Clinical Trial Details
— Status: Completed
Administrative data
| NCT number |
NCT03409510 |
| Other study ID # |
Dpigment2010 |
| Secondary ID |
|
| Status |
Completed |
| Phase |
N/A
|
| First received |
January 10, 2018 |
| Last updated |
January 29, 2018 |
| Start date |
November 1, 2010 |
| Est. completion date |
June 25, 2011 |
Study information
| Verified date |
January 2018 |
| Source |
Bispebjerg Hospital |
| Contact |
n/a |
| Is FDA regulated |
No |
| Health authority |
|
| Study type |
Interventional
|
Clinical Trial Summary
Skin pigmentation (melanin) absorbs ultra violet type B (UVB) radiation found in sunlight and
is believed to be responsible for darker-skinned persons' generally low 25(OH)D status. This
phenomenon is found in immigrants living in Northern countries and their 25(OH)D responses to
UVB-irradiation seem low.
We hypothesized that objectively measured skin pigmentation and/or pigment genes influence
UVB-induced 25(OH)D increase significantly in combination with other influential parameters.
The influence of objectively measured constitutive and facultative skin pigmentation on
UVB-induced 25(OH)D increase over time was investigated together with other possible
influential parameters. These other influential parameters include sex, age, weight, height,
BMI, number of fatty fish meals per week, Fitzpatrick Skin Type and 25(OH)D start level. The
genetic parameters include 33 Vitamin D receptor and pigment SNPs.
This is a single-centre, open and non-blinded clinical trial. No randomisation was used, as
the participants were allocated into two groups based on their Fitzpatrick Skin type and
ethnic origin. The light-skinned group included participants with Fitzpatrick Skin type II-IV
and were of Northern origin (Denmark, the Faroe Islands and the UK). The darker-skinned
included Fitzpatrick Skin Types V-VI originating from countries located at latitudes below 50
degrees N. Thus, it could be ensured that the participants represented a wide range of skin
pigmentation. The light-skinned (N = 22) and the darker-skinned subjects (N = 18) were
exposed to identical UVB doses on identical body areas over nine weeks with weekly
measurements of 25(OH)D. The UVB-induced 25(OH)D synthesis was investigated in
summer-pigmented skin with melanin throughout the epidermis and during winter when ambient
UVB exposure is negligible. Demographic data (gender, age, weight, height, Fitzpatrick Skin
Type, measured constitutive and facultative skin pigmentation (PPF)) was collected/measured
and registered in prior to study start. The number of daily consumed fatty fish meals was
recorded in a questionnaire. Serum 25(OH)D was analysed weekly.
Description:
Background. Melanin molecules (pigment) in skin, absorb UVB and presumably reduce the
photo-conversion of 7-dehydrocholesterol. Epidemiological studies have reported poor vitamin
D status in relatively darker-skinned immigrants residing in countries located at high
latitudes. It is therefore commonly believed that skin pigmentation is a major influencer of
vitamin D status. However, controlled UVB intervention studies have yielded conflicting
results regarding the influence of skin pigmentation on UVB induced vitamin D increase.
Study hypothesis. The investigators hypothesized that objectively measured skin pigmentation
(assessed as Pigment Protection Factor) and/or pigment genes influence UVB-induced
25-hydroxyvitamin D (25(OH)D) increase significantly in combination with other influential
parameters. These other influential parameters include sex, age, weight, height, BMI, number
of fatty fish meals per week, Fitzpatrick Skin Type and 25(OH)D start level. The genetic
parameters include 33 Vitamin D receptor- and pigment-SNPs.
Study design. This is a single-centre, open and non-blinded clinical trial.
Intervention. The study was planned so that all included participants would receive identical
UVB doses gradually increasing over nine weeks from November to January. The total UVB dose
during the nine weeks was planned to be 26 kilo Joules/m2 (56 standard erythema doses).
Irradiation time would be determined and regulated by measuring UV intensity with a
Sola-Hazard spectroradiometer at study start, after five weeks and at the end of the exposure
period. To avoid erythema, the standard erythema dose must be below the lowest measured UVB
tolerance in the participants included. Based on previous experience of measurement on
light-skinned individual, the UVB treatment dose was set to a maximum of 1.4 kilo Joules/m2
(3 standard erythema doses).
