Vitamin D production depends on ultraviolet-b dose but not on dose rate: A randomized controlled trial

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1 DOI: /j x Original Article Vitamin D production depends on ultraviolet-b dose but not on dose rate: A randomized controlled trial Morten K.B. Bogh 1, Anne V. Schmedes 2, Peter A. Philipsen 1, Elisabeth Thieden 1 and Hans Christian Wulf 1 1 Department of Dermatology, Copenhagen University Hospital, Bispebjerg, Denmark; 2 Department of Clinical Biochemistry, Lillebaelt Hospital, Vejle, Denmark Correspondence: Morten K.B. Bogh, MD, Department of Dermatology D92, Copenhagen University Hospital, Bispebjerg, Bispebjerg Bakke 23, DK-2400 Copenhagen NV, Denmark, Tel.: , Fax: , bogh@dadlnet.dk The work was performed at Copenhagen University Hospital, Bispebjerg, Denmark. Abstract: Ultraviolet-B (UV-B) radiation increases serum vitamin D level expressed as 25-hydroxyvitamin D 3 (25(OH)D), but the dose response relationship and the importance of dose rate is unclear. Of 172 fair-skinned persons screened for 25(OH)D, 55 with insufficient baseline 25(OH)D 50 nm (mean 31.2 nm) were selected and randomized to one of 11 groups of five participants. Each group was exposed to one of four different UV-B doses: 0.375, 0.75, 1.5 or 3.0 standard erythema dose (SED) for 1, 5, 10 or 20 min. All participants had four UV-B sessions with 2- to 3-day interval with 24% of their skin exposed. Skin pigmentation and 25(OH)D were measured before and after the irradiations. The increase in 25(OH)D after UV-B exposure (adjusted for baseline 25(OH)D) was positively correlated with the UV-B dose (P = 0.001; R 2 = 0.176) but not to dose rate (1 20 min). 25(OH)D increased in response to four UV-B treatments of 3 SED with 24.8 nm on average and 14.2 nm after four UV-B treatments of just SED. In conclusion, the increase in 25(OH)D after UV-B exposure depends on the dose but not on the dose rate (1 20 min). Further, a significant increase in 25(OH)D was achieved with a very low UV-B dose. Key words: dose rate UV-B dose vitamin D Accepted for publication 9 September 2010 Introduction Solar ultraviolet (UV) radiation of the skin often provides 90 95% of the vitamin D requirements expressed as 25-hydroxyvitamin-D 3 (25(OH)D) (1,2). Only ultraviolet-b (UV-B) radiation ( nm) initiates vitamin D synthesis, but it is also mainly UV-B that causes skin cancer (3 5). Nevertheless, sun exposure has been recommended to avoid vitamin D insufficiency (6,7). The UV-B dose necessary for sufficient production of vitamin D should be established to minimize the risk of skin cancer by avoiding unnecessary UV radiation. In addition, many photobiological UV effects like photo-carcinogenesis and erythema are independent of dose rate over a wide range of irradiances, which means that the reciprocity is obeyed (8 10); however, it is not known whether this is applicable for the UV-B-induced vitamin D production. Although there are several human ex vivo studies exploring the dose response relationship between vitamin D and UV-B radiation (11 14), there are only a few human in vivo studies (15 17). The majority of these studies (15,16) used minimal erythema dose (MED) where they treated the lighter skin tones with lower doses of UV-B than those for darker skin tones, assuming that skin pigmentation is of major importance for UVinduced vitamin D production. However, a newly published study calls into question whether skin pigmentation is of importance for vitamin D production (18). Consequently, it is difficult to draw firm conclusions from previous MED studies. In addition, no work has been published showing the relation between dose rate Abbreviations: 25(OH)D, 25-hydroxyvitamin D 3 ; D 25(OH)D, the increase in 25(OH)D after UVB; UVB, ultraviolet-b; SED, standard erythema dose; PPF, pigment protection factor. and vitamin D production. We therefore sought to define the importance of the UV-B dose and dose rate for vitamin D production in the skin. Materials and methods Design A randomized, controlled trial was conducted at Copenhagen University Hospital, Bispebjerg, Denmark, 56 N, in January March 2008, when the ambient UV-B radiation is negligible, and the low outdoor temperature prohibits exposure apart from the hands and face. The study is registered at ClinicalTrials.Gov and its unique ID registration number is NCT Study objective The aims of the study were (i) to determine the dose response relationship between UV-B