south Florida (25°N), Levis et al. found
that 39% of the study participants were < 50nmol/L in March (17). Living in low
latitudes and sunny climates does not prevent against vitamin D deficiency or
insufficiency. Thus, vitamin D deficiency is a problem throughout the United States.

285
Unfortunately, many studies (16-20) use non-validated recall methods to assess
sun exposure and therefore, sun exposure can't be adequately quantified. The objectives
of our study were to validate our week and month sun exposure recall questionnaires as
useful tools for measuring individual sun exposure and to validate our week and month
vitamin D-specific food frequency questionnaires (FFQ) as useful tools for measuring
vitamin D intake. These sun exposure and vitamin D intake recall measurements, along
with skin reflectance, were used to predict vitamin D status and compared to a prediction
equation using daily sun exposure logs (and PS badges), food records, and skin
reflectance, to determine if the recall methods could predict vitamin D status as well as
the reference methods could. Furthermore, this study is the first to use the method of
triads to investigate vitamin D from intake and sun exposure.
4.3 a Subjects
Subjects were students or employees recruited from the UC Davis (UCD)
campus. Flyers were posted around campus seeking volunteers with both high and low
levels of outdoor activity to participate in the study. E-mails were sent to different
student organizations/associations to recruit subjects of different ethnicities to represent
the range of skin pigmentation seen in the US population. However, because our
recruitment was limited to the UCD campus, our study sample may not be a
representative sample of the population. Subjects were screened for eligibility and
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included if they had a BMI between 18.5-30 and were 19-39 years of age. Subjects were
excluded if they reported consistent sunscreen use on all or most exposed skin, frequent
use of tanning beds or use of high-dose vitamin D supplements (excluding RDA-level
multiple vitamin and mineral supplements) within the past 2 months. These exclusion
criteria were selected because of difficulty in assessing sunscreen application/use and the
significant contribution of tanning-bed use or high-dose vitamin D supplements to
vitamin D status. Subjects were also excluded if they reported pregnancy, or any disease
or underlying condition requiring treatment that may affect vitamin D
absorption/metabolism or use of medications that affect vitamin D metabolism.
4.3 b Study Design
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asked to maintain their typical routines.
The subjects came to the WHNRC at weeks 0,4 and 8 (7 for Fall) to get their
blood drawn, have their skin reflectance measured and to fill out the sun exposure recall
questionnaires and the vitamin D-specific FFQ. Subjects met with the study coordinator
weekly to exchange PS badges and records. The study coordinator also e-mailed
reminders for wearing the PS badge and keeping food records.
4.3 c USDA UV-B Monitoring and Research Program (UVMRP)
Information from the USDA UV-B Monitoring Station at the UC Davis Climate
Center (Davis, CA, 38.5 N) was used to determine ambient UV-B levels in the
environment. The station uses a UV-B Broadband Pyranometer (Yankee Environmental
Systems, Inc., Turners Falls, MA) which measures global irradiance on a continual basis
in the UV-B spectral range of 280-330 nm (21). There are 34 climatologic sites around
the United States including one climate station at the UCD campus. The UV-B radiation
detected by the instrument is converted to visible light and this signal is measured in
W/m2 every 15 seconds and repeated in 3 minute averages. We downloaded the
erythemally-weighted (ie. erythemal-producing UV-B spectrum) measurements from the
USDA UV-B Network website (http://uvb.nrel.colostate.edu/uvb/home_page.html) and
averaged the values for each hour from 7am-7pm (21). We then converted the hourly
averages to J/m2 to make them comparable to the PS badge measurements and to

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determine joules from the daily sun exposure logs and from the week sun exposure recall
questionnaires.
4.3 d Daily Sun Exposure Logs
Sun exposure was assessed using daily sun exposure logs pre-labeled with the
date. Subjects kept daily sun exposure logs from 7am-7pm each cohort/season which
totaled an average of 53 days per subject. The daily sun exposure log collected
information about time of day when subjects were outside, location and how many
minutes that they were in the direct sun or shade which was greater than 5 minutes.
They also recorded their outdoor activity for those minutes. Then they had to record
what they were wearing using a key provided to estimate body surface area (BSA)
exposed to the sun during each time period. The key divided the body into 4 sections,
(A) head, (B) torso, (C) legs, (D) feet and is shown in Table 4.1. Then within each
section, A-D, the body is progressively covered up and described using numbers which
correspond to percent BSA exposed to the sun (Table 4.1). The percent BSA exposed
was determined using the "rule of nines" commonly used for burn victims (22) with
confirmation from estimating the BSA exposed for different clothing using a male and
female volunteer and a measuring tape. The subjects also had to record if they were
using sunscreen, what SPF was used and where the sunscreen was applied using the
same key for BSA exposed. They were trained on how to fill out the sun logs and asked
to keep track of their sun exposure as they go and not to change their normal behavior or
use their memory later. They were also asked to time themselves using a watch or cell
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phone for accuracy. Subjects periodically turned in their completed sun exposure logs to
check for compliance and to clarify any questions or concerns.
The daily sun exposure logs were used to calculate a daily value in joules for
each subject. This was accomplished by including the average UV-B number in J/m2 for
each hour that the person was outside using the ambient measurements from the UV-B
Monitoring Station and adjusting this value for minutes outside and BSA exposed (m ).
This gave a UV-B value in joules for each time they reported being outside and the
joules were then totaled for every time they were outside to obtain their daily joules.
Depending on where the subject was located the closest UV-B Monitoring
Station measurements were used to determine their ambient UV-B exposure. Ambient
UV-B measurements were adjusted appropriately for altitude because UV-B radiation
increases with the decreasing amount of UV scattering and absorbing that occurs in
higher altitudes (23). The UV-B measurements were also adjusted for shade versus
direct sun and shade UV-B was estimated to be 50% of direct sun UV-B. Consequently,
the personal UV-B measurements reflected location (latitude and altitude), time of day,
minutes in the direct sun or shade and %BSA exposed to the sun.
Subjects wore polysulphone (PS) dosimeter badges (Kimlin, Australian Sun and
Health Research Lab, Queensland University of Technology, Brisbane, Qld 4059
Australia) once a week as an objective measure of sun exposure in joules and they were
used to validate the sun exposure logs and sun exposure recall questionnaires in joules,
all adjusted for clothing.

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4.3 e Sun Exposure Recall Questionnaires
Sun exposure was also assessed using a non-validated recall questionnaire.
When subjects came to the WHNRC for their bloods (week 0, 4, 7 or 8) they were also
asked to recall their sun exposure over the past week and month. The week recall
questionnaire included the same questions as the daily log such as time of day, location,
minutes in the direct sun, clothing worn and sunscreen use. Total UV-B exposure in
joules for each day was calculated using the week recall questionnaire the same way as
calculated for the daily sun exposure log. The sun exposure recall questionnaire joules
were then compared to the joules from the daily sun exposure logs on the same days.
After subjects filled out the week recall, they were asked to recall their sun
exposure over the past month. The month recall questionnaire included the 3 weeks
preceding the week recall for a total of 4 weeks. However, this questionnaire was not as
detailed as the week recall and only asked about total minutes in each time period (7am-
7pm) for each week, not each day, along with location, clothing and sunscreen worn.
For this questionnaire, minutes and the sun exposure index (SEI = minutes x BSA) (20)
was used to estimated sun exposure because an exact value in joules could not accurately
be calculated without daily information. The month sun exposure recall in minutes and
the SEI was compared to the daily sun exposure log minutes and SEI for the same
weeks.
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4.3 f Dietary Assessment using Food Records
consecutive 4-day food records (the Fall cohort had one week with a 3-day food record
instead of 4 days) which were pre-labeled with the date. Records were kept every other
week for a total of 16 records (15 records for Fall). Food records have previously been
validated as an accurate tool to measure intake through observational studies (24). The
4-day food records included a weekend day to capture typical intake and days included
rotated from Wednesday-Saturday to Sunday-Wednesday. Subjects were asked to
record information of time and place of food consumption, brand names of products,
restaurants where meals were consumed and recipes used. Subjects were also asked
about consumption of vitamin D-fortified foods and supplement use. The subjects were
trained on how to keep accurate food records by a Registered Dietitian (RD) and
provided with detailed instructions and a list of serving size descriptions to visualize
portions. They were also asked to follow their typical food and supplement habits and to
keep track as foods were consumed and not use their memory later. Food records were
periodically turned in and reviewed by the RD for completeness in the level of detail in
food descriptions and preparation. The food records were analyzed for vitamin D
content using the Nutrition Data System for Research (NDSR) Program (2006,
University of Minnesota, Minneapolis, MN) and a daily intake in IU was determined.

292
4.3 g Vitamin D-Specific Food Frequency Questionnaire (FFQ) to Assess
Vitamin D Intake
Vitamin D intake was also assessed using a modified quantitative food frequency
questionnaire designed specifically for assessing only vitamin D intake and administered
by the RD. FFQs typically have a list of foods with the frequency of consumption and
quantity. Vitamin D is not found in a wide variety of foods (1) and therefore, the
questionnaire included only foods containing vitamin D which were chosen from a food
composition table. When the subjects came into the WHNRC for their blood draws
(week 0,4, 7 or 8) they were asked to recall their intake over the past week and month in
regard to foods containing vitamin D. The foods included in the questionnaire were milk
(soy, chocolate, etc), items made with milk (pudding/flan, etc), ice cream, whipped
cream/cool-whip, yogurt (brand specific), cheese, butter/margarine, eggs, fish (salmon,
mackerel, tuna, sardines, catfish, cod liver oil, other), liver, ready-to-eat cereals (brand
specific), Ensure/Slimfast supplemental shakes, vitamin-D fortified orange juice (OJ) or
other vitamin-D fortified foods (Table 4.2). For each of these foods the subjects were
asked how many times they had eaten the food in the past week/month and serving sizes
consumed. The RD used measuring tools (cups, spoons, a ruler, etc.) to help participants
with serving sizes. They were also asked about use of multivitamin supplements and
calcium supplements which could contain vitamin D. The vitamin D-specific FFQs
were analyzed for vitamin D content using the Nutrition Data System for Research
(NDSR) Program (2006, University of Minnesota, Minneapolis, MN) and a daily
average intake in IU was determined for the week and month recalls. These amounts
were compared to a daily average IU from the food records (reference) to determine if
the vitamin D-specific FFQ is a useful tool for measuring vitamin D intake.
4.3 h Skin Reflectance (Pigment) Assessment
4.3 i Covariates
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Weight and height were measured at week 0, 4, and 7 or 8 without shoes using a
4.3 j Serum 25-Hydroxyvitamin D (2SOHD)
Human Nutrition Research Center (WHNRC) per subject per blood draw (week 0, 4, 7
measured on the same day was 5.0% (range, 0.04-10.0%) and for duplicate samples
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WHNRC participates in the Vitamin D External Quality Assessment Scheme (DEQAS)
9.0, Systat Inc, Pt. Richmond, CA) and outliers were removed based on visual
inspection. Statistical Analysis Software (SAS version 9.1.3, Stat Corp, College Station,
TX) was used to perform all statistical analyses. Continuous data was tested for
normality using the Kolmogorov-Smirnov test and variables that were not normally
distributed (p < 0.05) were transformed using Box-Cox transformations. If a variable
couldn't be transformed to a normal distribution then a nonparametric test was
associations between variables and to estimate the correlation between the recall
measurements, record measurements and biochemical measurements for vitamin D from
intake and sun exposure. The correlations between each of the three methods were used
to calculate the validity coefficient using the method of triads (28). Correlations were
evaluated as poor (< 0.20), moderate (0.20-0.6), or good (> 0.60) (29). All p values <
0.05 were considered statistically significant.

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4.4 a The Method of Triads
The method of triads was used to calculate the validity coefficient from a
triangular comparison between the vitamin D-specific FFQ, the food records (reference)
and serum 250HD (28). The method of triads is typically used in dietary validation
studies (25-29) but we also calculated validity coefficients between the sun exposure
recall, the daily sun exposure logs (reference) and serum 250HD. The equation for
calculating the validity coefficient uses the correlations between each of the three
methods, as such: PQT = V (rojB x (rQR/rBR)), where PQT is the validity coefficient for the
recall questionnaire, TQB is the correlation between the recall and biochemical
measurement, TQR is the correlation between the recall and record, and TBR is the
correlation between the biochemical and record measurement (29). The method of triads
assumes that the measurements are linearly related to the true value and that they have
independent random errors (28). The random errors in the biochemical marker, serum
250HD, are likely independent of the recall and record measurements but the recall and
record measurements may have a real positive correlation between their random errors.
If this correlation is the only violation of the model assumptions, then the method of
triads will overestimate the validity coefficients of the recall and record measurement
and this can be interpreted as the upper limit for the true validity coefficient (30). The
lower limit is defined as the correlations between the biochemical measurement and the
recall measurement and the biochemical measurement and the record measurement (30).
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The biochemical measurement as an additional reference allows for the estimation of the
validity coefficient while validating the recall method as a useful tool to use.
4.4 b Multiple Linear Regression Analysis
groups each season. The sun exposure and diet recall measurements were also used in
the model to predict serum 250HD. Pitman's test was used to determine if the sun
exposure and diet recall measurements in the model were statistically similar predictors
of serum 250HD as the records for sun exposure and diet.
4.5 Results
Approximately two-thirds (n= 45) of the subjects reported their usual sun exposure as
"low" and one-third (n= 27) as "high" upon entering the study. The average age of the
subjects was 24 ± 4 years and the average BMI was 23 ± 3.

