Vitamin D4 in mushrooms and yeast - Boston University D4 in mushrooms and yeast Williams, Jennifer...
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Boston University
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Theses & Dissertations Boston University Theses & Dissertations
2013
Vitamin D4 in mushrooms and
yeast
Williams, Jennifer
Boston University
https://hdl.handle.net/2144/12248
Boston University
BOSTON UNIVERSITY
SCHOOL OF MEDICINE
Thesis
VITAMIN D4 IN MUSHROOMS AND YEAST
by
JENNIFER WILLIAMS
B.S., Lock Haven University of Pennsylvania, 2011
Submitted in partial fulfillment of the
requirements for the degree of
Master of Arts
2013
Approved by
First Reader
Michael F. Holick, Ph.D./M.D.
Professor of Medicine, Physiology and Biophysics and Molecular
Medicine
Second Reader
Richard J. Rushmore, Ph.D.
Assistant Professor of Anatomy and Neurobiology
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ACKNOWLEDGEMENTS
I would like to thank everyone in Dr. Holick’s lab for their assistance with my
research, with special thanks to Dr. Michael Holick, Lorrie Butler, Jaimee Bogusz, Cindy
Piao, Tony Juncaj, Bryon Curletto, Zhiren Lu, Kelly Persons, and Matthias Wacker. I
would also like to thank Dr. Jarrett Rushmore for his guidance as my academic advisor.
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VITAMIN D4 IN MUSHROOMS AND YEAST
JENNIFER WILLIAMS
Boston University School of Medicine, 2013
Major Professor: Michael F. Holick, Ph.D./M.D., Professor of Medicine, Physiology
and Biophysics and Molecular Medicine
ABSTRACT
Vitamin D deficiency is a pandemic that is now one of the most common
nutritional deficiencies worldwide. Vitamin D deficiency leads to reduced calcium
absorption from our diet, which causes hyperparathyroidism. The increase in parathyroid
hormone results in a defective mineralization of our skeleton, leading to the development
of rickets in children and osteomalacia in adults. It also increases bone reabsorption
resulting in a decrease in bone mineral density. During the winter months there is
decreased or complete absence of the production of vitamin D in the skin; therefore
finding natural dietary sources of vitamin D becomes important. Some mushroom
species exposed to ultraviolet radiation produce vitamin D2 as well as vitamin D4. The
goal of this project was to use high performance liquid chromatography with a
photodiode ultraviolet absorbance detector to identify which provitamin Ds and vitamin
Ds were present in various edible mushrooms species including skiitake (Lentinus
edodes), oyster (Pleurotus ostreatus), portabella (Agaricus bisporus), crimini (Agaricus
bisporus), and white button (Agaricus bisporus); and the yeast species Saccharomyces
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cerevisiae (Baker’s yeast). Provitamin Ds and vitamin Ds from the mushroom powder
(Monterey Mushroom), mushroom samples, and the yeast sample were extracted with
methanol and run on a Zorbax CN column. All the provitamin D samples were analyzed
with reverse phase HPLC on a Zorbax ODS column along with standards for provitamin
D2 (ergosterol), provitamin D3 (7-dehydrocholesterol), and provitamin D4 (22,23-
dihydroergosterol). The collected vitamin D samples were run on a Vydac C18 column
along with standards for vitamin D2 (ergocalciferol), vitamin D3 (cholecalciferol) and
vitamin D4. Provitamin D4 and vitamin D4 isolated and collected from the mushroom
powder sample were used as standards. Provitamin D4 was identified in every mushroom
species as well as the yeast sample. Vitamin D4 was identified in three of the UV
irradiated mushroom species including; white button, shiitake and oyster, as well as the
yeast sample. Provitamin D3 was identified in a shiitake mushroom. The ultraviolet
absorption spectra for compounds identified as vitamin D2, vitamin D4, provitamin D2,
provitamin D3 and provitamin D4 in all samples matched the UV absorption spectra of a
5,6-cis-triene of vitamin D and a 5,7-diene of provitamin D standards. These results
demonstrate that in addition to vitamin D2, some mushroom species and the yeast
Saccharomyces cerevisiae can also produce vitamin D4. In addition, shiitake mushrooms
contain provitamin D3, and thus has the ability to produce vitamin D3 after exposure to
UV radiation. Mushrooms and yeast are therefore a natural dietary source of multiple
vitamin Ds.
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TABLE OF CONTENTS
Title i
Reader’s Approval Page ii
Acknowledgements iii
Abstract iv
Table of Contents vi
List of Figures vii
List of Abbreviations x
Introduction 1
Materials and Methods 28
Samples 28
Irradiation 28
Extraction 29
Chromatography 31
Results 33
Mushrooms 33
Yeast 56
Discussion 66
Bibliography 70
Curriculum Vitae 79
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LIST OF FIGURES
Figure Title Page
1 Photosynthetic production of vitamin D3 and its photobyproducts from 3
7-dehydrocholesterol (7-DHC) in the skin.
2 Bioactivation of vitamin D3 (cholecalciferol) to its active form 1α,25(OH)2D3. 7 3 Six known forms of vitamin D and their corresponding precursors. 20
4 Photosynthetic production of vitamin D2 and its photobyproducts from 21
ergosterol (provitamin D2) in mushrooms.
5 Bioactivation of vitamin D2 (ergocalciferol) to its active form 1α,25(OH)2D2. 22
6 Characteristic UV absorbance spectrum of provitamin D. 25
7 Characteristic UV absorbance spectrum of vitamin D. 25
8 Characteristic UV absorbance spectra of previtamin D photobyproducts 26
9 UV irradiation of a portabella mushroom. 29
10 Shiitake mushroom biopsied 6 times. 30
11 Mushroom powder capsule. 31
12 Straight phase HPLC of white button mushroom extract and UV spectra. 35
13 Straight phase HPLC of shiitake mushroom extract and UV spectra. 36
14 Straight phase HPLC of oyster mushroom extract and UV spectra. 37
15 Straight phase HPLC of crimini mushroom extract and UV spectra. 38
16 Straight phase HPLC of portabella mushroom extract and UV spectra. 39
17 Reverse phase HPLC of white button mushroom vitamin D and UV spectra. 40
18 Reverse phase HPLC of shiitake mushroom vitamin D and UV spectra. 41
19 Reverse phase HPLC of oyster mushroom vitamin D and UV spectra. 42
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20 Reverse phase HPLC of crimini mushroom vitamin D and UV spectra. 43
21 Reverse phase HPLC of portabella mushroom vitamin D and UV spectra. 44
22 Reverse phase HPLC of vitamin D2 standard and UV spectrum ran on the same 45
day as the mushroom samples.
23 Reverse phase HPLC of vitamin D4 standard and UV spectrum ran on the same 46
day as the mushroom samples.
24 Reverse phase HPLC of vitamin D3 standard and UV spectrum ran on the same 47
day as the mushroom samples.
25 Reverse phase HPLC of white button mushroom provitamin D and UV spectra. 48
26 Reverse phase HPLC of shiitake mushroom provitamin D and UV spectra. 49
27 Reverse phase HPLC of oyster mushroom provitamin D and UV spectra. 50
28 Reverse phase HPLC of crimini mushroom provitamin D and UV spectra. 51
29 Reverse phase HPLC of portabella mushroom provitamin D and UV spectra. 52
30 Reverse phase HPLC of provitamin D2 standard and UV spectrum ran on the 53
Same day as the mushroom samples.
31 Reverse phase HPLC of provitamin D4 standard and UV spectrum ran on the 54
Same day as the mushroom samples.
32 Reverse phase HPLC of provitamin D3 standard and UV spectrum ran on the 55
Same day as the mushroom samples.
33 Straight phase HPLC of yeast extract and UV spectra. 57
34 Reverse phase HPLC of yeast vitamin D and UV spectra. 58
35 Reverse phase HPLC of vitamin D2 standard and UV spectrum ran on the same 59
day as the yeast sample.
36 Reverse phase HPLC of vitamin D4 standard and UV spectrum ran on the same 60
day as the yeast sample.
37 Reverse phase HPLC of vitamin D3 standard and UV spectrum ran on the same 61
Day as the yeast sample.
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38 Reverse phase HPLC of yeast provitamin D and UV spectra. 62
39 Reverse phase HPLC of provitamin D2 standard and UV spectrum ran on the 63
same day as the yeast sample.
