Photobiology of vitamin D in mushrooms and its bioavailability in humans

Abstract

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

Prehistoric Overview

It is theorized that the origins of vitamin D came from Earth’s earliest life forms over 900 million years ago. Primitive life forms were bombarded by the sun’s UV (UV) radiation; this caused damage to their UV-sensitive DNA, RNA and proteins.¹ Provitamin D absorbs UV radiation between 240 and 320 nm and thus could act as a sunscreen. Phytoplankton and zooplankton that produced provitamin D₂ (ergosterol) were likely to have their DNA and RNA protected from UV radiation and were able to pass along their genes to their progeny.^1,2 Five hundred million years ago phytoplankton such as Emiliania huxleyi (E. huxleyi) were producing provitamin D₂.^1,2 Upon exposure to UVB radiation, provitamin D₂ was converted to previtamin D₂, which then thermally isomerized to vitamin D₂ (Fig. 1). It is believed that animals higher on the food chain acquired their vitamin D from phytoplankton and zooplankton.¹

Figure 1. Schematic showing the structures of the provitamin D (side chains labeled R) ergosterol (red), 7-dehydrocholesterol (7-DHC) (blue), 22,23-dihydroergosterol (provitamin D₄) (black). Upon UV irradiation, the provitamin D (orange) is converted to previtamin D (green), which can thermally convert to vitamin D (purple)_. Previtamin D is converted to photobyproducts tachysterol (dark gray) and lumisterol (brown) (Holick, copyright 2012, reproduced with permission).

Land vertebrates require vitamin D to maintain adequate serum calcium levels and bone metabolism. Poikilothermic animals have been studied and were found to contain several provitamin D’s in their skin, with the major provitamin D identified as 7-dehydrocholesterol (7-DHC, provitamin D₃).¹ Northern grass frogs and anolis lizards were found to have 1 to 2 orders of magnitude greater concentrations of provitamin D₃ in their skin compared with humans. Therefore, some poikilotherms have a tremendous capability to produce vitamin D₃ in their skin.¹ This finding suggests that the cutaneous production of vitamin D may have played a major evolutionary role in poikilothermic land vertebrates.

Historic Overview

Vitamin D deficiency has been implicated with several bone pathologies, one of the earliest being rickets. In the early 19th century, rickets was considered an epidemic affecting Europe and the United States, particularly in the northern industrialized cities. Autopsy studies conducted in Boston and Leiden, The Netherlands in the late 19th century found 80–90% of children had rickets. Rickets can be caused by vitamin D deficiency, calcium deficiency, acquired and inherited disorders metabolism of vitamin D, calcium and phosphorus. Specific signs and symptoms include, growth retardation, muscle weakness, skeletal deformities, stunted growth and bowed legs.^3,4

By the late 18th and early 19th century, rickets was a common condition for children. In 1822, the Polish physician Sniadecki noticed that children living in Warsaw, Poland, a densely populated city, had a higher incidence of rickets compared with children living in the countryside where sun exposure was more common. Sniadecki was the first to hypothesize the importance of sunlight to bone health.³ In 1861, Dr. Trousseau of France postulated the etiology of rickets was a lack of sun exposure and diet, and successfully treated patients with cod-liver oil.⁴

In the early 20th century, the German physician Huldschinsky treated patients suffering from rickets with exposure to a mercury-vapor quartz lamp that emitted UVB radiation.³ After six weeks of treatment, the children had radiologic improvement in their condition, demonstrated by an increase in mineralization in the children’s X-ray. In 1921, Hess and Unger observed that exposing children to direct sunlight three to four times a week improved their clinical and radiological manifestations of rickets.⁵ Hess and Unger also observed that cod liver oil acted as a preventative measure and treatment for rickets in adolescents.³

