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UV and Vitamin D with in vitro model – PhD WJ Olds 2010

William James Olds
BAppSc (Hons) (Physics) QUT, GradCert Res Commercialisation QUT
For the degree of Doctor of Philosophy, 2010.
School of Public Health Faculty of Health
Institute of Health and Biomedical Innovation Queensland University of Technology Kelvin Grove, Queensland, 4059, Australia.

Abstract

Ultraviolet radiation (UV) is the carcinogen that causes the most common malignancy in humans - skin cancer. However, moderate UV exposure is essential for producing vitamin D in our skin. Vitamin D increases the absorption of calcium from the diet, and adequate calcium is necessary for the building and maintenance of bones. Thus, low levels of vitamin D can cause osteomalacia and rickets and contribute to osteoporosis. Emerging evidence also suggests vitamin D may protect against falls, internal cancers, psychiatric conditions, autoimmune diseases and cardiovascular diseases. Since the dominant source of vitamin D is sunlight exposure, there is a need to understand what is a "balanced" level of sun exposure to maintain an adequate level of vitamin D but minimise the risks of eye damage, skin damage and skin cancer resulting from excessive UV exposure.

There are many steps in the pathway from incoming solar UV to the eventual vitamin D status of humans (measured as 25-hydroxyvitamin D in the blood), and our knowledge about many of these steps is currently incomplete. This project begins by investigating the levels of UV available for synthesising vitamin D, and how these levels vary across seasons, latitudes and times of the day. The thesis then covers experiments conducted with an in vitro model, which was developed to study several aspects of vitamin D synthesis. Results from the model suggest the relationship between UV dose and vitamin D is not linear. This is an important input into public health messages regarding 'safe' UV exposure: larger doses of UV, beyond a certain limit, may not continue to produce vitamin D; however, they will increase the risk of skin cancers and eye damage. The model also showed that, when given identical doses of UV, the amount of vitamin D produced was impacted by temperature. In humans, a temperature-dependent reaction must occur in the top layers of human skin, prior to vitamin D entering the bloodstream. The hypothesis will be raised that cooler temperatures (occurring in winter and at high latitudes) may reduce vitamin D production in humans. Finally, the model has also been used to study the wavelengths of UV thought to be responsible for producing vitamin D. It appears that vitamin D production is limited to a small range of UV wavelengths, which may be narrower than previously thought.

Together, these results suggest that further research is needed into the ability of humans to synthesise vitamin D from sunlight. In particular, more information is needed about the dose-response relationship in humans and to investigate the proposed impact of temperature. Having an accurate action spectrum will also be essential for measuring the available levels of vitamin D-effective UV. As this research continues, it will contribute to the scientific evidence-base needed for devising a public health message that will balance the risks of excessive UV exposure with maintaining adequate vitamin D.