exposure and vitamin D production and (ii) to determine the effect of dose rate on the vitamin D production after UV-B exposure. Participants and procedure We screened 172 healthy fair-skinned volunteers (101 women and 71 men, mean age 29.7, range years) for their 25(OH)D level in January Out of the 172, 56 participants were selected with a relatively homogenous baseline 25(OH)D level; a total of 55 participants completed the study, one dropped out because of personal reasons (Fig. S1). All were irradiated four times with 2- to 3-day interval on the chest and back [24% body surface area (19)] with either 0.375, 0.75, 1.5 or 3.0 standard erythema dose (SED) during 1, 5, 10 or 20 min. The data from the groups who received 3 SED were also used in our recent study (18). The baseline 25(OH)D varied from 16 to 50 nm. These limits were chosen as recent studies showed that baseline 25(OH)D influences the production of 25(OH)D after UV-B exposure (18,20 22). 14 ª 2010 John Wiley & Sons A/S, Experimental Dermatology, 20, 14 18

2 Vitamin D increase after UVB All participants responded to a questionnaire that ascertained demographic characteristics, chronic diseases, medication, use of dietary supplements, sun habits, sun holidays and solarium use since childhood. Weight and height were recorded from which the body mass index (BMI) and the total body area in square metres were calculated (Table 1) (23). Inclusion criteria Inclusion criteria were as follows: (i) age years; (ii) avoidance of sun-bed exposure; (iii) not travelling south of 45 North latitude 3 months before the study; (iv) avoiding vitamin D supplementation 2 months before the study and during the study period. Exclusion criteria Participants were ineligible for the study if they had (i) skin disease; (ii) psychiatric diseases; (iii) drug addiction; (iv) intake of medication that could cause photosensitive skin; (v) intake of cholesterol-lowering medicine; or (vi) if they were pregnant. Randomization The participants were randomized to receive 0.375, 0.75, 1.5 or 3.0 SED for 1, 5, 10 or 20 min to investigate the importance of UV-B dose and dose rate (Table 2). We used a computer-generated randomization list for numbered sealed envelopes containing notes with the four different UV-B doses and the four different exposure times. The envelopes were used consecutively as the participants entered the study. Neither the patient nor the investigator knew in advance which treatment was to be given. Ethics The ethics committee approved the study protocol (H-B ), and the study was carried out in accordance with the Declaration of Helsinki. All participants gave written informed consent. Skin type, skin pigmentation and skin redness To follow skin response to the UV-B radiation, redness per cent and pigment protection factor (PPF, measuring range ) Table 1. Baseline characteristics 1 among the 55 participants and the relation between the baseline factors and the increase in 25(OH)D (D 25(OH)D) after UV-B [not adjusted for UV-B dose ( SED)] Baseline factors Baseline P-value Sex: men women (%) 22 (40%) 33 (60%) 0.69 Weekly fish meals 1.5 ± 1.5 (0 7) 0.94 PPF buttocks ± 1.5 ( ) Redness per cent buttocks ± 6.7 ( ) 0.23 PPF upper body ± 1.6 ( ) 0.15 Redness per cent upper body ± 4.9 ( ) 0.34 Age 31.8 ± 8.6 (18 51) 0.12 Weight, kg 74.9 ± 14.0 (50 116) 0.58 Height, m 1.75 ± 0.1 ( ) 0.94 BMI, kg m ± 3.8 ( ) 0.42 Total body area (m 2 ) ± 0.22 ( ) 0.60 S-25(OH)D, nm 31.2 ± 8.3 (16 50) S-PTH, pm 3.8 ± 1.8 ( ) 0.77 Ca 2+,mm 1.22 ± 0.03 ( ) 0.42 S-Alk. phos., U l 64.7 ± 13.5 (21 92) 0.42 S-Total chol., mm 4.8 ± 0.9 ( ) (OH)D, 25-hydroxyvitamin-D 3 ; PPF, pigment protection factor; BMI, body mass index; PTH, parathyroid hormone; alk. phos., alkaline phosphatase; total chol, total cholesterol; UV-B, ultraviolet-b; SED, standard erythema dose. 1 Mean ± SD (range). 2 Constitutive objectively measured skin type. 3 P = 0.34 when adjusted for UV-B dose ( SED). 4 Objectively measured redness per cent among all the participants. 5 Facultative objectively measured skin type. 6 Total body area (in square metres) = height 0.40 weight 0.54 (23). 