40 Reverse phase HPLC of provitamin D4 standard and UV spectrum ran on the 64
same day as the yeast sample. 41 Reverse phase HPLC of provitamin D3 standard and UV spectrum ran on the 65 same day as the yeast sample.
x
ABBREVIATIONS 1,25(OH)2D 1,25-dihydroxyvitamin D
1,25(OH)2D2 1,25-dihydroxyvitamin D2
7-DHC 7-dehydrocholesterol
25(OH)D 25-hydroxyvitamin D
°C degrees Celsius ACN acetonitrile cm centimeters cZc 5, 6-cis-cis dH2O distilled H2O
FDA Food and Drug Administration FNB Food and Nutrition Board HPLC high performance liquid chromatography
IPA isopropyl alcohol
IU international units MA Massachusetts mAU miliAbsorbance Units mL milliliters mm millimeters N North N2 nitrogen
ng nanograms
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PTH parathyroid hormone RA rheumatoid arthritis RDAs recommended dietary allowances
RPM revolutions per minute
tZc 5, 6-trans-cis US United States UVB ultraviolet B VDR vitamin D receptor VDREs vitamin D response elements
1
INTRODUCTION Evolutionary Perspective
Evolutionarily, vitamin D is known to have been produced by phytoplankton that
have inhabited the oceans for more than 500 million years (Holick, 2004a). The
phytoplankton species Emiliania huxleyi, which has remained unchanged for over 750
million years, was found to contain the precursor to vitamin D2; the provitamin D
ergosterol. Upon exposure to ultraviolet B [UVB] solar radiation this provitamin D2 was
converted to previtamin D2, and subsequently to vitamin D2 through a thermo-
isomerization (Holick, 2003). These organisms used calcium from their ocean
environment to support vital life functions including signal transduction, metabolism and
the development of exoskeletons. As organisms in the oceans began to evolve into
vertebrates, calcium from the ocean was used to mineralize the pre-existing collagen
matrix to form the hard endoskeleton. Vertebrates that eventually evolved into land-
dwellers were challenged with the loss of the calcium rich ocean environment. These
vertebrates developed the ability to absorb calcium through their small intestine. It is
theorized that the production of vitamin D3 in the skin upon sun exposure helped these
vertebrates to enhance their ability to utilize calcium from plant food sources (Holick,
2004a). The relationship between this photosynthetic vitamin D3 product and regulation
of calcium metabolism may be the reason that vitamin D has remained so significant
throughout evolution, allowing ocean life forms to survive when they evolved and moved
onto land. This relationship between calcium and vitamin D is present in humans today
(Holick, 2004b).
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Photobiology of Vitamin D
For the majority of people, their main source of vitamin D comes from day-to-day
exposure to the sun. Upon exposure to UVB radiation (290-315 nm) from sunlight,
vitamin D3’s precursor 7-dehydrocholesterol [7-DHC] (provitamin D3) found in our
bodies’ skin cells is converted to previtamin D3 (Holick, 2004b). The previtamin D3 then
converts to vitamin D3 through a temperature-dependent isomerization. With additional
UVB exposure previtamin D3 is converted to the photobyproducts lumisterol and
tachysterol. Previtamin D3 and vitamin D3 are converted to other photobyproducts that
include suprasterols and toxisterols (Holick, 2004b) (Figure1). Due to this formation of
photobyproducts, over production of vitamin D3 in the skin does not occur. The
provitamin D3 molecule contains 4 ring structures, and when exposed to UVB radiation,
the double bonds in the B ring rearrange and this ring opens, producing previtamin D3
(Holick, 2004a). There are two conformers of previtamin D3 that exist. The more
thermodynamically unstable form, a 5, 6-cis, cis [cZc] conformer, can convert to vitamin
D. In a test tube, the cZc conformer will rapidly convert to the more stable 5, 6-trans, cis
[tZc] conformer. This more stable tZc conformer is not able to convert to vitamin D3.
The provitamin D3 is present in the triglycerides of the lipid bilayer of the dermal and
epidermal skin cells, stuck in between its polar head groups and non-polar fatty acid side
chains. As a result of this, in the skin, the previtamin D3 that is formed cannot convert to
the more stable tZc conformer, and therefore the cZc conformer rapidly converts to
vitamin D3 (Holick, Tian, & Allen, 1995). This membrane enhanced catalytic process
explains why the conversion of previtamin D3 to vitamin D3 is 10 times faster in the skin
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compared to its conversion in an organic solvent. It has been hypothesized that formation
of a less rigid provitamin D compound in the plasma membrane causes an increase in the
permeability of the membrane to certain ions, one of which being calcium (Holick,
2004a). Research validating this idea would help to explain how vitamin D is able to
affect calcium ion movement into and out of the cell. Once vitamin D3 is produced, it is
ejected from the plasma membrane into the extracellular space. Vitamin D3 is brought
into circulation by a vitamin D binding protein located on the dermal capillary surface
that remains bound to vitamin D3 until its delivery to the liver (Haddad, Matsuoka, Hollis,
Hu, & Wortsman, 1993).
Figure 1: Photosynthetic production of vitamin D3 and its photobyproducts from 7-
dehydrocholesterol (7-DHC) in the skin.
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There are many factors that can affect the amount of vitamin D3 made in the skin;
including skin pigmentation, time of day, latitude, season, and aging. The UVB photons
from the sun that are absorbed by provitamin D during the production of vitamin D in the
skin can also be absorbed by the earth’s ozone layer. The zenith angle, or the angle at
which sunlight hits the earth’s surface, determines how much vitamin D is produced in
our skin. This is why in the early morning, late afternoon, and winter we produce little, if
any, vitamin D in our skin. During these times, more of the UVB photons are absorbed
by the ozone layer because the zenith angle is so oblique that the sunlight must pass
through an increased amount of the ozone (Webb, Kline, & Holick, 1988). Webb et al.,
(1988) demonstrated the effects of season and latitude on the cutaneous production of
vitamin D3 by exposing 7-DHC to sunlight on cloudless days at various latitudes. They
found that in Boston, MA (42.2 degrees N), vitamin D3 was not produced from November
through February, and in Edmonton (52 degrees N) this time frame was from October
through March. This realization places importance on the need for dietary sources of
vitamin D when it cannot be produced in our skin, to avoid becoming deficient. Skin
type has an effect on the cutaneous production of vitamin D because the melanin pigment
in our skin can absorb UVB photons. Darker skin types have an increased amount of
melanin, which decreases how much vitamin D3 is produced in their skin compared to
individuals with lighter skin types. A study by Chen et al., (2007) demonstrated this by
exposing different skin types to the same amount of sunlight and then measured the
percent of 7-DHC converted to previtamin D3, resulting in the lighter skin types
producing a greater amount of vitamin D3. Due to the fear of developing skin cancer,
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many people have begun to take the advice that you should never be exposed to direct
sunlight by wearing protective clothing or applying a sunscreen. Sunscreens also absorb
UVB photons from the sun, therefore decreasing the conversion of 7-DHC to previtamin
D3 by as much as 99%. These are all factors weighing on the importance of discovering
new natural sources of vitamin D in foods.
Other sources of Vitamin D
The majority of people produce most of their vitamin D from the sun, and for an
extended period of time throughout the year in regions 35° N of the equator the sun’s rays
are unable to cause this cutaneous production of vitamin D. Therefore, it is especially
important for people to increase their ingestion of vitamin D during the winter months.
This presents a challenge because there are very few foods that naturally contain vitamin
D. Vitamin D3 can be found in oily fish, including sardines, salmon and mackerel, as
well as cod liver oil (Holick, 2007). Vitamin D is also naturally found in eggs and pork,
but only at a small fraction of the recommended daily dose. There are a number of foods
in the United States that are fortified with vitamin D including milk and other dairy
products, bread, cereal and orange juice (Holick, 2011). In the 1930s, vitamin D fortified
milk was produced by irradiating it after the addition of ergosterol. Ergosterol is the
provitamin D precursor of vitamin D2. During this time period, when a large percentage
of children living in industrialized cities in Boston, New York, and various countries in
northern Europe suffered from rickets caused by vitamin D deficiency, the fortification of
milk with vitamin D led to the eradication of this disease (Holick, 2011).
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Metabolism of Vitamin D
Vitamin D3 made in the skin, also known as cholecalciferol, is not biologically
active. Two hydroxylations must first occur in order for vitamin D to cause its
physiological effects. In the liver the enzyme 25-hydroxylase (CYP2R1) adds a hydroxyl
group to carbon 25 of vitamin D. The 25-hydroxyvitamin D [25(OH)D] then travels to
the kidneys where the enzyme 1-alpha-hydroxylase (CYP27B1) adds a hydroxyl group to
carbon 1 producing 1,25-dihydroxyvitamin D [1,25(OH)2D], the active form of vitamin
D3 (Figure 2). The catabolism of 1,25(OH)2D and its precursor 25(OH)D is carried out
by the mitochondrial enzyme complex 24-hydroxylase (CYP24A1) through an initial
hydroxylation on either carbon 23 or 24 (Sakaki et al., 2000).
Measuring the blood concentration of 25(OH)D can indicate the amount of
vitamin D that is ingested or produced in the skin because its production is not tightly
regulated in the liver. The circulating half-life of 25(OH)D is 2-3 weeks and is the major
circulating form of vitamin D, reflecting a person’s vitamin D reserves (Holick et al.,
2011). This is the appropriate clinical test to determine a person’s vitamin D status. On
the other hand, the production of the active form of vitamin D is closely regulated in the
kidneys, and its half-life in circulation is less than 4 hours, thus making it useless for
determining a person’s vitamin D status (Holick, 2007).
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Figure 2: Bioactivation of vitamin D3 (cholecalciferol) to its active form 1α,25(OH)2D3.
The Vitamin D Receptor Upon binding of 1,25(OH)2D to its intracellular vitamin D receptor [VDR], this
ligand-receptor complex dimerizes with a retinoid-X-receptor. This heterodimer-receptor
complex goes on to bind regions of the DNA known as vitamin D response elements
[VDREs], and changes the expression of certain genes, either activating or suppressing
transcription of these genes (Sutton & MacDonald, 2003). The VDREs are gene
sequences in or near the target gene’s promoter region (Sundar & Rahman, 2011).