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4.5 a Sun Exposure Recall Versus Daily Sun Exposure Logs
The week sun exposure recall questionnaire was administered at week 0, 4 and 8
(7 for Fall) to assess sun exposure over the previous week before the subjects' blood
draw. The measurements in joules from the week 4 and 7/8 recalls were compared to the
subjects' daily sun exposure logs, as the reference, in joules for the same period. No
daily logs were available prior to week 0. The week 4 and 7/8 measurements were
highly correlated to the logs (r = 0.83, p < 0.001, Figure 4.2, Table 4.4) validating the
week sun exposure recall questionnaire as a useful tool for assessing sun exposure.
However, since the week recalls administered at week 4 and week 7/8 covered a
period during which daily records were kept, we examined the regression of the week 0,
4, and 7/8 recall data with daily log data for the average of the entire 7-8 week period to
determine if the week 0 recall performed as well as the week 4 and week 7/8 recalls.
This was done to confirm that the week 4 and 7/8 coefficients of determination (R2) were
not unusually large because keeping the daily logs during those periods could have
prompted better recall at the end of those weeks. As seen in Figure 4.3, all 3 of the
week recalls were significant predictors of average sun exposure in joules for the entire
7-8 week period. The week 0 measurement in joules explained 77% of the variation (R2
= 0.77, p < 0.0001) in average sun exposure and was equally as good of a predictor as
the week 4 and 7/8 joules (Figure 4.3). However, the average of the 3 week recalls was
a significantly better predictor of average sun exposure, as would be expected, because
the average covered 3 weeks providing more information and a better estimate of the
whole time period.

299
The month sun exposure recall questionnaire was also administered at week 0,4
and 8 (7 for Fall) to assess sun exposure over the previous month before the subjects'
blood draw. The measurements in time (minutes) from the week 4 and 7/8 month recalls
were compared to the subjects' daily sun exposure logs in minutes for the same period as
the reference. The recall measurements were highly correlated to the logs (r = 0.91, p <
0.001, Figure 4.4, Table 4.4) validating the month sun exposure recall questionnaire as
a useful tool for assessing sun exposure.
To see if the month sun recall at week 0 could also predict average time as well
as the other months, the minutes from each of the three month recalls and the average of
the month recalls were used to predict the average minutes from the daily sun exposure
logs (reference) along with dummy variables for cohort. As seen in Figure 4.5, they
were all significant predictors of average sun exposure in minutes. However, the week 0
month sun recall measurement in minutes only explained 45% of the variation (R =
0.45, p < 0.0001) in average sun exposure and wasn't as good a predictor as the month
recall at week 4 and 7/8 (Figure 4.5). However, the month recall at week 0 is before the
daily sun log time period (weeks 0-8) and could reflect different sun exposure
patterns/behavior in the pervious season.
The month sun exposure recall questionnaire was also used to calculate a sun
exposure index (SEI) which uses time adjusted for BSA exposed. Therefore, the SEI
measurements from the week 4 and 7/8 month recalls were compared to the subjects'
daily sun exposure logs SEI measurements for the same period as the reference. The
recall measurements were more highly correlated to the logs (r = 0.95, p < 0.001, Figure
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4.6, Table 4.4) than time alone validating the month sun exposure recall questionnaire,
using the SEI, as a useful tool for assessing sun exposure.
To see if the month sun recall at week 0 could also predict the average SEI as
well as the other months, the SEI from each of the three month recalls and the average of
the month recalls were used to predict the average SEI from the daily sun exposure logs,
as the reference, along with dummy variables for cohort. As seen in Figure 4.7, they
were all significant predictors of average sun exposure using the SEI. However, the
week 0 month sun recall SEI measurement only explained 64% of the variation (R2 =
0.64, p < 0.0001) in average sun exposure and wasn't as good of a predictor as the
month recall at week 4 and 7/8 (Figure 4.7). There may be a training effect from
keeping daily sun exposure logs, reflected in the accuracy of the week 4 and 7/8 month
recall. However, the week 0 month recall is before the subjects started keeping daily sun
exposure logs and may reflect different sun exposure behavior which may differ from
the average SEI in the logs.
4.5 b Sun Exposure Recall Association with Serum 2SOHD
Sun exposure contributes to serum 250HD and to examine the association with
serum 250HD at each week along with the sun recall at each week a correlation matrix
was constructed for the week and month sun recalls (Table 4.5, Table 4.6, Table 4.7).
The serum 250HD measurements at week 0,4, 7/8 and the average were highly
correlated to each other (r = 0.89 to r = 0.97, p < 0.0001) (Table 4.5, Table 4.6, Table
4.7). For the week sun recall, the average joules were highly correlated to the week 0,4
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and 7/8 measurements (r = 0.80 to r = 0.95, p < 0.0001, Table 4.5). The lowest
correlation for joules was between the week 0 joules and the week 7/8 joules although
still highly significant (r = 0.47, p < 0.0001, Table 4.5). For the month sun recall in
time, the average minutes were also highly correlated to the month recalls at week 0, 4
and 7/8 (r = 0.75 to r = 0.87, p < 0.0001, Table 4.6) with the lowest correlation for
minutes between the month recall at week 0 and week 7/8 (r = 0.46, p < 0.0001, Table
4.6). For the month sun recall using the SEI, the average SEI was also highly correlated
to the month recalls at week 0, 4 and 7/8 (r = 0.84 to r = 0.92, p < 0.0001, Table 4.7)
with the lowest correlation for the SEI between the month recall at week 0 and week 7/8
(r = 0.63, p < 0.0001, Table 4.7). Thus, the serum 250HD measurements at the
different time points were highly correlated and the sun recall measurements at the
different time points were moderately to highly correlated.
Correlations between the sun recall measurements and serum 250HD at the same
time points were also examined to see how well they correlated with each other. The
week 0 sun recall in joules and the week 0 serum 250HD measurement (r = 0.39, p =
0.0006), as well as, the week 4 sun recall and week 4 250HD (r = 0.34, p = 0.0038) and
the week 7/8 sun recall and week 7/8 250HD were significantly correlated to each other
(r = 0.31, p = 0.0107) (Figure 4.8, Table 4.4). The average of the joules for the week
recalls and the average of the 3 serum levels had the highest correlation as expected (r =
0.42, p = 0.0003, Figure 4.8, Table 4.4). Thus, sun exposure recalled from the week
prior to the blood draw is significantly correlated to serum 250HD. For the month sun
recall in minutes the correlation between the individual weeks and their respective serum
250HD level ranged from r = 0.27 (p = 0.0234) to r = 0.43 (p = 0.0002) (Figure 4.9,
Table 4.4). However, the month sun recalls had higher correlations using the sun
exposure index, presumably because both time and BSA exposed are taken into
consideration. The month recall at week 0 using the SEI and the week 0 serum 250HD
measurement (r = 0.49, p < 0.0001), as well as, the week 4 month recall and week 4
250HD (r = 0.48, p < 0.0001) and the week 7/8 month recall and week 7/8 250HD were
significantly correlated to each other (r = 0.40, p = 0.0005) (Figure 4.10, Table 4.4).
The average SEI for the month sun recalls and the average of the 3 serum levels had the
highest correlation as expected (r = 0.51, p < 0.0001, Figure 4.10 Table 4.4).
Therefore, having subjects recall sun exposure, including time and BSA exposed, over
the month prior to their blood draw is most useful for examining the relationship
between sun exposure and serum 250HD.
4.5 c The Method of Triads for Sun Exposure Recall
Spearman correlation coefficients between each of the three sun exposure
measurements were used to calculate the validity coefficient for the sun exposure recall
questionnaires using the method of triads approach (28) and the results are shown in
Table 4.8. The validity coefficients for the week sun recall in joules, the month sun
recall in time and the month sun recall using the SEI were 0.84, 0.90 and 0.94,
respectively (Table 4.8). Overall, the validity coefficients confirmed the usefulness of
the sun recall questionnaires in predicting sun exposure and serum 250HD.
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4.5 d Vitamin D-Specific Food Frequency Questionnaire (FFQ) Versus Food
Records for Vitamin D Intake
A week recall using the vitamin D-specific FFQ was administered at week 0,4
and 8 (7 for Fall) to assess vitamin D intake over the previous week before the subjects'
blood draw. The average vitamin D intake calculated from the week 4 and 7/8 recall
questionnaires were compared to the vitamin D calculated from the subjects' food
records, over the same period, as the reference. The measurements were highly
correlated (r = 0.77, p < 0.001, Figure 4.11, Table 4.4) validating the week recall
vitamin D-specific FFQ as a useful tool for assessing vitamin D intake.
As with sun exposure, we wanted to see if the week 0 recall could predict
average vitamin D intake as well as the other weeks, therefore, the average vitamin D
intake from each of the three week recalls and the average of the week recalls were used
to predict the average intake from the food records (reference) along with dummy
variables for cohort. As seen in Figure 4.12, all 3 of the week recalls were significant
predictors of average vitamin D intake. The week 0 measurement explained 54% of the
variation (R2 = 0.54, p < 0.0001) in average vitamin D intake and was equally as good of
a predictor as the week 4 and 7/8 measurement (Figure 4.12). However, the average of
the 3 week recalls was a significantly better predictor of average vitamin D intake, as
would be expected, because the average provides more information and a better estimate
of the whole time period.
A month recall using the vitamin D-specific FFQ was also administered at week
0, 4 and 8 (7 for Fall) to assess vitamin D intake over the previous month before the
304
subjects' blood draw. The average vitamin D intake calculated from the month recall at
week 4 and 7/8 was compared to the vitamin D calculated from the subjects' food
records, over the same period, as the reference. The measurements were highly
correlated (r = 0.69, p < 0.0001, Figure 4.13, Table 4.4) validating the month recall
vitamin D-specific FFQ as a useful tool for assessing vitamin D intake.
As with the week recall, we wanted to see if the month recall at week 0 could
predict average vitamin D intake as well as the other weeks, therefore, the average
vitamin D intake from each of the three month recalls and the average of the month
recalls were used to predict the average intake from the food records (reference) along
with dummy variables for cohort. As seen in Figure 4.14, all of the month recall
measurements were significant predictors of average vitamin D intake. The month recall
at week 0 explained 49% of the variation (R = 0.49, p < 0.0001) in average vitamin D
intake and was equally as good of a predictor as the week 4 and 7/8 month
measurements (Figure 4.14). Vitamin D intake seems to remain somewhat consistent
overtime and is easier to predict using the vitamin D-specific FFQ.
4.5 e Vitamin D-Specific FFQ Association with Serum 250HD
Vitamin D from the diet contributes to circulating levels of 250HD. To examine
the association with serum 250HD at each week along with vitamin D intake from the
vitamin D-specific FFQ at each week, a correlation matrix was constructed for the week
and month diet recalls (Table 4.9, Table 4.10). For the week recall using the vitamin D-
specific FFQ, the average vitamin D intake was highly correlated to the week 0, 4 and
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7/8 measurements (r = 0.84 to r = 0.88, p < 0.0001, Table 4.9). The lowest correlation
for vitamin D intake was between the week 0 and the week 7/8 measurements although
still highly significant (r = 0.59, p < 0.0001, Table 4.9). For the month recall, using the
vitamin D-specific FFQ, the average vitamin D intake was also highly correlated to the
month recalls at week 0, 4 and 7/8 (r = 0.88 to r = 0.93, p < 0.0001, Table 4.10) with the
lowest correlation for intake between the month recall at week 0 and week 7/8 (r = 0.70,
p < 0.0001, Table 4.10). Thus, the week and month recall measurements using the
vitamin D-specific FFQ at the different time points were moderately to highly correlated
to each other.
Correlations between the week recall measurements using the vitamin D-specific
FFQ and serum 250HD at the same time points were also examined to see how well
they correlated with each other. The week 0 recall and the week 0 serum 250HD
measurement (r = 0.34, p = 0.0037), as well as, the week 4 recall and week 4 250HD (r
= 0.35, p = 0.0029) and the week 7/8 recall and week 7/8 250HD were significantly
correlated to each other (r = 0.33, p = 0.0046) (Figure 4.15, Table 4.4). The average of
the vitamin D intake from the week recalls and the average of the 3 serum levels had the
highest correlation as expected (r = 0.36, p = 0.0017, Figure 4.15, Table 4.4). Thus,
vitamin D intake recalled from the week prior to the blood draw is significantly
correlated to serum 250HD. For the month recall, using the vitamin D-specific FFQ, the
correlation between the individual weeks and their respective serum 250HD level
ranged from r = 0.33 (p = 0.0046) to r = 0.39 (p = 0.0007) (Figure 4.16, Table 4.4).
Therefore, significant moderate correlations were found between serum 250HD and
vitamin D intake using the vitamin D-specific FFQ.

