Dermato-Endocrinology

ISSN: (Print) 1938-1980 (Online) Journal homepage: www.tandfonline.com/journals/kder20

Photobiology of vitamin D in mushrooms and its

bioavailability in humans

Raphael-John H. Keegan, Zhiren Lu, Jaimee M. Bogusz, Jennifer E. Williams &

Michael F. Holick

To cite this article: Raphael-John H. Keegan, Zhiren Lu, Jaimee M. Bogusz, Jennifer E. Williams
& Michael F. Holick (2013) Photobiology of vitamin D in mushrooms and its bioavailability in
humans, Dermato-Endocrinology, 5:1, 165-176, DOI: 10.4161/derm.23321

To link to this article: https://doi.org/10.4161/derm.23321

Copyright © 2013 Landes Bioscience

Published online: 01 Jan 2013.

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 Report Report
Dermato-Endocrinology 5:1, 165–176; January/February/March 2013; © 2013 Landes Bioscience

Photobiology of vitamin D in mushrooms

and its bioavailability in humans
Raphael-John H. Keegan,1 Zhiren Lu,1 Jaimee M. Bogusz,1 Jennifer E. Williams1 and Michael F. Holick1,*
1
Department of Medicine; Section of Endocrinology, Nutrition, and Diabetes; Vitamin D, Skin and Bone Research Laboratory;
Boston University Medical Center; Boston, MA USA

Keywords: vitamin D, vitamin D2, mushrooms, 25-hydroxyvitamin D, ultraviolet radiation

Abbreviations: 25(OH)D, 25-hydroxyvitamin D; 7-DHC, 7-dehydrocholesterol; cZc, 5,6-s-cis-s-cis previtamin D; E. huxleyi,

©2013 Landes Bioscience. Do not distribute.

Emiliania huxleyi; HPLC, high performance liquid chromatograph; IU, international units; LCMS/MS, liquid chromatography
tandem mass spectroscopy; S. cerevisiae, Saccharomyces cerevisiae; tZc, 5,6-s-trans-s-cis previtamin D; UV, ultraviolet;
UVB, ultraviolet B

Mushrooms exposed to sunlight or UV radiation are an excellent source of dietary vitamin D2 because they contain high
concentrations of the vitamin D precursor, provitamin D2. When mushrooms are exposed to UV radiation, provitamin
D2 is converted to previtamin D2. Once formed, previtamin D2 rapidly isomerizes to vitamin D2 in a similar manner that
previtamin D3 isomerizes to vitamin D3 in human skin. Continued exposure of mushrooms to UV radiation results in
the production of lumisterol2 and tachysterol2. It was observed that the concentration of lumisterol2 remained constant
in white button mushrooms for up to 24 h after being produced. However, in the same mushroom tachysterol2
concentrations rapidly declined and were undetectable after 24 h. Shiitake mushrooms not only produce vitamin D2
but also can produce vitamin D3 and vitamin D4. A study of the bioavailability of vitamin D2 in mushrooms compared
with the bioavailability of vitamin D2 or vitamin D3 in a supplement revealed that ingestion of 2000 IUs of vitamin D2 in
mushrooms was as effective as ingesting 2000 IUs of vitamin D2 or vitamin D3 in a supplement in raising and maintaining
blood levels of 25-hydroxyvitamin D which is a marker for a person’s vitamin D status. Therefore, mushrooms are a rich
source of vitamin D2 that when consumed can increase and maintain blood levels of 25-hydroxyvitamin D in a healthy
range. Ingestion of mushrooms may also provide the consumer with a source of vitamin D3 and vitamin D4.

Prehistoric Overview grass frogs and anolis lizards were found to have 1 to 2 orders of
magnitude greater concentrations of provitamin D3 in their skin
It is theorized that the origins of vitamin D came from Earth’s compared with humans. Therefore, some poikilotherms have
earliest life forms over 900 million years ago. Primitive life a tremendous capability to produce vitamin D3 in their skin.1
forms were bombarded by the sun’s UV (UV) radiation; this This finding suggests that the cutaneous production of vitamin
caused damage to their UV-sensitive DNA, RNA and proteins.1 D may have played a major evolutionary role in poikilothermic
Provitamin D absorbs UV radiation between 240 and 320 nm land vertebrates.
and thus could act as a sunscreen. Phytoplankton and zooplank-
ton that produced provitamin D2 (ergosterol) were likely to Historic Overview
have their DNA and RNA protected from UV radiation and
were able to pass along their genes to their progeny.1,2 Five hun- Vitamin D deficiency has been implicated with several bone
dred million years ago phytoplankton such as Emiliania huxleyi pathologies, one of the earliest being rickets. In the early 19th
(E. huxleyi) were producing provitamin D2.1,2 Upon exposure to century, rickets was considered an epidemic affecting Europe
UVB radiation, provitamin D2 was converted to previtamin D2, and the United States, particularly in the northern industrial-
which then thermally isomerized to vitamin D2 (Fig. 1). It is ized cities. Autopsy studies conducted in Boston and Leiden, The
believed that animals higher on the food chain acquired their Netherlands in the late 19th century found 80–90% of children
vitamin D from phytoplankton and zooplankton.1 had rickets. Rickets can be caused by vitamin D deficiency, cal-
Land vertebrates require vitamin D to maintain adequate cium deficiency, acquired and inherited disorders metabolism of
serum calcium levels and bone metabolism. Poikilothermic ani- vitamin D, calcium and phosphorus. Specific signs and symp-
mals have been studied and were found to contain several provi- toms include, growth retardation, muscle weakness, skeletal
tamin D’s in their skin, with the major provitamin D identified deformities, stunted growth and bowed legs.3,4
as 7-dehydrocholesterol (7-DHC, provitamin D3).1 Northern