Examples of the effects 1,25(OH)2D has on gene transcription include the positive effect
1,25(OH)2D has on the level of circulating fibroblast growth factor-23 (Hu et al., 2012),
as well as the regulation of human immune responses by controlling the expression of the
FOXP3 gene in CD4(+)T cells (Kang et al., 2012). Studies in mouse and human models
have identified VDREs near the klotho gene, a gene whose expression is regulated by
vitamin D and codes for a protein that helps regulate reabsorption of calcium and
phosphate in the kidney (Forster et al., 2011).
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In the intestine, 1,25(OH)2D causes an increase in calcium absorption, but the
mechanism behind this increase is still in question. Binding of 1,25(OH)2D3 to the
intestinal VDR has been shown to up-regulate the production of calbindin, a calcium
binding protein that assists in the transport of calcium across the intestinal epithelium. It
has also been suggested that 1,25(OH)2D3 stimulates the active transport of calcium
across the basolateral membrane of the intestine by stimulating synthesis of a plasma
membrane calcium pump (Christakos, 2012). A recent study suggests that 1,25(OH)2D3
up-regulates the expression of specific claudin proteins, which are thought to form tight
junctions between enterocytes, assisting in the paracellular transport of calcium ions
across the intestinal epithelium (Fujita et al., 2008). The question still remains to whether
or not there is a calcium channel present that is allowing for the increase in calcium
absorption due to increased levels of 1,25(OH)2D. The regulatory effect of 1,25(OH)2D
on gene transcription plays an important role in its function, causing diverse effects
throughout the body.
Vitamin D’s Importance to Bone Health
There is a substantial amount of research to support the idea that vitamin D is
essential for maintaining overall health. Vitamin D’s importance starts with its effect on
our ability to absorb intestinal calcium from our diet. If a person is deficient in vitamin
D, that person will have less efficient intestinal calcium absorption. Physiologically,
adequate levels of calcium are needed for proper cellular signaling, cardiovascular
function, and blood clotting. Without the ability to absorb calcium from our diet, the
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body would take from its calcium stores within bone, leading to a weak skeleton prone to
fracture. In osteoblasts, binding of 1,25(OH)2D to its VDR causes the expression of a
nuclear ligand whose actions lead to the maturation of pre-osteoclasts in the bone. These
newly formed osteoclasts then work to mobilize calcium from bone to sustain the normal
concentration of blood calcium (Holick, 2007). When concentrations of ionized calcium
in the serum drop below normal, in association with vitamin D deficiency, the
parathyroid glands sense this and as a result increase the secretion of parathyroid
hormone [PTH]. This increase in PTH causes an increase in there absorption of calcium
in the kidney (Holick, 2004a). Elevated PTH levels in a vitamin D deficient person will
also lead to a decrease in both reabsorption of phosphorus in the kidney and absorption of
phosphorus in the intestine. The resulting low or low-normal blood phosphorus
concentration in combination with the low-normal blood calcium concentration results in
an inadequate calcium-phosphate product, leading to a defect in the mineralization of the
skeleton. This inadequate mineralization process is the underlying problem in children
diagnosed with rickets and adults suffering from osteomalacia (Holick, 2007). This
secondary hyperparathyroidism also leads to increased levels of 1,25(OH)2D. Therefore,
measuring the active form of vitamin D is of no value for determining a person’s vitamin
D status.
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Vitamin D’s Importance to Overall Health
The VDR is found in a multitude of cells and organs in the body including
activated T and B lymphocytes, osteoblasts, pancreatic islet cells, mononuclear cells, the
colon, small intestine, brain, breast, prostate, gonads, skin, and the heart (Holick, 2004a).
This widespread presence of the VDR throughout the body poses the question of the
effects of vitamin D in each of these areas. In the pancreas, previous studies have found
that 1,25(OH)2D3 caused a stimulation of insulin secretion. A study that measured the
effect of 1,25(OH)2D3 in a rat model showed that it decreased beta cell growth (S. Lee,
Clark, Gill, & Christakos, 1994). These two effects are contradictory, warranting further
investigation into testing the role of vitamin D in the pancreas. In addition to vitamin D’s
effect on calcium absorption in the small intestine, it may also help maintain our
digestive system’s health. In a study testing the effects of 1,25(OH)2D3 on disease status
of mice with symptoms of inflammatory bowel disease, Cantorna, Munsick, Bemiss, &
Mahon, (2000) observed that supplementation with vitamin D alleviated the symptoms
and stopped the advancement of the disease, compared to mice who were vitamin D
deficient. Inflammatory bowel diseases arise due to an autoimmune reaction, but the
exact underlying cause is still unknown. Vitamin D could play an important role in the
prevention of these diseases. Previous research has also demonstrated that vitamin D has
the potential to effectively decrease the likelihood of developing various cancers. A
study using human myeloid leukemia cells demonstrated that 1,25(OH)2D had a dose-
dependent effect on inhibiting the proliferation and inducing the maturation of these cells
(Miyaura et al., 1981). 1,25(OH)2D3 has an anti-proliferative effect on various malignant
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cancer cells. The actions underlying these anticancer effects involve 1,25(OH)2D3’s
ability to stimulate apoptosis, arrest the cell cycle, and inhibit angiogenesis, invasion and
metastasis. A study recently shed light on the mechanism behind this effect on estrogen-
receptor positive breast cancer cells, showing that 1,25(OH)2D3 affects the expression of
multiple proteins and enzymes leading to an end result of decreased estrogen synthesis
and decreased inflammation. It is theorized that because breast cancer cells have local
25-hydroxyvitamin D-1α-hydroxylase (CYP27B1) activity, increased dietary vitamin D
may have the potential to prevent breast cancer by controlling differentiation and
proliferation of the cancer cells (Krishnan, Swami, & Feldman, 2012). This local 25-
hydroxyvitamin D-1α-hydroxylase activity has also been found in prostate and colon
cancer cells (Friedrich et al., 2006). Additionally, Sha et al., (2013) demonstrated that
apoptosis of prostate cancer cells could be induced by 1,25(OH) 2D3.
One limitation that may be placed on this anti-carcinogenic property is the
inevitable effect 1,25(OH)2D3 has on circulating calcium concentrations, resulting in
hypercalcemia. One method used to solve this problem is the use of analogs of the active
form of vitamin D. A recent study by Park et al., (2012) showed that 19-nor-1,25-
dihydroxyvitamin D2 (paricalcitol), an analog of 1,25(OH)2D3 known to have fewer
calcemic effects, had antitumor and anti-inflammatory effects on gastric cancer cells.
This study also demonstrated that in mice treated with paricalcitol, intraperitoneal
metastases growth and tumor volume was reduced.
A clinical application that is currently able to utilize the strong anti-proliferative
activity of 1,25(OH) 2D3 is in the treatment of psoriasis (Smith, Pincus, Donovan, &
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Holick, 1988). People with psoriasis suffer from red, itchy and often times dry patches of
skin, brought on by an increase in the proliferation of skin cells known as keratinocytes,
leading to an increase in the skin’s thickness (A.D.A.M., 2011). Smith, Walworth, &
Holick, (1986) demonstrated that through binding to its VDR in keratinocytes,
1,25(OH)2D3 was able to significantly inhibit growth, and also induce differentiation of
these skin cells. A clinical study comparing the treatment of psoriasis with 1,25(OH)2D3
to an already established topical method showed that not only did the treatment with
1,25(OH)2D3 prove to be as effective, but it was also preferred over the alternative by
patients who participated in the study (Kowalzick, 2001).
The importance of vitamin D to our overall health can also be attributed to its
potential to treat and/or prevent a number of autoimmune diseases including type 1
diabetes mellitus, rheumatoid arthritis, multiple sclerosis (Ponsonby, McMichael, & Van
der Mei, 2002), and Crohn’s disease (Koutkia, Lu, Chen, & Holick, 2001). The
modulatory effects of vitamin D on the immune system are in part explained by the fact
that activated T (Bhalla, Amento, Clemens, Holick, & Krane, 1983) and B (Provvedini,
Tsoukas, Deftos, & Manolagas, 1986) lymphocytes have VDRs. In a cohort study,
Hyppönen, Läärä, Reunanen, Järvelin, & Virtanen, (2001) studied the correlation
between vitamin D supplementation in children during the first year of life and the
subsequent prevalence of type-1 diabetes during the first 31years of life. In this study,
those children who received a consistent recommended dose of vitamin D (2000
international units [IU] per day) had an 88% reduction in the risk of being diagnosed with
type-1 diabetes later in life. Kostoglou-Athanassiou, Athanassiou, Lyraki, Raftakis, &
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Antoniadis, (2012) measured the 25(OH)D levels of people suffering from rheumatoid
arthritis [RA], and compared this to the levels of 25(OH)D in people without the disease.
This study revealed that those patients with RA had low levels of 25(OH)D compared to
the disease free group.
There is a plethora of research describing the role of vitamin D in cardiovascular
health. It has been reported that vitamin D has the ability to suppress the renin-
angiotensin system through VDR activation, suggesting a possible mechanism for its
ability to treat hypertension associated with cardiovascular disease (Li et al., 2002). A
study testing this theory exposed individuals with mild hypertension to UVB radiation
and then measured their 25(OH)D and blood pressure levels, showing a significant rise in
25(OH)D and a drop in both diastolic and systolic blood pressure (Krause, Bühring,
Hopfenmüller, Holick, & Sharma, 1998). In addition, Carrara et al., (2013) demonstrated
that patients who were both vitamin D deficient and hypertensive initially, and were
treated with vitamin D3 supplementation, saw a decrease in their renin and aldosterone
levels. With decreased aldosterone levels, the levels of sodium and water reabsorbed by
the kidneys would decrease, presumably leading to a decrease in blood pressure in
hypertensive individuals. All the research mentioned has provided the evidence that
vitamin D may have various significant benefits to our health. It has become obvious that
proper bone health is just one of many vital biological functions vitamin D can help to
maintain.
