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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

iii

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.

iv

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

v

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.

vi

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

vii

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

viii

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.





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.





33 Straight phase HPLC of yeast extract and UV spectra. 57

34 Reverse phase HPLC of yeast vitamin D and UV spectra. 58


day as the yeast sample.


day as the yeast sample.


Day as the yeast sample.

ix

38 Reverse phase HPLC of yeast provitamin D and UV spectra. 62


same day as the yeast sample.


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

xi

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).

2

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

3

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.

4

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,

5

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).

6

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).

7

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).

8

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

9

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.

10

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

11

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, &

12

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, &

13

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.

14

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).

15

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


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


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



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



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



(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



(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


at 280 nm. (B) UV spectrum of provitamin D2 standard peak (7.05 min).

54





55





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


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


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


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


same day as the yeast sample. (A) Chromatogram of provitamin D4 standard on a Zorbax


mAU at 280 nm. (B) UV spectrum of provitamin D4 standard peak (8.78 min).

65


same day as the yeast sample. (A) Chromatogram of provitamin D3 standard on a Zorbax


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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