*Correspondence to: Michael F. Holick; Email: [email protected]

Submitted: 12/06/12; Accepted: 12/08/12
http://dx.doi.org/10.4161/derm.23321

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 ©2013 Landes Bioscience. Do not distribute.
Figure 1. Schematic showing the structures of the provitamin D (side chains labeled R) ergosterol (red), 7-dehydrocholesterol (7-DHC) (blue), 22,23-di-
hydroergosterol (provitamin D4) (black). Upon UV irradiation, the provitamin D (orange) is converted to previtamin D (green), which can thermally
convert to vitamin D (purple). Previtamin D is converted to photobyproducts tachysterol (dark gray) and lumisterol (brown) (Holick, copyright 2012,
reproduced with permission).

By the late 18th and early 19th century, rickets was a common in humans and animals. It was originally assumed that endog-
condition for children. In 1822, the Polish physician Sniadecki enously produced vitamin D was the same as vitamin D pro-
noticed that children living in Warsaw, Poland, a densely popu- duced by irradiated yeast. However, chickens fed vitamin D from
lated city, had a higher incidence of rickets compared with chil- irradiated yeast had minimal antirachitic affects, while cod liver
dren living in the countryside where sun exposure was more oil reversed the effects of rickets.1-3
common. Sniadecki was the first to hypothesize the importance It was then concluded that vitamin D produced from the skin
of sunlight to bone health.3 In 1861, Dr. Trousseau of France was different than vitamin D produced by irradiated yeast. To
postulated the etiology of rickets was a lack of sun exposure and distinguish the two forms, they were named vitamin D2 (ergocal-
diet, and successfully treated patients with cod-liver oil.4 ciferol) from yeast and vitamin D3 (cholecalciferol) from human
In the early 20th century, the German physician Huldschinsky and animal skin.3 Vitamin D2 and vitamin D3 are structurally
treated patients suffering from rickets with exposure to a mer- very similar. The only exceptions are that vitamin D2 has a dou-
cury-vapor quartz lamp that emitted UVB radiation.3 After six ble bond between carbons 22 and 23 and a methyl group on car-
weeks of treatment, the children had radiologic improvement in bon 24 (Fig. 1).
their condition, demonstrated by an increase in mineralization
in the children’s X-ray. In 1921, Hess and Unger observed that Photobiology of Vitamin D2 in Mushrooms
exposing children to direct sunlight three to four times a week
improved their clinical and radiological manifestations of rick- Mushrooms are fungi and belong to the division Basidiomycota.
ets.5 Hess and Unger also observed that cod liver oil acted as a The vitamin D benefits of edible mushrooms has been known
preventative measure and treatment for rickets in adolescents.3 since 1994 when Mattilla et al. extracted provitamin D2 from
wild mushrooms.9 White button mushrooms (Agaricus bisporus)
Vitamin D in Food are grown in the dark and therefore contain negligible concentra-
tions of vitamin D2. White button mushrooms were examined
In 1918, Mellanby first showed that feeding puppies cod liver and found to contain 56.3 μg/100 g fresh weight of provitamin
oil could prevent rickets from occurring.6 In 1924, Steenbock D2, and 0.11 μg/100 g fresh weight of vitamin D2.10
and Black observed that UV irradiated foods had an antirachitic When exposed to UV radiation, mushrooms become an abun-
effect when consumed by animals.7 Later, in 1925, Cowell dem- dant source of vitamin D2.11 Mushroom producers have recently
onstrated that bovine milk exposed for 20 min under a mercury begun exposing mushrooms to UV radiation in order to have
vapor lamp could be used to treat rickets when consumed by their product contain vitamin D2. The photobiology of vitamin
adolescents.8 Within two decades, a wide variety of foods and D3 has been well studied in poikilothermic animals and human
beverages were fortified with vitamin D.3 These discoveries lead skin; however, little is known about how vitamin D2 is produced
to the identification of vitamin D as having an antirachitic effect in irradiated mushrooms.