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Vitamin D as a Nutritional Deficiency
Vitamin D deficiency is a pandemic that is now one of the most common
nutritional deficiencies worldwide (Holick & Chen, 2008). In addition to the immense
amount of research suggesting the multiple benefits of maintaining adequate levels of
vitamin D, there is also research showing how vitamin D deficiency can cause a number
of health risks. If a person has a 25(OH)D level of <20 ng/ml they are considered to be
vitamin D deficient, and with a level of 21-29 ng/ml they are considered to be insufficient
(Malabanan, Veronikis, & Holick, 1998). According to the Institute of Medicine, the
current recommended dietary allowances [RDAs] of vitamin D set by the Food and
Nutrition Board [FNB] are; 400 IU for children younger than a year, 600 IU for children
and adults under the age of 70, and 800 IU for adults over the age of 70 (Ross et al.,
2011). The toxicity of vitamin D has been studied and research has shown that it is very
rare for a person to become unintentionally intoxicated through vitamin D
supplementation (Holick, 2011). It has been proposed that the current daily RDAs are
inadequate and should be raised in order for vitamin D to have a maximum benefit to our
overall health (Standing Committee on the Scientific Evaluation of Dietary Reference
Intakes, Food and Nutrition Board, Institute of Medicine, 1997). In a study where infants
under the age of one year were regularly supplemented 2000 IU per day, these infants did
not experience vitamin D toxicity. Additionally, toxicity did not occur in children ages 1-
12 ingesting 5000 UI a day, or in adults and teenagers ingesting 10,000 IUs per day
(Holick, 2011).
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In the beginning of the 1900s, vitamin D deficient rickets was a widespread
problem among children living in cities such as Boston and New York because their
exposure to sunlight became sparse as a result of the industrialized build-up in these
cities. This disease was also prevalent in children throughout inner cities in Europe, and
was described as a disease that retarded growth of bones while causing skeletal
deformities. Symptoms of this disease known as rickets include skeletal deformities;
such as bony projections on the rib cage, short stature due to bowed knees or knocked
knees, asymmetrical or odd shaped skull, dental deformities, and weak muscles lacking
tone (Holick, 2006). Children deficient in vitamin D can also suffer from stunted growth
and weak bones that easily break (―Kids and Vitamin D Deficiency, n.d.). Studies have
shown that when children and adults are deficient in vitamin D their ability to absorb
calcium from their diet is reduced by 85-90% (Holick, 2011). In adults vitamin D
deficiency can often times lead to osteomalacia, a disease whose symptoms include
muscle weakness and chronic widespread bone pain (Holick, 2011). Plotnikoff &
Quigley, (2003) measured 25(OH)D levels in 150 patients with nonspecific
musculoskeletal pain and found 93% of them to be deficient in vitamin D. These results,
along with the fact that skeletal muscles have VDRs, suggest that the musculoskeletal
weakness associated with osteomalacia is due to the low levels of vitamin D in these
patients.
Many studies have reported on the number of people suffering from vitamin D
deficiency. A recent study by Reis, Von Mühlen, Miller, Michos, & Appel, (2009)
measuring the 25(OH)D levels of adolescents ages 12-19 found that on average these
16
children were vitamin D insufficient (24.8 ng/mL). This study also found that the low
25(OH)D levels in these adolescences correlated with high blood pressures (Reis et al.,
2009). Infants who are only breast fed and are not given vitamin D supplementation are
at a higher risk of developing vitamin D deficiency (Merewood et al., 2012). A study by
S. H. Lee et al., (2012) found a negative correlation between 25(OH)D levels and the
prevalence of obesity and metabolic syndrome in Korean children, demonstrating the
notion that vitamin D deficiency is a risk factor for these diseases. Taha, Dost, &
Sedrani, (1984) measured 25(OH)D levels in women and their babies in Saudi Arabia and
found that due to their lack of sun exposure from wearing clothes that cover most of the
body, a prevalence of vitamin D deficiency was seen in these women during pregnancy.
This study showed that vitamin D deficiency can have adverse effects on infants’ calcium
levels, with 59% of newborns having low plasma calcium concentrations. Individuals
living in New Zealand have been found to have insufficient levels of vitamin D during
the winter months; with an associated increased PTH level, which is suggested to have
negative effects on bone health (Rockell, Skeaff, Venn, Williams, & Green, 2008).
People of every age should make it a priority to ensure their 25(OH)D levels are
maintained. Elderly people who spend less time outdoors are particularly at risk for
vitamin D deficiency. This risk is even greater in black elderly people due to their skin’s
increased production of melanin; one study found over 80% of this population suffering
from vitamin D deficiency in Boston, MA at the end of the summer (Holick, 2004b).
Additionally, results from this study also found 32% of adults up to the age of 29 were
vitamin D deficient.
17
Associated Health Risks of Vitamin D Deficiency
An interesting observation was made for the first time in 1941 by Apperly, (1941)
who reported on the incidence of cancer mortality among people in the United States and
Canada. He reported that at locations closer to the equator that received more solar
radiation, an overall decline in cancer mortality was seen. An explanation to this could
be the fact that at locations closer to the equator, more UVB photons from the sun reach
the earth, and can therefore cause an increased production of vitamin D in our skin. It is
believed that the major risk factor for developing melanoma is exposure to ultraviolet
[UV] radiation. Therefore, many people avoid sun exposure. It was interestingly
observed by Berwick et al., (2005) in a study on patients who were diagnosed with
melanoma that those who had a history of more severe sun exposure were found to have
an increased survival rate. Vitamin D is thought to take part in this positive correlation
between amount of sun exposure and survival rate (Kricker & Armstrong, 2006). In a
case control study by Kennedy, Bajdik, Willemze, DeGruijl, & Bouwes Bavinck, (2003)
a negative correlation was found between lifetime sun exposure and the risk of
developing malignant melanoma. Similarly, an inverse relationship has been observed
between exposure to UV radiation and mortality rates from prostate cancer (Schwartz &
Hanchette, 2006) and to an even greater extend in African Americans (Schwartz, 2005).
Research validating this observation has found receptors for vitamin D in human prostate
cells that cause differentiation, and oppose proliferation and metastasis in these cells
when bound by 1,25(OH)D (Schwartz, 2005). A study published in 2003 that compared
a group of men diagnosed with prostatic adenocarcinoma and another group of men with
18
benign prostatic hypertrophy determined that men whose average lifetime exposure to
UV radiation was no greater than approximately 2 hours per day had a three-fold greater
risk of developing prostate cancer (Bodiwala et al., 2003). Garland et al., (1989)
measured circulating levels of 25(OH)D and found a three-fold decrease in the risk of
developing colon cancer in people with 25(OH)D levels of 20 ng/mL or more. Further
research has demonstrated that colon cancer cells that express VDRs exhibited reduced
cellular growth and increased differentiation when dosed with 1,25(OH) 2D3 (Holick,
2008). A recent study by Lin et al., (2012) measured the solar UV radiation of specific
geographical areas by the use of ozone mapping; and in conjunction with the National
Institutes of Health Diet and Health Study data, found total UV radiation exposure to
have a negative correlation with the risk of developing various cancers including non-
Hodgkin’s Lymphoma, prostate, lung, bladder and kidney. The risk of developing a
number of autoimmune diseases is linked to vitamin D deficiency and decreased
exposure to sunlight. Munger et al., (2004) analyzed data from the Nurses’ Health
Studies and found that in women, an increased risk of developing multiple sclerosis was
associated with decreased vitamin D supplementation. Multiple studies have shown that
across the world, latitude plays a critical role in disease frequency of multiple sclerosis,
with a decrease in prevalence in locations as you move toward the equator (Sellner et al.,
2011). In addition, the association between vitamin D status and the risk of developing
RA has been previously discussed. People with this chronic inflammatory autoimmune
disease often also have osteoporosis and are susceptible to bone fracture, a result of
having low 25(OH)D levels. It has been hypothesized that gene polymorphisms in the
19
VDR of women with osteoporosis leads to a decrease in bone mass density, and is a
probable cause of osteoporosis in women with RA (Hussien et al., 2012). Studies have
been successful in treating mouse models predisposed to autoimmune diseases including
multiple sclerosis, RA and type-1 diabetes with 1,25(OH) 2D3 at the beginning of life,
leading to the prevention of these diseases (Holick, 2004b). Research linking disease and
vitamin D deficiency is extensive and puts great importance on maintaining our levels of
vitamin D through natural food sources and supplementation when exposure to sunlight is
not possible.
Vitamin D in Fungi
There are currently six known forms of vitamin D, all of which are formed when
their corresponding precursors are exposed to UVB radiation (Figure 3). In all forms, the
B-ring of the prosecosteroid hormone, upon UVB irradiation, is photolyzed to form
previtamin D, and a subsequent isomerization produces vitamin D.