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4.5 f The Method of Triads for the Vitamin D-Specific FFQ
Spearman correlations between each of the three dietary measurements were
used to calculate the validity coefficient for the vitamin D-specific FFQ using the
method of triads approach (28) and the results are shown in Table 4.8. Using the
vitamin D-specific FFQ, the validity coefficient for the week recall was 0.91 and the
validity coefficient for the month recall was 0.90 (Table 4.8) confirming the usefulness
of the recall questionnaires in predicting vitamin D.
4.5 g Model Predicting Vitamin D Status using Sun Exposure Recall
Methods
Multiple linear regression analysis was used to predict vitamin D status using
continuous variables for sun exposure, vitamin D intake and skin reflectance. The
reference model used joules from the daily sun exposure logs, vitamin D intake from the
food records, skin reflectance and cohort. This model explained 53% of the variation in
serum 250HD, with sun exposure as the most significant predictor of status then skin
reflectance and then vitamin D intake (Table 4.11). In comparison, the sun exposure
recall measurements were used in the model instead of the daily sun exposure logs. In
Table 4.11, the week sun recall in joules for week 0 was used to predict serum 250HD
at week 0, along with the skin reflectance measurement at week 0 and the food records at
week 0. This model explained 41% of the variation in serum 250HD and the week sun
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recall was the most significant predictor of status. The week 4, 7/8 recalls and an
average of the week 0, 4 and 8 sun recalls, are also shown in Table 4.11, and these
models explained 47%, 49% and 51% of the variation in serum 250HD, respectively.
The week sun exposure recall was the most significant predictor of status in all of the
models. Thus, the week sun exposure recall is a useful tool to use to predict vitamin D
status.
The month sun exposure recall was also used in the model first as minutes
(Table 4.12) and then as the SEI (Table 4.13). For the full model using time, the
reference model used average time outside from the daily sun exposure logs and
explained 47% of the variation in serum 250HD (Table 4.12). The month sun exposure
recall in minutes was put into the week 0 model, week 4 model, week 7/8 model and an
average was used for all the weeks (Table 4.12). The week 0 model explained 39% of
the variation in serum 250HD while the week 4, week 7/8 and the average model
explained 46%, 40% and 45% of the variation in serum 250HD, respectively (Table
4.12). For the full model using the SEI, the reference model used the average SEI from
the daily sun exposure logs and explained 53% of the variation in serum 250HD (Table
4.13). The month sun exposure recall using the SEI was put into week 0 model, week 4
model, week 7/8 model and an average was used for all the weeks (Table 4.13). The
week 0 model explained 47% of the variation in serum 250HD while the week 4, week
7/8 and the average model explained 54%, 48% and 55% of the variation in serum
250HD, respectively (Table 4.13). The average of the 3 weeks explained more of the
variation in serum 250HD than the reference model (55% vs. 53%, respectively, Table
4.13). Perhaps, incorporating the month recall at week 0, adds valuable information over
a wider time period making the average more useful for predicting status. In all of the
models, the month recall using the SEI was the most significant predictor of status.
Consequently, the month sun exposure recall, especially using the SEI, is a useful tool
for predicting vitamin D status.
For comparison, the SEI was calculated from the subjects' screening
questionnaires upon entry into the study. Subjects were asked about typical minutes
outside and typical attire for the season in which they were entering the study. From this
information, an average SEI was calculated for each subject at baseline. In Table 4.13,
the SEI from the screening questionnaire, was included in the model for sun exposure.
This model explained 40% of the variation in serum 250HD and although the SEI was
significant, skin reflectance had a higher standard coefficient (Table 4.13). Thus, the
sun exposure recall measurements were better at predicting status and should be used to
assess sun exposure instead of one measurement at baseline.
4.5 h Model Predicting Vitamin D Status using Vitamin D Intake Recall
Methods (Vitamin D-Specific FFQ)
The vitamin D-specific FFQ measurements were included in the model as
continuous variables for average vitamin D intake, along with joules from the daily sun
exposure logs, skin reflectance and dummy variables for cohort to predict vitamin D
status. The reference model used vitamin D intake from the food records, joules from
the daily sun exposure logs, skin reflectance and cohort and explained 53% of the
variation in serum 250HD (Table 4.14). In comparison the week recall using the
vitamin D-specific FFQ, instead of using the food records in the model, explained 54%
of the variation in serum 250HD and using the month vitamin D-specific FFQ
measurements explained 55% of the variation in serum 250HD (Table 4.14). Thus, the
FFQ methods worked as well as the reference methods in predicting vitamin D status.
Thus, the vitamin D intake recall methods are useful tools when assessing vitamin D
status.
4.5 i Model Predicting Vitamin D Status using Both Sun Exposure Recall
and Vitamin D Intake Recall Methods
Multiple linear regression analysis was used to predict vitamin D status using the
recall methods and the reference methods for comparison. The reference model used
joules from the daily sun exposure logs, vitamin D intake from the food records, skin
reflectance and cohort. This model explained 53% of the variation in serum 250HD,
with sun exposure as the most significant predictor of status then skin reflectance and
then vitamin D intake (Table 4.15). In comparison the first model in Table 4.15, used
the average week sun exposure recall measurements (joules) instead of the daily sun
exposure logs (joules) and the average week recall for vitamin D intake using the
vitamin D-specific FFQ instead of the food records. This model explained 52% of
variation in serum 250HD and sun exposure was still the most significant predictor of
status. The second model in Table 4.15, used the average month sun exposure recall
measurements (minutes) instead of the daily sun exposure logs (minutes) and the
average month recall for vitamin D intake using the vitamin D-specific FFQ instead of
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the food records. This model explained 47% of the variation in serum 250HD. The
third model in Table 4.15, used the average month sun exposure recall measurements
(SEI) instead of the daily sun exposure logs (SEI) and the average month recall for
vitamin D intake using the vitamin D-specific FFQ instead of the food records. This
model explained 56% of the variation in serum 250HD, and was significantly better at
predicting serum 250HD than the model using time for the month sun exposure recall
measurement (Figure 4.17). The model using the week recall measurements for sun
(joules) and intake and the model using the month recall measurement for sun (SEI) and
intake were equally as good at predicting vitamin D status as the references models
(Figure 4.17). Thus, the recall methods in the model, both for sun exposure and vitamin
D intake, are useful tools for predicting vitamin D status.
4.6 Discussion
The objectives of this study were to determine the validity of our vitamin D-
specific FFQ and our sun exposure recall questionnaires. This study is the first to use
the method of triads to investigate vitamin D from intake and sun exposure recall
methods. Thus, the method of triads was used to calculate the validity coefficient from
a triangular comparison between the recall method, the reference method and serum
250HD (28). The validity coefficients for the sun recall questionnaires ranged from
0.84 to 0.94 (Table 4.8). The validity coefficients for the vitamin D-specific FFQ
ranged from 0.91 to 0.90 (Table 4.8). Both of the sun and intake recall methods had
high validity coefficients validating them as useful tools for assessing vitamin D. It must
311
be kept in the mind that serum 250HD is affected by both intake and sun exposure, and
therefore, correlations between intake and 250HD and sun exposure and 250HD, may
be lower than other dietary compounds and their serum counterpart. However, serum
250HD levels are a good biomarker for vitamin D because they are not homeostatically
controlled and are largely dependent on dietary intake and production from sun exposure
(4). Biomarkers are useful for validating methods because they are considered objective,
meaning that they cannot be altered by the subject and they do not rely on the subject's
memory or compliance with record keeping (30). They are not subject to the same type
of measurement errors as other dietary assessment methods, such as errors in measuring
portion sizes, estimating actual consumption from the portion and errors associated with
the use of food composition databases (31). However, even biomarkers are not perfectly
correlated with dietary intake due to other factors related to bioavailability, metabolism,
the environment, genetics and lifestyle factors (32). Overall, the validity coefficients
reflect the recall questionnaires ability to adequately assess vitamin D status.
Several sun exposure questionnaires have been used but have not been validated.
Lips et al. developed a questionnaire using two categories of reported time outdoors
(little or frequent) and two categories of reported sun exposure level (low or high) to get
a sunshine score of low, intermediate or high to categorize people (16). Salamone et al.
used this categorization with another questionnaire that asked for recalled time spent
outside and sunscreen use over the past week (17). Lawson et al. used a 14-point scale
assigned to the number of reported times a week that the subject went shopping, visiting,
gardening or on holiday (18). The scores were used to assess the relative variation in
potential UV exposure and unfortunately, not an actual amount of time spent outdoors
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(18). Sowers et al. used a sun exposure score to rank women based on self-reported time
outside adjusted for percent body surface area exposed considering sunscreen use but not
the time of day of exposures (19). Not only was sun exposure based on recalled data,
they also assessed dietary intake from only one 24-hour recall (19). Barger-Lux and
Heaney used a sun exposure index (SEI) by multiplying usual body surface area (BSA)
exposed by the reported average sun exposure per week without sunscreen (20). Thus,
they used averages reported by the subjects for both time and clothing worn (20). For
comparison, we calculated a SEI, at baseline, using subjects' reported average time
outside and BSA exposed in the model to predict vitamin D status. This sun exposure
measurement in the model explained less of the variation (R2 = 0.40, Table 4.13) in
status than the model using the sun exposure month recall questionnaire (SEI) (R2 =
0.55, Table 4.13). Consequently, the recall questionnaires solicited more information
and not just an estimate of typical sun exposure. However, studies have depended on
recalled information for sun exposure without validating their data collection method to
a standard reference such as the PS badges or prospective sun records.
The reproducibility of sun exposure questionnaires has been tested in several
studies, from 6 months to 5 years after the initial questionnaire was given (37, 38).
However, they did not examine the association with vitamin D status. These
reproducibility studies showed similar results in which outdoor exposure that was
occupational was more reproducible than recreational exposure, sunburns were
moderately reproducible, and residential history was most reproducible (37, 38).
Chodick et al. compared 7-day records to recalls mailed out six months later which
asked the participants to recall the same 7 days (39). They found that agreement was
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significantly higher for reported time outdoors during weekdays than for weekends (p <
0.05). Typical weekly patterns are probably easier to recall than intermittent sun
exposure on the weekends.
Our study captured sun exposure during the week and weekends using 7-day and
one-month recall questionnaires. In our study spearman correlations were used to
correlate the recall methods to the references methods. The measurements in joules
from the week sun exposure recall were highly correlated (r = 0.83, p < 0.001, Figure
4.2, Table 4.4) to the daily sun exposure log joules. Similarly, the measurements in time
(minutes) from the month recalls were highly correlated (r = 0.91, p < 0.001, Figure 4.4,
Table 4.4) to the daily sun exposure logs in minutes. Furthermore, the SEI
measurements from the month recalls were also highly correlated (r = 0.95, p < 0.001,
Figure 4.6, Table 4.4) to the SEI from the daily sun exposure logs. Consequently, the
sun exposure recall methods are useful tools for assessing sun exposure.
The average vitamin D intake calculated from the week recall questionnaires
were highly correlated (r = 0.77, p < 0.001, Figure 4.11, Table 4.4) to the vitamin D
measurements from the food records. Similarly, the average vitamin D intake calculated
from the month recall questionnaires were also highly correlated (r = 0.69, p < 0.001,
Figure 4.13, Table 4.4) to the vitamin D measurements from the food records. Thus,
the week and month recall for vitamin D intake using the vitamin D-specific FFQ were
useful tools for assessing vitamin D intake.
All the recall measurements at each week were also significantly correlated to
serum 250HD at the same time points and the average of the recall measurements were
significantly correlated to the average serum 250HD (Table 4.4). However, having
314
subjects recall sun exposure, including time and BSA exposed, over the month prior to
their blood draw was most useful for examining the relationship between sun exposure
and serum 250HD. Thus, time and BSA exposed (SEI) needs to be taken into account
not just time.
Multiple linear regression analysis was used to predict vitamin D status using the
recall methods and the reference methods for comparison. The model using the week
recall measurements for sun (joules) and intake and the model using the month recall
measurement for sun (SEI) and intake were equally as good at predicting vitamin D
status as the references models (Figure 4.17). Thus, the recall methods in the model,
both for sun exposure and vitamin D intake, were useful tools for predicting vitamin D
status.
The strengths of the study include longitudinal data over each season while
participants went about their usual daily activities and measurement of their daily sun
exposure. The study also included a wide range of skin pigmentation and sun exposure.
Some of the limitations of the study include a small sample size and a limited range of
dietary intake.
Our study has shown that the contribution of sun exposure and vitamin D intake
to vitamin D status can be assessed using simple recall methods, along with skin
reflectance, in free-living adults in future population-bases studies.