7 P = 0.02 (R 2 = 0.238) when adjusted for UV-B dose ( SED). were measured on the back, abdomen (facultative skin pigmentation) and buttocks (constitutive skin pigmentation) at baseline and before every UV-B session using a skin reflectance meter (UV-Optimize; Scientific, Chromo-light, Espergaerde, Denmark) (24). PPF is a direct measure of melanin in the skin (25) and is objectively measured number of SED to MED by skin reflectance measurements (26). The individual number of MED is the given number of SED divided by the measured PPF (27). The correlation between clinical measured MED and PPF is highly significant (28). A typical fair-skinned person has a PPF value of 4 5 on the buttocks and up to 8 12 on other body locations, depending on previous sun exposure (29). The redness per cent (0 100%) is an objectively skin reflectance measure of haemoglobin in the skin. Zero per cent skin redness is the reflectance from a blood-drained skin area, and 100% skin redness is fixed to the reflectance in the most affected person in a group of persons with facial dark-blue nevus flammeus. There is linearity between the clinical scale of skin redness generated by UV lamp irradiation and the objectively measure of skin redness by reflectance (27). Self-reported skin type according to Fitzpatrick was registered at baseline (30). There were five participants with Fitzpatrick skin type I; 18 with skin type II; 21 with skin type III; and 11 with skin type IV. Irradiation A broadband UV-B light source consisting of a flat bank of Philips TL 12 tubes ( nm; Philips, Eindhoven, the Netherlands) was used. It could irradiate all body surfaces with equal irradiance. Four UV-B exposures were given to the chest and back (24% body surface area) (19) with 2- to 3-day interval. To obtain the different UV-B doses during a given time, the distance between the UV source and the subjects was varied. The UV-irradiance was checked weekly during the treatment period using a Sola-Hazard spectroradiometer (Solatell, Cornwall, UK) to monitor the stability of the light source during the trial period. The radiation dose was measured in the erythema weighted international standard SED (31). One SED is defined as 100 J m 2 at 298 nm using CIE erythema action spectrum, and it is the UV dose that elicits just perceptible erythema in the most sensitive person in a group of very sun-sensitive healthy persons (32). Biochemical analyses Blood samples were analysed for 25(OH)D, parathyroidea hormone, ionized calcium, alkaline phosphatase and total cholesterol Table 2. D 25(OH)D (nm) for the 11 groups exposed to different UV-B doses for different times. Number of participants in each UV protocol is given in square brackets 1 UV-B exposure dose ( 4) Dose rate (exposure time 1 20 min) 1 min 5 min 10 min 20 min Mean (SD) 3.0 SED 29.4 [5] 19.8 [5] 25.7 [5] 23.9 [5] 24.8 (10.1) 1.5 SED 16.6 [5] 22.8 [5] 16.2 [5] 18.6 (7.8) 0.75 SED 19.0 [5] 20.7 [5] 19.9 (6.4) SED 11.6 [5] 16.8 [5] 14.2 (7.7) UV-B, ultraviolet-b. 1 The 55 participants were randomized to four UV-B exposures of 0.375, 0.75, 1.5 or 3.0 standard erythema dose (SED) every second or third day. The exposure time was 1, 5, 10 or 20 min to obtain the UV-B doses in the table. The statistical analyses were performed including the entire data sample of 55 participants and not on the single groups of five participants. ª 2010 John Wiley & Sons A/S, Experimental Dermatology, 20,

3 Bogh et al. at baseline and 2 days after the fourth and last UV-B session. The blood samples were centrifuged (5000 g in 10 min) within 2 h of sampling. All the analyses have been described previously (18). Definition of vitamin D levels In this study, the definition of vitamin D sufficiency is defined as 25(OH)D levels above 50 nm (20 ng ml), vitamin D insufficiency as 25(OH)D levels below 50 nm (20 ng ml), and vitamin D deficiency as 25(OH)D levels below 25 nm (10 ng ml). Increase in vitamin D found in our study compared to the increase after sunlight The effective vitamin D producing radiation from our UV-B source (TL 12) was converted to the same effective vitamin D producing radiation from sunlight given the same erythema effective dose (SED) (Table 3) (5). Building on the same conversion sun exposure times for production of different amounts of 25(OH)D on different latitudes is presented in Table 4. The time to reach the stated vitamin D after four exposures with 2- to 3-day interval on 24% body surface area with direct incident sunlight is estimated from the regression line in Fig. 1. Notably, the time to reach 3 SED for the TL 12 UVB light source is increased by a factor of 1.6 (erythema weighted) if all irradiation below 295 nm (which is not present in sunlight) is removed from the calculations presented in Table 3. The time to reach the same vitamin D weighted dose is increased by