166 Dermato-Endocrinology Volume 5 Issue 1

 Photoproduction of Previtamin D2 in Mushrooms Photoproduction of Lumisterol2 and Tachysterol2

White button mushrooms were irradiated and studied in White button mushrooms were exposed to UV radiation for 1,
order to elucidate the mechanism of vitamin D2 production. 2, 3, 4, 5 and 10 min at 25°C (Fig. 4). After 5 min of exposure
Provitamin D2 dissolved in methanol (50 μg/mL) in borosili- to UV radiation a peak that migrated at 7.3 min and had a UV
cate ampoules were irradiated and used as a positive control as absorption spectrum λmax 272 nm, consistent with lumisterol2
previously described.12 Mushrooms and ampoules were irradi- (Fig. 2A) and a peak with retention time 11.7 min that had a
ated for 5 min, 3.5 inches from a RC-500B Pulsed UV Curing UV absorption spectrum λmax 281 nm consistent with tachys-
System (Xenon Corporation). After irradiation mushroom sam- terol2 was detected in the irradiated white button mushrooms
ples were obtained with a brass 0.5 cm 2 cork borer to a depth (Fig. 2B). A similar result was observed in irradiated ampoules;
of 0.1 cm at various times for up to 96 h. The samples were previtamin D2, lumisterol2 and tachysterol2 were detected (Fig.
homogenized in 6.0 mL of 100% methanol. The samples were 2C and D) and confirms a previous report.13

©2013 Landes Bioscience. Do not distribute.

centrifuged and the liquid layer was pipetted off and dried with Previtamin D2 and lumisterol2 were only observed after expo-
N2. The dried samples were dissolved in either 0.3% or 0.8% sure to 1 min of UV radiation. After 3 min of UV exposure, previ-
isopropanol in hexane and chromatographed on a high perfor- tamin D2, vitamin D2, lumisterol2 and tachysterol2 were detected
mance liquid chromatograph stacked Agilent 1100 (HPLC) (Fig. 4). Photoequilibrium of the previtamin D2 photobyprod-
attached to a photodiode detector. Two different columns were ucts was reached between 4 and 5 min of UV exposure. Although
used as a stationary phase (5 μm spherical silica gel) to sepa- the ratio of previtamin D2 and photoproducts did not change
rate provitamin D2 from its photoproducts and vitamin D2. A after 5 min of exposure, continued exposure increased the total
Zorbax RX-SIL column with 0.8% isopropanol in hexane was amount of previtamin D2 and its photoproducts. Mushrooms
used to separate lumisterol 2 from previtamin D2 and the Zorbax that were not exposed to UV radiation did not contain detectable
CN column with 0.3% isopropanol in hexane was used to sepa- amounts of previtamin D2, its photobyproducts or vitamin D2.
rate tachysterol 2 from vitamin D2 (Fig. 2A and B). The concen-
trations of photobyproducts were calculated using a conversion Stability of Lumisterol2 and Tachysterol2 in
factor obtained from a standard curve. Mushrooms
As can be seen in Figure 2A, immediately after exposure to 5
min of UV radiation provitamin D2 was converted to a product A 24 h time course was conducted to study the stability of the
that migrated at 6.7 min and had a UV absorption spectrum photobyproducts of previtamin D2 in white button mushrooms.
λmax 260 nm, consistent with previtamin D2. The peak with White button mushrooms and provitamin D2 ampoules were
retention time 10.6 min had a UV absorption λmax at 265 nm irradiated for 5 min; mushroom samples were collected every 2 h
and was consistent with vitamin D2 (Fig. 2B). These findings for 24 h. As expected, previtamin D2 and vitamin D2 decreased
in irradiated mushrooms are similar to what was observed in and increased, respectively, in a time dependent manner (Fig. 3).
irradiated ampoules containing provitamin D2 and confirms An extended time course of 336 h was conducted to study the
the previous observations by Kalaras et al.13 (Fig. 2C and D). stability of lumisterol2 and tachysterol2 in methanol that were
A 96 h time course was conducted to examine the conversion recovered from the irradiation of provitamin D2. Lumisterol2 and
of previtamin D2 to vitamin D2 in UV irradiated white button tachysterol2 remained stable in methanol for more than 300 h
mushrooms and ampoules. Immediately after irradiation and at (Fig. 5A). The amount of lumisterol2 remained constant over time
various times, the samples were chromatographed to determine in irradiated mushrooms. Though, the amount of tachysterol2
the percent conversion of previtamin D2 and vitamin D2 (Fig. remained constant in irradiated ampoules, in irradiated white
3). There was a time dependent increase in the amount of vita- button mushrooms, tachysterol2 rapidly declined to undetectable
min D2. At 6 h after irradiation, 24% of the previtamin D2 in levels within 24 h (Fig. 5B). The same rapid disappearance of
white button mushroom had converted to vitamin D2 whereas tachysterol2 was also observed in oyster (Pleurotus ostreatus) and
only 10% of the previtamin D2 in the irradiated provitamin D2 shiitake ((Lentinula edodes) mushrooms (data not shown).
ampoules converted to vitamin D2.
After 11.5 h, 50% of the previtamin D2 present in white but- Vitamin D4 in Mushrooms
ton mushrooms had converted to vitamin D2 compared with
only 19% of the previtamin D2 converted to vitamin D2 in the Vitamin D4 is a form of vitamin D that is structurally similar to
ampoules (Fig. 3). These results mimic what was observed in vitamin D3 and was first produced by Windaus and Trautmann
ampoules containing 7-DHC in methanol and lizard skin after in 1937.15 Vitamin D4 has a methyl group on carbon 24 of the
exposure to UV radiation and kept at 25°C (Fig. 3). The results vitamin D3 side chain (Fig. 1). Vitamin D4 is produced from the
demonstrate that the kinetics for previtamin D2 conversion to UV irradiation of its precursor, 22,23-dihydroergosterol (provi-
vitamin D2 in mushrooms was enhanced compared with the tamin D4) (Fig. 1). Vitamin D4 was reported to be about 60% as
conversion in an organic solvent, similar to lizard skin.14 active as vitamin D3 in healing rickets in rats.16
Philips et al. reported the presence of provitamin D4 in several
mushroom species including crimini (Agaricus bisporus), porta-
bella (Agaricus bisporus), enoki (Flammulina velutipes), shiitake,

