Abundance of Free and Conjugated Ergosterol in Various Regions of Mushrooms

Belle Lin1, Tyler Kalajian2, Kelly Persons2, Michael F. Holick2

Results

References

Acknowledgements

Conclusions

Discussion Methods

Introduction

•  A total of 3 types of mushroom, including Crimini (Agaricus bisporus), Shiitake (Lentinula edodes), and Portobello (Agaricus bisporus), were tested.

•  Mushroom samples were systematically obtained using a core borer which has a diameter of 12 millimeters, then sliced into three samples—top, middle, and bottom. In addition, the stem was also tested.

•  Samples were homogenized using a mortar and pestle with methanol. •  Crimini and Shiitake mushroom samples were left to settle overnight, while the

Portobello mushroom samples were left for an hour. •  Then, the samples were centrifuged and dried off with nitrogen gas. •  To determine the presence of conjugated ergosterol, samples were saponified. •  The solution was analyzed using the high-performance liquid chromatography (HPLC)

in 0.8% isopropyl alcohol in hexane at a rate of 1.5 ml per minute.

Eight percent of the United States population aged one year and greater was at risk of vitamin D deficiency from 2001 to 2006.1 A lack of vitamin D has been linked with various life-threatening diseases, such as cardiovascular disease, cancer, osteomalacia, and numerous others.2 People can get vitamin D through natural sunlight or dietary intake, including egg yolks, cheese, fatty fish, beef liver, mushrooms, and fortified foods. To fulfill the dietary requirements, a person only needs to absorb15 µg of vitamin D. There are a total of 5 different forms of vitamin D, including mixture of ergocalciferol and lumisterol, ergocalciferol, cholecalciferol, 22-dihydroergocalciferol, and sitocalciferol. Mostly, the human body makes use of ergocalciferol (vitamin D2) and cholecalciferol (vitamin D3). Mushrooms have been found to produce vitamin D3, vitamin D4, and exceptionally huge amounts of vitamin D2.3 After being exposed to UV radiation, ergosterol (provitamin D2) will be converted to preergocalciferol (previtamin D2), which is then converted to ergocalciferol (vitamin D2). Previous studies have shown that numerous mushroom species contain differing huge amounts of ergosterol. A study done by Jasinghe and Perera of National University of Singapore found that when the gills instead of the cap of are directed towards the UV source, there is a greater amount of vitamin D2 conversion in various mushrooms, including Shiitake, Enoki, Button, Oyster, and Abalone.4 Objectives: 1.  To determine the abundance of ergosterol in three

species of mushroom, including Crimini, Portobello, and Shiitake

2.  To determine the presence and abundance of conjugated ergosterol

3.  To determine the specific area of greatest ergosterol concentration

1.  Looker, A. C., Johnson, C. L., Lacher, D. A., Pfeiffer, C. M., Schleicher, R. L., Sempos, C. T. Vitamin D status: United states, 2001–2006. NCHS data brief, 2000, 59(59), 1-8.

2.  Kalaras, M. D., Beelman, R. B., Holick, M. F., Elias, R. J. Generation of potentially bioactive ergosterol-derived products following pulsed ultraviolet light exposure of mushrooms (Agaricus bisporus). Food chemistry, 2012, 135, 396-401.

3.  Keegan, R.-J. H., Lu, Z., Bogusz, J. M., Williams, J. E., & Holick, M. F. (2013). Photobiology of vitamin D in mushrooms and its bioavailability in humans. Dermato-Endocrinology, 5(1), 165–176. http://doi.org/10.4161/derm.23321

4.  Jasinghe, V. J., Perera, C. O. Distribution of ergosterol in different tissues of mushrooms and its effect on the conversion of ergosterol to vitamin D 2 by UV irradiation. Food chemistry, 2005, 92, 541-546.

Mushrooms contain significant amounts of ergosterol (provitamin D2), which is converted into previtamin D2 upon UV exposure and then to vitamin D2. While ergosterol has been identified in past research, the specific region in the mushroom in which ergosterol is the most abundant has not. Crimini (Agaricus bisporus), Shiitake (Lentinula edodes), and Portobello (Agaricus bisporus) mushrooms were tested in this experiment. Samples were obtained using a core borer, diameter of 12 mm, and split into four parts—top, middle, bottom, and stem. Then, they underwent either methanol extraction or saponification and followed by hexane extraction and were analyzed by straight phased high-performance liquid chromatography (HPLC) to determine the absence of conjugated ergosterol. The results show that Crimini mushrooms had the greatest amount of ergosterol (22.9 ± 1.7 µg/g), followed by Portobello mushrooms (12.5 ± 1.3 µg/g) and Shiitake mushrooms (8.9 ± 5.1 µg/g). These three types of mushrooms differ significantly (p=0.029). After saponification, Crimini, Portobello, and Shiitake mushrooms have 7.1 ± 1.9, 4.4 ± 1.6, and 3.8 ± 1.0 µg/g, respectively, of ergosterol, showing that ergosterol in mushrooms exists in the free form and when converted will be bioavailable. Secondly, it was discovered that the bottom portion of the mushroom has significantly more ergosterol than the top and middle layers. The top, middle, and bottom portion were found to be statistically significantly different with p values of 0.012, 0.012, and 0.038, respectively. But, the stem was insignificant among the mushrooms. In the future, the bottom portion of the mushroom and the stem could be used for dietary supplements for more concentrated vitamin D2. This information will also be useful for future research on vitamin D2 production in mushrooms.