20
Figure 3: Six known forms of vitamin D and their corresponding precursors. Provitamin D2 (ergosterol) and vitamin D2 (ergocalciferol) (purple), provitamin D3 (7-
dehydrocholesterol) and vitamin D3 (cholecalciferol) (green), provitamin D4 (22,23-
dihydroergosterol) and vitamin D4 (22-dihydroergocalciferol) (blue), provitamin D5 (7-
dehydrositosterol) and vitamin D5 (sitocalciferol) (red), provitamin D6 (7-
dehydrostigmasterol) and vitamin D6 (pink), provitamin D7 (7-dehydrocampesterol) and
vitamin D7 (orange).
Various types of edible mushrooms have the potential to produce vitamin D2
similar to the way in which we produce vitamin D3 in our skin. Upon UVB irradiation,
mushrooms convert the provitamin D2 (ergosterol) to previtamin D2 (Kalaras, Beelman,
Holick, & Elias, 2012) (Keegan, R. J. H., Lu, Z., Bogusz, J. M., Williams J. E., & Holick,
M. F., 2012). Through a temperature-dependent isomerization previtamin D2 is
converted to vitamin D2 (ergocalciferol). The only structural differences between vitamin
D2 and vitamin D3 is vitamin D2 has a double bond between carbons 22 and 23, and a
methyl group on carbon 24 (Figure 3). Upon UVB exposure, provitamin D2 also coverts
to a number of photobyproducts, including lumisterol2 and tachyterol2 (Keegan et al.,
21
2012) (Figure 4). The production of these photobyproducts as a function of amount of
UVB irradiation has been quantitatively studied in white button mushrooms (Agaricus
bisporus) (Kalaras et al., 2012).
Figure 4: Photosynthetic production of vitamin D2 and its photobyproducts from
ergosterol (provitamin D2) in mushrooms.
UVB irradiated mushrooms are an important natural dietary source of vitamin D
for vegans and other individuals who do not consume foods fortified with vitamin D, or
take vitamin D supplementation. This importance increases during the winter months
when sun exposure is not adequate enough to produce vitamin D3 in the skin (Holick,
2004a). The vitamin D2 content of various edible mushrooms types was recently
analyzed using high performance liquid chromatography [HPLC] and mass spectroscopy.
Mushrooms found to contain vitamin D2 included white button, crimini, portabella, enoki,
shiitake, oyster, morel, chanterelle and maitake (Phillips et al., 2011). Studies measuring
22
the bioavailability of vitamin D2 contained in edible mushrooms have shown that vitamin
D2 from mushrooms is as effective as vitamin D2 supplementation at raising and
maintaining levels of 25(OH)D in the blood (Outila, Mattila, Piironen, & Lamberg-
Allardt, 1999) (Urbain, Singler, Ihorst, Biesalski, & Bertz, 2011) (Keegan et al., 2012).
The bioavailability of vitamin D2 from irradiated mushrooms has been demonstrated in a
rat model to increase serum 25(OH)D levels, and have a positive effect on bone mineral
density and serum calcium concentrations (Jasinghe, Perera, & Barlow, 2005). Vitamin
D2 is metabolized in the liver and kidneys the same way vitamin D3 is, to produce the
active form 1,25-dihydroxyvitamin D2 [1,25(OH)2D2] (Outila et al., 1999) (Figure 5).
Figure 5: Bioactivation of vitamin D2 (ergocalciferol) to its active form 1α,25(OH)2D2.
A number of mushroom suppliers in the US commercially produce vitamin D
enhanced mushrooms by UVB-treatment. A number of factors can affect the amount of
vitamin D produced in mushrooms as a result of UVB-treatment including intensity, time
of exposure, level of moisture and wavelength of radiation. This process has been
standardized to produce mushrooms that contain a nutritiously significant amount of
vitamin D to benefit individuals that consume mushrooms to maintain their 25(OH)D
levels (3.83 μg/g) (Roberts, Teichert, & McHugh, 2008).
23
Similar to how vitamin D2 is made in mushrooms, it is also produced in yeast.
Yeast also contains the provitamin D2 ergosterol, that when exposed to UVB irradiation
converts to previtamin D2 (Keegan et al., 2012). The process of isolating ergosterol from
yeast to produce vitamin D2 has played a major role in the treatment of vitamin D
deficiency throughout the world; through its use as an over-the-counter supplement and
the fortification of foods including milk, orange juice and bread (Holick, 2007). This
posed the question of whether or not the vitamin D2 added to orange juice and bread is
bioavailable and effective in raising a person’s vitamin D status. Biancuzzo et al., (2010)
compared the effectiveness of orange juice fortified with vitamin D2 or vitamin D3 to
vitamin D2 and vitamin D3 supplementation in raising 25(OH)D levels. This study
demonstrated that vitamin D2 and vitamin D3 have the same bioavailability in orange juice
and this bioavailability is comparable to vitamin D2 and vitamin D3 in capsule form.
Holick et al., (2008) also studied the effectiveness of vitamin D2 versus vitamin D3 from
supplementation on maintaining total 25(OH)D levels and found that the two forms had
an equal effect. Subjects who were supplemented with vitamin D2 or vitamin D3 for 11
weeks also sustained their total 1,25(OH)2D levels, in addition to a raising their total
25(OH)D levels (Biancuzzo, Clarke, Reitz, Travison, & Holick, 2013). Bread is fortified
with yeast rich in vitamin D2 or with vitamin D3 made synthetically from cholesterol
obtained from lanolin. A study using a rat model demonstrated that vitamin D2 in bread
fortified with yeast increased bone density (Hohman et al., 2011). In vitamin D deficient
rats fed bread rich in vitamin D2 from yeast, 25(OH)D levels increased in a dose
dependent fashion. A study in humans measured the effectiveness of bread fortified with
24
vitamin D3 at raising 25(OH)D levels in women and found it to be comparable to the
effect of vitamin D3 supplementation (Natri et al., 2006). Surveys have reported the
average consumption of vitamin D rich bread in the US is 3 slices (79 grams) per day
(Jasinghe et al., 2005). The current FDA allowance of vitamin D in bread is 400 IU per
100 grams. These findings support the indication that bread could be an important source
of vitamin D for individuals who do not take vitamin D supplementation.
Qualitative and Quantitative Identification of Vitamin D Compounds
HPLC is a common technique used to identify the presence of vitamin D in food
samples, including yeast and mushrooms (Keegan et al., 2012). Normal (straight) phase
HPLC uses a polar silica column and a polar mobile phase to separate vitamin D from its
precursors, previtamin D and provitamin D; as well as from the photobyproducts
lumisterol and tachysterol. Reverse phase HPLC uses a nonpolar stationary phase and a
polar mobile phase to separate different provitamin Ds and vitamin Ds.
UV absorbance spectral analysis with photodiode detection is also used to identify
these compounds in conjunction with their retention time on HPLC. Provitamin D
compounds have a UV absorbance spectrum characteristic for the 5,7-diene within their
molecular structure (Figure 6). Vitamin D compounds have a characteristic UV
absorbance spectrum at λmax 265 nm characteristic for the 5,6-cis-triene within their
molecular structure (Figure 7). Previtamin D compounds have a UV absorbance
spectrum that is similar to vitamin D, but with a λmax of 260 nm. The photobyproducts
lumisterol and tachysterol each also have characteristic UV spectra (Figure 8).
25
Figure 6: Characteristic UV absorbance spectrum of provitamin D.
Figure 7: Characteristic UV absorbance spectrum of vitamin D. λmax at 265 nm.
26
Figure 8: Characteristic UV absorbance spectra of previtamin D photobyproducts. (A)
lumisterol (B) tachysterol.
Wavelength (nm)
Abso
rban
ce (
mA
U)
Wavelength (nm)
A
Abso
rban
ce (
mA
u)
27
Specific Aims
Although it has been demonstrated that mushrooms and yeast have the ability to
produce vitamin D2 upon UVB irradiation, there is limited research investigating the
presence of other forms of vitamin D in these fungi. The purpose of the study is to: 1)
extract and isolate from various edible mushroom species as well as the yeast
Saccharomyces cerevisiae (baker’s yeast) vitamin Ds and provitamin Ds, 2) identify them
with reverse phase HPLC, and 3) determine if mushrooms and yeast can produce vitamin
D2, vitamin D3, and vitamin D4 (22-dihydroergocalciferol) after exposure to UV radiation.
28
MATERIALS AND METHODS
Samples Five mushroom species were analyzed for the presence of various provitamin D
and vitamin D compounds, including; skiitake (Lentinula edodes), oyster (Pleurotus
ostreatus), portabella (Agaricus bisporus), crimini (Agaricus bisporus), and white button
(Agaricus bisporus). Mushroom samples were obtained from a supermarket in Boston,
MA. The yeast sample (Eagle Baker’s Yeast) was obtained from Lallemand Inc.
(Montreal, Quebec).
Irradiation For each mushroom species, one mushroom sample was used for analyzing the
presence of provitamin Ds and vitamin Ds. To analyze the presence of various vitamin
Ds in the mushroom species, each mushroom sample was first irradiated for 5 minutes at
a distance of 4 inches under a Xenon RC-500 pulsed UV lamp (Xenon Corporation,
Woburn, MA) (Figure 9). This sample was then placed in an incubation chamber for 24
hours at 37°C in order to enhance the temperature-dependent conversion of previtamin D
to vitamin D.
29
Figure 9: UV irradiation of a portabella mushroom. Xenon RC-500 pulsed UV lamp
(Xenon Corporation, Woburn, MA).