315
and Dr. Aronov for running the LC-MS. I would also like to thank Dr. Slusser for
helping us with the USDA UV-B Monitoring Station, the UCD Department of Land Air
Water Resources (LAWR) (Dr. Mata) for giving us access to the station. I would like to
thank Dr. Kimlin and his staff for donating the PS badges and analyzing them. I would
also like to thank Thuan Nguyen and Jan Peerson for their advice on statistics. I am
grateful for all the help and guidance I received.
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patterns. Public Health Nutr 2001;4:73-8.
32. Hunter D. Biochemical indicators of dietary intake. In: Willett WC, ed.
Nutritional Epidemiology. New York: Oxford Press, 1998:174-243.
4.9 Figures
ure 4.1: Study Design

^n=72
n=17 n=17 n=20 n=18
Each Cohort:


5000 i
i 1 1 1 1 1
0 1000 2000 3000 4000 5000
Week Recall (Erythemal Spectrum Joules)
Figure 4.2: Correlation of the Week 4 and 81 Sun Exposure Recall in Joules2 versus the
Same Weeks Calculated using Daily Sun Exposure Log Joules1
*r = Spearman Correlation Coefficient (!Week 7 for Fall)

Significant p-value < 0.05 N= 144 (2 recalls/subject)
Regression Equation: Log JA0.1 = 0.27158 + 0.79738*Recall JA0.1 (R2 = 0.67, p <
0.0001, neither are normally distributed)
2
Joules were determined (from ambient erythemally-weighted UV-B levels adjusted for
minutes outside and body surface area (BSA) exposed) for the week 4 & 7 or 8 recall
and for the daily logs on the same days for comparison.
320
i.u -
4)
a,b a
§ 0.8-
b
Predicting Daily SunlExp
<3>
o o
- ; t ••:?V:' :v' :
1 0.2-
~k
0.0 J i " '"i "" i
WeekO Week 4 Week 7 or 8 Average
Week Sun Recall (J)
Figure 4.3: Comparison of R2 for the Weekly Sun Exposure Recall Joules1 to Predict
Average Sun Exposure for the Total 7-8 Week Period from the Daily Sun Exposure
Logs Joules1
Different letters represent a significant difference (p < 0.05) between the different weeks
and the average of the week sun exposure recall (J) to predict average sun exposure (J).
Joules were determined (from ambient erythemally-weighted UV-B levels adjusted for
minutes outside and body surface area (BSA) exposed). Week recall was conducted at
week 0, 4, and 8 (7 for Fall).
Regression Equations:
Ave. Daily Log (Eryth)A0.1 - 0.92998 + 0.20090*WkO RecallA0.2 + 0.16047*Winter +
0.26817*Spring + 0.25175*Summer (R2= 0.77, p < 0.0001 for recall, Fall as reference)
Ave. Daily Log (Eryth)A0.1 = 0.97923 + 0.19783*Wk4 RecallA0.2 + 0.04296*Winter +

Ave. Daily Log (Eryth)A0.1 = 1.10958 + 0.15758*Wk8 RecallA0.2 - 0.09471 *Winter +

0.12852*Spring + 0.2261 l*Summer (R2= 0.67, p < 0.0001 for recall, Fall as reference)
Ave. Daily Log (Eryth)A0.1 = 0.06441 + 0.95616*Ave Wk RecallA0.1 +

0.01071*Winter + 0.03548*Spring + 0.06852*Summer (R2= 0.93, p < 0.0001 for recall,
Fall as reference)
8000 i
O
T 1 , 1 ,
0 2000 4000 6000 8000
Month Recall Time (mins)
Figure 4.4: Correlation of Month Sun Exposure Recall at Week 4 and 81 in Minutes
versus Daily Sun Exposure Logs in Minutes for the Same Months

Significant p-value < 0.05 ^ e e k 7 for Fall
Regression Equation: Log minsA0.1 = 0.84986 + 0.17033*nl Recall mins (R2 = 0.82, p <
0.0001)
322
i.u -
b
a
CO
b b
© 0.8 -
a
N
a
s
ce 0.6 -
98 a
©
S
0.4 -
4)
CM
U
0.2 -
0.0 - " • i
L
--L—i —' 'i^ "• " "i •
Week 0 Week 4 Week 7 or 8 Average

Month Sun Recall (Time)
Figure 4.5: Comparison of R2 for the Monthly Sun Exposure Recalled in Time1 to
Predict Average Sun Exposure for the Total 7-8 Week Period from the Daily Sun
Exposure Logs in Time
Different letters represent a significant difference (p < 0.05) between the different time
points and the average of the month sun exposure recall (time) to predict average sun
exposure (time).
îme recalled in minutes per month. Month recall was conducted at week 0, 4, and 8 (7
for Fall)
nl Ave Log (Time) = 1.20353 + 0.55678*Mo 0 Time RecallA0.2 + 0.03288*Winter +
0.35208*Spring - 0.05513*Summer (R2= 0.45, p < 0.0001 for recall, Fall as reference)
nl Ave Log (Time) = -1.68601 + 0.77531*nl Mo 4 Time Recall + 0.16467*Winter +

nl Ave Log (Time) = -1.09802 + 0.72084*nl Mo 8 Time Recall - 0.15816*Winter -

nl Ave Log (Time) = -2.13103 + 0.82512*nl Ave Mo Time Recall - 0.00449*Winter +

0 2000 4000 6000 8000
Month Recall SEI (mins x BSA)

Figure 4.6: Correlation of Month Sun Exposure Recall at Week 4 and 81 using a Sun
Exposure Index1 versus Daily Sun Exposure Logs For the Same Time Period using a
Sun Exposure Index1

!
Sun Exposure Index (SEI) = mins x body surface area (BSA) exposed
Regression Equation: nl Log SEI - 15.54 - 17.09*Recall SEIA-0.1 (R2 = 0.90, p <
0.0001)
324
i.u - b
b b
2
a 0.8 -
o
D.
X a
w
a
s
VI 0.6 -
«
o
8 0.4 -
a
(X
s-
.o 0.2 -
*
J
0.0 i 1 r L
- l

Month Sun Recall (SEI)
Figure 4.7: Comparison of R2 for the Monthly Sun Exposure Recalled (SEI)1 to Predict
Average Sun Exposure for the Total 7-8 Week Period from the Daily Sun Exposure
Logs (SEI) l
points and the average of the month sun exposure recall (SEI) to predict average sun
exposure (SEI).
'SEI (sun exposure index) = mins x body surface area (BSA) exposed. Month recall was
conducted at week 0, 4, and 8 (7 for Fall).
nl Ave Log (SEI) = -2.02243 + 0.67020*nl Mo 0 SEI Recall + 0.26341*Winter +
nl Ave Log (SEI) = -2.64302 + 0.87673*nl Mo 4 SEI Recall + 0.18672*Winter +

nl Ave Log (SEI) = -2.26167 + 0.87097*nl Mo 8 SEI Recall - 0.32533*Winter -

nl Ave Log (SEI) = -3.19383 + 0.92926*nl Ave Mo SEI Recall + 0.07526*Winter +

250 -I 250 -i
Week 0 Snn Recall (Erythema! JoulesA0.2) Week 4 Sun Recall (Erythemal JoulesA0.2)
0 1 2 3 4 0.8 1.0 1.2 1.4 1.6 1.8 2.0
Week 81 Snn Recall (Erythemal JoulesA0.2) Average Week Sun Recall (Erythemal JoulesA0.1)
Figure 4.8: Correlation of the Week Recall for Sun Exposure and Serum 250HD (LC-
MS Data) at Week 0, 4, 7 or 8, and an Average of the 3 Time Points

Boxed circles = outliers removed
!
Week 7 for the Fall Cohort
326
r = 0.43* o
p - 0.0002 0 ^,
0 ^ '^^
-*-'
s* ^
1« o
^ ^ ^-^ ^-^ 0 ^ - - '
o *°o ^<_^r -_-~--'
0
° ID O J>T •""'•'*'• ^*b o
u
°i$.$yZ>^-~ --O
8 O
^^mfô
• / > * „8
O
O
^ o
8 o
0 2000 4000 6000 8000 1000 2000 3000 4000 5000 6000 7000
Month Sun Recall at Week 0 (mins) Month Sun Recall at Week 4 (mins)
200 ^
3©
s
I
A 150
JS
« 100
on
2
4)
0 1000 2000 3000 4000 5000 6000 7000 1000 2000 3000 4000 5000 6000 7000
Month Sun Recall at Week 8 (mins) Average Month Sun Recall (Total mins)
Figure 4.9: Correlation of the Month Recall for Sun Exposure in Time (mins) and
Serum 250HD (LC-MS Data) at Week 0,4, 7 or 8, and an Average of the 3 Time Points

!
Week 7 for the Fall Cohort
3 4 5 6 ? 3 4 6 6 7
Month Son Recall at Week 0 (ill SEI) Month Sun Recall at Week 4 (nl SEI)
r = 0.40*
p = 0.0005
E
& 150
Month Sun Recall at Week 8 (nl SEI) Average Month Sun Recall (Total nl SEI)
Figure 4.10: Correlation of the Month Recall for Sun Exposure using the Sun Exposure
Index (SEI)2 and Serum 250HD (LC-MS Data) at Week 0,4, 7 or 8, and an Average of
the 3 Time Points

'Week 7 for the Fall Cohort
2
SEI = mins x body surface area (BSA) exposed
0 200 400 600 800 1000
Week Recall Vit D FFQ (IU)
Figure 4.11: Week 4 and 8 1 Recall for Vitamin D-Specific Food Frequency
Questionnaire (FFQ) versus Food Records for Vitamin D Intake Over the Same Time
Period

Boxed circle = outlier removed
Regression Equation: Food Record (IU) A 0.2 = 0.91785 + 0.69597*Vit D FFQ (IU) A 0.2
(R2 = 0.58, p < 0.0001)
329
1.0
n/
.3
« 0.8 -
in D Tnl
a
a
a o.6 - a
W
>
1 0.4 -
Predi
f 0.2-
U.U i i i i
Week 0 Week 4 Week 7 or 8 Average
WeekD ietRe call (IU)
Figure 4.12: Comparison of R2 for the Weekly Vitamin D-Specific Food Frequency
Questionnaires (FFQ) l to Predict Average Vitamin D Intake (IU) from the Food
Records
Different letters represent a significant difference (p < 0.05) between the different weeks
and the average of the week vitamin D-specific FFQ to predict average vitamin D intake.
*Week vitamin D-FFQ recall was conducted at week 0, 4, and 8 (7 for Fall).
nl Ave Diet Record = 2.32537 + 0.57071*nl WkO Diet Recall - 0.14201*Winter +
0.04777*Spring + 0.00778*Summer (R2= 0.54, p < 0.0001, Fall as reference)
nl Ave Diet Record = 3.39700 + 0.41953*Wk4 Diet RecallA0.3 - 0.13295*Winter +

nl Ave Diet Record - -0.76880 + 3.65175*Wk8 Diet RecallA0.1 + 0.02906*Winter +

nl Ave Diet Record = 1.18211 + 0.79599*nl Ave Wk Diet Recall - 0.04705* Winter +
1000 i
0 200 400 600 800 1000
Month Recall Vit D FFQ (IU)
Figure 4.13: Month Recall at Week 4 and Sôr Vitamin D-Specific Food Frequency
Questionnaire (FFQ) versus Food Records for Vitamin D Intake Over the Same Time
Period

Significant p-value < 0.05 *Week 7 for Fall
Boxed circle = outlier removed
Regression Equation: Food Record (IU)A0.2 = -0.85077 + 2.22629* Vit D FFQ (HJTO.l
(R2 = 0.57, p < 0.0001)
331
u.e -
a,b
&
i-E a
5 0.6-
© a •^ • v . ••' •
a
S
^ 0.4 -
.s
u
-3
:
' / ' • • •:•. •• • •
•'v""'.V.' ; " : ' ;

S 0.2-
i-l:
fc
0.0 - ' • • • • • • - • ;
v r
- '•'"" " r ::::
• • i i

Month Diet Recall (IU)
Figure 4.14: Comparison of R2 for the Monthly Vitamin D-Specific Food Frequency
Questionnaires (FFQ) l to Predict Average Vitamin D Intake (IU) from the Food
Records
points and the average of the month vitamin D-specific FFQ to predict average vitamin
D intake.
'Month vitamin D-FFQ recall was conducted at week 0, 4, and 8 (7 for Fall).
nl Ave Diet Record = -0.47696 + 3.43117*Mo 0 Diet RecallA0.1 - 0.21441*Winter +
nl Ave Diet Record = -0.59916 + 3.60271 *Mo 4 Diet RecallA0.1 - 0.21320*Winter

