Bioavailability of vitamin D2 from UV-B-irradiated button mushrooms in healthy adults deficient in serum 25-hydroxyvitamin D: A randomized controlled trial

Abstract

Mushrooms contain very little or any vitamin D(2) but are abundant in ergosterol, which can be converted into vitamin D(2) by ultraviolet (UV) irradiation. Our objective was to investigate the bioavailability of vitamin D(2) from vitamin D(2)-enhanced mushrooms by UV-B in humans, and comparing it with a vitamin D(2) supplement.
Fresh mushrooms were irradiated with an UV-B dose of 1.5 J/cm(2), increasing vitamin D(2) content from <1 to 491 μg/100 g and made to an experimental soup. In this 5-week, single-blinded, randomized, placebo-controlled trial, 26 young subjects with serum 25-hydroxyvitamin D (25OHD) ≤ 50 nmol/l were randomly assigned into three groups ((a) mushroom, (b) supplement and (c) placebo). They received during winter (a) 28,000 IU (700 μg) vitamin D(2) via the experimental soup, or (b) 28,000 IU vitamin D(2) via a supplement or (c) placebo, respectively.
After 2 weeks, serum 25OHD was significantly higher in the mushroom than in the placebo group (P=0.001). The serum 25OHD concentrations in the mushroom and supplement groups rose significantly and similarly over the study period by 3.9 nmol/l (95% confidence interval (95% CI): 2.9, 4.8) and by 4.7 nmol/l per week (95% CI: 3.8, 5.7), respectively.
We are the first to demonstrate in humans that the bioavailability of vitamin D(2) from vitamin D(2)-enhanced button mushrooms via UV-B irradiation was effective in improving vitamin D status and not different to a vitamin D(2) supplement. This trial was registered at http://germanctr.de as DRKS00000195.

Full text

1
TITLE PAGE
Bioavailability of vitamin D
2
from UV-B-irradiated button mushrooms in healthy adults
deficient in serum 25-hydroxyvitamin D: a randomized controlled trial
1-3
Paul Urbain, Fabian Singler , Gabriele Ihorst , Hans-Konrad Biesalski, and Hartmut Bertz
1
From the Department of Haematology/Oncology (HB), Section of Nutrition (PU, FS),
University Medical Center Freiburg, Freiburg, Germany; Institute of Medical Biometry,
Medical Informatics and Center of Clinical Trials, University of Freiburg, Freiburg, Germany
(GI); and Department of Biological Chemistry and Nutrition, University of Hohenheim,
Stuttgart, Germany (PU, FS, HKB).
2
Supported by AFR PhD grant from the Luxemburg Ministry of Culture, Higher Education
and Research (PU). Financial support for the irradiation experiments by the Dr.Heinrich-
Kircher Foundation, University of Freiburg, Freiburg, Germany. Mushrooms donated by
Schlossbergpilz GmbH, Freiburg, Germany.
3
Address correspondence to P Urbain, University Medical Center, Department of
Hematology/Oncology, Section of Nutrition, Hugstetter Str 55, D-79106 Freiburg, Germany.
Phone: +49-761 2703360; Fax: +49-761 2703334; E-mail: paul.urbain@uniklinik-freiburg.de.