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 ©2013 Landes Bioscience. Do not distribute.

Figure 2. For figure legend, see page 5.

168 Dermato-Endocrinology Volume 5 Issue 1

 Figure 2. HPLC chromatographs of extracts from white button mushrooms and ampoules irradiated for 5 min under the Xenon RC-500 pulsed UV
lamp. The X-axis is retention time in minutes; Y-axis is miliAbsorbance Units (mAU) at 265 nm. A Zorbax RX-SIL column was used in 0.8% isopropanol
(IPA) in hexane to separate lumisterol2 (L2) from previtamin D2 (PreD2) (A and C). A Zorbax CN column in 0.3% IPA in hexane was used to separate
tachysterol2 (T2) from vitamin D2 (D2) (C and D). Irradiated white button mushroom (A and B). Irradiated ampoule containing 50 μg/mL of ergosterol
(C-E).

©2013 Landes Bioscience. Do not distribute.

Figure 3. Conversion of previtamin D2 to vitamin D2 at 25°C in irradiated white button mushrooms (◆), irradiated ampoules containing 50 μg/mL of
ergosterol in methanol (●), 7-dehydrocholesterol (7-DHC) in ampoules (■) and lizard skin (*) at 25°C. Inset is percent conversion of previtamin D2 to
vitamin D2 and previtamin D3 to vitamin D3 between 0 and 12 h.

maitake (Grifola frondose), oyster, morel (Morchella spp) and yeast using the reverse phase HPLC system with a Zorbax ODS
chanterelle (Cantharellus cibarius).17 column to separate the various provitamin Ds revealed all sam-
We examined crimini, oyster, portabella, shiitake, white ples contained provitamin D2 and vitamin D2. In addition the
button mushrooms, white button mushroom power (Monterey peak with retention time at 9.6 min and a UV absorbance spec-
Mushrooms, Inc.) and Saccharomyces cerevisiae (S. cerevisiae) yeast trum of a 5,7-diene consistent with provitamin D4 (Fig. 6A).15
(Lallemand Inc.) for the presence of provitamin D2, vitamin D2, Provitamin D4 obtained from white button mushroom powder
provitamin D4, vitamin D4 as well as other potential provitamin was dissolved in methanol (4 μg/mL) and placed in a borosilicate
Ds and vitamin Ds. Mushroom samples were obtained in a simi- ampoule and irradiated for 10 min using a RC-500B Pulsed UV
lar manner that was described for white button mushrooms. One Curing System. After irradiation the provitamin D4 ampoule was
gram of S. cerevisiae was extracted with 5 mL of methanol for dried with N2 gas and prepared for straight phase HPLC. HPLC
1 min and sonicated with a sonic disrupter (Teledyne Tekmar). analysis revealed peaks with retention times that had UV absorp-
After being centrifuged for 5 min, the liquid layer was removed, tion spectra consistent with provitamin D4, previtamin D4,
and the extraction was repeated 5 additional times. The extracts lumisterol4 and tachysterol4, and were used as our standards (Fig.
were combined and taken to dryness under N2 gas. The dried 6B and C). HPLC analysis of S. cerevisiae yeast revealed peaks
samples were dissolved in methanol, ethanol and dH2O (32:8:1 that also co-eluted with standard provitamin D4 and vitamin
ratio) and chromatographed on a reverse phase HPLC using a D4. Oyster mushrooms were irradiated for 5 min and examined
Zorbax ODS column. for the presence of vitamin D4. The dried samples were dis-
Analysis of crimini, oyster, portabella, shiitake, white button solved in 20% methanol in acetonitrile and prepared for reverse
mushrooms, white button mushroom powder and S. cerevisiae phase HPLC, using a Vydac C18 column to separate the various

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 ©2013 Landes Bioscience. Do not distribute.
Figure 4. Photobyproducts in irradiated white button mushrooms with various exposure times to UV radiation. Samples dissolved in either 0.3% or
0.8% isopropanol in hexane were chromatographed on a high performance liquid chromatograph, and were analyzed for previtamin D2 (blue), lumis-
terol2 (red), vitamin D2 (green) and tachysterol2 (purple). Mean ± SEM.