+0.12425*Spring + 0.02003*Summer (R2= 0.61, p < 0.0001, Fall as reference)
nl Ave Diet Record = -0.93587 + 3.74069*Mo 8 Diet RecallA0.1 - 0.00810*Winter +

nl Ave Diet Record = 1.67589 + 0.70901 *nl Ave Mo Diet Recall - 0.15079*Winter +
332
r = 0.34*
p = 0.0037
200 400 600 800 1000 1200 100 200 300 400 500 600 700
Week 0 Vitamin D FFQ (IU) Week 4 Vitamin D FFQ (IU)
200-
r = 0.33*
I
S
p » 0.0046
S 150
i
3 100
>8°° °
100 200 300 400 500 600 700 200 400 600
Week 81 Vitamin 0 FFQ (HI) Average Vitamin D FFQ (IU)
Figure 4.15: Correlation of the Week Recall Vitamin-D Specific Food Frequency
Questionnaire (FFQ) for Vitamin D Intake and Serum 250HD (LC-MS Data) at Week 0,
4, 7 or 8, and an Average of the 3 Time Points

*Week 7 for the Fall Cohort
333
a
& 150
200 400 600 800 1000 100 200 300 400 500 600
Month Vitamin D FFQ at Week 0 (IU) Month Vitamin D FFQ at Week 4 (IU)
200 400 600 200 400

Month Vitamin D FFQ at Week 81 (IU) Average Month Vitamin D FFQ (IU)
Figure 4.16: Correlation of the Month Recall Vitamin-D Specific Food Frequency
Questionnaire (FFQ) for Vitamin D Intake and Serum 250HD (LC-MS Data) at Week 0,
4, 7 or 8, and an Average of the 3 Time Points

*Week 7 for the Fall Cohort
334
0.6 -i b
a,b a,b
a,b
0.5 - a
Model1
o
% 0.3 -
fa
for
c*Pd 0.2-
0.1 -
0.0 - '" • i i i • — i —
1
— i "
&
&
4*
xSfP
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rf^» < < X >*
^ **X
4* ^
J& ^
&
^
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a^ ^ S ^•°'
^
Figure 4.17: Comparison of R2 for the Different Full Models1 to Predict Average Serum
250HD
Different letters represent a significant difference (p < 0.05) between the full models to
predict week 7 or 8 serum 250HD using the LC-MS data.
Full models (continuous variables for sun exposure, vitamin D intake, skin reflectance
and dummy variables for cohort) including: Model with Week Recall (J) = Continuous
variables for sun exposure using week recall (J), vitamin D-specific week FFQ for
vitamin D intake, skin reflectance and dummy variables for cohort. Model with Month
Recall (Time) = Continuous variables for sun exposure using month recall (time),
vitamin D-specific month FFQ for vitamin D intake, skin reflectance and dummy
variables for cohort. Model with Month Recall (SEI) = Continuous variables for sun
exposure using month recall (SEI), vitamin D-specific month FFQ for vitamin D intake,
skin reflectance and dummy variables for cohort. Model with Daily Sun Logs (J) =
Continuous variables for sun exposure using daily sun exposure logs (J), food records
for vitamin D intake, skin reflectance and dummy variables for cohort. Model with PS
Badges (J) = Continuous variables for sun exposure using weekly PS badges (J), food
records for vitamin D intake, skin reflectance and dummy variables for cohort.
J (joules) = ambient UV-B adjusted for time outside (mins) and body surface area (BSA)
exposed. SEI (sun exposure index) = mins x BSA exposed.
4.10 Tables
Table 4.1: Percent Body Surface Area (BSA) Exposed*

Baseball Cowboy
A Nothing Beanie/helmet cap hat Neck Hands
Head 4% 3% 2% 1% 2% 4%
Quarter
Bikini top/ length Long sleeves/
B Nothing Sports bra Tank top Tee shirt shirt Coat
Torso 47% 42% 18% 10% 3% 0%
Bikini Short shorts/ Shorts to
C bottom Skirt the knee Pants
Legs 38% 24% 8% 0%
Tennis
D Nothing Sandals shoes
Feet 2% 1.5% 0%
*Clothing key for the daily sun exposure logs, BSA based on "rule of nines" for burn
patients (22).
Table 4.2: Categories Included in the Vitamin D-Specific Food
Frequency Questionnaire (FFQ) in Bold and Examples of Foods1
Food or Beverage Serving Size Vit D1 (II
MILK
Milk 8FO 100
Milk, chocolate 8FO 100
Soy milk, ready-to-drink, regular, fortified 8FO 100
Cream, half and half (10-12% fat) 1TB 2.4
ITEMS MADE WITH MILK
Flan 4 0Z 48.4
Pudding, homemade 4 0Z 43.4
Potatoes, mashed 1/2 CP 6.6
Sauce, cream 1/4 CP 28.4
Whipped cream or dessert topping, aerosol, dairy, regular 1/4 CP 4.8
YOGURT / ICE CREAM
Yogurt, frozen 1/2 CP 0.4
Yogurt, fruited, regular, unknown % fat 6 0Z 0.8
Yogurt, Yoplait Original Yogurt - fruit flavors 6 0Z 80
Ice cream and frozen desserts, regular (11% fat) 1/2 CP 25.2
CHEESE/BUTTER
Cheddar cheese, natural 10Z 3.6
Mozzarella cheese, unknown type 10Z 2
American cheese, process 10Z 2.4
Parmesan cheese, dry (grated), regular 1TB 2
Cottage cheese, regular or creamed (4% fat) 1/2 CP 1.2
Cream cheese, regular (approx 30% fat), brick 1 TB, solid 1.6
Butter, regular, salted 1TB 8
EGGS / FISH / LIVER
Eggs, whole, cooked 1 large 25.6
Salmon, cooked from fresh or frozen, unknown kind 3 OZ, edible portion 296
Mackerel, cooked from fresh or frozen, unknown kind 3 OZ, edible portion 183.6
Tuna, canned, water pack, regular, drained - not rinsed 3 0Z 136
Sardines, canned in water - drained 3 0Z 156.4
Catfish 3 0Z 483.6
Cod liver oil ITS 454
Shrimp, cooked from fresh or frozen 3 OZ, edible portion 121.6
Sashimi - raw fish 1 TB, solid 30.4
Sushi, with fish and vegetables in seaweed 5 piece 9.2
Liver, unknown type 3 OZ, after cooking 13.6
CEREAL
Cereal, ready-to-eat, Cheerios (General Mills) 1CP 40
Cereal, ready-to-eat, Frosted Flakes (Kellogg's) 1CP 53.2
OTHER / SUPPLEMENTS
Ensure Liquid 8FO 100
Slim-Fast - ready-to-drink can, Creamy Milk Chocolate 11 FO 140
Orange, juice, fortified with calcium and vitamin D 8 FO, without ice 100
Vitamins, multi-vitamin, regular 1 tablet 400
Foods analyzed for vitamin D (IU) content using the Nutrition Data System for Research (NDSR)
Program (2006, University of Minnesota, Minneapolis, MN).
337
Table 4.3:
1
Ethnicity/Ancestry
Hispanic 1 2 1 0 4
N. Asian 1 3 2 6 12
S. Asian 5 3 6 1 15
African 0 4 5 6 15
Gender
Female 14 11 16 12 53
Male 3 6 4 6 19
Reported Sun Exposure
Low 10 13 8 14 45
High 7 4 12 4 27
3
Age (x ± SD) 23 ± 3 27 ± 5 23 ± 4 22 ± 3 24 ±
4
BMI (x ± SD) 23 ± 2 24 ± 2 23 ± 2 22 ± 3 23 ±
'Self-reported ethnicity/ancestry.
2
3
4
Body mass index (kg/m2) averaged over wk 0,4 and 7 or 8.
Table 4.4: Correlations Between Sun and Diet Recall and Reference
Measurements and Serum 250HD 1
Spearman Correlations p-value
Week Recall for Sun Exposure:
Daily Sun Log (J) & Week 4,8 Recall (J) 0.83 <0.0001*
Wk 0 250HD & Wk 0 Week Recall (J) 0.39 0.0006*
Wk 4 250HD & Wk 4 Week Recall (J) 0.34 0.0038*
Wk 8 250HD & Wk 8 Week Recall (J)** 0.31 0.0107*
Ave. 250HD & Ave. Week Recall (J) 0.42 0.0003*
Month Recall for Sun Exposure:
Daily Log (Time) & Month (wk 4,8) Recall (Time) 0.91 O.0001*
Wk 0 250HD & Wk 0 Month Recall (Time) 0.38 0.0010*
Ave. 250HD & Ave. Month Recall (Time) 0.40 0.0004*
Daily Log (SEI) & Month (wk 4,8) Recall (SEI) 0.95 O.0001*
Wk 0 250HD & Wk 0 Month Recall (SEI) 0.49 O.0001*
Wk 4 250HD & Wk 4 Month Recall (SEI) 0.48 O.0001*
Wk 8 250HD & Wk 8 Month Recall (SEI) 0.40 0.0005*
Ave. 250HD & Ave. Month Recall (SEI) 0.51 O.0001*
Week Recall for Vitamin D Intake:
Food Record (IU) & Week 4,8 FFQ (IU)** 0.77 O.0001*
Wk 0 250HD & Wk 0 Week Recall (IU) 0.34 0.0037*
Ave. 250HD & Ave. Week Recall (IU) 0.36 0.0017*
Month Recall for Vitamin D Intake:
Food Record (IU) & Month (wk 4,8) FFQ (IU)** 0.69 O.0001*
Wk 0 250HD & Wk 0 Month Recall (IU) 0.33 0.0046*
Ave. 250HD & Ave. Month Recall (IU) 0.37 0.0012*
'Using LC-MS Data for Serum 250HD and week 7 for Fall
r = Spearman Correlation Coefficient

*Significant p-value < 0.05 **Outliers removed from correlation
Table 4.5: Correlation* Matri x o f t h e Wfeek Sun Exposure Recall in Joules (J) and
Serum 2 5 0 H D (LC-MS Data ) at Week 0, 4, 8 1 and Averag e of the 3 Weeks
WkO Wk4 Wk8 Ave WkO J Wk4J Wk8J Ave
250HD 250HD 250HD 2SOHD WkJ
WkO 1 0.92 0.91 0.97 0.41 0.32 0.11 0.36
250HD <0001 <0001 <.0001 0.0005 0.0073 0.3573 0.0028
Wk4 1 0.89 0.96 0.41 0.38 0.18 0.40
250HD <.0001 <.0001 0.0005 0.0015 0.1526 0.0007
Wk8 1 0.96 0.43 0.44 0.31 0.48
250HD <.0001 0.0002 0.0002 0.0107 <.0001
Ave 1 0.42 0.40 0.22 0.43
250HD 0.0003 0.0007 0.0762 0.0007
1 0.77 0.47 0.80
WkO J <.0001 <.0001 <.0001
1 0.77 0.95
Wk4J <.0001 <0001
1 0.86
Wk8J <.0001
AveWk 1
J
*Week 7 for Fall, n:=68 (4 out iers remxy/ed)

340
Table 4.6: Correlation* Matrix of the Month Sun Exposure Recall in Time (mins)
and Serum 2 5 0 H D (LC-MS Data) at Week 0 , 4 , 8 and Average of the 3 Weeks
Ave
WkO Wk4 Wk8 Ave Month Month Month Mo
250HD 250HD 250HD 250HD OTime 4 Time 8 Time Time
WkO 1 0.93 0.91 0.97 0.38 0.36 0.13 0.35
250HD <.0001 <.0001 <0001 0.001 0.0016 0.2772 0.0026
Wk4 1 0.90 0.97 0.36 0.43 0.19 0.37
250HD <0001 <.0001 0.0022 0.0002 0.1178 0.0013
Wk8 1 0.97 0.40 0.46 0.27 0.45
250HD <.0001 0.0005 <.0001 0.0234 <.0001
Ave 1 0.39 0.42 0.20 0.40
250HD 0.0007 0.0002 0.0847 0.0004
Month 1 0.56 0.46 0.87
OTime <.0001 <.0001 <.0001
Month 1 0.73 0.79
4 Time <.0001 <.0001
Month 1 0.75
8 Time <.0001
Ave Mo 1
Time
1
Week 7 for Fall

341
Table 4.7: Correlation* Matrix of the Month Sun Exposure Recall using the Sun
Exposure Index (SEI) and Serum 250HD (LC-MS Data) at Week 0,4, 81 and
Average of the 3 Weeks
Ave
250HD 250HD 2SOHD 250HD OSEI 4 SEI 8 SEI SEI
WkO 1 0.93 0.91 0.97 0.49 0.41 0.26 0.46
250HD <.0001 <.0001 <.0001 <.0001 0.0003 0.028 <.0001
Wk4 1 0.90 0.97 0.46 0.48 0.31 0.47
250HD <.0001 <.0001 <0001 <.0001 0.0086 <.0001
Wk8 1 0.97 0.50 0.50 0.40 0.53
250HD <.0001 <.0001 <.0001 0.0005 <.0001
Ave 1 0.50 0.48 0.34 0.51
250HD <.0001 <.0001 0.0034 <.0001
Month 1 0.72 0.63 0.92
OSEI <.0001 <.0001 <.0001
Month 1 0.83 0.88
4 SEI <.0001 <.0001
Month 1 0.84
8 SEI <.0001
Ave Mo 1
SEI
^ e e k 7 for Fall