2
ABSTRACT
Background/Objectives: Mushrooms are almost free of vitamin D
2
but abundant in
ergosterol, which can be converted into vitamin D
2
by UV irradiation. Our objective was to
investigate the bioavailability of vitamin D
2
from UV-treated and vitamin D
2
-enhanced
mushrooms in humans, and comparing it with a vitamin D
2
supplement.
Subjects/Methods: Fresh mushrooms were irradiated with an UV-B dose of 1.5 J/ cm²,
increasing vitamin D
2
content from <1 to 491 µg/100 g, and made to an experimental soup. In
this 5-week, single-blinded, randomized, placebo-controlled trial, 26 young subjects with
serum 25-hydroxyvitamin D (25OHD) ≤50 nmol/L were randomly assigned into 3 groups [a)
mushroom, b) supplement, c) placebo]. They received during winter a) 700 µg (28 000 IU)
vitamin D
2
via the experimental soup, or b) 700 µg vitamin D
2
via a supplement, or c)
placebo, respectively.
Results: After two weeks, serum 25OHD was significantly higher in the mushroom than in
the placebo group (P = 0.001). The serum 25OHD concentrations in the mushroom and
supplement groups rose significantly and similarly over the study period by 3.9 nmol/L per
week (95% CI: 2.9, 4.8) and by 4.7 nmol/L per week (95% CI: 3.8, 5.7), respectively.
Conclusions: We are the first to demonstrate in humans that the bioavailability of vitamin D
2
from vitamin D
2
-enhanced button mushrooms via UV-B irradiation was as effective as a
vitamin D
2
supplement in treating vitamin D deficiency. This trial was registered at
germanctr.de as DRKS00000195.

3
TEXT
1. Introduction
Vitamin D deficiency, defined as a serum 25-hydroxyvitamin D (25OHD) concentration <50
nmol/L (Holick and Chen, 2008; Malabanan et al., 1998), is a public health issue prevalent
worldwide (Chapuy et al., 1997; Hintzpeter et al., 2008; Tangpricha et al., 2002; van der
Wielen et al., 1995), particularly in regions with a big seasonal shift in solar altitude, as the
major source of vitamin D for humans is sunlight-induced dermal synthesis (Engelsen et al.,
2005; Webb et al., 1988). Other criteria, like dark skin (Armas et al., 2007; Clemens et al.,
1982), old age (Need et al., 1993), and immobility (Semba et al., 2000) further reduce the
endogenous vitamin D synthesis. In addition to that, few foods contain vitamin D in
noteworthy concentrations; those that do are fish liver oils, fatty fish, vitamin D-fortified
margarine and egg yolk (Mattila et al., 1992; Mattila et al., 1995).
Naturally, the vitamin D
2
content of cultivated mushrooms is almost nil [<0.1 µg/100 g fresh
weight (fw)], yet they are very rich in ergosterol (Jasinghe and Perera, 2005; Mattila et al.,
2002). Ergosterol is the principal sterol in fungi, and several studies have reported that
mushrooms can be greatly enhanced with vitamin D
2
by ultraviolet (UV) irradiation,
resembling the dermal synthesis of vitamin D
3
in humans (Ko et al., 2008; Mau et al., 1998).
The conversion rate of ergosterol to vitamin D
2
under UV irradiation depends on the UV
spectrum (UV-A, -B or -C), irradiation dose, moisture content, and the mushrooms´
orientation toward the UV source (Jasinghe and Perera, 2005, 2006; Roberts et al., 2008).
Outila et al. (1999) were the first to demonstrate that vitamin D
2
was well absorbed from
lyophilized and homogenized mushrooms in humans. Jasinghe et al (2005, 2006) were the
first to publish in vivo studies on the bioavailability of vitamin D
2
from UV-irradiated
mushrooms, showing as others (Koyyalamudi et al., 2009) that vitamin D
2
from vitamin D
2
-

4
enhanced mushrooms is well absorbed, is metabolized in rodents, and that it improves bone
mineralization.
Recently the case history of a patient with vitamin D deficiency and secondary
hyperparathyroidism was published, who refused to take supplements, but self-treated his
deficiency by consuming mushrooms daily, which he had exposed to UV-B irradiation
(Ozzard et al., 2008).
To the best of our knowledge, this is the first report on the bioavailability of vitamin D
2
from
UV-treated mushrooms in humans. Hence the primary objective of this randomized controlled
trial was to demonstrate the possibility of improving the 25OHD status with this natural food
source in terms of a higher serum 25OHD concentration in young vitamin D-deficient adults 4
weeks after a weekly vitamin D
2
dose of 700 µg (28 000 IU) compared to placebo. A
secondary objective was to compare the bioavailability of vitamin D
2
from UV-B treated
mushrooms with a vitamin D
2
supplement.
2. Subjects and Methods
• Subjects
Subjects were recruited from employees of the University Medical Center Freiburg by
advertising. The study protocol was approved by our Ethics Commission. Exclusion criteria
included kidney stones, pregnancy, anticonvulsant or steroid therapy in any form, frequenting
a tanning salon, or residence in the mountains or southern countries right before or during the
study. The subjects were not allowed to take vitamin D supplements or fish liver oils, and
were asked to eat fish no more than once a week during the study period.
Caucasian adults in good general health, younger than 45 years with a body mass index (BMI)
between 18.5-26 kg/m², and not fulfilling any exclusion criterion were eligible to provide
blood specimens for further testing after having signed a written consent form. Out of 49