Methodological aspects.
Skin pigmentation. The content of melanin in epidermal biopsies is significantly correlated
to measurement of pigment protection factor (PPF). This measurement of PPF can be performed
non-invasively with a skin reflectance meter (UV-Optimize Scientific, Chromo-light, Denmark).
In the following, PPF is therefore used as a measure of skin pigmentation and referred to as
such. The measuring range of PPF is 1 to 25, meaning that a UVB dose between 1 standard
erythema dose and 25 standard erythema doses is needed to elicit a just perceptible erythema.
The constitutive PPF was measured on the buttocks, a body area not normally exposed to UVB.
The facultative PPF was assessed as a mean of measurements on the chest, midriff, back of
shoulder, and the medial and lateral sides of the arm. These are body areas influenced by
prior lifelong solar exposure. Both constitutive PPF and facultative PPF were measured at
study start, after 4½ weeks, and at study end. Mean values at these time-points for each
parameter were used for further investigation. Furthermore, subjects self-reported their skin
photo-type according to Fitzpatrick's criteria was assessed.
Blood analysis. Serum 25(OH)D was analyzed on a liquid chromatography tandem mass
spectrometer (LC-MS/MS), which is the current most accurate method for this analysis. This
method has been cross checked against another clinical laboratory and proven reliable.
Analytical variability. To minimize analysis variance, at least triplet analyses (technical
replicates) were performed of PPF measurement and 25(OH)D analysis. All 25(OH)D samples from
the same subject were analyzed in one batch. An internal standard solution with 25(OH)D was
used in each run.
The total relative standard deviation (SD) varied between 4.9% at 20 nmol/l and 14.1% at 222
nmol/l reflecting experimental variability.
Biological variability. By including 10 measuring time points for serum 25(OH)D per
participant, the intra-individual variability was reduced. Serum 25(OH)D is known to variate
with season and solar exposure. This study was therefore conducted during winter half-year,
when ambient UVB radiation and solar-exposed body areas are negligible at this time of the
year (17).
Genetic parameters. SNP genotyping. A total of 33 SNPs were investigated. SNPs located in the
Vitamin D Receptor (VDR) gene. The influence of the vitamin D receptor gene was investigated
by genotyping the two single nucleotide polymorphisms (SNP), rs1544410 (BsmI) and rs2228570
(FokI), located in the gene (ENSG00000111424, Chromosome 12q13) as previously described.
These two SNPs were selected due to their association with bone diseases as well as many
other diseases, thereby indicating that polymorphisms in those regions of VDR were important
for the functioning of VDR. These two SNPs were investigated together with the other
investigated demographic parameters.
SNPs located in genes with influence on pigment. Thirty-one SNPs were selected according to
potential influence on pigment.
All SNPs were genotyped. SNPs with genotype subgroups containing less than five subjects were
merged with other allele-sharing subgroups. Heterozygote subgroups with less than five
subjects was merged with an allele-sharing subgroup displaying insignificant (P > 0.05)
difference in influence on the 25(OH)D increase rate. SNPs with no allele dose effect or
dominant allele effect on 25(OH)D were excluded. For SNPs with dominant allele effect,
subgroups with no significant differences in influence on 25(OH)D increase rate were merged
according to allele sharing.
Selection of participants. Participants were included and allocated into a light-skinned
group and a darker-skinned group based on Fitzpatrick Skin Type (self-reported skin
photo-type) and ethnic origin. The light-skinned included Fitzpatrick Skin Types I-IV
originating from Northern countries (Denmark, the Faroe Islands and the UK). The
darker-skinned included Fitzpatrick Skin Types V-VI originating from countries located at
latitudes below 50 degrees N. Thus, it could be ensured that the participants represented a
wide range of skin pigmentation. All participants must reside in Denmark. Furthermore, an
equal sex distribution was attempted.
Statistical considerations.
Randomization. No randomization was used, as the participants were allocated into two groups
based on other criteria at inclusion. Both groups received identical intervention and
blinding was therefore not used.