See also VitaminDWiki


Table of Contents

2 Ultraviolet Radiation and its Impacts on Health
2.1 Introduction..... 45
2.2 Solar UV....... 46
2.2.1 Factors influencing ambient levels of solar UV. 48
2.3 Artificial UV Sources 51
2.4 Personal Exposure to Solar UV .... 51
2.5 Skin Cancer ..... 53
2.5.1 Non-melanoma skin cancer (NMSC) ...... 55
2.5.2 Cutaneous malignant melanoma (CMM).... 57
2.5.3 Incidence rates for skin cancer 59
2.5.4 Prevention of skin cancer... 60
2.6 Other Skin-Damaging Effects of UV . 61
2.6.1 Sunburn ... 61
UV dose received by the skin
2.6.2 Photoageing of the skin .... 62
2.6.3 Photodermatoses ........ 62
2.7 UV-Induced Eye Damage ....... 63
Photokeratitis ...... 63
Cataracts ......... 63
Pterygium ........ 63
Squamous cell carcinoma of the cornea and conjunctiva . . 63
2.8 Immune Suppression 63
2.9 Vitamin D Production ......... 64
2.9.1 Photobiology of vitamin D production in skin . 64
2.9.2 Factors influencing production of vitamin D in the skin...... 65
Skin pigmentation ... 65
Concentration of 7-DHC in skin ..... 65
2.9.3 Dietary sources of vitamin D . 67
2.9.4 Activation of vitamin D.... 68
2.9.5 Assessment of vitamin D status 69
2.9.6 Definition of vitamin D deficiency........ 69
2.9.7 Prevalence of vitamin D deficiency........ 70
2.9.8 Vitamin D and bone health.. 74
Bone formation and remodelling ..... 74
Osteomalacia, rickets and osteoporosis . 76
2.9.9 Vitamin D and fractures .... 79
2.9.10 Vitamin D, falls and muscle function ....... 82
2.9.11 Non-calcemic actions of vitamin D ........ 83
Cancer 83
Autoimmune diseases . 90
Cardiovascular disease . 92
Schizophrenia and psychiatric disorders 92
Infections ........ 93
Other hypothesised effects of vitamin D . 94
2.10 Relating UV to Health Outcomes Using Action Spectra 95
2.10.1 Action spectra for NMSC and CMM....... 96
2.10.2 Erythemal action spectrum .. 97
Minimal erythemal dose (MED) ..... 99
UV index ......... 99
2.10.3 Previtamin D action spectrum 100
2.11 Chapter Summary .. 103
3 Preliminary Investigations 107
3.1 Introduction ..... 107
3.2 Objectives ...... 109
3.3 Methods ....... 109
3.3.1 Computer modelling ...... 109
3.3.2 Ground-level measurement of ambient UV using Brewer spectroradiometers ......... 110
3.4 Results ........ 111
3.4.1 Effect of the time of day .... 111
3.4.2 Effect of the season.......112
3.4.3 Effect of latitude ........ 114
3.4.4 Effect of latitude: verification using ground-level Brewer spectroradiometer data ....... 117
3.5 Discussion ...... 119
3.6 Identification of Knowledge Gaps...121
3.6.1 Dose-response relationship for UV dose and vitamin D production121
3.6.2 The difficulty of producing vitamin D at mid- and high latitudes during winter 125
3.6.3 Action spectrum for previtamin D ........ 126
4 Aims and Objectives 129
5 Methodology: Vitamin D In Vitro Model 135 5.1 Introduction.....135
5.2 Approaches to Investigating Vitamin D Synthesis ... 136
5.3 Overview of the Vitamin D In Vitro Model........ 137
5.3.1 Strengths of the model ..... 138
5.3.2 Limitations of the model .... 139
5.4 7-DHC Solution...141
5.4.1 Chemicals and solvents .... 141
5.4.2 Optimising the concentration . 141
5.4.3 Stock solution longevity .... 143
5.4.4 Controls ... 144
5.5 Cuvettes ....... 144
5.6 Exposing the Samples to UV ...... 146
5.6.1 Spectral UV exposures: solar simulator ..... 146
5.7 Incubating the Samples ......... 150
5.8 High-Performance Liquid Chromatography (HPLC) .. 151
5.8.1 Principle of HPLC ....... 151
5.8.2 Method .... 151
5.8.3 Detection ... 155
5.8.4 Determining retention times . 155
5.8.5 Optimising flow rate......156
5.8.6 Optimising detection wavelengths ........ 156
5.8.7 Selecting mobile phase solvents 158
5.8.8 Standard operating parameters 158
5.8.9 Calibration protocol ...... 159
5.8.10 Batch analysis 161
5.8.11 Limits of detection and quantitation ....... 162
5.9 Proof-of-Concept Using Solar-Simulated UV ...... 163
5.9.1 Aim ...... 163
5.9.2 Method .... 163
5.9.3 Results and discussion ..... 163
5.10 Proof-of-Concept Using Solar UV ... 164
5.10.1 Aims ..... 164
5.10.2 Method .... 164
5.10.3 Results and discussion ..... 165
Comparison of the results for solar-simulated UV and solar UV .... 166
5.11 Ethanol is Not Affected By UV ....166
5.12 Chapter Summary .. 166
6 Dose-Response Relationship of UV and Vitamin D 169
6.1 Introduction ..... 169
6.2 Aim and Objectives.169
6.3 Methods ....... 170