a factor of 1.2 if all irradiation below 295 nm is removed. Statistical analysis Before the study, following presumptions were assumed: given a significance level of 5% and a assumed standard deviation of 9 nm Table 3. The effective vitamin D producing radiation in our UV-B source (TL 12) and in sunlight given the same erythema effective dose (SED) Erythema weighted Vitamin D weighted (J m 2 ) SED J m 2 TL12 1 Copenhagen 2 Malaga 2 Equator 2 for the 25(OH)D analysis at the 50 nm level, the study was designed to show at least a difference of 16 nm between the groups with a power of 80% if at least five subjects per group completed the study. The data were statistically handled using SPSS 18.0 for Windows (SPSS, Chicago, IL, USA). The correlations between the increase in 25(OH)D (D 25(OH)D) after UV-B exposure and sex, age, height, weight, BMI, UV-B dose, dose rate, baseline 25(OH)D level, fish meals per week, body surface area in square metres, biochemical parameters, facultative skin pigmentation (PPF upper body), constitutive skin pigmentation (PPF buttocks) and redness per cent were examined using analysis of variance (ANOVA) both individually and with the UV-B dose included for all variables in every analysis. For all significant analyses, linear regression was performed. The statistical analyses were performed including the entire subject sample of 55 participants and not on the single groups of five participants. The significance limit was P < Before calculations, D 25(OH)D was adjusted for baseline 25(OH)D level using adjusted D 25(OH)D = D 25(OH)D + {0.223 [baseline 25(OH)D] ) mean baseline 25(OH)D} (18). Mean baseline 25(OH)D was 31.2 nm, Table 1. Results Personal and biochemical characteristics at baseline are shown in Table 1 for the 55 participants. We found a significant, positive correlation between D 25(OH)D after UV-B exposure [adjusted for baseline 25(OH)D] and the UV-B dose (P = 0.001; R 2 = 0.176, Fig. 1). 25(OH)D increased in response to the four UV-B treatments with 24.8 nm on average after a total of 12 SED (4 3 SED) and 14.2 nm after 1.5 SED ( SED) in total (Table 2). Additionally, we found a significant, negative correlation between D 25(OH)D and D parathyroid hormone (PTH) (P = 0.012; R 2 = 0.113). There was no significant correlation between the D 25(OH)D and the dose rate (1 20 min) (P = 0.8 without adjustments for UV-B dose and P = 0.4 with adjustments for UV-B dose). No significant relations were found between UV-B, ultraviolet-b; SED, standard erythema dose. 1 The vitamin D weighted dose was calculated using the UV spectrum of the TL 12 UV-B light source weighted by the CIE previtamin D 3 action spectrum. 2 The vitamin D weighted dose was calculated using typical solar spectra on clear sky days in summertime at noon, weighted by the CIE previtamin D 3 action spectrum. Solar UV index is presented in Table 4. Table 4. Building on the conversion factors in Table 3, this table illustrates the total sun exposure times for production of different amounts of 25(OH)D on different latitudes. The time to reach the stated vitamin D after four exposures with 2-to 3-day interval on 24% body surface area with direct incident sunlight is estimated from the regression line in Fig. 1 Solar UV index 1 and location 6.2 Denmark 9.3 Malaga 11.8 Equator 25(OH)D increase, nm Min:s Min:s Min:s :03 14:09 11: :02 7:05 5: :31 3:32 2: :45 1:46 1:23 1 At zenith in summertime, equivalent to 3 h in the middle of the day. Figure 1. Relationship between the increase in vitamin Dà (D 25(OH)D, nm) and the UVB dose (SED*) illustrated by the solid regression line (Y = 3.5X ; P = 0.001; R2 = 0.176). The dotted line is a suggested extrapolation through origin. à: Adjusted for baseline 25(OH)D (18). *: 1 SED is defined as 100 J/m2 at 298 nm using CIE erythema action spectrum (32). 16 ª 2010 John Wiley & Sons A/S, Experimental Dermatology, 20, 14 18