Extraction
A total of 6 biopsies were taken from the cap of each mushroom sample, each
biopsy had an area of 0.5 cm2 and a depth of 1-2 mm (Figure 10). The fresh mushroom
sample was first homogenized with a glass hand homogenizer in 1 mL of methanol, and
after centrifugation for 5 minutes (3200 RPM), the methanol layer was extracted. The
methanol layer was dried with a stream of nitrogen (N2) gas. The dried mushroom extract
was ready for HPLC analysis after re-dissolving it in 0.3% isopropyl alcohol [IPA] in
hexane.
30
Figure 10: Shiitake mushroom biopsied 6 times.
For the analysis of provitamin Ds and vitamin Ds in the yeast sample, 1 gram of
yeast was sonicated with a Tekmar sonic disruptor in 5 mL of methanol for 1 minute, and
then put on a shaker for 5 minutes. The sample was centrifuged for 5 minutes (3200
RPM) and the methanol layer was extracted. This process was repeated 2 additional
times for the initial 1 gram of yeast. This extraction process was then repeated with a
separate additional 1 gram of yeast. The two methanol samples were combined, and
dried with a stream of N2 gas until the total volume equaled 25 mL. From this final
sample, 10 mL was dried with a stream of N2 gas. This dried yeast extract was ready for
HPLC analysis after re-dissolving it in 0.3% IPA in hexane.
In order to obtain standard compounds for provitamin D4 and vitamin D4, these
compounds were extracted from a mushroom powder sample (Monterey Mushrooms)
that was previously analyzed with HPLC and confirmed to contain these compounds
(Figure 11). To extract these compounds, 1 capsule (approximately 0.33 grams) was
combined with 5 mL of methanol and put on a shaker for 5 min. This sample was then
centrifuged for 5 minutes (3200 RPM), and the methanol layer was extracted. The
31
methanol was dried with a stream of N2 gas. This dried mushroom powder sample was
ready for HPLC analysis after re-dissolving it in 0.3% IPA in hexane.
Figure 11: Mushroom powder capsule.
Chromatography The provitamin D4 and vitamin D4 compounds were isolated from the mushroom
powder sample by dissolving the dried extracts in 0.3% IPA in hexane and
chromatographing them on a Zorbax CN column. This straight phase HPLC system was
used to separate and collect the vitamin D and provitamin D compounds from the
mushroom powder sample. The collected vitamin D and provitamin D samples were
dried with a stream of N2 gas. The provitamin D sample was dissolved in methanol,
ethanol, and distilled water [dH2O] (32:8:1 ratio) and chromatographed on a reverse
phase HPLC system using a Zorbax ODS column. This reverse phase HPLC system was
used to separate provitamin D2 from provitamin D4. The isolated provitamin D4 was
32
collected and used as a standard for the analysis of provitamin D in the mushroom and
yeast samples. The dried extracts that contained vitamin D were dissolved in 20%
methanol in acetonitrile [ACN] and chromatographed on a Vydac C18 column. This
reverse phase HPLC system was used to separate vitamin D2 from vitamin D4. The
isolated vitamin D4 was collected and used as a standard for the analysis of vitamin D in
the mushroom and yeast samples.
For the analysis of provitamin D and vitamin D content in the mushroom and
yeast samples, the dried extracts were dissolved in 0.3% IPA in hexane in preparation for
straight phase HPLC analysis. These samples were chromatographed on a Zorbax CN
column in order to separate and collect the provitamin D and vitamin D compounds. The
collected provitamin D and vitamin D samples were dried with N2 gas. The provitamin D
samples were dissolved in methanol, ethanol, and dH2O (32:8:1 ratio) and
chromatographed on a reverse phase HPLC system using a Zorbax ODS column, along
with standards for provitamin D2, provitamin D3 and provitamin D4. The vitamin D
samples were dissolved in 20% methanol in ACN. These samples were chromatographed
on a reverse phase HPLC system using a Vydac C18 column, along with standards for
vitamin D2, vitamin D3 and vitamin D4.
33
RESULTS
Mushrooms Using high performance liquid chromatography with UV photodiode absorbance
detection [HPLC-UV] provitamin D4, provitamin D2, and vitamin D2 were found in every
mushroom species that was exposed to UV radiation, including; white button, oyster,
skiitake, portabella and crimini. Vitamin D4 was identified in three of the five mushroom
species, including; shiitake, white button and oyster. All standard provitamin D and
vitamin D compounds chromatographed for comparison were done so under identical
HPLC conditions. Every compound identified as provitamin D had a UV absorbance
spectrum that matched the UV absorbance spectrum of a 5,7-diene (Figure 6). Every
compound identified as vitamin D had a UV absorbance spectrum that matched the UV
absorbance spectrum of a 5,6-cis-triene (Figure 7). The straight phase HPLC
chromatograms for each mushroom species are seen in Figures 12-16.
For all the mushroom species, the provitamin D and vitamin D collected during
straight phase analysis were chromatographed on reverse phase HPLC. Reverse phase
HPLC analysis revealed 2 peaks, identified as vitamin D2 and vitamin D4 for white button,
skiitake, and oyster mushroom species (Figures 17-19). For the crimini and portabella
mushroom samples one vitamin D peak was observed and identified as vitamin D2
(Figures 20, 21). Standards of vitamin D2 and vitamin D4 eluted at the same retention
time as the identified vitamin D2 and vitamin D4 peaks, 9.7 minutes and 11.3 minutes,
respectively (Figure 22, 23). A standard of vitamin D3 eluted with a retention time of
34
10.8 minutes (Figure 24). No detectable vitamin D3 was observed in any of the
mushroom samples.
Reverse phase HPLC analysis revealed 2 peaks that were identified as provitamin
D2 and provitamin D4 in every mushroom species (Figures 25-29). Standards of
provitamin D2 and provitamin D4 eluted at the same retention time as the identified
vitamin D2 and vitamin D4 peaks, 7.1 minutes and 8.3 minutes, respectively (Figure 30,
31). The shiitake mushroom sample revealed a peak identified as provitamin D3 (7.9
minutes) (Figure 26). A provitamin D3 standard eluted at the same retention time as this
identified provitamin D3 peak (Figure 32). No detectable provitamin D3 was found in any
of the other mushroom samples.
35
Figure 12: Straight phase HPLC of white button mushroom extract and UV spectra. (A)
Chromatogram of white button mushroom extract on a Zorbax CN column using a 0.3%
IPA in hexane mobile phase. Y-axis is miliAbsorbance units (mAU) at 280 nm. (B) UV
spectrum of vitamin D peak (11.96 min). Y-axis is mAU at 265 nm. (C) UV spectrum of
provitamin D peak (15.43 min).
Wavelength (nm)
Ab
sorb
ance
(m
AU
)
C
Wavelength
(nm)
Abso
rban
ce (
mA
U)
B
36
Figure 13: Straight phase HPLC of shiitake mushroom extract and UV spectra. (A) Chromatogram of shiitake mushroom extract on a Zorbax CN column using a 0.3% IPA
in hexane mobile phase. Y-axis is mAU at 265 nm. (B) UV spectrum of vitamin D peak
(10.48 min). Y-axis is mAU at 265 nm. (C) UV spectrum of provitamin D peak (13.11
min).
Wavelength (nm)
Ab
sorb
ance
(mA
U)
C
Wavelength (nm)
Abso
rban
ce (
mA
U)
B
37
Figure 14: Straight phase HPLC of oyster mushroom extract and UV spectra. (A)
Chromatogram of oyster mushroom extract on a Zorbax CN column using a 0.3% IPA in
hexane mobile phase. Y-axis is mAU at 265 nm. (B) UV spectrum of vitamin D peak
(12.50 min). Y-axis is mAU at 265 nm. (C) UV spectrum of provitamin D peak (16.12
min).
C
Wavelength (nm)
Abso
rban
ce (
mA
U)
B
Ab
sorb
ance
(m
AU
)
16
.12
9
Wavelength (nm)
Provitamin D
12.5
06
PVitamin D
A
Time (minutes)
Abso
rban
ce (
mA
u)
38
Figure 15: Straight phase HPLC of crimini mushroom extract and UV spectra. (A)
Chromatogram of crimini mushroom extract on a Zorbax CN column using a 0.3% IPA
in hexane mobile phase. Y-axis is mAU at 265 nm. (B) UV spectrum of vitamin D peak
(11.80 min). Y-axis is mAU at 265 nm. (C) UV spectrum of provitamin D peak (15.08
min).
C
Wavelength (nm)
Abso
rban
ce (
mA
U)
Time (minutes)
Provitamin D
Ab
sorb
ance
(m
AU
)
A
1
5.0
82
11
.80
1
Vitamin D
B
Wavelength (nm)
Ab
sorb
ance
(m
Au
)
39
Figure 16: Straight phase HPLC of portabella mushroom extract and UV spectra. (A)
Chromatogram of portabella mushroom extract on a Zorbax CN column using a 0.3%
IPA in hexane mobile phase. Y-axis is mAU at 265 nm. (B) UV spectrum of vitamin D
peak (12.59 min). Y-axis is mAU at 265 nm. (C) UV spectrum of provitamin D peak
(16.26 min).
Wavelength (nm)
Ab
sorb
ance
(m
AU
)
C
Wavelength (nm)
Abso
rban
ce (
mA
U)
Provitamin D Vitamin D
Time (minutes)
Ab
sorb
ance
(m
AU
)
A
12.5
90
16.2
63
B
40
Figure 17: Reverse phase HPLC of white button mushroom vitamin D and UV spectra.