5
female and male volunteers we randomized 27 vitamin D-deficient (25OHD ≤50 nmol/L)
subjects with normal serum calcium concentrations (2.2-2.7 mmol/L) to enter the study.
• Study design
This study was a 5-week, prospective, randomized, 3-arm, single-blind, placebo-controlled
trial to investigate the bioavailability of vitamin D
2
from UV-B-irradiated button mushrooms
and vitamin D
2
supplement, respectively.
The four first weekly visits (weeks 0, 1, 2, 3) constituted the interventional part of the study
and the two last visits (weeks 4, 5) served as follow-up. The primary objective and further
endpoints were analyzed till week 4, because that is when we expected the strongest
interventional effect. At the initial, baseline visit (week 0), weight and height were
documented. Each subsequent weekly visit at the same time of day consisted of a self-
developed 7-day dietary questionnaire, sun exposure was recorded and blood drawn. The
study was performed during the winter from late January till early March 2010, when a) a
vitamin D deficiency in healthy subjects is most likely, and b) solar UV-B radiation is
minimal to avoid the confounding effect of cutaneous vitamin D
3
synthesis on our intake-
response evaluation.
Our 27 subjects were randomly assigned into 3 equal groups [a) mushroom, b) supplement, c)
placebo] using a computer-generated sequence to receive 4 times at weekly intervals either a)
700 µg (28 000 IU) vitamin D
2
via the experimental soup containing the UV-B-irradiated
mushrooms (vitamin D
2
content of 191.8 µg/100 g) and placebo, or b) 1.5 µg (6 IU) vitamin
D
2
via a conventional mushroom soup and 700 µg (28 000 IU) vitamin D
2
via a supplement,
or c) 1.5 µg (6 IU) vitamin D
2
by a conventional mushroom soup and placebo, respectively.
The liquid supplement consisted of an ethanol formulation of vitamin D
2
[2 000 000 IU/100
ml, ethanol 90% (V/V)] (Stérogyl, Desma Pharma, Paris, France) and was dissolved in

6
orange juice. The placebo consisted of pure orange juice. The supplement or placebo were
served shortly before the soup.
• Blood sample analysis
The blood samples were stored for coagulation about 30-60 min in the dark at room
temperature previous to centrifugation (2.000 rpm for 7 min). The serum samples were frozen
at -78 °C until weekly analysis by MVZ Clotten (Freiburg, Germany). Serum 25OHD
2
and
serum 25OHD
3
were measured combined as 25OHD by a radioimmunoassay (RIA)
purchased from DiaSorin Inc. (Stillwater, MN, USA). The quality and accuracy of the serum
25OHD analysis were monitored by interlaboratory tests evaluated by INSTANT e.V.
(Duesseldorf, Germany). The detection limit for the RIA assay was 10 nmol/L, inter- and
intraassay coefficients of variation for 25OHD were 11.1% and 10.1%. Serum intact
parathyroid hormone (iPTH) was measured by non-competitive immunoassay on the Roche
Modular Analytics E170. Serum calcium was measured using a photometric color test with
Olympus calcium Arsenazo III OSR60117. The reference ranges were 50-175 nmol/L, 11.3-
42.5 ng/l and 2.2-2.7 mmol/l, for 25OHD, iPTH and serum calcium, respectively. Serum
25OHD and calcium were measured weekly, serum iPTH was measured twice at week 0 and
week 4.
• Raw material
We used fresh brown button mushrooms (Agaricus bisporus) provided by a local mushroom
producer (Schlossbergpilze, Freiburg, Germany) on the day of harvest in this study. They had
a moisture content of 91.4%, determined by the vacuum oven method.
• Irradiation of mushrooms