Table 4.8: Method of Triads* Validation of Recall Questionnaires
Validity
1
Correlation coefficients coefficients2
Recall Method Q vs. B 4 Q vs. R5 Bvs. R
3
Qvc
Sun Recall (Week Joules) 0.31 0.83 0.36 0.84
Sun Recall (Month Time) 0.34 0.91 0.38 0.90
Sun Recall (Month SEI) 0.44 0.95 0.47 0.94
Vit D FFQ (Week IU) 0.33 0.77 0.31 0.91
Vit D FFQ (Month IU) 0.36 0.69 0.31 0.90
1
Spearman Correlation Coefficients
2
Validity Coefficients for recall questionnaires using Method of Triads
3
Q = Average week 4 and 7 or 8 recall questionnaire measurement (Sun recall using
questionnaires or vitamin D intake recall using vitamin D-specific food frequency
questionnaire (Vit D FFQ))
4
B = Average biochemical measurement (Serum 250HD using LC-MS data)
5
R = Average reference measurement for 7-8 weeks (Daily sun exposure logs for sun
recall and food records for Vit D FFQ)
*Method of Triads Equation = Qvc = V (QvsB) x ((QvsR)/(BvsR)) (29)
343
Table 4.9: Correlation* Matrix of the Week Vitamin D-Specific FFQ (IU) and
Serum 25QHD (LC-MS Data) at Week 0,4, 81 and Average of the 3 Weeks
Ave
WkO Wk4 Wk8 Ave WkO Wk4 Wk8 Wk
250HD 250HD 250HD 250HD Diet Diet Diet Diet
WkO 1 0.93 0.91 0.97 0.34 0.27 0.26 0.32
250HD <.0001 <.0001 <.0001 0.0037 0.0232 0.0263 0.0061
Wk4 1 0.90 0.97 0.37 0.35 0.37 0.40
2SOHD <.0001 <.0001 0.0012 0.0029 0.0015 0.0005
Wk8 1 0.97 0.32 0.30 0.33 0.34
250HD <.0001 0.0063 0.0103 0.0046 0.0033
Ave 1 0.36 0.31 0.32 0.36
250HD 0.0019 0.0087 0.0055 0.0017
WkO 1 0.66 0.59 0.84
Diet <.0001 <0001 <.0001
Wk4 1 0.69 0.88
Diet <.0001 <.0001
Wk8 1 0.86
Diet <.0001
AveWk 1
Diet
^ e e k 7 for Fall

344
Table 4.10: Correlation* Matrix of the Month Vitamin D-Specific FFQ (IU) and
Serum 25QHD (LC-MS Data) at Week 0, 4, 81 and Average of the 3 Weeks
Ave
250HD 250HD 250HD 250HD ODiet 4 Diet 8 Diet Diet
WkO 1 0.93 0.91 0.97 0.33 0.30 0.36 0.35
250HD <.0001 <.0001 <.0001 0.0046 0.0103 0.0022 0.0028
Wk4 1 0.90 0.97 0.36 0.39 0.41 0.40
250HD <.0001 <.0001 0.0016 0.0007 0.0004 0.0005
Wk8 1 0.97 0.33 0.30 0.36 0.34
250HD <.0001 0.0042 0.0093 0.0018 0.0032
Ave 1 0.35 0.34 0.38 ' 0.37
250HD 0.0022 0.0035 0.0009 0.0012
Month 1 0.78 0.70 0.90
ODiet <.0001 <.0001 <.0001
Month 1 0.81 0.93
4 Diet <.0001 <.0001
Month 1 0.88
8 Diet <.0001
Ave Mo 1
Diet
êekTforFall

Table 4.11: Multiple Linear Regression Model using Sun Exposure Recalled Over the Past Week at
Week 0, 4 and 81 to Predict Serum 25QHD2 in the Full Model
Model ri Est. SE Est. p-value R2
Full Model at Week 0 (Wk Recall) 72 0.41
Joules (Erythemal Spectrum) 0.11855 0.03618 0.41704 0.0017*
VitD Intake (IU) 0.00885 0.00789 0.10974 0.2660
Fall Ref
Winter -0.13125 0.06544 -0.25326 0.0491*
Spring -0.16132 0.06497 -0.32830 0.0156*
Summer -0.11166 0.06801 -0.21969 0.1055
Full Model at Wk 4 (Week Recall) 72 0.47
Vit D Intake (IU) 0.03084 0.00980 0.28840 0.0025*
Fall Ref
Winter -0.04984 0.01885 -0.29666 0.0103*
Spring -0.05993 0.02347 -0.37625 0.0130*
Summer -0.03476 0.02322 -0.21100 0.1392
Full Model at Wk 8 (Week Recall) 68 0.49
Vit D Intake (IU) 0.13089 0.04135 0.29991 0.0024*
Fall Ref
Winter -0.28829 0.13973 -0.28001 0.0434*
Spring -0.17319 0.17719 -0.17700 0.3322
Summer -0.22809 0.16344 -0.21702 0.1679
Full Model Ave. of Wk 0,4, 8
(Week Recall) 72 0.51
Joules (Erythemal Spectrum) 0.18027 0.03905 0.59808 O.0001*
Vit D Intake (IU) 0.01959 0.00964 0.18196 0.0463*
Fall Ref
Winter -0.05115 0.01776 -0.31615 0.0054*
Spring -0.07056 0.02329 -0.45989 0.0035*
Summer -0.05689 0.02308 -0.35851 0.0163*
Full Model** with Daily Sun Log 72 0.53
Joules (Erythemal Spectrum) 0.18255 0.03619 0.64429 <0.0001*
Vit D Intake (IU) 0.02067 0.00938 0.19198 0.0311*
Fall Ref
Winter -0.05230 0.01736 -0.32323 0.0037*
Spring -0.07380 0.02257 -0.48101 0.0017*
Summer -0.06626 0.02316 -0.41755 0.0057*
•Significant p-value < 0.05 Ref = Reference dummy variable in the model. 'Week 7 for Fall
Joules = mean joules includes ambient UV-B, time & BSA exposed per subject either from week recall or
daily logs (reference). Forehead = mean forehead L* skin reflectance per subject.
IU = mean vitamin D intake from food records including supplements per subject. 2LC-MS data
**Reference Model.
Table 4.12: Multiple Linear Regression Model using Time Recalled Over the Past Month at Week 0,4
and 81 to Predict Serum 250HD2 in the Full Model
Full Model at Wk 0 (Month Recall) 72 0.39
Time (mins) 0.09001 0.03026 0.31528 0.0041*
VitD Intake (IU) 0.01059 0.00795 0.13128 0.1871
Fall Ref
Winter -0.19499 0.06237 -0.37626 0.0026*
Spring -0.12741 0.06353 -0.25928 0.0491*
Summer -0.05699 0.06465 -0.11211 0.3813
Time (mins) 0.03084 0.00907 0.35219 0.0012*
Vit D Intake (IU) 0.02763 0.01009 0.25841 0.0080*
Fall Ref
Winter -0.04363 0.01915 -0.25973 0.0260*
Spring -0.02956 0.02005 -0.18557 0.1452
Summer 0.00032 0.01992 0.00196 0.9872
Time (mins) 0.12827 0.05702 0.24242 0.0279*
VitD Intake (IU) 0.09513 0.04244 0.22254 0.0284*
Fall Ref
Winter -0.13717 0.13051 -0.12975 0.2971
Spring 0.10544 0.14140 0.10519 0.4585
Summer 0.12145 0.13477 0.11713 0.3708
Full Model Ave. of Wk 0, 4, 8
(Month Recall) 72 0.45
Time (mins) 0.03444 0.00978 0.36453 0.0008*
Vit D Intake (IU) 0.02389 0.01009 0.22191 0.0208*
Fall Ref
Winter -0.04473 0.01867 -0.27646 0.0195*
Spring -0.02415 0.01987 -0.15741 0.2286
Summer -0.00634 0.01971 -0.03995 0.7488
Full Model** with Time (Daily Logs) 72 0.46
Time (mins) 0.03671 0.00981 0.37596 0.0004*
VitD Intake (IU) 0.02241 0.01002 0.20812 0.0287*
Fall Ref
Winter -0.04422 0.01848 -0.27332 0.0196*
Spring -0.02344 0.01950 -0.15276 0.2339
Summer 0.00178 0.01915 0.01123 0.9262
*Significant p-value < 0.05 Ref = Reference dummy variable in the model. 'Week 7 for Fall
Time = mean minutes per subject either from month recall or daily logs (reference).
IU = mean vitamin D intakefromfood records including supplements per subject. 2LC-MS data
**Reference Model.
347
Table 4.13: Multiple Linear Regression Model using a Sun Exposure Index (SEI) Recalled Over the
Past Month at Week 0, 4 and 81 to Predict Serum 250HD 2 in the Full Model
SEI (mins x BSA) 0.07291 0.01656 0.44018 O.0001*
Vit D Intake (IU) 0.00974 0.00744 0.12071 0.1950
Fall Ref
Winter -0.16210 0.05906 -0.31278 0.0078*
Spring -0.14083 0.05949 -0.28659 0.0209*
Summer -0.08716 0.06127 -0.17147 0.1597
Full Model at Wk 4 (Month Recall) 72
SEI (mins x BSA) 0.02688 0.00552 0.49091 O.0001* 0.54
Vit D Intake (IU) 0.02138 0.00957 0.19996 0.0290*
Fall Ref
Winter -0.04165 0.01779 -0.24795 0.0223*
Spring -0.04106 0.01895 -0.25779 0.0339*
Summer -0.01788 0.01922 -0.10854 0.3555
SEI (mins x BSA) 0.14427 0.03606 0.41565 0.0002*
Vit D Intake (IU) 0.09236 0.03948 0.21605 0.0224*
Fall Ref
Winter -0.15527 0.12126 -0.14687 0.2049
Spring -0.02122 0.13630 -0.02117 0.8768
Summer -0.02098 0.13223 -0.02023 0.8744
Full Model Ave. of Wk 0,4, 8
(Month Recall) 72 0.55
SEI (mins x BSA) 0.02951 0.00550 0.51319 O.0001*
Vit D Intake (IU) 0.01993 0.00922 0.18510 0.0343*
Fall Ref
Winter -0.03913 0.01697 -0.24185 0.0243*
Spring -0.03340 0.01807 -0.21769 0.0691
Summer -0.02248 0.01842 -0.14167 0.2266
Full Model with SEI
(Screening Questionnaire WkO) 71 0.40
SEI (mins x BSA) 0.10433 0.03948 0.29250 0.0103*
Vit D Intake (IU) 0.01755 0.01092 0.16170 0.1130
Fall Ref
Winter -0.04605 0.01979 -0.28245 0.0232*
Spring -0.02170 0.02078 -0.14329 0.3004
Summer -0.00054 0.02018 -0.00345 0.9787
Full Model** with SEI
(Daily Sun Log) 72 0.53
SEI (mins x BSA) 0.02757 0.00546 0.49568 O.0001*
Vit D Intake (IU) 0.01933 0.00941 0.17955 0.0440*
Fall Ref
Winter -0.04174 0.Q1727 -0.25797 0.0185*
Spring -0.03593 0.01867 -0.23422 0.0587
Summer -0.02254 0.01884 -0.14206 0.2358
Table 4.13 Continued:
'Week 7 for Fall
2
LC-MS data
Sun Exposure Index (SEI) = mean SEI calculated from minutes outside x body surface
area (BSA) exposed per subject either from month recall, screening questionnaire or
daily logs (reference).
IU = mean vitamin D intake from food records including supplements per subject.
**Reference Model.
Table 4.14: Multiple Linear Regression Model using Vitamin D-Specific Food Frequency
Questionnaire (FFQ), to Assess Vitamin D Intake, Recalled Over the Past Week or Month at Week 0,
4 and 81 to Predict Serum 250HD2 in the Full Model
Full Model using Week FFQ
(AveofWkO,4,8) 72 0.54
Vit D Intake from FFQ (IU) 0.02213 0.00862 0.22919 0.0125*
Fall Ref
Winter -0.05238 0.01715 -0.32374 0.0033*
Spring -0.06391 0.02282 -0.41654 0.0067*
Summer -0.05779 0.02327 -0.36413 0.0156*
Full Model using Month FFQ
(AveofWkO,4, 8) 72 0.55
Fall Ref
Winter -0.05552 0.01703 -0.34310 0.0018*
Spring -0.06274 0.02269 -0.40895 0.0074*
Summer -0.05602 0.02319 -0.35303 0.0185*
Full Model** with Food Records 72 0.53
Vit D Intake from FR (IU) 0.02067 0.00938 0.19198 0.0311*
Fall Ref
Winter -0.05230 0.01736 -0.32323 0.0037*
Spring -0.07380 0.02257 -0.48101 0.0017*
Summer -0.06626 0.02316 -0.41755 0.0057*
•Significant p-value < 0.05 Ref = Reference dummy variable in the model. 'Week 7 for Fall
Joules = mean joules includes ambient UV-B, time & BSA exposed per subject either from daily sun
exposure logs.
IU = mean week or month vitamin D intake from vitamin D-specific food frequency questionnaire (FFQ)
or mean vitamin D intake from food records (FR) (reference) per subject.
2
LC-MS data "Reference Model.
Table 4.15: Multiple Linear Regression Models using Recalled Sun Exposure and a Vitamin D-
Specific FFQ or Daily Sun Exposure Logs and Food Records to Predict Serum 25QHD1
Model Est. SE Est. p-value R
Full Model using
Week Vit D FFQ & Sun Recall 72 0.52
Fall Ref
Winter -0.05123 0.01752 -0.31662 0.0048*
Spring -0.06050 0.02361 -0.39436 0.0127*
Summer -0.04878 0.02320 -0.30739 0.0394*
Full Model using
Month Vit D FFQ & Sun Recall 72 0.47
Time (mins) 0.03011 0.00975 0.31867 0.0030*
Fall
Winter -0.04908 0.01828 -0.30333 0.0092*
Spring -0.01504 0.01967 -0.09805 0.4473
Summer 0.00059 0.01939 0.00370 0.9759
Full Model using
Month Vit D FFQ & Sun Recall 72 0.56
SEI (mins x BSA) 0.02730 0.00555 0.47471 O.0001*
Fall Ref
Winter -0.04318 0.01674 -0.26689 0.0122*
Spring -0.02587 0.01806 -0.16861 0.1568
Summer -0.01597 0.01832 -0.10066 0.3864
Full Model** using
Food Records & Daily Sun Logs 72 0.53
Vit D Intake from FR (IU) 0.02067 0.00938 0.19198 0.0311*
Fall Ref
Winter -0.05230 0.01736 -0.32323 0.0037*
Spring -0.07380 0.02257 -0.48101 0.0017*
Summer -0.06626 0.02316 -0.41755 0.0057*
*Significant p-value < 0.05 Ref = Reference dummy variable in the model. 'LC-MS Date
Joules = mean joules includes ambient UV-B, time & BSA exposed per subject either from week sun
recall or daily sun exposure logs (reference). Time = mean minutes per subject from month sun recall.
SEI (Sun Exposure Index) = mean SEI calculated from minutes outside x body surface area (BSA)
exposed per subject from month recall. Forehead = mean forehead L* skin reflectance per subject.
IU = mean week or month vitamin D intake from vitamin D-specific food frequency questionnaire (FFQ)
or mean vitamin D intake from food records (FR) (reference) per subject. **Reference Model.
351
Appendix A:
Study Consent Form