4 Vitamin D increase after UVB D 25(OH)D and sex, number of fish meals per week, constitutive or facultative pigmentation, skin redness, age, weight, height, BMI, total body area in square metres, baseline 25(OH)D, PTH, ionized calcium and alkaline phosphatase or total cholesterol (Table 1). As expected, the facultative skin pigmentation increased (mean (SD)) significantly from 5.1 (2.0) to 7.1 (1.7) during the course of UV-B treatments (P < 0.001) for participants who received 3.0 SED per session, whereas no increase in pigmentation was induced by the lower UV-B doses. There were no significant changes in the skin redness per cent upper body [mean (SD) 23.6% (4.9) before and 24.5% (5.1) after] or the PTH [mean (SD) 3.8 pm (1.8) before and 3.7 pm (1.8) after] for all participants. Erythema was experienced by 20% of the participants who received the highest UV-B dose of 3.0 SED. None of the participants in the other groups with lower UV-B doses experienced any side effects. Discussion Synthesis of vitamin D in the skin is a positive effect of UV-B exposure. A negative effect is DNA damage, resulting in skin cancer. In addition, too much UV-B radiation compromises skin appearance and function (33,34). This dilemma is the reason why dermatologists are under growing pressure to re-evaluate dermatological recommendations on sun protection and health campaigns for skin cancer prevention to guarantee a sufficient vitamin D status. Our aim was to enlighten that dilemma by defining the smallest UV-B dose needed to give a significant increase in 25(OH)D. We found that 25(OH)D increased in response to the four UV-B treatments by 24.8 nm on average after a total UV-B dose of 12 SED (4 3 SED) and, perhaps more notably, we found an increase of 14.2 nm after a total UV-B dose of just 1.5 SED ( SED) (Table 2). Further, we found that the increase in 25(OH)D production is obtained rapidly. The linear regression model (Fig. 1, solid line) is based on the present data although the true line must go through origin (0.0). We therefore suggest that a very small UV-B dose will result in significant 25(OH)D production (Fig. 1, dotted line), whereas higher UV-B doses result in a less efficient 25(OH)D production and a proportionally greater risk of skin cancer. A recent study (35) reports a 25(OH)D increase by 26 nm (mean) after a total UV-B dose of approximately 24 SED (three times UV-B of 1.3 SED per week for 6 weeks) to 35% body surface area. Notably, the 25(OH)D increase were almost identical with the 25(OH)D increase found in our study on 24.8 nm after a total UV-B dose of 12 SED to 24% body surface area. These findings suggest that a state of saturation occurs for the higher UV-B doses and body surface areas. We converted our UV-B radiation data into sunlight data of vitamin D effective radiation (Table 3). By using the data in Fig. 1, it is possible to calculate the sun exposure times necessary for a certain vitamin D increase on different latitudes (Table 4). Notably, a person needs only four sun exposures (with 2- to 3-day interval) each of a few minutes to have effective 25(OH)D production in Denmark in the summertime and only four sun exposures of approximately 80 s on the Equator all the year round. However, as stated in a paper in press (36), people lying in the sun are irradiated only from above lying supine or prone, why it would require a sunbathing subject to be exposed for around twice the exposure times given on four separate occasions to achieve the 25(OH)D increase stated in Table 4. A few human in vivo studies exist of the dose response relationship between UV-B and vitamin D (15,16). The studies used MED, an individual biological dose, as they assumed that skin pigmentation reduces the vitamin D production in the skin after UV-B. The fair-skinned participants were therefore treated with lower doses of UV-B than were the dark-skinned participants. Consequently, it is difficult to interpret the results as it is debated whether skin pigmentation reduces vitamin D production (18,37,38). Further, recent studies have shown that baseline 25(OH)D influences the production of 25(OH)D after UV-B exposure (18,20 22,39). We therefore adjusted D 25(OH)D for baseline 25(OH)D according to the mean baseline 25(OH)D level (31.2 nm, Table 1) in the analysis of the importance of UV-B dose, using the relation found in our recent published study (18). Another recent human study with only two different UV-B doses also reports elevated production of 25(OH)D for the higher UV-B doses (17). We found that the dose rate was not important to the increase in 25(OH)D. Despite the basic photo-biological fact that physiological UV effects in photo-carcinogenesis are known not to depend on dose rate over a wide range (8 10), this study is to our knowledge the first study exploring this relationship with vitamin D production after UV-B exposure as endpoint. We chose only relatively short exposure times (1 20 min) as relevant exposure doses are likely to be obtained in the sun within this time frame (Table 4). To strengthen