7
To produce vitamin D
2
-enhanced mushrooms, they were placed completely separated from
each other on a 2 cm meshed grid, and each side (caps and gills) was irradiated
simultaneously with UV-B (306 nm) at an irradiation dose of 1.5 J/cm
2
after 25 min at
ambient temperature (22 °C). The custom-made UV unit was equipped with 8 UV-B lamps
176 cm in length (UV21, Waldmann, Villingen-Schwenningen, Germany). The total
irradiation area was 0.72 m
2
with a homogeneous intensity of UV-B. The radiation dose was
measured by a radiometer (UV34, PCE Group, Meschede, Germany).
• Soup preparation and analytic method
Directly after irradiation, the mushrooms were diced and used in a puréed mushroom soup.
All the experimental and conventional mushroom soups needed for the study were portioned
out and stored in a freezer at -20 °C. Soup ingredients were water, button mushrooms, soy
cream, flour, olive oil and spices [51.2 kcal/100 g; 4.2% fat, 0.8% protein, 2.9%
carbohydrates (weight/weight)]. To calculate the exact amount of soup needed to fulfil the
study requirements, one portion of each soup was shipped on dry ice to SGS Institut Fresenius
(Berlin, Germany) for vitamin D
2
analysis, as well as all other mushroom samples. The assay
is based on semipreparative HPLC purification followed by analytical reversed-phase HPLC.
• Sample size calculation and statistics
The study was designed to detect a difference of 20 nmol/L in 25OHD serum concentrations
between the mushroom and placebo groups with a power of 80% with a one-sided t-test at a
significance level of 5%. The assumed standard deviations (SD) were 13.0 in the placebo and
14.7 in the mushroom groups, derived from previous investigations. The resulting sample size
was 7 per group. The study was analyzed using SPSS version 16.0 (SPSS Inc, Chicago, IL,
USA) and SAS version 9.2 (SAS Institute Inc, Cary, NC, USA).

8
For descriptive data analyses, values are presented as means ± SDs. Group comparisons were
made using two sample t-tests and one-way ANOVA with post hoc Tukey tests. P-values will
be provided for the comparisons of secondary objectives and should be regarded as
exploratory. Results with P <0.05 will be denoted as significant. The development of 25OHD
concentrations during the study was investigated using a random effects model (SAS proc
mixed) with 25OHD as dependent variable, different time slope parameters for each treatment
group (i.e. time*treatment interaction) and subject specified as a random effect. Treatment
differences during the study can thus be investigated by testing the differences in time slopes.
Results will be given in terms of estimates for regression slope parameters which represent
the 25OHD increase per week in each group and their accompanying 95% confidence
intervals.
3. Results
• Baseline characteristics of subjects
Of the 27 subjects recruited for the study, one from the mushroom group dropped out because
of pregnancy before the first visit, while 26 completed the interventional phase and were used
for the further evaluation. One subject had to miss the last follow-up blood withdrawal (week
5).
Characteristics of the study population are summarized in Table 1. Mean age and BMI of the
subjects were 30.8 ± 5.8 y and 22.1 ± 2.5 kg/m
2
, respectively. There were no significant
differences in these parameters among the 3 study groups at baseline. In addition, the 3 study
groups were similar with regard to initial serum concentrations of 25OHD, iPTH, and calcium
(Table 2).
• Mushrooms´ vitamin D
2
content