Abstract

Figure 1. Three regions of mushroom—top, middle, and bottom

Figure 2. Overnight Settling Sample

Figure 3. Saponification with sodium hydroxide, water, and ethanol

Figure 4. Two separating layers based on polarity

Figure 5. Drying Samples with nitrogen gas

Ergosterol Ergocalciferol OH OH

0 2 4 6 8

10 12

Crimini Portobello Shiitake

Con

cent

ratio

n of

E

rgos

tero

l (µg

/g)

Types of Mushroom

Top Middle Bottom Stem

Figure 8. Average concentration of ergosterol in various regions of Crimini, Shiitake, and Portobello mushrooms After splitting the mushrooms into four regions, the concentration of ergosterol was quantified and compared.

Figure 7. Ergosterol concentration of methanol extracted compared to lipid extraction after saponification Samples that underwent methanol extraction reveal the abundance of free ergosterol. Those that underwent saponification indicates the abundance of both free and conjugated ergosterol.

The ergosterol levels of different types of mushroom that underwent methanol extraction were quantified. In decreasing order of concentration, Crimini, Portobello, and Shiitake mushrooms had 22.9 ± 1.7, 12.5 ± 1.3, 8.9 ± 5.1 µg/g of ergosterol, respectively. The Kruskal Wallis Test showed that compared to Crimini and Portobello mushrooms, Shiitake mushrooms had significantly less ergosterol (p=0.029) . The smaller amounts of ergosterol after saponification of Crimini, Portobello, and Shiitake mushrooms (7.1 ±1.9, 4.4 ± 1.6, and 3.8 ± 1.0 µg/g) reveal that all ergosterol is free, since the saponified samples did not have a much larger concentration. There are no ergosterol that is bound to other molecules. This shows that all converted vitamin D2 is free and bioavailable to the human body. The chromatograms reveal that there are greater amounts of ergosterol in the bottom layer in all three mushrooms, contrary to what was previously thought. Portobello mushrooms appears to have an upward trend of ergosterol concentration from the top of the mushroom to the stem. Crimini mushrooms, as well, have an upward tread, but instead their stem has relatively less ergosterol compared to the bottom layer. The top layer, bottom layer, and stem are inversely proportional to the concentration of vitamin D2 in Shiitake mushrooms. Statistical analysis of the data showed that in each of the three species of mushrooms the bottom and stem had significantly more ergosterol than the top and middle regions. The Kruskal Wallis Test confirmed that the top, middle, and bottom layers of each type of mushroom is different from each other.

Previous studies have found mushrooms to be a good source of vitamin D2. This study further reinforces the concept of obtaining sufficient amounts of vitamin D from mushrooms. As all converted vitamin D2 is bioavailable in mushrooms, irradiated mushrooms are a good source of vitamin D. The results reveal that in the future as opposed to the other regions of the mushroom, the bottom layer and stem can be used for dietary supplement and experiments, since the bottom regions of the mushroom are more concentrated in ergosterol. Future studies can be done to determine the particular region with the greatest vitamin D2 conversion by irradiating the samples and quantifying the abundance of vitamin D2 produced. This will further show which regions of mushrooms should be utilized in supplements to ensure the greatest yield of vitamin D.

Thank you to everyone in the Vitamin D, Skin, and Bone Research Lab: Dr. Holick, Kelly Persons, Tyler Kalajian, and Arash Hossein for making this summer educational and memorable.

Ergosterol Vitamin D

Figure 6. HPLC chromatograph and UV absorption spectrums Each peak represents a particular substance. The peak on the far right indicates ergosterol, while the one next to it is vitamin D. The UV absorption spectrums show each compound. By identifying each substance, the abundance can be calculated by multiplying the area under the curve with a conversion factor (0.4 for vitamin D and 0.99 for ergosterol.)

Time (minutes)

Abs

orba

nce

Abs

orba

nce

Abs

orba

nce

Syosset High School, 70 S Woods Rd, Syosset, NY 11791 1, Vitamin D, Skin, and Bone Research Laboratory, Department of Medicine, Boston University School of Medicine, Boston, MA 02118 2

Wavelength (nm)

Wavelength (nm)

Figure 9. Statistical analysis of the layers and types of mushrooms The Kruskal Wallis Test shows that the types of mushroom was significant different (p=0.029). The test further reveals that the top (p=0.012), middle (p=0.012), and bottom (p=0.038) regions were significantly different between each type of mushrooms. On the other hand, the stem (p=0.208) was found to be insignificant.

0 5 10 15 20 25

Crimini

Portobello

Shiitake

Concentration of Ergosterol (µg/g)

Type

s of M

ushr

oom

s

Saponification Methanol Extraction

Types of Mushroom Portobello Crimini Shiitake

95%

Con

fiden

ce In

terv

al

Abu

ndan

ce (n

g/g)

Slices Top Middle Bottom Stem

12500.000

10000.000

7500.000

5000.000

2500.000

.000

Where can I get the most vitamin D2 from mushrooms?