6.4 Results ........ 172
6.4.1 Time-dependence of vitamin D formation .... 174
6.4.2 Dose-response relationship of UV and vitamin D production . . . 177
6.4.3 Effect of initial 7-DHC concentration on vitamin D production . . 179
6.5 Chapter Summary .. 179
7 Temperature and Vitamin D Production
7.1 Introduction ..... 181
7.2 Aim and Objectives.182
7.3 Determining the Temperature Range . 182
7.3.1 Location of vitamin D formation in the skin ... 183
7.3.2 Epidermal temperature .... 183
7.3.3 The effect of ambient temperature on skin temperature ...... 183
7.4 Methods ....... 186
7.4.1 In vitro model 186
7.4.2 Human skin temperature measurements .... 187
7.5 Results ........ 187
7.5.1 In vitro model 187
7.5.2 Human skin temperatures ... 191
7.6 Chapter Summary .. 191
8 Action Spectrum for Vitamin D 193
8.1 Introduction ..... 193
8.2 Aim and Objectives.194
8.3 General Methods .. 194
8.3.1 Exposure to monochromatic UV
Explanation of the irradiation monochromator (IM)
Explanation of the slit width of the IM .. 197
Calibration of the IM .. 198
Determining exposure times for a set dose.........200
Adjusting exposure times for cuvette transmittivity .... 201
8.3.2 7-DHC solution and samples.202
8.3.3 Incubation .. 202
8.3.4 HPLC analysis ......... 202
8.4 Pilot Study......203
8.4.1 Aims ..... 203
8.4.2 Methods ... 203
8.4.3 Data analysis 206
8.4.4 Results .... 206
8.4.5 Conclusions from the pilot study and directions for the main study 208
Operating procedures . 208
Wavelengths of interest 208
UV dose ......... 208
Slit width ........ 209
8.4.6 Optimising the UV dose....209
8.4.7 Optimising the FWHM and slit width of the IM211
8.5 Vitamin D Action Spectrum (Main Study)........212
8.5.1 Aims ..... 212
8.5.2 Methods ... 212
8.5.3 Results .... 214
8.5.4 Data analysis 214
8.5.5 Vitamin D action spectrum .. 216
8.6 Comparison of the QUT and CIE Action Spectra .... 219
8.6.1 Confirmation of the QUT action spectrum....223
8.7 7-DHC Depletion Spectrum......225
8.7.1 Aim ...... 225
8.7.2 Methods ... 225
8.7.3 Results .... 225
8.8 Chapter Summary .. 232
9 Discussion 235
9.1 Introduction ..... 235
9.2 Summary of the Results of the Project 236
9.2.1 Ambient levels of previtamin D UV and erythemal UV (Chapter 3) 236
9.2.2 In vitro model (Chapter 5) ...238
9.2.3 Dose-response relationship (Chapter 6)......238
9.2.4 Skin temperature and vitamin D (Chapter 7)...240
9.2.5 Action spectrum for vitamin D (Chapter 8)....242
9.3 Discussing the Results in the Context of Prior Research 243
9.3.1 Ambient levels of previtamin D UV and erythemal UV ...... 243
9.3.2 In vitro model 245
9.3.3 Dose-response relationship .. 246
9.3.4 Skin temperature and vitamin D ......... 249
9.3.5 Action spectrum for vitamin D 251
9.4 Limitations and Strengths of the Study255
9.4.1 Limitations . 256
9.4.2 Strengths ... 259
10 Implications 261
10.1 Introduction ..... 261
10.2 Towards a Balanced Public Health Message ....... 262
10.3 What Doses, How Often and When?.265
10.4 Vitamin D Production in Different Climates ....... 267
10.5 Vitamin D Production at Different Body Sites......272
10.6 Which Body Sites to Expose? .....274
10.7 Vitamin D, Latitude and the Boston Paradox ...... 275
11 Conclusions 279
A Procedure for the In Vitro Model 281
B Dose-Response Relationship (Suppl.) 319
C Skin Temperature and Vitamin D (Suppl.) 333
C.1 Standard Procedure . 333
C.2 Exhaustive Dataset . 335
D Vitamin D Action Spectrum (Suppl.)
D.1 Calibration of the IM 349
D.1.1 Overview .. 349
D.1.2 Measuring the output of the primary standard . 350
D.1.3 Measuring the output of the irradiation monochromator ..... 352
D.1.4 Dark count analysis ...... 354
D.1.5 Transferring the calibration .. 357
D.2 Determining Exposure Times at Each Wavelength ... 358
D.2.1 Adjusting exposure times for cuvette transmittivity ........ 359
D.3 Detection of Low Concentration Samples by HPLC .. 360
E Calculations Used for Implications Chapter 365
E.1 Calculating Vitamin D Production From UV Index ... 365
E.2 Incorporating the Impact of Ambient Temperature ... 367
E.2.1 Uncertainty analysis ...... 370
E.3 Vitamin D Production at Different Body Sites ...... 372
E.3.1 Uncertainty analysis ...... 373
E.4 Weighting the Boston Solar UV Spectrum ........ 375
E.4.1 Uncertainty analysis ...... 376
F Posters 377
F.1 Poster 1 ........ 377
F.2 Poster 2 ........ 377
Bibliography



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