vitamin Ds. UV irradiated mushrooms revealed the presence of cap. UV irradiated shiitake mushrooms were examined for the
previtamin D4 with a retention time of 6.2 min in straight phase; presence of vitamin D3. Shiitake mushrooms were irradiated
its presence was confirmed by chromatography after its conver- with the gills facing up for 5 min using a RC-500B Pulsed UV
sion to vitamin D4. The analysis revealed peaks with retention Curing System. Mushroom samples were obtained in a similar
times of 10.8 and 13 min that had a UV absorbance spectra with manner that was described previously. The samples were chro-
λmax 265 nm for the 5,6-cis-triene system consistent with vita- matographed on reverse phase HPLC using a C18 column. UV
min D2 and vitamin D4, respectively (Fig. 6D). Provitamin D2 irradiated mushrooms revealed the presence of previtamin D3
and provitamin D4 were detected in the unexposed mushrooms with a retention time of 6.3 min in straight phase; its presence
(data not shown). For comparison white button mushroom pow- was confirmed by chromatography after its conversion to vita-
der contained ~88% vitamin D2 and ~12% vitamin D4, whereas min D3. Peaks with retention times of 5.1 and 6.1 min had UV
S. cerevisiae yeast sample contained ~99% vitamin D2 and ~1% absorbance spectra with λmax 265 nm consistent with vitamin
vitamin D4. D2 and vitamin D3, respectively (Fig. 7B).

Vitamin D3 in Mushrooms Bioavailability of Vitamin D2 in Mushrooms

It has been assumed that mushrooms only produce vitamin D2 Various foods and beverages have been fortified with vitamin
when irradiated. However, since some mushrooms can produce D in the United States for more than 70 y since there are few
vitamin D4 we reevaluated, by HPLC, mushroom extracts for that contain vitamin D naturally. Milk, orange juice, bread,
the possibility of identifying other provitamin Ds. Evaluation other dairy products and some cereals have been fortified with
of extracts from shiitake mushrooms revealed by reverse phase this essential vitamin.18 Mushrooms exposed to sunlight or UV
HPLC a peak with a retention time of 11.5 min that was identi- radiation are a good source of vitamin D2. Several clinical stud-
fied as provitamin D4, and a peak with a retention time of 10.8 ies have been conducted to determine the bioavailability of vita-
min with an identical UV absorption spectrum consistent with min D2 and vitamin D3 in fortified foods and beverages and
a 5,7-diene (Fig. 7A). Co-chromatography studies revealed that the efficacy of fortification in increasing 25-hydroxyvitamin
this peak was 7-DHC. 7-DHC was found to migrate between D [25(OH)D] levels; the measure for determining a person’s
provitamin D2 and provitamin D4 that had retention times vitamin D status.18 Fortification of foods with vitamin D2 or
of 10.8, 9.6 and 11.5 min, respectively. Concentrations of the vitamin D3 has been shown to be a safe and effective way to
7-DHC and provitamin D4 were 5.8 and 2.6 greater in the gills increase 25(OH)D levels in children and adults.18
of the mushroom compared with the surface of the mushroom

170 Dermato-Endocrinology Volume 5 Issue 1

 Biancuzzo et al. found that there was no difference
in total 25(OH)D levels in healthy adults who con-
sumed orange juice that was fortified with 1000 IU of
vitamin D2 or 1000 IUs of vitamin D3 compared with
consuming a supplement containing either 1000 IU
vitamin D2 or 1000 IU vitamin D3.19 It was also found
that there was no difference in the increase of 25(OH)
D3 levels between subjects who consumed vitamin
D3-fortified orange juice or vitamin D3 supplements or
in 25(OH)D2 levels of subjects who consumed vitamin
D2-fortified orange juice or vitamin D2 supplements.
Additionally, Natri et al. found that bread fortified with

©2013 Landes Bioscience. Do not distribute.