our findings, we included detailed information on lifestyle factors, collected information on fish meals per week, included two serum samples from each subject; measured pigmentation; and conducted the study when the ambient UV radiation was negligible and the blood concentration of vitamin D was considered as the year s lowest in Denmark 56 N. UV-B radiation was used instead of artificial sunlight because the spectrum of the sun varies according to the time of day and the time of year; artificial sun delivers only one of these spectra, and it is difficult to obtain uniform irradiation of large body surface areas with artificial sunlight. Baseline 25(OH)D influences the vitamin D production after UV-B (18,20 22,39); to narrow the concentration range, we therefore screened the 25(OH)D level before inclusion of participants and included participants with a relatively homogeneous baseline 25(OH)D between 16 and 50 nm (Table 1). Further, we have adjusted D 25(OH)D for baseline 25(OH)D. Notably, we found no significant relations between D 25(OH)D and constitutive or facultative pigmentation after UV-B exposure despite including participants with PPF upper body values between 1.7 and 10.0 (Fitzpatrick I IV). This supports the results from our recent published study where participants with a broader range in skin pigmentation were included but no correlation between 25(OH)D production and skin pigmentation was found (18). We used a population with a narrow baseline 25(OH)D, and we did not divide the participants into subgroups according to their previous sun exposure, as we did in our recent study (18). This may explain the lack of relation between D 25(OH)D and total cholesterol in this study. A limitation was the variance in the liquid chromatography-mass spectrometry (LC-MS MS) analysis of 25(OH)D of approximately 8.5%, despite including two serum samples from each subject and doing each 25(OH)D analysis twice. The fact that our study was made in the laboratory with broadband UVB lamps (TL 12) and not with lamps emitting natural sunlight represents a ª 2010 John Wiley & Sons A/S, Experimental Dermatology, 20,

5 Bogh et al. limitation. Another limitation is that our UVB lamps (TL 12) emits radiation below 295 nm, and therefore the exposure times stated in Table 4 should be multiplied by a factor of 1.2 if the radiation below 295 nm (which is not present in sunlight) is negligible in terms of vitamin D production. One might also speculate that the UVA part in natural sunlight would degrade the UV-B-induced vitamin D. Further, a recent paper raises doubts about the validity of the CIE vitamin D action spectrum (40). Our study shows that fair-skinned persons without any underlying medical condition should be able to produce sufficient vitamin D from a few low doses of UV-B to approximately 24% of body surface area. However, because of its skin-carcinogenic effect, we would not recommend the general population to increase UV- B exposure as a source of vitamin D because sufficient vitamin D levels can be obtained with vitamin D supplements (e.g. tablets) (41,42). Further studies are needed to test whether UV-B irradiation of smaller body surface areas is sufficient for optimal vitamin D production, as significant production of 25(OH)D is already obtained after a very low UV-B dose. In conclusion, we found a significant correlation between UV-B dose and the increase in 25(OH)D, giving an increase of 25(OH)D with 14.2 nm on average after just four UV-B sessions with SED, which correlates with a 4-min sun exposure around noon during the summer day in Denmark. Further, we found that the dose rate was not associated with the increase in 25(OH)D after UV-B exposure. Acknowledgements We thank the participants for their contribution to the study. We also thank Henrik Jørgensen, MD, PhD, Department of Biochemistry, Bispebjerg Hospital, and the laboratory technicians, Department of Biochemistry, Bispebjerg Hospital and Lillebaelt Hospital, for their help in the project. Paul Eriksen, Danish Meteorological Institute, is thanked for the solar spectra. 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Am J Clin Nutr 2009: 88: Supporting Information Additional Supporting Information may be found in the online version of this article: Figure S1. CONSORT flowchart to show the flow of the participants. Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article. 18 ª 2010 John Wiley & Sons A/S, Experimental Dermatology, 20, 14 18