Vitamin D In Food

In 1918, Mellanby first showed that feeding puppies cod liver oil could prevent rickets from occurring.⁶ In 1924, Steenbock and Black observed that UV irradiated foods had an antirachitic effect when consumed by animals.⁷ Later, in 1925, Cowell demonstrated that bovine milk exposed for 20 min under a mercury vapor lamp could be used to treat rickets when consumed by adolescents.⁸ Within two decades, a wide variety of foods and beverages were fortified with vitamin D.³ These discoveries lead to the identification of vitamin D as having an antirachitic effect in humans and animals. It was originally assumed that endogenously produced vitamin D was the same as vitamin D produced by irradiated yeast. However, chickens fed vitamin D from irradiated yeast had minimal antirachitic affects, while cod liver oil reversed the effects of rickets.^1-3

It was then concluded that vitamin D produced from the skin was different than vitamin D produced by irradiated yeast. To distinguish the two forms, they were named vitamin D₂ (ergocalciferol) from yeast and vitamin D₃ (cholecalciferol) from human and animal skin.³ Vitamin D₂ and vitamin D₃ are structurally very similar. The only exceptions are that vitamin D₂ has a double bond between carbons 22 and 23 and a methyl group on carbon 24 (Fig. 1).

Photobiology of Vitamin D₂ in Mushrooms

Mushrooms are fungi and belong to the division Basidiomycota. The vitamin D benefits of edible mushrooms has been known since 1994 when Mattilla et al. extracted provitamin D₂ from wild mushrooms.⁹ White button mushrooms (Agaricus bisporus) are grown in the dark and therefore contain negligible concentrations of vitamin D₂. White button mushrooms were examined and found to contain 56.3 μg/100 g fresh weight of provitamin D₂, and 0.11 μg/100 g fresh weight of vitamin D₂.¹⁰

When exposed to UV radiation, mushrooms become an abundant source of vitamin D_2.¹¹ Mushroom producers have recently begun exposing mushrooms to UV radiation in order to have their product contain vitamin D₂. The photobiology of vitamin D₃ has been well studied in poikilothermic animals and human skin; however, little is known about how vitamin D₂ is produced in irradiated mushrooms.

Photoproduction of Previtamin D₂ in Mushrooms

White button mushrooms were irradiated and studied in order to elucidate the mechanism of vitamin D₂ production. Provitamin D₂ dissolved in methanol (50 μg/mL) in borosilicate ampoules were irradiated and used as a positive control as previously described.¹² Mushrooms and ampoules were irradiated for 5 min, 3.5 inches from a RC-500B Pulsed UV Curing System (Xenon Corporation). After irradiation mushroom samples were obtained with a brass 0.5 cm² cork borer to a depth of 0.1 cm at various times for up to 96 h. The samples were homogenized in 6.0 mL of 100% methanol. The samples were centrifuged and the liquid layer was pipetted off and dried with N₂. The dried samples were dissolved in either 0.3% or 0.8% isopropanol in hexane and chromatographed on a high performance liquid chromatograph stacked Agilent 1100 (HPLC) attached to a photodiode detector. Two different columns were used as a stationary phase (5 μm spherical silica gel) to separate provitamin D₂ from its photoproducts and vitamin D₂. A Zorbax RX-SIL column with 0.8% isopropanol in hexane was used to separate lumisterol₂ from previtamin D₂ and the Zorbax CN column with 0.3% isopropanol in hexane was used to separate tachysterol₂ from vitamin D₂ (Fig. 2A and B). The concentrations of photobyproducts were calculated using a conversion factor obtained from a standard curve.

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

As can be seen in Figure 2A, immediately after exposure to 5 min of UV radiation provitamin D₂ was converted to a product that migrated at 6.7 min and had a UV absorption spectrum λ_max 260 nm, consistent with previtamin D₂. The peak with retention time 10.6 min had a UV absorption λ_max at 265 nm and was consistent with vitamin D₂ (Fig. 2B). These findings in irradiated mushrooms are similar to what was observed in irradiated ampoules containing provitamin D₂ and confirms the previous observations by Kalaras et al.¹³ (Fig. 2C and D).