9
Our study´s brown button mushrooms cultivated in the dark had very low concentrations of
vitamin D
2
(0.18 µg/100 g fw). The conversion of ergosterol to vitamin D
2
under UV-B
irradiation in this study was very high, and we achieved concentrations of 491 µg/100g fw
(56.8 µg vitamin D
2
/g dry solids (ds)).
• Time course of the main serum parameters
The time course data of serum 25OHD concentrations for each study group over the 5-week
period are presented in Table 2, Figure 1.
The primary objective was to test the efficacy of the vitamin D
2
-enhanced mushrooms to
improve the 25OHD status. The data show that one week after the last consumption of such
mushrooms at week 4, the mushroom group´s serum 25OHD was significantly higher than
that in the placebo group (P <0.0001) (Table 2).
A secondary objective consisted of testing the bioavailability of vitamin D
2
from the UV-B
treated mushrooms compared to a common vitamin D
2
supplement. When modelling the
development of 25OHD concentrations as a linear function of time (mixed regression model),
we found that the mushroom and supplement groups increased their 25OHD concentrations
significantly over the study period by 3.9 nmol/L per week (95% CI: 2.9, 4.8; P <0.0001) and
by 4.7 nmol/L per week (95% CI: 3.8, 5.7; P <0.0001) (Figure 1). Regression slopes of the
concentrations of the serum 25OHD in the mushroom and supplement groups did not
significantly differ from one another (P = 0.20).
Further analysis showed that already two weeks after the first consumption of the vitamin D
2
-
enhanced mushrooms, the two interventional groups´ serum 25OHD concentrations were
significantly higher than in the placebo group (P = 0.001) (Table 2).
The development of hypercalcemia (defined as serum calcium >2.7 mmol/L) was the main
safety criteria for the vitamin D
2
administration. Serum calcium remained within the reference
range at all time points. No physical symptoms were reported during the study.

10
Neither at baseline nor week 4 did the serum concentrations in iPTH and calcium differ
significantly among the three study groups (Table 2).
Furthermore, the data reveal significant within-subject changes in serum 25OHD in all three
study groups already at week 1. During that period, serum 25OHD rose significantly (P
<0.001) by 33.1% and 46.1% in the mushroom and supplement groups, respectively. In
addition, serum 25OHD decreased significantly (P = 0.03) by - 6.5% in the placebo group.
Serum 25OHD decreased significantly in the placebo group from baseline to wk 4 (from 38.7
nmol/L to 24.5 nmol/L) altogether by an unexpectedly high percentage of 36.8% (P <0.0001).
4. Discussion
Here we describe for the first time in humans that UV irradiation of mushrooms creates an
excellent source of vitamin D
2
which has equivalent bioavailability as a vitamin D
2
supplement.
The rapid serum 25OHD increase in our mushroom group is a clear demonstration that
ingesting 700 µg (28 000 IU) vitamin D
2
once a week for 4 weeks via UV-B-irradiated and
vitamin D
2
-enhanced mushrooms is effective in treating vitamin D deficiency in young,
healthy adults. Already one week after their first ingestion of UV-B-irradiated mushrooms,
serum 25OHD rose significantly. Furthermore, it was significantly higher than in placebo
group one week after the second administration of enhanced mushrooms (week 2).
Most studies on the bioavailability of vitamin D in humans have been conducted using
supplements, not natural food sources. Consistent with our observation of equivalent vitamin
D
2
bioavailability from a soup prepared with UV-irradiated mushrooms and supplement, an
earlier study (Outila et al., 1999) demonstrated the same efficiency with non-irradiated, but
lyophilized and homogenized mushrooms in humans. Both findings disprove the hypothesis
of Van-den-Berg (1997), who maintained that the bioavailability of vitamin D from natural