vitamin D3 increased total serum 25(OH)D levels in
women as effectively as a vitamin D3 supplement.20
To date, two groups have reported on the bioavail-
ability of vitamin D2 in UV irradiated mushrooms.
Urbain et al. conducted a five-week single-blinded,
randomized, placebo-controlled trial in 26 healthy
Caucasian adults with 25(OH)D levels below 20 ng/
mL.21 These subjects were randomized to three groups
and assigned to receive either 28,000 IU vitamin D2
from UV irradiated mushrooms in a soup and placebo,
60 IU vitamin D2 in soup that contained non-UV-
irradiated mushrooms and 28,000 IU vitamin D2 in a
liquid supplement, or 60 IU vitamin D2 in a non-UV-
irradiated mushroom soup and placebo supplement four
times a week for four weeks. After four weeks, serum
25(OH)D levels increased significantly and consuming
Figure 5. Stability of lumisterol2 and tachysterol2 in irradiated ampoules and
vitamin D2 from UV-irradiated mushrooms was equally mushrooms. Ampoules and mushroom samples were exposed to 5 min of UV
as effective at raising 25(OH)D levels as ingesting the radiation. Samples were taken over time. Samples were dissolved in either 0.3% or
same amount of vitamin D2 as a supplement. 0.8% isopropanol in hexane and chromatographed on a high performance liquid
Stephenson et al. conducted a similar study where chromatograph at 25°C. Mean ± SEM. (A) Lumisterol2 (◆) and tachysterol2 (■),
in irradiated ampoules. (B) Lumisterol2 (◆) and tachysterol2 (■), in white button
subjects were randomized to consume one serving
mushroom samples.
of mushrooms with a standard meal each day for six
weeks.22 Four groups received either one serving of
non-UV-irradiated mushrooms plus meal (control), UV irradi- D3 supplement at raising and maintaining total serum 25(OH)
ated mushrooms containing 352 IU vitamin D2 with a meal, D levels.19,25 Furthermore, taking 50,000 IU vitamin D2 once a
UV irradiated mushrooms containing 684 IU vitamin D2 with a week for eight weeks and every other week thereafter for up to
meal or a supplement containing 1,128 IU vitamin D2 with non- six years increased serum total 25(OH)D levels and is considered
UV-irradiated mushrooms. At the end of six weeks, 25(OH)D2 to be effective for the treatment and prevention of vitamin D
levels were higher in all groups except the control group. They deficiency.26 Similarly, Demetriou reported that 50,000 IU vita-
observed a significant decrease in serum 25(OH)D3 in the group min D2 repletion and maintenance therapy in vitamin D defi-
receiving 684 IU vitamin D2 in UV irradiated mushrooms and cient patients significantly increased serum 25(OH)D2 and total
in the group receiving the vitamin D2 supplement. There was a 25(OH)D despite a decrease in serum 25(OH)D3 levels.27
mean decrease of 25(OH)D3 of 0.32 ng/mL that was offset by an We conducted a clinical study to determine if ingestion of
increase of 0.40 ng/mL in 25(OH)D2. Overall, however, there vitamin D2 in a dried white button mushroom extract (Monterey
was a small decrease in total 25(OH)D levels in the groups con- Mushrooms, Inc.) was as effective at increasing and maintain-
suming UV irradiated mushrooms that contained vitamin D2. ing vitamin D status as supplemental vitamin D3 and vitamin
This observation contributed to the controversy surround- D2. Thirty healthy adults were enrolled in the study (6 male, 19
ing the efficacy of maintaining total serum 25(OH)D levels female, mean age 35.2 y) and were randomized to ingest cap-
after taking supplemental or dietary vitamin D2 vs. vitamin D3. sules containing 2000 IU vitamin D2, 2000 IU vitamin D3 or
Some reports have suggested that vitamin D3 was more effec- 2000 IU mushroom vitamin D2 once a day for three months
tive than vitamin D2 at maintaining total serum 25(OH)D during the winter. Vitamin D concentrations were verified to be
levels.23,24 In contrast, Holick et al., similar to Biancuzzo et al., within 10% by HPLC. Twenty-five subjects completed the study.
found that the daily ingestion of a 1000 IU vitamin D2 supple- Fourteen subjects were randomized to the mushroom vitamin D2
ment was as effective as the daily ingestion of a 1000 IU vitamin group, eight subjects to the supplemental vitamin D2 group and 3

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 ©2013 Landes Bioscience. Do not distribute.

Figure 6. For figure legend, see page 9.

172 Dermato-Endocrinology Volume 5 Issue 1

 Figure 6. HPLC chromatograms of extracts from white button mushrooms. (A) An extract of white button mushroom chromatographed on reverse
phase on a Zorbax ODS column. Methanol, ethanol and dH2O (32:8:1 ratio) to separate the provitamin Ds. Y-axis is miliAbsorbance Units (mAU) at 280
nm. Ergosterol (E), provitamin D4 (ProD4). (B) An ampoule containing provitamin D4 (ProD4) irradiated for 10 min and chromatographed on a Zorbax
RX-SIL column 0.8% isopropanol (IPA) in hexane to separate lumisterol4 (L4) from previtamin D4 (PreD4). Y-axis is mAU at 265 nm. Tachysterol4 (T4), vita-
min D4 (D4). (C) An ampoule containing provitamin D4 (ProD4) irradiated for 10 min chromatographed on a Zorbax CN column in 0.3% IPA in hexane to
separate tachysterol4 (T4), from vitamin D4 (D4). Y-axis is mAU at 265 nm. Previtamin D4 (PreD4), lumisterol4 (L4). (D) An extract from an oyster mushroom
irradiated for 5 min and chromatographed on a C18 column in 20% methanol in acetonitrile to separate vitamin Ds. Y-axis is mAU at 265 nm. Tachys-
terol2 (T2), vitamin D2 (D2), vitamin D4 (D4).

©2013 Landes Bioscience. Do not distribute.