A 96 h time course was conducted to examine the conversion of previtamin D₂ to vitamin D₂ in UV irradiated white button mushrooms and ampoules. Immediately after irradiation and at various times, the samples were chromatographed to determine the percent conversion of previtamin D₂ and vitamin D₂ (Fig. 3). There was a time dependent increase in the amount of vitamin D₂. At 6 h after irradiation, 24% of the previtamin D₂ in white button mushroom had converted to vitamin D₂ whereas only 10% of the previtamin D₂ in the irradiated provitamin D₂ ampoules converted to vitamin D₂.

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

After 11.5 h, 50% of the previtamin D₂ present in white button mushrooms had converted to vitamin D₂ compared with only 19% of the previtamin D₂ converted to vitamin D₂ in the ampoules (Fig. 3). These results mimic what was observed in ampoules containing 7-DHC in methanol and lizard skin after exposure to UV radiation and kept at 25 °C (Fig. 3). The results demonstrate that the kinetics for previtamin D₂ conversion to vitamin D₂ in mushrooms was enhanced compared with the conversion in an organic solvent, similar to lizard skin.¹⁴

Photoproduction of Lumisterol ₂ and Tachysterol ₂

White button mushrooms were exposed to UV radiation for 1, 2, 3, 4, 5 and 10 min at 25 °C (Fig. 4). After 5 min of exposure to UV radiation a peak that migrated at 7.3 min and had a UV absorption spectrum λ_max 272 nm, consistent with lumisterol₂ (Fig. 2A) and a peak with retention time 11.7 min that had a UV absorption spectrum λ_max 281 nm consistent with tachysterol₂ was detected in the irradiated white button mushrooms (Fig. 2B). A similar result was observed in irradiated ampoules; previtamin D₂, lumisterol₂ and tachysterol₂ were detected (Fig. 2C and D) and confirms a previous report.¹³

Figure 4. Photobyproducts in irradiated white button mushrooms with various exposure times to UV radiation. Samples dissolved in either 0.3% or 0.8% isopropanol in hexane were chromatographed on a high performance liquid chromatograph, and were analyzed for previtamin D₂ (blue), lumisterol₂ (red), vitamin D₂ (green) and tachysterol₂ (purple). Mean ± SEM.

Previtamin D₂ and lumisterol₂ were only observed after exposure to 1 min of UV radiation. After 3 min of UV exposure, previtamin D₂, vitamin D₂, lumisterol₂ and tachysterol₂ were detected (Fig. 4). Photoequilibrium of the previtamin D₂ photobyproducts was reached between 4 and 5 min of UV exposure. Although the ratio of previtamin D₂ and photoproducts did not change after 5 min of exposure, continued exposure increased the total amount of previtamin D₂ and its photoproducts. Mushrooms that were not exposed to UV radiation did not contain detectable amounts of previtamin D₂, its photobyproducts or vitamin D₂.

Stability of Lumisterol ₂ and Tachysterol ₂ in Mushrooms

A 24 h time course was conducted to study the stability of the photobyproducts of previtamin D₂ in white button mushrooms. White button mushrooms and provitamin D₂ ampoules were irradiated for 5 min; mushroom samples were collected every 2 h for 24 h. As expected, previtamin D₂ and vitamin D₂ decreased and increased, respectively, in a time dependent manner (Fig. 3). An extended time course of 336 h was conducted to study the stability of lumisterol₂ and tachysterol₂ in methanol that were recovered from the irradiation of provitamin D₂. Lumisterol₂ and tachysterol₂ remained stable in methanol for more than 300 h (Fig. 5A). The amount of lumisterol₂ remained constant over time in irradiated mushrooms. Though, the amount of tachysterol₂ remained constant in irradiated ampoules, in irradiated white button mushrooms, tachysterol₂ rapidly declined to undetectable levels within 24 h (Fig. 5B). The same rapid disappearance of tachysterol₂ was also observed in oyster (Pleurotus ostreatus) and shiitake ((Lentinula edodes) mushrooms (data not shown).

Figure 5. Stability of lumisterol₂ and tachysterol₂ in irradiated ampoules and mushrooms. Ampoules and mushroom samples were exposed to 5 min of UV radiation. Samples were taken over time. Samples were dissolved in either 0.3% or 0.8% isopropanol in hexane and chromatographed on a high performance liquid chromatograph at 25 °C. Mean ± SEM. (A) Lumisterol₂ (♦) and tachysterol₂ (■), in irradiated ampoules. (B) Lumisterol₂ (♦) and tachysterol₂ (■), in white button mushroom samples.