11
food sources is probably lower than from supplements. We believe that the absorption rate of
vitamin D
2
from mushrooms is about 55 to 99%, equal to the recommended values for
supplements (van den Berg, 1997).
Serum 25OHD rose by 52% and 68% in the mushroom and supplement groups, respectively,
after the 4-week supplementation of 700 µg (28 000 IU) vitamin D
2
weekly. The rise in
25OHD observed in our study resembles the results by Mastaglia et al (2006), where serum
25OHD rose by 52% in the 125 µg (5 000 IU) vitamin D
2
/d group after a 4-week
supplementation. In turn, Malabanan et al (1998) showed that a weekly oral supplement of
1250 µg (50 000 IU) vitamin D
2
for 8 weeks raised serum 25OHD by 101%. Contradictory
results were reported by Tjellesen et al (1986), where after a 4-week supplement of 100 µg (4
000 IU) vitamin D
2
daily, the total serum 25OHD remained constant due to a significant
increase in 25OHD
2
and an incomprehensible, significant decrease in 25OHD
3
.
Other authors showed that a single dose of 1250 µg (50 000 IU) of vitamin D
2
or vitamin D
3
produced similar increases in serum 25OHD over the first 3 days, but serum 25OHD began to
fall immediately thereafter in the vitamin D
2
group until, by day 14, it reached baseline
concentrations (Armas et al., 2004). In contrast, serum 25OHD concentrations continued to
rise until day 14 in the vitamin D
3
group, then falling slowly over the following 14 days.
Armas et al (2004), and other investigators have suggested that that the potency of vitamin D
2
is much less than that of vitamin D
3
(Tjellesen et al., 1986; Trang et al., 1998). Their results
have shown that the biological activities of vitamin D
2
and vitamin D
3
are no longer
considered equal in humans. In contrast, Rapuri et al (2004) reported that vitamin D
2
and
vitamin D
3
supplements appear to be equally capable of improving the serum 25OHD status
in elderly women. In addition, Holick et al (2008) showed recently that both vitamin D forms
were equivalent in maintaining 25OHD status. This issue remains controversial, and it is still
not entirely clear which vitamin D form is preferable for treating 25OHD deficiency.

12
We did not compare the potencies of these two types of vitamin D, but we could not confirm
the reported short initial increase followed by a rapid fall in serum 25OHD after vitamin D
2
supplementation (Armas et al., 2004). When adjusted for a concomitant increase in serum
25OHD in the placebo group in the last week (due to the confounding effect of cutaneous
vitamin D
3
synthesis), the high serum 25OHD concentrations in both interventional groups
achieved remained constant during the follow-up period.
The vitamin D
2
dose used in this study was on average 100 µg/d (4 000 IU/d) in a weekly
dose of 700 µg (28 000 IU/d) for a short period of 4 weeks. This is 20 times higher than the
current recommended daily allowance for adults in Germany [5 µg/d (20 IU/d)]. But
according to the current state of knowledge, an intake of 100 µg (4 000 IU) vitamin D/d in
healthy subjects is safe (Hathcock et al., 2007; Vieth et al., 2001) and vitamin D
2
is
particularly low in toxicity (Pepper et al., 2009; Stephenson and Peiris, 2009). As anticipated,
we observed no cases of hypercalcemia or any adverse events in this study.
Our results demonstrate for the first time that the bioavailability of vitamin D
2
from vitamin
D
2
-enhanced button mushrooms via UV-B irradiation was effective in treating vitamin D
deficiency in young, healthy adults. Furthermore, the absorption rate and metabolism of
vitamin D
2
from UV-B-irradiated mushrooms were capable of raising circulating serum
25OHD concentrations as effectively as a vitamin D
2
supplement. In conclusion, as the
vitamin D
2
enhancement of mushrooms boosts their nutraceutical value, it would be a
worthwhile means of fighting widespread vitamin D deficiency in the general population.
Further research and development are required to find solutions for making such vitamin D
2
-
enhanced mushrooms commercially available in a safe and affordable manner.