Figure 7. Reverse phase HPLC chromatograms of extracts from shiitake mushrooms. (A) An extract from a shiitake mushroom chromatographed on
a Zorbax ODS column in methanol, ethanol, and dH20 (32:8:1 ratio) to separate the provitamin Ds. Y axis is miliAbsorbance Units (mAU) at 280 nm. Er-
gosterol (E), provitamin D3 (ProD3), provitamin D4 (ProD4). (B) An extract from a shiitake mushroom irradiated for 5 min and chromatographed on a C18
column in 20% methanol in acetonitrile to separate vitamin Ds. Y axis is miliAbsorbance Units (mAU) at 265 nm. Vitamin D2 (D2), vitamin D3 (D3).

subjects to the supplemental vitamin D3 group. Serum concentra- Total serum 25(OH)D levels significantly increased from 19.4 ±
tions of 25(OH)D2, 25(OH)D3 and 25(OH)D were measured 2.3 ng/mL to 29.2 ± 2.0 ng/mL (p < 0.01) (Fig. 8B).
once a week for 12 weeks by liquid chromatography tandem mass Subjects in the supplemental vitamin D3 group had a mean
spectroscopy (LCMS/MS) as previously described.28 baseline serum of 25(OH)D3 of 16.3 ± 0.6 ng/mL with a final
Subjects in the mushroom vitamin D2 group had a mean base- baseline serum level of 34.4 ± 1.3 ng/mL (Fig. 8C). Total
line serum 25(OH)D2 of 0.6 ± 0.3 ng/mL that increased signifi- 25(OH)D increased from 17.1 ± 1.4 ng/mL to 34.4 ± 1.3 ng/mL
cantly to 18.6 ± 1.4 ng/mL at the end of 12 weeks (p < 0.0001). (p < 0.05). The discrepancy in mean baseline serum levels is due
Total serum 25(OH)D levels increased from 20.6 ± 2.4 ng/mL to to some detectable 25(OH)D2 of 0.8 ng/mL (Fig. 8C).
30.1 ± 2.6 ng/mL (p < 0.001) (Fig. 8A). Baseline serum total 25(OH)D levels were not significantly
Subjects in the supplemental vitamin D2 group had a mean different between the groups; 17.1 ± 1.2, 19.4 ± 2.3 and 20.6 ±
baseline serum 25(OH)D2 of 1.5 ± 1.2 ng/mL that increased sig- 2.4 ng/mL for the supplemental D3 and vitamin D2, and mush-
nificantly to 13.3 ± 2.0 ng/mL at the end of 12 weeks (p < 0.001). room vitamin D2 groups respectively. Serum 25(OH)D levels

www.landesbioscience.com Dermato-Endocrinology 173

 gradually increased and plateaued at seven weeks and were main-
tained for the following five weeks. At the end of 12 weeks, serum
total 25(OH)D levels were not statistically significantly different
in all three groups: 34.4 ± 1.1, 29.2 ± 2.0 and 30.1 ± 2.6 ng/mL
for supplemental vitamin D3, D2 and mushroom D2 respectively.
These results demonstrate ingestion of mushrooms contain-
ing D2 was as effective at increasing and maintaining total serum
25(OH)D levels as supplemental vitamin D2 and vitamin D3.

Conclusion

Vitamin D deficiency is a pandemic. It has increased the risk

©2013 Landes Bioscience. Do not distribute.