Vitamin D ₄ in Mushrooms

Vitamin D₄ is a form of vitamin D that is structurally similar to vitamin D₃ and was first produced by Windaus and Trautmann in 1937.¹⁵ Vitamin D₄ has a methyl group on carbon 24 of the vitamin D₃ side chain (Fig. 1). Vitamin D₄ is produced from the UV irradiation of its precursor, 22,23-dihydroergosterol (provitamin D₄) (Fig. 1). Vitamin D₄ was reported to be about 60% as active as vitamin D₃ in healing rickets in rats.¹⁶

Philips et al. reported the presence of provitamin D₄ in several mushroom species including crimini (Agaricus bisporus), portabella (Agaricus bisporus), enoki (Flammulina velutipes), shiitake, maitake (Grifola frondose), oyster, morel (Morchella spp) and chanterelle (Cantharellus cibarius).¹⁷

We examined crimini, oyster, portabella, shiitake, white button mushrooms, white button mushroom power (Monterey Mushrooms, Inc.) and Saccharomyces cerevisiae (S. cerevisiae) yeast (Lallemand Inc.) for the presence of provitamin D₂, vitamin D₂, provitamin D₄, vitamin D₄ as well as other potential provitamin Ds and vitamin Ds. Mushroom samples were obtained in a similar manner that was described for white button mushrooms. One gram of S. cerevisiae was extracted with 5 mL of methanol for 1 min and sonicated with a sonic disrupter (Teledyne Tekmar). After being centrifuged for 5 min, the liquid layer was removed, and the extraction was repeated 5 additional times. The extracts were combined and taken to dryness under N₂ gas. The dried samples were dissolved in methanol, ethanol and dH₂O (32:8:1 ratio) and chromatographed on a reverse phase HPLC using a Zorbax ODS column.

Analysis of crimini, oyster, portabella, shiitake, white button mushrooms, white button mushroom powder and S. cerevisiae yeast using the reverse phase HPLC system with a Zorbax ODS column to separate the various provitamin Ds revealed all samples contained provitamin D₂ and vitamin D₂. In addition the peak with retention time at 9.6 min and a UV absorbance spectrum of a 5,7-diene consistent with provitamin D₄ (Fig. 6A).¹⁵ Provitamin D₄ obtained from white button mushroom powder was dissolved in methanol (4 μg/mL) and placed in a borosilicate ampoule and irradiated for 10 min using a RC-500B Pulsed UV Curing System. After irradiation the provitamin D₄ ampoule was dried with N₂ gas and prepared for straight phase HPLC. HPLC analysis revealed peaks with retention times that had UV absorption spectra consistent with provitamin D₄, previtamin D₄, lumisterol₄ and tachysterol₄, and were used as our standards (Fig. 6B and C). HPLC analysis of S. cerevisiae yeast revealed peaks that also co-eluted with standard provitamin D₄ and vitamin D₄. Oyster mushrooms were irradiated for 5 min and examined for the presence of vitamin D₄. The dried samples were dissolved in 20% methanol in acetonitrile and prepared for reverse phase HPLC, using a Vydac C18 column to separate the various vitamin Ds. UV irradiated mushrooms revealed the presence of previtamin D₄ with a retention time of 6.2 min in straight phase; its presence was confirmed by chromatography after its conversion to vitamin D₄. The analysis revealed peaks with retention times of 10.8 and 13 min that had a UV absorbance spectra with λ_max 265 nm for the 5,6-cis-triene system consistent with vitamin D₂ and vitamin D₄, respectively (Fig. 6D). Provitamin D₂ and provitamin D₄ were detected in the unexposed mushrooms (data not shown). For comparison white button mushroom powder contained ~88% vitamin D₂ and ~12% vitamin D₄, whereas S. cerevisiae yeast sample contained ~99% vitamin D₂ and ~1% vitamin D₄.