13
ACKNOWLEGMENTS
We are grateful to Carole Cürten for proofreading this manuscript, to Peter Metzger from the
Schlossbergpilz GmbH, Germany for donating the button mushrooms used in this study, to
Daniela Klein and Elmar Maier for their help preparing the soups in the university’s kitchen,
and to Weber GmbH for technical support with the construction of the UV unit.
The authors’ responsibilities were as follows-PU: study concept and design, obtaining
funding, statistical analysis, data interpretation, writing the manuscript; FS: construction of
UV-unit, collection, assembly and interpretation of data; GI: study design, statistical advice
and analysis; HKB: critical review and contribution to the final draft; HB: supervision of the
study, critical review and contribution to the final draft. All authors have read and approved
the final manuscript. None of the authors expresses a conflict of interest.
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observed adverse effect level. Am J Clin Nutr 73, 288-94.
Webb AR, Kline L and Holick MF (1988). 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 Endocrinol Metab 67, 373-8.
TABLES

15
TABLE 1
Baseline characteristics of subjects and dropouts (n = 26)
1
Variables Mushroom
group (n = 8)
Supplement group
(n = 9)
Placebo group
(n = 9)
male:female (n) 3:5 4:5 2:7
Age (y) 28.6 ± 4.3
2
31.1 ± 6.7 32.4 ± 6.0
BMI (kg/m
2
) 22.0 ± 1.7 23.7 ± 2.1 20.6 ± 2.6
study dropout 1 at week 0 1 at week 5 -
1
There were no significant differences between the groups at baseline.
2
Means ± SDs (all such values).

16
TABLE 2
Laboratory values in the three study arms
1
Serum
parameter and
time (week)
Mushroom
group
(n = 8)
Supplement
group
(n = 9)
Placebo group
(n = 9)
P
2
25OHD (75-175 nmol/L)
3
0 34.0 ± 11.0
6
28.7 ± 10.0
6
38.7 ± 14.2
4
0.160
1 45.2 ± 7.0 42.0 ± 9.2
4
35.0 ± 13.0 0.125
2 47.2 ± 8.0
a
46.2 ± 8.0
a, 4
31.2 ± 9.7
b, 4
0.001
3 51.0 ± 11.2
a
50.7 ± 7.7
a
27.5 ± 7.7
b, 4
< 0,0001
4 51.5 ± 7.7
a, 5
48.2 ± 8.7
a, 4
24.5 ± 7.2
b, 4
< 0,0001
5 56.7 ± 7.2
a
58.0 ± 11.2
a
28.7 ± 8.7
b
< 0,0001
iPTH (11.3-42.5 ng/L)
0 31.7 ± 9.5
44.0 ± 14.7 40.4 ± 18.4 0.241
4 30.2 ± 12.7 40.0 ± 11.4 35.6 ± 11.9 0.265
Calcium (2.2-2.7 mmol/L)
0 2.53 ± 0.08 2.47 ± 0.08 2.48 ± 0.09
6
0.327
4 2.43 ± 0.07 2.40 ± 0.13 2.37 ± 0.09
0.442
1
Mean ± SD (all such values). iPTH, intact parathyroid hormone.
2
Values at each time of measurement with different superscript letters are significantly
different from each other (one-factor ANOVA and post hoc analysis with Tukey´s test).
3
Reference range.
4-6
Significantly different from the following mean in the same column (paired t test):
4
P
<0.05,
5
P <0.01,
6
P <0.001.

17
FIGURE LEGEND PAGE
FIGURE 1 Time course of the mean changes in serum 25OHD over the 5-week study period
in subjects who consumed four times (weeks 0, 1, 2, 3) mushrooms enhanced with vitamin D
2
via UV-B irradiation (mushroom group, n = 8, ●) or vitamin D
2
-containing supplements
(supplement group, n = 9, ▲) or placebo (placebo group, n = 9, ■) at the end of the winter.
Error bars are 2 SE. At week 5 one subject dropped out of the supplement group.
Concentrations were significantly different (ANOVA, Tukey´s test) between the mushroom
and placebo groups, and between the supplement and placebo groups from week 2 onward
(wk 2: P = 0.002; P = 0.004, respectively; weeks 3, 4, 5: P <0.0001). Time courses for serum

18
25OHD over the study period in the mushroom and supplement groups did not differ
significantly.