(A) Chromatogram of collected white button mushroom vitamin D on a Vydac C18
column using a 20% methanol in ACN mobile phase. Y-axis is mAU at 265 nm. (B) UV
spectrum of vitamin D2 peak (9.57 min). (C) UV spectrum of vitamin D4 peak (11.39
min).
Ab
sorb
ance
(m
AU
)
Wavelength (nm)
C
Wavelength (nm)
Abso
rban
ce (
mA
U)
B
Time (minutes)
Ab
sorb
ance
(m
AU
)
A
Vitamin D2
9.5
77
Vitamin D4
11.3
99
41
Figure 18: Reverse phase HPLC of shiitake mushroom vitamin D and UV spectra. (A)
Chromatogram of collected shiitake mushroom vitamin D on a Vydac C18 column using
a 20% methanol in ACN mobile phase. Y-axis is mAU at 265 nm. (B) UV spectrum of
vitamin D2 peak (9.51 min). (C) UV spectrum of vitamin D4 peak (11.18 min).
Abso
rban
ce (
mA
U)
11.1
81
Vitamin D4
Vitamin D2
9.5
11
Time (minutes)
42
Figure 19: Reverse phase HPLC of oyster mushroom vitamin D and UV spectra. (A)
Chromatogram of collected oyster mushroom vitamin D on a Vydac C18 column using a
20% methanol in ACN mobile phase. Y-axis is mAU at 265 nm. (B) UV spectrum of
vitamin D2 peak (9.40 min). (C) UV spectrum of vitamin D4 peak (11.15 min).
43
Figure 20: Reverse phase HPLC of crimini mushroom vitamin D and UV spectra. (A)
Chromatogram of collected crimini mushroom vitamin D on a Vydac C18 column using a
20% methanol in ACN mobile phase. Y-axis is mAU at 265 nm. (B) UV spectrum of
vitamin D2 peak (9.19 min).
44
Figure 21: Reverse phase HPLC of portabella mushroom vitamin D and UV spectra. (A)
Chromatogram of collected portabella mushroom vitamin D on a Vydac C18 column
using a 20% methanol in ACN mobile phase. Y-axis is mAU at 265 nm. (B) UV
spectrum of vitamin D2 peak (9.26 min).
45
Figure 22: Reverse phase HPLC of vitamin D2 standard and UV spectrum ran on the
same day as the mushroom samples. (A) Chromatogram of vitamin D2 standard on a
Vydac C18 column using a 20% methanol in ACN mobile phase. Y-axis is mAU at 265 nm. (B) UV spectrum of vitamin D2 standard peak (9.66 min).
46
Figure 23: Reverse phase HPLC of vitamin D4 standard and UV spectrum ran on the
same day as the mushroom samples. (A) Chromatogram of vitamin D4 standard on a
Vydac C18 column using a 20% methanol in ACN mobile phase. Y-axis is mAU at 265
nm. (B) UV spectrum of vitamin D4 standard peak (11.32 min).
47
Figure 24: Reverse phase HPLC of vitamin D3 standard and UV spectrum ran on the
same day as the mushroom samples. (A) Chromatogram of vitamin D3 standard on a
Vydac C18 column using a 20% methanol in ACN mobile phase. Y-axis is mAU at 265
nm. (B) UV spectrum of vitamin D3 standard peak (10.80 min).
48
Figure 25: Reverse phase HPLC of white button mushroom provitamin D and UV
spectra. (A) Chromatogram of collected white button mushroom provitamin D on a
Zorbax ODS column using a methanol, ethanol and dH2O (32:8:1 ratio). Y-axis is mAU
at 280 nm. (B) UV spectrum of provitamin D2 peak (7.06 min). (C) UV spectrum of
provitamin D4 peak (8.33min).
49
Figure 26: Reverse phase HPLC of shiitake mushroom provitamin D and UV spectra. (A)
Chromatogram of collected shiitake mushroom provitamin D on a Zorbax ODS column
using a methanol, ethanol and dH2O (32:8:1 ratio). Y-axis is mAU at 280 nm. (B) UV
spectrum of provitamin D2 peak (7.10 min). (C) UV spectrum of provitamin D3 peak
(7.87 min). (D) UV spectrum of provitamin D4 peak (8.36 min).
Abso
rban
ce (
mA
u)
Wavelength (nm)
B
Abso
rban
ce (
mA
U)
Wavelength (nm)
C
Wavelength (nm)
Abso
rban
ce (
mA
u)
D
50
Figure 27: Reverse phase HPLC of oyster mushroom provitamin D and UV spectra. (A)
Chromatogram of collected oyster mushroom provitamin D on a Zorbax ODS column
using a methanol, ethanol and dH2O (32:8:1 ratio). Y-axis is mAU at 280 nm. (B) UV
spectrum of provitamin D2 peak (7.13 min). (C) UV spectrum of provitamin D4 peak
(8.38 min).
Ab
sorb
ance
(m
AU
)
Wavelength (nm)
C
Abso
rban
ce (
mA
U)
B
Time (minutes)
A
7.1
32
8.3
84
Provitamin D2
Provitamin D4
Wavelength (nm)
Abso
rban
ce (
mA
u)
51
Figure 28: Reverse phase HPLC of crimini mushroom provitamin D and UV spectra. (A) Chromatogram of collected crimini mushroom provitamin D on a Zorbax ODS column
using a methanol, ethanol and dH2O (32:8:1 ratio). Y-axis is mAU at 280 nm. (B) UV
spectrum of provitamin D2 peak (7.11 min). (C) UV spectrum of provitamin D4 peak
(8.35 min).
52
Figure 29: Reverse phase HPLC of portabella mushroom provitamin D and UV spectra.
(A) Chromatogram of collected portabella mushroom provitamin D on a Zorbax ODS
column using a methanol, ethanol and dH2O (32:8:1 ratio). Y-axis is mAU at 280 nm. (B)
UV spectrum of provitamin D2 peak (7.12 min). (C) UV spectrum of provitamin D4 peak
(8.37 min).
Ab
sorb
ance
(m
AU
)
Wavelength (nm)
C
Wavelength (nm)
Abso
rban
ce (
mA
U)
B
A
bso
rban
ce (
mA
U)
A
Provitamin D4
7.1
25
Provitamin D2
8.3
72
Time (minutes)
53
Figure 30: Reverse phase HPLC of provitamin D2 standard and UV spectrum ran on the
same day as the mushroom samples. (A) Chromatogram of provitamin D2 standard on a
Zorbax ODS column using a methanol, ethanol and dH2O (32:8:1 ratio). Y-axis is mAU
at 280 nm. (B) UV spectrum of provitamin D2 standard peak (7.05 min).
54
Figure 31: Reverse phase HPLC of provitamin D4 standard and UV spectrum ran on the
same day as the mushroom samples. (A) Chromatogram of provitamin D4 standard on a
Zorbax ODS column using a methanol, ethanol and dH2O (32:8:1 ratio). Y-axis is mAU
at 280 nm. (B) UV spectrum of provitamin D4 standard peak (8.33 min).
55
Figure 32: Reverse phase HPLC of provitamin D3 standard and UV spectrum ran on the
same day as the mushroom samples. (A) Chromatogram of provitamin D3 standard on a
Zorbax ODS column using a methanol, ethanol and dH2O (32:8:1 ratio). Y-axis is mAU
at 280 nm. (B) UV spectrum of provitamin D3 standard peak (7.57 min).
56
Yeast
Provitamin D2, provitamin D4, vitamin D2 and vitamin D4 were detected in the
yeast Saccharomyces cerevisiae that had been exposed to UV radiation. All standard
provitamin D and vitamin D compounds chromatographed for comparison were done so
under identical HPLC conditions. Every compound identified as vitamin D had a UV
absorbance spectrum that matched the UV absorbance spectrum of a 5,6-cis-triene
(Figure 7). The yeast vitamin D compound chromatographed on straight phase HPLC
had a retention time of 11.6 minutes (Figure 33). Analysis of the vitamin D peak with
reverse phase HPLC revealed 2 peaks. The compound that eluted with a retention time of
8.5 minutes was identified as vitamin D2 (Figure 34). A standard for vitamin D2 eluted at
the same retention time as this identified vitamin D2 peak (Figure 35). The compound
that eluted with a retention time of 10.1 minutes was identified as vitamin D4 (Figure 34).
A standard for vitamin D4 eluted at the same retention time as this identified vitamin D4
peak (Figure 36). A vitamin D3 standard had a retention time of 9.4 minutes (Figure 37).
No detectable vitamin D3 was observed in the yeast sample.
Every compound identified as provitamin D had a UV absorbance spectrum that
matched the UV absorbance spectrum of a 5,7-diene (Figure 6). The yeast provitamin D
compound that was chromatographed on straight phase HPLC had a retention time of
14.8 minutes (Figure 33). Analysis of the provitamin D peak with reverse phase HPLC
revealed 2 peaks. The compound that eluted with a retention time of 7.4 minutes was
identified as provitamin D2 (Figure 38). A standard for provitamin D2 eluted at the same
retention time as this identified provitamin D2 peak (Figure 39). The compound that
57
eluted with a retention time of 8.7 minutes was identified as provitamin D4 (Figure 38).
A standard for provitamin D4 eluted at the same retention time as this identified
provitamin D4 peak (Figure 40). A provitamin D3 standard had a retention time of 8.0
minutes (Figure 41). No detectable provitamin D3 was observed in the yeast sample.