of skeletal and chronic diseases associated with vitamin D defi-
ciency worldwide. Therefore, obtaining vitamin D from sensible
sun exposure, foods that naturally contain vitamin D, and from
supplementation with vitamin D is imperative to maintain a
healthy lifestyle.
Phytoplankton have been producing vitamin D2 from provita-
min D2 for over 500 million years. The phytoplankton E. huxlei
contained 1.0 μg/g wet weight of provitamin D2 and has likely
been a source of vitamin D2 for the oceanic food chain for mil-
lions of years.2 Due to their large quantities of provitamin D2,
fungi like phytoplankton have a huge capacity to produce vita-
min D2 when exposed to UV irradiation.
It is now known that in mushrooms, provitamin D2 is con-
verted to previtamin D2 upon UV irradiation. Previtamin D2
can absorb UVB radiation resulting in the production of the
photobyproducts, lumisterol 2 and tachysterol 2.13 An evaluation
of the thermal isomerization of previtamin D2 to vitamin D2 in
mushrooms revealed that it was rapidly converted to vitamin D2
most likely by a non-enzymatic membrane-enhanced catalytic
mechanism similar to what was observed in lizard and human
skin.14,29 The planar structure of 7-DHC and provitamin D2 can
fit in between the triglyceride side chains and polar head groups.
Upon exposure to UVB radiation the 5,7-diene absorbs the radi-
ation, converting to its respective previtamin D. Previtamin D
exists as two conformers cis-cis (cZc) and cis-trans (cZt). In an
organic solvent, ~90% exists in the thermodynamically favored
cZt while only ~10% exists in the cZc state. Although more
thermodynamically stable, the cZt conformer of previtamin D
is unable to isomerize to vitamin D; only its less stable cZc can.
Thus it takes several days at 25°C for the cZt conformer to con-
vert to cZc, which in turn converts to the thermodynamically
stable vitamin D3. Our observation that previtamin D2 more
Figure 8. Mean (± SEM) 25-hydroxyvitamin D2 (◆), 25-hy- rapidly converts to vitamin D2 in mushrooms than in methanol
droxyvitamin D3 (■) and total 25-hydroxyvitamin D (●) suggests that this mechanism of a membrane-enhanced conver-
concentrations over time after oral administration of (A)
2000 IU of mushroom vitamin D2 in capsules (n = 14), (B)
sion of previtamin D2 to vitamin D2 has existed for hundreds of
2000 IU of supplemental vitamin D2 in capsules (n = 8) millions of years.
and (C) 2000 IU of supplemental vitamin D3 in capsules Tachysterol3 and lumisterol3 produced in human skin have
(n = 3). The change in total serum 25-hydroxyvitamin D no biological function on calcium metabolism.30 Therefore, the
concentrations from baseline to final visits in each group physiologic significance of mushrooms producing lumisterol2 and
was statistically significant as was the change in serum
25-hydroxyvitamin D2 concentrations from baseline to
tachysterol2 is unknown. During our investigation on the time
final visit in the mushroom vitamin D2 group and the dependent conversion of previtamin D2 to vitamin D2 in white
supplemental vitamin D2 group. (*p < 0.05, **p < 0.01, button mushrooms we observed that the concentration of tachys-
***p < 0.001). No statistically significant difference was terol2 began to decline and was undetectable after 24 h. To be cer-
observed between final total 25-hydroxyvitamin D tain that this decline was not due to tachysterol2 being unstable,
concentrations between all three groups.

174 Dermato-Endocrinology Volume 5 Issue 1

we conducted a stability evaluation of tachysterol2 in methanol adults who ingested either 2000 IU supplement containing vita-
at room temperature for more than one week and found it to be min D2 or vitamin D3. These results confirm other studies that
stable. We also observed that this phenomenon also occurred in have demonstrated that ingesting vitamin D2 either from forti-
oyster and shiitake mushrooms suggesting that mushrooms are fied orange juice,19 a supplement20 or a pharmaceutical formula-
utilizing in some manner the tachysterol2. This observation may tion12,26 were all capable of increasing total circulating 25(OH)D
provide insight as to a possible biologic function of tachysterol concentrations for at least 3 mo and up to 6 y. Therefore ingesting
not only in mushrooms but also in human skin. mushrooms containing vitamin D2 can be an effective strategy to
It is known that plants and poikilothermic animals contain enhance the vitamin D status of the consumer. The observation
several different forms of provitamin D.1,31 Similarly mushrooms that some mushrooms when exposed to UVB radiation also pro-
are capable of producing more than one provitamin D. It had duce vitamin D3 and vitamin D4 can also provide the consumer
always been assumed that UV irradiated mushrooms were only with at least two additional vitamin Ds.
capable of producing vitamin D2. Our study confirms that some

©2013 Landes Bioscience. Do not distribute.

mushrooms do contain provitamin D4 and we now also report Disclosure of Potential Conflicts of Interest
that shiitake mushrooms contain 7-DHC. Therefore during UV This work was supported by The Mushroom Council and from
irradiation some mushrooms are capable of producing vitamin the National Institutes of Health Clinical Translational Science
D2, vitamin D3 and vitamin D4. Institute Grant UL1-TR000157. The funders had no role in
Mushrooms exposed to UVB radiation contain a significant study design, data collection and analysis, decision to publish
amount of vitamin D2 and therefore is an excellent alternative or preparation of the manuscript; ClinicalTrials.gov Identifier:
food source for vitamin D, especially for vegans. However, there NCT01815437.
continues to be concern that vitamin D2 not only is less effective
than vitamin D3 in maintaining total serum 25(OH)D concen- Acknowledgments
trations but that the ingestion of vitamin D2 can ultimately result We would like to thank the Xenon Corporation for supplying the
in a decrease in total 25(OH)D concentrations.23,24 Stephenson lamp used for irradiation studies. We are grateful to Joe D’Amico
et al.22 reported that ingesting UVB irradiated mushrooms con- of To-Jo Mushrooms for supplying us with mushroom growing
taining vitamin D2 resulted in a small decrease in total 25(OH) kits for some of our studies, Monterey Mushrooms Inc. for sup-
D levels. In our study, healthy adults who ingested daily for 3 mo plying us with the vitamin D2 mushroom powder capsules used
2000 IUs of vitamin D2 from mushrooms were able to raise and in our clinical trial and Lallemand Inc. for supplying the yeast
maintain their total 25(OH)D concentrations similar to healthy samples.
11. Jasinghe VJ, Perera CO, Barlow PJ. Bioavailability of 19. Biancuzzo RM, Young A, Bibuld D, Cai MH, Winter
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176 Dermato-Endocrinology Volume 5 Issue 1