Size of study. SD was 14.1 nmol/l for high-end 25(OH)D samples. On the basis of a previous
study lasting two weeks (10), the expected detectable difference was 15 nmol/l. The sample
size corresponding to number of subjects required to complete the study was 16 subjects in
each group, given a significance level of 5% and 80 % power to detect a difference of 15
nmol/l. Due to the relatively long study period, a minimum of 21 subjects were included in
each group allowing five drop-outs. Three subjects dropped out from the darker-skinned group,
and none from the light-skinned group.
Data collection. All participants were assigned with a unique study number prior to study
start. Demographic data (gender, age, weight, height, Fitzpatrick Skin Type, measured
constitutive and facultative skin pigmentation (PPF)) was collected/measured and registered
in prior to study start. The number of daily consumed fatty fish meals was recorded in a
questionnaire. All forms were marked with the participants unique study number. 25(OH)D
samples were analyzed after study end. The participant study number linked to participant
contact details were held in a separate sheet kept securely and in line with the regulation
of the Danish Data Protection Agency.
Background diet and change in diet during intervention. The number of daily consumed fatty
fish meals was recorded in a questionnaire during the study period as this parameter
potentially can influence the primary outcome measure, 25(OH)D increase over time. The
questionnaire was examined for errors weekly and to ensure compliance.
Background health status and lifestyle, and changes in health status and lifestyle during
intervention.
The health status of the participants and start of new medication was monitored weekly
throughout the study.
Adverse events. As the intervention included UVB radiation, all participants were assessed
for and requested to report skin erythema.
Compliance. To optimize compliance in this long-term study, the participants were requested
to schedule weekly intervention dates and complete all planned intervention throughout the
study period. Furthermore, it was required that all participants had weekly blood samples and
measurement of skin pigmentation three times during the study period. Also it was required
that a diet questionnaire was completed and checked for errors on a weekly basis. All
participants were informed thoroughly of the study requirements prior to inclusion. The
acceptable level of compliance was defined at a completion of the entire intervention
planned, assessment of all objective measurements and completion of all questionnaires.
Statistical analysis. Descriptive statistics of participants by group will be presented to
characterize the participants included in the study. Individual data were tested with the
Kolmogorov-Smirnov test to assess whether the data were normally distributed. Normally
distributed data will be tested by 2-tailed t test. Otherwise non-parametric tests will be
used such as 2-tailed Mann-Whitney's test or Kruskal-Wallis test. Comparison of 25(OH)D
levels at different time points will be performed using paired t test (2-tailed). Categorical
data will be tested with Fisher's exact test or Pearson's Chi-square test (2-tailed).
As the light-skinned group and the darker-skinned group may differ, these two groups will
initially be investigated separately to examine whether there is basis to merge these two
groups for common analysis. Therefore, the increase in 25(OH)D over time will be investigated
separately in the light-skinned group and the darker-skinned group the following models:
linear, inverse, quadratic, cubic, power, sigmoid and exponential. The derivate function was
defined as the average daily change in 25(OH)D between two sample time-points (Δ25(OH)D per
day). Selection of the best suitable model will be performed based on the accordance between
the investigations of the 25(OH)D increase over time (individual- and group-based) and the
derivate function.
The inter-individual variations in the combined group in a linear transformation of the
increase of 25(OH)D over time will be explored by comparing general linear models (GLMs)
with: (1) common increase rate (25(OH)D increase per day and slope) and common 25(OH)D start
level (intercept), (2) common increase rates and individual intercepts (measured 25(OH)D
start levels) and (3) individual increase rates (i.e. an individual constant) and measured
25(OH)D start levels.
The influence of parameters (secondary outcome measures) on the variation of the 25(OH)D
increase rate will be examined separately. As the influence of separate significant
parameters may not be independent, separate significant parameters will subsequently be
investigated by a stepwise backward elimination of a combined GLM according to P-value (25).
Data will be statistically analyzed using SPSS 24.0 for Windows (SPSS Inc., Chicago, U.S.A.).
P < 0.05 will be considered significant. No interim analysis will be performed.