352
EXPERIMENTAL SUBJECT'S BILL OF RIGHTS

Social and Behavioral Studies
The rights below are the rights of every person who is asked to be in a research
study. As an experimental subject, you have the following rights:
1. To be told what area, subject, or issue the study is trying to find out about.
2. To be told what will happen to you and what the procedures are.
3. To be told about the risks or discomforts, if any, of the things that will happen to you
for research purposes.
4. To be told if you can expect any benefit from participating and, if so, what the
benefit might be.
5. To be allowed to ask any questions concerning the study, both before agreeing to be
involved and during the course of the study.
6. To be told what sort of medical treatment is available if any complications or injuries

arise.
7. To refuse to participate or to change your mind about participating after the study is
started.
8. To receive your signed and dated copy of this form and the consent form.
9. To be free of pressure when considering whether you wish to agree to be in the

study.
If you have other questions, please ask the researcher or research assistant. In
addition, you may contact the Institutional Review Board, which is concerned with
protecting volunteers in research projects. You may reach the IRB office by calling
(916) 703-9151, from 8:00 a.m. to 5:00 p.m., Monday through Friday, or by writing
to the Institutional Review Board, CRISP Bldg., Suite 1400, Rm. 1429, 2921
Stockton Blvd., Sacramento, California 95817.
Signature of Subject Date

353
UNIVERSITY OF CALIFORNIA, DAVIS

CONSENT TO PARTICIPATE IN A RESEARCH STUDY
Investigator's Name: Laura Hall, RD, PhD Graduate Student

Department/Telephone: Nutrition Department, UC Davis, (530) 754-9266
Study Title: SUN EXPOSURE, SKIN REFLECTANCE AND VITAMIN D

INTAKE TO PREDICT VITAMIN D STATUS
WHAT IS THE PURPOSE OF THIS STUDY?

You are being asked to participate in our research study because you are a
healthy young adult who has self-identified as having either a high or low level of
outdoor activity. We know that vitamin D is produced in the skin with sun
exposure and we also know that we get vitamin D from the foods we eat. However,
there is not a lot of information on how sun exposure during normal outdoor
activities contributes to a person's level of vitamin D in their blood (vitamin D
status).
In this study we hope to learn about how much sun exposure people get in
Davis throughout the year and how this contributes to vitamin D in the blood. We
are also interested in how much vitamin D people get in their diets. This
information will help us know more about how to get enough vitamin D from sun
exposure and in the diet.
WHAT WILL HAPPEN IF I TAKE PART IN THIS STUDY AND HOW MANY
PEOPLE WILL PARTICIPATE?
The study will last 8 weeks beginning the second week of the quarter with a
total of 20-30 participants. If you decide to participate we will ask that you have
your skin type determined, wear a personal UVB dosimeter/badge, fill out
morbidity (illness) questionnaires, keep sun exposure logs and diet records, recall
sun exposure and diet information and have your blood drawn for analysis. The
study will be repeated four times throughout the year to account for seasonal
differences in sun exposure. Each time we will recruit a new group of 20-30
participants.
In order for you to participate you will be required to attend an educational
meeting (approximately one hour in duration) in which we will teach you how to
wear the personal UVB badge and how to keep sun exposure logs and food records.
At the end of each week, you will need to turn in your logs/questionnaires and
badges. Your records will be reviewed for clarity and this will take 15 minutes.
You will be required to wear a personal UVB badge once a week for 8 weeks
(8 times), keep daily sun exposure logs for 8 weeks (56 total) and keep food records
for 4 days every other week for 8 weeks (16 total). We will also have you recall
your sun exposure and diet information three times during the study (weeks 0, 4
and 8) to see how your memory compares to your detailed records and to assess sun
354
exposure before the study began. This will occur during the visits for the blood
draws for your convenience. You will also be asked to keep track of your
morbidity (illnesses) each week (8 total) to see how this relates to your vitamin D
status.
The UVB badge that you will be asked to wear is the size of a small postage
stamp and will be worn on your wrist on a wristband. You will keep the badge
inside of it's light-proof bag until you are asked to wear it. You will be asked to
wear it from 7am-7pm and then place it back into it's bag.
Your blood will be drawn at the beginning of the study, in the middle of the
study (after 4 weeks) and at the end of the study (after 8 weeks) and analyzed for
vitamin D, its metabolites, and other indicators of vitamin D status (including
parathyroid hormone, which is involved in regulation of vitamin D activity, and
cathelicidin, a natural "antibiotic" protein found in the blood). You will need to
fast from fat (not eat fat) 4 hours before each blood draw. Twenty milliliters (ie.
four teaspoons) of blood will be drawn at each visit.
After each blood draw we will measure your skin type (reflectance) to
determine how easily vitamin D is be made in your skin because people with lighter
skin make more vitamin D after the same sun exposure than people with darker
skin. These measurements will be taken, one on the inside of the upper arm, on the
back of the hand and on the middle of the forehead in a private examining room
using a hand-held device. Each measurement takes about 10 seconds. A small (the
size of a dime) window on the instrument touches your skin, flashes light like a
camera flash, and measures the light reflected from your skin.
During these blood draws we will also measure your height and weight and
ask if any information from the screening questionnaire has changed since the
screening questionnaire was first given (ie. medication use). The 3 blood draws will
therefore take approximately 1 hour in duration each time.
WHAT OTHER CHOICES DO I HAVE IF I DO NOT TAKE PART IN THIS

STUDY?
You do not have to participate in this study.
WHAT RISKS CAN I EXPECT FROM BEING IN THIS STUDY?

There are minimal risks involved with having your blood drawn, such as,
bleeding/bruising and infection but the chances of this occurring are low. There is
no risk or pain involved from having your skin type measured or wearing a UVB
badge.
ARE THERE BENEFITS TO TAKING PART IN THIS STUDY?

It is possible that you will not benefit directly by participating in this study.
However, you will be given information about your vitamin D status. You will get a
printout of your UV/sun exposure and you will be given information on your diet.
WILL MY INFORMATION BE KEPT PRIVATE?

We will do our best to keep your information confidential. However, we
cannot guarantee total privacy. You will be assigned a study identification number
355
that will identify you instead of your name. Personal identifiers, such as name,
phone number and address, will be kept in a locked file cabinet in a separate,
secure location from study data forms and from the main study database. No
personal identifiers will be included on computer files. AH computers will be secure
with password and firewall protection. Only the study investigators will have
access to this information. Results from the study may be presented or published
but your identity will not be included.
WILL I BE COMPENSATED FOR BEING IN THIS STUDY?

You will get $25 after the first blood draw, $75 after the second blood draw
(after 4 weeks) and then another $100 after the last blood draw (after 8 weeks).
You will be compensated a total of $200.
WHAT ARE THE COSTS OF TAKING PART IN THIS STUDY?

There is no cost to you beyond the time and effort required to complete the
procedure(s) described above. We will schedule meetings and blood draws around
your schedule.
WHAT HAPPENS IF I AM INJURED BECAUSE I TOOK PART IN THIS

STUDY?
If you are injured as a direct result of research procedures, you will receive
reasonably necessary medical treatment at no cost. The University of California
does not provide any other form of compensation for injury. In the case of injury
resulting from this study, you do not lose any of your legal rights to seek payment
by signing this form.
CAN I STOP BEING IN THIS STUDY?

You may refuse to participate in this study. You may change your mind
about being in the study and quit after the study has started.
WILL SPECIMENS (Blood) TAKEN FROM ME BE USED FOR FUTURE

RESEARCH PURPOSES?
During the course of the research blood samples will be collected. We
would like to keep some of the blood for future research purposes. The research
that may be done with your specimen(s) will not benefit you directly. Any reports
about the research, done with your specimen(s), will not be shared with you.
Your name, address, phone number and any other identifying information will be
taken off anything associated with your specimen before it is given to the
researcher. This would make it very difficult for any research results to be linked
to you or your family. Also, people outside the research process will not have
access to results about any one person which will help to protect your privacy.
There are very few risks to you. We will protect your records so that your name,
address, and phone number will be kept private. Please read each question below
and think about your choice. After reading each question, initial next to "YES"
or "NO". If you have any questions, please discuss this with the researcher.
356
1. My blood may be kept for use in future research: YES

NO
2. Someone may contact me in the future to ask me to use my blood

specimens in future research:
YES NO
For further information on the use of specimens for future research purposes and
your rights as a research participant, please visit:
http://research.ucdavis.edu/IRBAdmin/Participants
WHO CAN ANSWER MY QUESTIONS ABOUT THIS STUDY?

If you have any questions about this research project please contact:
Laura Hall or Dr. Charles Stephensen directly at (530) 754-9266 or call the
study line at (530) 752-5177 ext. 7 and leave a message. You will be called back as
soon as possible and no later than 24 hours.
If you have any questions regarding your rights and participation as a research
subject, please contact the IRB Administration at (916) 703-9151 or write to IRB
Administration, CRISP Building, Suite 1400, Rm. 1429,2921 Stockton Blvd.,
Sacramento, CA 95817. The IRB Administration has also developed a web site
designed to make you familiar with your rights. The web site discusses your basic
rights as a research participant, an explanation of the informed consent process,
the basic requirement that written consent be in a language understandable to you,
and suggested sample questions to ask the research investigator regarding your
participation in the study. This web site can be accessed at:
www.research.ucdavis.edu/IRBAdmin
My signature below will indicate that I have decided to participate in this study as a
research subject. I have read and understand the information above. I understand
that I will be given a signed and dated copy of this consent form and the Bill of
Rights.
Signature of Subject or Legal Representative Print Name
Date Time
Signature of Investigator Print Name
Date Time
357
Appendix B:
Polysulphone (PS) Badge Instructions and Blood Draw Instructions

Personal UVB Badge Instructions:
-Your badge will be kept wrapped in foil (free from light) and kept in an envelope.
-You will be assigned to wear the badge on a specific day once a week (assigned on
your calendar, on its envelope and you will receive a reminder e-mail the day
before).
-On the day assigned, carefully open the envelope & unwrap the badge out of the
foil.
-Attach the badge to a wrist band (number side up) and wear it on your right wrist
from 7am (or when you wake up) to 7pm (or when the sun goes down).
-Do not cover badge with clothing (ie. put badge over sleeve or roll up sleeve).
-After 7pm/sun down, wrap the badge back in foil and seal it in its envelope (please
tape the envelope back together).
-Note the actual time worn on your daily sun exposure log.
-Treat the badge like a CD, do not touch the film!
-Do not get the badge wet! When you are swimming, please place the badge in the
sun away from the pool where it won't be damaged. (A few rain drops is okay)
-Please note any damage to the badge on its envelope (ie, scratches, water, etc.).
-The badge will be collected at our scheduled time to meet and you will be given
new badges.
-Even if you don't plan on going outside, please wear it anyway indoors.
Blood Draw Instructions:

-DO NOT eat any fat 4 hours before your scheduled blood draw.
-Meal ideas: toast with jam, juice, fruit, nonfat yogurt, salad with nonfat
dressing, carrots, celery, nonfat crackers
-Please e-mail Laura if you need help with meal planning before blood draws
-Drink 1-2 glasses of water before your blood draw.
-Bring your binder with all your logs and your latest badge(s).
-Arrive early!
359
Appendix C:
Sun Exposure Log Instructions and Key

360
Instructions for keeping a daily sun exposure log:

-Please keep track (as you go) on the day specified and do not try to use your memory
later. The accuracy of the results depends on you!
-Note if you are wearing the UVB badge today and when you actually wore it.
-Note if you took a multivitamin/mineral supplement and how much vitamin D was in it
(please give brand name).
-Note if you took a supplement with vitamin D in it (not a multivitamin), ie. Calcium
supplements typically have vitamin D in them.
-Note if you're wearing sunscreen (or makeup with sunscreen) and what SPF it is. Note
whether you reapplied it.
-Record your location (Please note if you are not in Davis)
-Record your outdoor activity next to the appropriate time period
-For example: walk, bike, run, sit, stand, swim, garden
-Not: talk on phone, study, eat lunch (please put sit or stand for these)
-Record how long you were outdoors during each time period if it is greater than 5
mins. (Please note if you are in the direct sun or the shade)
-For example: If you were outside from 1:33pm to 1:55pm, note 22 mins. in the
time period from 1pm-1:59pm.
If half of this time was in the shade, then note 1 lmin (direct) and 1 lmin (shade).
-Shade would be from a tree, awning/canopy, or an umbrella.
-If you are biking in and out of shade, please consider that direct sun.
-You can also divide the time periods for different activities or clothes
-Do not keep track of time in a car, unless it is a convertible with the top down.
-Record which parts of your body were exposed to the sun (or covered with sunscreen)
using the key provided.
-The key divides the body into 4 areas: A (head), B (torso), C, (legs), D (feet)
and helps describe how much of your skin is exposed to the sun (Please see key).
For example:
SUN EXPOSURE LOG Day of the week:_Mon

Date: 1-1-07
Are you wearing the UV badge today? |Y]/N Time: 8:45am-7:00pm
Did you take a multivit/mineral today? Y / |N|, Supplement w/D? Y / [N]
Amt of D Are you wearing sunscreen today? JY] / N SPF 15
Location Body parts with

(Note if Outdoor Time spent outdoors Yes (Y) Yes (Y) Body parts exposed sunscreen
outside activity in minutes for neck for to the sun (use key) (use key)
Time of day Davis) Direct sun Shade covered gloves A B C D A B C D
7am-7:59am
8am-8:59am Biked 15 Y 1 4 4 2
9am-9:59am
10a-10:59am Ran 20 3 3 2 3 1 3
11a-11:59am Ran 10 3 3 2 3 1 3
12p-12:59pm
1pm-1:59pm
2pm-2:59pm Sat 40 Y 1 4 4 2
3pm-3:59pm
4pm-4:59pm
5pm-5:59pm Biked 15 Y Y 1 6 4 2
6pm-6:59pm
Total time outdoors today: _60 4( [
362
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363
Appendix D:
Sun Exposure Recall Questionnaires

364
Study ID #:
Date:
Week#:
Interviewer:
SUN EXPOSURE in the last week:
1. Approximately how many minutes did you spend in the sun in the past week?
Date:
-7 days -6 days -5 days -4 days -3 days -2 days Yesterday
7am-8am ABCD ABCD ABCD ABCD ABCD ABCD ABCD
Clothes:
Mins:
Clothes:
Mins:
Clothes:
Mins:
10am- ABCD ABCD ABCD ABCD ABCD ABCD ABCD
11am
Clothes:
Mins:
11am- ABCD ABCD ABCD ABCD ABCD ABCD ABCD
12pm
Clothes:
Mins:
12pm-lpm ABCD ABCD ABCD ABCD ABCD ABCD ABCD
Clothes:
Mins:
lpm-2pm ABCD ABCD ABCD ABCD ABCD ABCD ABCD
Clothes:
Mins:
2pm-3pm ABCD ABCD ABCD ABCD ABCD ABCD ABCD
Clothes:
Mins:
Clothes:
Mins:
Clothes:
Mins:
Clothes:
Mins:
Clothes:
Mins:
NOTE: Also indicate areas of body exposed in each grid square using the key provided.
365
2. Describe your hair (ie. is it shaved?) Does the top of your head burn?
Yes / No Does your hair typically cover your neck? Yes / No
3. Did you wear your UVB badge this week? Yes / No

a. Between which hours?
b. Did you take it off? Yes / No If yes, Why?
c. Did your badge get scratched or damaged in anyway?
4. What type of outdoor activities did you participate in this past week?
a. Length of time?
5. In the last week how many times did you apply sunscreen or cosmetics with
sunscreen in them? SPF?
Typically where? Indicate when on the table above with a *.
6. Did you sun bathe in the last week? Yes / No

a. For how long?
b. Did you get a sunburn? Yes / No If yes, how bad? _________
7. Were you out of Davis this past week? Yes/No

a. Where?
b. How many miles away?
c. How long?
d. Time spent outdoors?
366
Study ID #:_
Date:
Week#:
Interviewer:
SUN EXPOSURE in the last four weeks:
Has your hair length changed in the past month that would affect sun exposure
on your head? Yes/No Describe:
Which days did you wear your badge? Any
problems/damage?
Approximately how many minutes did you spend in the sun in the past 4 wk,
summing across all 7 d of the week? (Max for each grid square would be 7 x 60 min = 420 min)
-4 wks -3 wks -2 wks Last week
7am-8am
8am-9am Information
from the 1
9am-10am wk recall
table will be
10am-11am used here
11 am-12pm
12pm-lpm
lpm-2pm
2pm-3pm
3pm-4pm
4pm-5pm
5pm-6pm
6pm-7pm
NOTE: Also indicate areas of body exposed in each grid square using the key provided.
4. In the last 4 weeks how many times did you apply sunscreen or cosmetics with
sunscreen? SPF?
Typically where? Indicate when on the table above with a *.
5. Did you sun bathe in the last 4 weeks? Yes / No
a. For how long?
b. Did you get a sunburn? Yes / No If yes, how bad?
Were you out of Davis this past month? Yes /No
a. Where? How many miles away?
b. How long? Time spent outdoors?
367
Appendix E:
Food Record Form

368
Study ID #:
$ FOOD RECORD Day of the week:
Date:
Week#
Time/Place Food & Beverage Brand Amount Fortified with Vit D?

Description Eaten Amount per serving?
Did you take a supplement with vit D in it today? Y / N, Amt. of Vit D?_
Brand:
Did you take a multivitamin/mineral with vit D in it today? Y / N, Amt.?_

Brand:
Was this a typical day for you? Y / N If no, what wasn't typical? (Higher?
Lower?)
369
Appendix F:
Vitamin D-Specific Food Frequency Questionnaires (FFQs)

370
Study ID #:
Date:
Week#:_
Interviewer:
DIET in the last week:
1. Did you take a multivitamin/mineral every day this past week? Yes / No
a. How many times?
b. Did you take any other supplements? Yes / No If yes, what?
2. How many times did you eat or drink this specific food or beverage in the past
week?
Food / Beverage Frequency/ Serving Comments

# of times sizes
Milk Don't forget milk in coffee
(Type of milk?) drinks and in
sauces/casseroles.
Chocolate milk
Pudding/Flan Which one?
Ice cream
Dessert toppings Whipped cream /Coolwhip?
Yogurt Brand:
Cheese Note type of cheese:
Butter
Margarine
Eggs Excluding egg whites.
Fish
Salmon
Mackerel
Tuna
Sardines
Catfish
Cod liver oil Note any fish oil sup.
Other
Liver
Ready-to-eat cereals Brand:
Bread Brand:
Ensure or slim fast Which beverage:
Vit D fortified OJ
Other vit D fortified
food/beverage
371
Study ID #:
Date:
Week#:_
Interviewer:
DIET in the last four weeks:
1. Did you take a multivitamin/mineral every day this past month? Yes / No
a. How many times?
b. Did you take any other supplements? Yes / No If yes, what?
2. How many times did you eat or drink this specific food or beverage in the past
month?
Food / Beverage Frequency/ Serving Comments

# of times sizes
Milk Don't forget milk in coffee
(Type of milk?) drinks and in
sauces/casseroles.
Chocolate milk
Pudding/Flan Which one?
Ice cream
Dessert toppings Whipped cream /Coolwhip?
Yogurt Brand:
Cheese Note type of cheese:
Butter
Margarine
Eggs Excluding egg whites.
Fish
Salmon
Mackerel
Tuna
Sardines
Catfish
Cod liver oil Note any fish oil sup.
Other
Liver
Ready-to-eat cereals Brand:
Bread Brand:
Ensure or slim fast Which beverage:
Vit D fortified OJ
Other vit D fortified
food/beverage

All About Vitamimin D

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All About Vitamimin D

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Sun Exposure, Skin Reflectance and

Vitamin D Intake to Predict Vitamin D Status

LAURA MARIE HALL

Submitted in partial satisfaction of the requirements for the degree of

OFFICE OF GRADUATE STUDIES

NCMHD Center of Excellence in Nutritional Genomics Grant, the Bristol-Myers Squibb

their advice and insight.

patience and encouragement. This is just the beginning!

To my loving family and

my husband, Jon- You are my sunshine

1 Vitamin D: A Review of the Literature 3

1.1 The History of Vitamin D 4

1.1a The Discovery of Vitamin D 4

L i b Vitamin D is not a Vitamin 5

1.1c Identification of Vitamin 5

L i d The Hormonal Form of Vitamin D 6

L i e The Physiological Functions of Vitamin D 6

1.1 f Discovery of the Vitamin D Receptor 8

1.2 Non-Calcemic Functions of Vitamin D 9

1.3 The Metabolism of Vitamin D 12

1.3 b The Photobiology of Vitamin D and its' Conversion in the Skin 13

1.3 c 25-Hydroxylase in the Liver 14

1.3 d la-Hydroxylase in the Kidney and Other Sites 15

1.4 Vitamin D Deficiency 16

1.4 a Prevalence of Vitamin D Deficiency 17

1.4 b Consequences of Vitamin D Deficiency 20

1.4 d Current Recommendations to Alleviate Vitamin D Deficiency 22

1.5 Factors Affecting Vitamin D Status (250HD) 22

1.5 a Seasonal Effects on Vitamin D Status 22

1.5 b Sun Exposure Effects on Vitamin D Status 23

1.5 c Sun Exposure and Dietary Vitamin D Contributions to Vitamin D 24

1.5 d Obesity Influencing Vitamin D Status 33

1.5 e Skin Pigment and Ethnicity Related to Vitamin D Status 33

1.5 f Sun Exposure and Dietary Vitamin D Contributions to Vitamin D 34

Status in High Sun Exposure Locations

1.6 Factors Affecting the Synthesis of Vitamin D in the Skin: Environmental 37

1.6 a The Influence of Season on Vitamin D Synthesis 37

1.6 b The Influence of Latitude on Vitamin D Synthesis 38

1.6 c The Influence of Time of Day on Vitamin D Synthesis 39

1.6 d The Influence of Altitude on Vitamin D Synthesis 40

1.6 e The Influence of Ozone and Aerosol on Vitamin D Synthesis 40

1.6 f The Influence of Clouds on Vitamin D Synthesis 41

1.6 g The Influence of Reflectance on Vitamin D Synthesis 42

1.7 Factors Affecting the Synthesis of Vitamin D in the Skin: 42

1.7 a The Effect of Melanin on Vitamin D Synthesis 43

1.7 c The Influence of Clothing, Glass and Plastics on Vitamin D 45

1.7 d The Effect of Aging on Vitamin D Synthesis 46

1.8 How Much Sun Exposure Do We Need? 47

1.8 a Risk and Benefit Considerations 49

1.9 Assessing Sun Exposure 54

1.9 b In Vitro Models of Vitamin D3 Synthesis and Human Skin Samples 56

1.9 c Sun Exposure Questionnaires 58

1.9 d Polysulphone (PS) Badges and Validations against Questionnaires 60

1.9 e Electronic Wrist Watch Dosimeters 63

1.9 f Ground-Based/On Ground UV Measurements 64

1.9 g The UV Index 66

1.9 h Web-Based Tools 67

1.9 i Simulated Solar Radiation 67

1.10 UV Doses Measured 70

1.10 a UV Doses in the United States 70

1.10b Personal UV Doses 71

1.10c UV Doses to the Body and UV Doses with Different Activities 73

1.11 Skin Pigmentation 78