Figure 6. HPLC chromatograms of extracts from white button mushrooms. (A) An extract of white button mushroom chromatographed on reverse phase on a Zorbax ODS column. Methanol, ethanol and dH₂O (32:8:1 ratio) to separate the provitamin Ds. Y-axis is miliAbsorbance Units (mAU) at 280 nm. Ergosterol (E), provitamin D₄ (ProD₄). (B) An ampoule containing provitamin D₄ (ProD₄) irradiated for 10 min and chromatographed on a Zorbax RX-SIL column 0.8% isopropanol (IPA) in hexane to separate lumisterol₄ (L₄) from previtamin D₄ (PreD₄). Y-axis is mAU at 265 nm. Tachysterol₄ (T₄), vitamin D₄ (D₄). (C) An ampoule containing provitamin D₄ (ProD₄) irradiated for 10 min chromatographed on a Zorbax CN column in 0.3% IPA in hexane to separate tachysterol₄ (T₄), from vitamin D₄ (D₄). Y-axis is mAU at 265 nm. Previtamin D₄ (PreD₄), lumisterol₄ (L₄). (D) An extract from an oyster mushroom irradiated for 5 min and chromatographed on a C18 column in 20% methanol in acetonitrile to separate vitamin Ds. Y-axis is mAU at 265 nm. Tachysterol₂ (T2), vitamin D₂ (D₂), vitamin D₄ (D₄).

Vitamin D₃ in Mushrooms

It has been assumed that mushrooms only produce vitamin D₂ when irradiated. However, since some mushrooms can produce vitamin D₄ we reevaluated, by HPLC, mushroom extracts for the possibility of identifying other provitamin Ds. Evaluation of extracts from shiitake mushrooms revealed by reverse phase HPLC a peak with a retention time of 11.5 min that was identified as provitamin D₄, and a peak with a retention time of 10.8 min with an identical UV absorption spectrum consistent with a 5,7-diene (Fig. 7A). Co-chromatography studies revealed that this peak was 7-DHC. 7-DHC was found to migrate between provitamin D₂ and provitamin D₄ that had retention times of 10.8, 9.6 and 11.5 min, respectively. Concentrations of the 7-DHC and provitamin D₄ were 5.8 and 2.6 greater in the gills of the mushroom compared with the surface of the mushroom cap. UV irradiated shiitake mushrooms were examined for the presence of vitamin D₃. Shiitake mushrooms were irradiated with the gills facing up for 5 min using a RC-500B Pulsed UV Curing System. Mushroom samples were obtained in a similar manner that was described previously. The samples were chromatographed on reverse phase HPLC using a C18 column. UV irradiated mushrooms revealed the presence of previtamin D₃ with a retention time of 6.3 min in straight phase; its presence was confirmed by chromatography after its conversion to vitamin D₃. Peaks with retention times of 5.1 and 6.1 min had UV absorbance spectra with λ_max 265 nm consistent with vitamin D₂ and vitamin D₃, respectively (Fig. 7B).

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

Bioavailability of Vitamin D₂ in Mushrooms

Various foods and beverages have been fortified with vitamin D in the United States for more than 70 y since there are few that contain vitamin D naturally. Milk, orange juice, bread, other dairy products and some cereals have been fortified with this essential vitamin.¹⁸ Mushrooms exposed to sunlight or UV radiation are a good source of vitamin D₂. Several clinical studies have been conducted to determine the bioavailability of vitamin D₂ and vitamin D₃ in fortified foods and beverages and the efficacy of fortification in increasing 25-hydroxyvitamin D [25(OH)D] levels; the measure for determining a person’s vitamin D status.¹⁸ Fortification of foods with vitamin D₂ or vitamin D₃ has been shown to be a safe and effective way to increase 25(OH)D levels in children and adults.¹⁸