Figure 33: Straight phase HPLC of yeast extract and UV spectra. (A) Chromatogram of
yeast extract on a Zorbax CN column using a 0.3% IPA in hexane mobile phase. Y-axis is
mAU at 280 nm. (B) UV spectrum of vitamin D peak (11.56 min). (C) UV spectrum of
provitamin D peak (14.79 min).
58
Figure 34: Reverse phase HPLC of yeast vitamin D and UV spectra. (A) Chromatogram of collected yeast vitamin D on a Vydac C18 column using a 20% methanol in ACN
mobile phase. Y-axis is mAU at 265 nm. (B) UV spectrum of vitamin D2 peak (8.55
min). (C) UV spectrum of vitamin D4 peak (10.12 min).
59
Figure 35: Reverse phase HPLC of vitamin D2 standard and UV spectrum ran on the
same day as the yeast sample. (A) Chromatogram of vitamin D2 standard on a Vydac C18
column using a 20% methanol in ACN mobile phase. Y-axis is mAU at 265 nm. (B) UV
spectrum of vitamin D2 standard peak (8.56 min).
60
Figure 36: Reverse phase HPLC of vitamin D4 standard and UV spectrum ran on the same day as the yeast sample. (A) Chromatogram of vitamin D4 standard on a Vydac C18 column using a 20% methanol in ACN mobile phase. Y-axis is mAU at 265 nm. (B) UV spectrum of vitamin D4 standard peak (10.00 min).
61
Figure 37: Reverse phase HPLC of vitamin D3 standard and UV spectrum ran on the
same day as the yeast sample. (A) Chromatogram of vitamin D3 standard on a Vydac
C18 column using a 20% methanol in ACN mobile phase. Y-axis is mAU at 265 nm. (B) UV spectrum of vitamin D3 standard peak (9.42 min).
62
Figure 38: Reverse phase HPLC of yeast provitamin D and UV spectra. (A)
Chromatogram of collected yeast provitamin D on a Zorbax ODS column using a
methanol, ethanol and dH2O (32:8:1 ratio) mobile phase. Y-axis is mAU at 280 nm. (B)
UV spectrum of provitamin D2 peak (7.39 min). (C) UV spectrum of provitamin D4 peak
(8.71 min).
63
Figure 39: Reverse phase HPLC of provitamin D2 standard and UV spectrum ran on the
same day as the yeast sample. (A) Chromatogram provitamin D2 standard on a Zorbax
ODS column using a methanol, ethanol and dH2O (32:8:1 ratio) mobile phase. Y-axis is
mAU at 280 nm. (B) UV spectrum of provitamin D2 standard (7.48 min).
64
Figure 40: Reverse phase HPLC of provitamin D4 standard and UV spectrum ran on the
same day as the yeast sample. (A) Chromatogram of provitamin D4 standard on a Zorbax
ODS column using a methanol, ethanol and dH2O (32:8:1 ratio) mobile phase. Y-axis is
mAU at 280 nm. (B) UV spectrum of provitamin D4 standard peak (8.78 min).
65
Figure 41: Reverse phase HPLC of provitamin D3 standard and UV spectrum ran on the
same day as the yeast sample. (A) Chromatogram of provitamin D3 standard on a Zorbax
ODS column using a methanol, ethanol and dH2O (32:8:1 ratio) mobile phase. Y-axis is
mAU at 280 nm. (B) UV spectrum of provitamin D3 standard peak (8.00 min).
66
DISCUSSION
Vitamin D4 is structurally identical to vitamin D3 with the exception that vitamin
D4 has an additional methyl group on carbon 24. Windaus and Trautmann were the first
to produce and patent vitamin D4 in 1937 (Bishop, Knutson, Moriarty, & Penmasta,
1998). De Luca, Weller, Blunt, & Neville, (1968) reported that the biological activity of
vitamin D4 in rats with rickets was approximately 60% as effective in the treatment of the
disease as compared to treatment using vitamin D3.
Previously, it was assumed that mushrooms could only produce vitamin D2. The
results of this study confirms recent research that has previously identified provitamin D4
and vitamin D4 in various edible mushroom species including white button, crimini,
portabella, enoki, shiitake, maitake, oyster, morel, and chanterelle (Phillips, Horst,
Koszewski, & Simon, 2012). The use of standards for various provitamin Ds and vitamin
Ds helped to assist in identifying the unknown vitamin D compounds as provitamin D4 or
vitamin D4. The use of the provitamin D2 and vitamin D2 standards identified the most
prominent provitamin D and vitamin D peak in all mushroom and yeast species as
provitamin D2 and vitamin D2. The use of a standard for provitamin D3 demonstrated
that shiitake mushrooms contain provitamin D3 (7-DHC). The presence of this
provitamin D3 indicates that shiitake mushrooms have the ability to produce vitamin D3
upon exposure to UV radiation. Thus, UV irradiated skiitake mushrooms are a natural
dietary source of not only vitamin D2, but also vitamin D3 and vitamin D4.
The widespread distribution of the VDR in tissues indicates that the compound
1,25(OH)2D3 has a plethora of biological actions throughout the body (Hu et al., 2012)
67
(Kang et al., 2012) (Forster et al., 2011). Keeping this in mind, the therapeutic potential
of 1,25(OH)2D3 is limited as a result of its potent effect on calcium levels. Levels of
1,25(OH)2D3 above normal physiological range can result in toxic side effects including
the calcification of soft tissue and renal stone formation. Therefore, designing or
discovering analogs of vitamin D that have the same beneficial overall effects without
causing hypercalcaemia is the important focus of many research groups. It is thought that
these vitamin D analogs could be used to prevent or treat various autoimmune diseases,
endocrine disorders such as hyperparathyroidism, and a multitude of cancers. In support
of this position, there have been breakthroughs in the treatment of psoriasis (Smith et al.,
1988) and secondary hyperparathyroidism in patients with kidney disease using current
analogs of 1,25(OH)2D3 (Brown & Slatopolsky, 2008). The fact that 1,25(OH)2D3 has
been shown to have anti-proliferative activity in a number of cell types by arresting
growth, promoting differentiation, and effecting the transcription of genes that control
apoptosis, angiogenesis, and autophagy, places importance on finding a vitamin D analog
for possible cancer prevention (Brown & Slatopolsky, 2008). Adorini et al., (2007)
studied the effects of elocalcitol (BXL-628), a vitamin D3 analog in patients diagnosed
with benign prostatic hyperplasia, and found that it diminished prostate growth. Overall,
these studies support the need for additional sources of vitamin D analogues.
Given the potential biological benefits of vitamin D4, analysis of foods for the
presence of this compound is needed. Provitamin D4 has previously been reported in
other organisms including the algae species Chollera (Patterson, 1969). Various fungal
and yeast species have previously been found to contain provitamin D4 (Weete, Abril, &
Blackwell, 2010) (McCorkindale, Hutchinson, Pursey, Scott, & Wheeler, 1969). An in
68
vivo study using a rat model observed that the metabolism of 1α,25-dihydroxyvitamin D4
is similar to the metabolism of 1α,25-dihydroxyvitamin D2 (Tachibana & Tsuji, 2001).
The biological activity of 1,25-dihydroxyvitamin D4 has been studied and found to have a
greater affinity for the vitamin D binding protein as well as increased anti-proliferation
and cell-differentiation activity in human promyelocytic leukemic HL-60 cells (Tsugawa
et al., 1999). Additional research on the actions of vitamin D4 and its presence in natural
food sources may uncover benefits comparable to the already demonstrated benefits of
vitamin D2 and vitamin D3.
The fact that vitamin D deficiency is a worldwide pandemic puts great importance
on finding natural food sources of vitamin D. Mushrooms and yeast have been shown to
contain significant levels of vitamin D2 upon UV irradiation. Taking advantage of this
process may help people throughout the world reach a vitamin D status that will help
them maintain overall better health. Concerns continue to be raised regarding the
efficacy of vitamin D2 compared to vitamin D3 in raising and maintaining total serum
25(OH)D levels (Trang et al., 1998) (Heaney, Recker, Grote, Horst, & Armas, 2011).
Stephensen et al., (2012) reported that small decreases in total 25(OH)D levels were seen
in individuals who ingested mushrooms that contained vitamin D2 as a result of UVB
irradiation. A study done in our lab compared the concentrations of total 25(OH)D in
healthy adults who ingested 2000 IU of vitamin D2 contained in UVB irradiated
mushrooms daily for three months, compared to healthy adults who ingested a
supplement containing either 2000 IU of vitamin D2 or vitamin D3. Results of this study
showed that the levels of total 25(OH)D at the end of the study were similar (Keegan et
69
al., 2012). These findings support the idea that UV irradiated mushrooms are a good
dietary source of vitamin D to raise and maintain serum levels of 25(OH)D. Studies need
to be conducted to determine if 25(OH)D4 is also present in the blood of people who
ingest UV exposed mushrooms that contain both vitamin D2 and vitamin D4.
The results of this research demonstrate that mushrooms have the ability to
produce vitamin D2, vitamin D3, and vitamin D4 upon UV exposure. Also, yeast has the
ability to produce vitamin D2 and vitamin D4 upon UV exposure. During times of the
year when the cutaneous production of vitamin D3 does not occur, the ingestion of
mushrooms, yeast, and other food sources that naturally contain vitamin D becomes
important to maintain healthy vitamin D levels.
70
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