Biancuzzo et al. found that there was no difference in total 25(OH)D levels in healthy adults who consumed orange juice that was fortified with 1000 IU of vitamin D₂ or 1000 IUs of vitamin D₃ compared with consuming a supplement containing either 1000 IU vitamin D₂ or 1000 IU vitamin D₃.¹⁹ It was also found that there was no difference in the increase of 25(OH)D₃ levels between subjects who consumed vitamin D₃-fortified orange juice or vitamin D₃ supplements or in 25(OH)D₂ levels of subjects who consumed vitamin D₂-fortified orange juice or vitamin D₂ supplements. Additionally, Natri et al. found that bread fortified with vitamin D₃ increased total serum 25(OH)D levels in women as effectively as a vitamin D₃ supplement.²⁰

To date, two groups have reported on the bioavailability of vitamin D₂ in UV irradiated mushrooms. Urbain et al. conducted a five-week single-blinded, randomized, placebo-controlled trial in 26 healthy Caucasian adults with 25(OH)D levels below 20 ng/mL.²¹ These subjects were randomized to three groups and assigned to receive either 28,000 IU vitamin D₂ from UV irradiated mushrooms in a soup and placebo, 60 IU vitamin D₂ in soup that contained non-UV- irradiated mushrooms and 28,000 IU vitamin D₂ in a liquid supplement, or 60 IU vitamin D₂ in a non-UV-irradiated mushroom soup and placebo supplement four times a week for four weeks. After four weeks, serum 25(OH)D levels increased significantly and consuming vitamin D₂ from UV-irradiated mushrooms was equally as effective at raising 25(OH)D levels as ingesting the same amount of vitamin D₂ as a supplement.

Stephenson et al. conducted a similar study where subjects were randomized to consume one serving of mushrooms with a standard meal each day for six weeks.²² Four groups received either one serving of non-UV-irradiated mushrooms plus meal (control), UV irradiated mushrooms containing 352 IU vitamin D₂ with a meal, UV irradiated mushrooms containing 684 IU vitamin D₂ with a meal or a supplement containing 1,128 IU vitamin D₂ with non-UV-irradiated mushrooms. At the end of six weeks, 25(OH)D₂ levels were higher in all groups except the control group. They observed a significant decrease in serum 25(OH)D₃ in the group receiving 684 IU vitamin D₂ in UV irradiated mushrooms and in the group receiving the vitamin D₂ supplement. There was a mean decrease of 25(OH)D₃ of 0.32 ng/mL that was offset by an increase of 0.40 ng/mL in 25(OH)D₂. Overall, however, there was a small decrease in total 25(OH)D levels in the groups consuming UV irradiated mushrooms that contained vitamin D₂.

This observation contributed to the controversy surrounding the efficacy of maintaining total serum 25(OH)D levels after taking supplemental or dietary vitamin D₂ vs. vitamin D₃. Some reports have suggested that vitamin D₃ was more effective than vitamin D₂ at maintaining total serum 25(OH)D levels.^23,24 In contrast, Holick et al., similar to Biancuzzo et al., found that the daily ingestion of a 1000 IU vitamin D₂ supplement was as effective as the daily ingestion of a 1000 IU vitamin D₃ supplement at raising and maintaining total serum 25(OH)D levels.^19,25 Furthermore, taking 50,000 IU vitamin D₂ once a week for eight weeks and every other week thereafter for up to six years increased serum total 25(OH)D levels and is considered to be effective for the treatment and prevention of vitamin D deficiency.²⁶ Similarly, Demetriou reported that 50,000 IU vitamin D₂ repletion and maintenance therapy in vitamin D deficient patients significantly increased serum 25(OH)D₂ and total 25(OH)D despite a decrease in serum 25(OH)D₃ levels.²⁷

We conducted a clinical study to determine if ingestion of vitamin D₂ in a dried white button mushroom extract (Monterey Mushrooms, Inc.) was as effective at increasing and maintaining vitamin D status as supplemental vitamin D₃ and vitamin D₂. Thirty healthy adults were enrolled in the study (6 male, 19 female, mean age 35.2 y) and were randomized to ingest capsules containing 2000 IU vitamin D₂, 2000 IU vitamin D₃ or 2000 IU mushroom vitamin D₂ once a day for three months during the winter. Vitamin D concentrations were verified to be within 10% by HPLC. Twenty-five subjects completed the study. Fourteen subjects were randomized to the mushroom vitamin D₂ group, eight subjects to the supplemental vitamin D₂ group and 3 subjects to the supplemental vitamin D₃ group. Serum concentrations of 25(OH)D₂, 25(OH)D₃ and 25(OH)D were measured once a week for 12 weeks by liquid chromatography tandem mass spectroscopy (LCMS/MS) as previously described.²⁸

Subjects in the mushroom vitamin D₂ group had a mean baseline serum 25(OH)D₂ of 0.6 ± 0.3 ng/mL that increased significantly to 18.6 ± 1.4 ng/mL at the end of 12 weeks (p < 0.0001). Total serum 25(OH)D levels increased from 20.6 ± 2.4 ng/mL to 30.1 ± 2.6 ng/mL (p < 0.001) (Fig. 8A).

Figure 8. Mean (± SEM) 25-hydroxyvitamin D₂ (♦), 25-hydroxyvitamin D₃ (■) and total 25-hydroxyvitamin D (●) concentrations over time after oral administration of (A) 2000 IU of mushroom vitamin D₂ in capsules (n = 14), (B) 2000 IU of supplemental vitamin D₂ in capsules (n = 8) and (C) 2000 IU of supplemental vitamin D₃ in capsules (n = 3). The change in total serum 25-hydroxyvitamin D concentrations from baseline to final visits in each group was statistically significant as was the change in serum 25-hydroxyvitamin D₂ concentrations from baseline to final visit in the mushroom vitamin D₂ group and the supplemental vitamin D₂ group. (*p < 0.05, **p < 0.01, ***p < 0.001) No statistically significant difference was observed between final total 25-hydroxyvitamin D concentrations between all three groups.

Subjects in the supplemental vitamin D₂ group had a mean baseline serum 25(OH)D₂ of 1.5 ± 1.2 ng/mL that increased significantly to 13.3 ± 2.0 ng/mL at the end of 12 weeks (p < 0.001). Total serum 25(OH)D levels significantly increased from 19.4 ± 2.3 ng/mL to 29.2 ± 2.0 ng/mL (p < 0.01) (Fig. 8B).

Subjects in the supplemental vitamin D₃ group had a mean baseline serum of 25(OH)D₃ of 16.3 ± 0.6 ng/mL with a final baseline serum level of 34.4 ± 1.3 ng/mL (Fig. 8C). Total 25(OH)D increased from 17.1 ± 1.4 ng/mL to 34.4 ± 1.3 ng/mL (p < 0.05). The discrepancy in mean baseline serum levels is due to some detectable 25(OH)D₂ of 0.8 ng/mL (Fig. 8C).

Baseline serum total 25(OH)D levels were not significantly different between the groups; 17.1 ± 1.2, 19.4 ± 2.3 and 20.6 ± 2.4 ng/mL for the supplemental D₃ and vitamin D₂, and mushroom vitamin D₂ groups respectively. Serum 25(OH)D levels gradually increased and plateaued at seven weeks and were maintained for the following five weeks. At the end of 12 weeks, serum total 25(OH)D levels were not statistically significantly different in all three groups: 34.4 ± 1.1, 29.2 ± 2.0 and 30.1 ± 2.6 ng/mL for supplemental vitamin D₃, D₂ and mushroom D₂ respectively.

These results demonstrate ingestion of mushrooms containing D₂ was as effective at increasing and maintaining total serum 25(OH)D levels as supplemental vitamin D₂ and vitamin D₃.

Conclusion

Vitamin D deficiency is a pandemic. It has increased the risk of skeletal and chronic diseases associated with vitamin D deficiency worldwide. Therefore, obtaining vitamin D from sensible sun exposure, foods that naturally contain vitamin D, and from supplementation with vitamin D is imperative to maintain a healthy lifestyle.

Phytoplankton have been producing vitamin D₂ from provitamin D₂ for over 500 million years. The phytoplankton E. huxlei contained 1.0 μg/g wet weight of provitamin D₂ and has likely been a source of vitamin D₂ for the oceanic food chain for millions of years.² Due to their large quantities of provitamin D₂, fungi like phytoplankton have a huge capacity to produce vitamin D₂ when exposed to UV irradiation.

It is now known that in mushrooms, provitamin D₂ is converted to previtamin D₂ upon UV irradiation. Previtamin D₂ can absorb UVB radiation resulting in the production of the photobyproducts, lumisterol₂ and tachysterol₂.¹³ An evaluation of the thermal isomerization of previtamin D₂ to vitamin D₂ in mushrooms revealed that it was rapidly converted to vitamin D₂ most likely by a non-enzymatic membrane-enhanced catalytic mechanism similar to what was observed in lizard and human skin.^14,29 The planar structure of 7-DHC and provitamin D₂ can fit in between the triglyceride side chains and polar head groups. Upon exposure to UVB radiation the 5,7-diene absorbs the radiation, converting to its respective previtamin D. Previtamin D exists as two conformers cis-cis (cZc) and cis-trans (cZt). In an organic solvent, ~90% exists in the thermodynamically favored cZt while only ~10% exists in the cZc state. Although more thermodynamically stable, the cZt conformer of previtamin D is unable to isomerize to vitamin D; only its less stable cZc can. Thus it takes several days at 25 °C for the cZt conformer to convert to cZc, which in turn converts to the thermodynamically stable vitamin D₃. Our observation that previtamin D₂ more rapidly converts to vitamin D₂ in mushrooms than in methanol suggests that this mechanism of a membrane-enhanced conversion of previtamin D₂ to vitamin D₂ has existed for hundreds of millions of years.

Tachysterol₃ and lumisterol₃ produced in human skin have no biological function on calcium metabolism.³⁰ Therefore, the physiologic significance of mushrooms producing lumisterol₂ and tachysterol₂ is unknown. During our investigation on the time dependent conversion of previtamin D₂ to vitamin D₂ in white button mushrooms we observed that the concentration of tachysterol₂ began to decline and was undetectable after 24 h. To be certain that this decline was not due to tachysterol₂ being unstable, we conducted a stability evaluation of tachysterol₂ in methanol at room temperature for more than one week and found it to be stable. We also observed that this phenomenon also occurred in oyster and shiitake mushrooms suggesting that mushrooms are utilizing in some manner the tachysterol₂. This observation may provide insight as to a possible biologic function of tachysterol not only in mushrooms but also in human skin.

It is known that plants and poikilothermic animals contain several different forms of provitamin D.^1,31 Similarly mushrooms are capable of producing more than one provitamin D. It had always been assumed that UV irradiated mushrooms were only capable of producing vitamin D₂. Our study confirms that some mushrooms do contain provitamin D₄ and we now also report that shiitake mushrooms contain 7-DHC. Therefore during UV irradiation some mushrooms are capable of producing vitamin D₂, vitamin D₃ and vitamin D₄.

Mushrooms exposed to UVB radiation contain a significant amount of vitamin D₂ and therefore is an excellent alternative food source for vitamin D, especially for vegans. However, there continues to be concern that vitamin D₂ not only is less effective than vitamin D₃ in maintaining total serum 25(OH)D concentrations but that the ingestion of vitamin D₂ can ultimately result in a decrease in total 25(OH)D concentrations.^23,24 Stephenson et al.²² reported that ingesting UVB irradiated mushrooms containing vitamin D₂ resulted in a small decrease in total 25(OH)D levels. In our study, healthy adults who ingested daily for 3 mo 2000 IUs of vitamin D₂ from mushrooms were able to raise and maintain their total 25(OH)D concentrations similar to healthy adults who ingested either 2000 IU supplement containing vitamin D₂ or vitamin D₃. These results confirm other studies that have demonstrated that ingesting vitamin D₂ either from fortified orange juice,¹⁹ a supplement²⁰ or a pharmaceutical formulation^12,26 were all capable of increasing total circulating 25(OH)D concentrations for at least 3 mo and up to 6 y. Therefore ingesting mushrooms containing vitamin D₂ can be an effective strategy to enhance the vitamin D status of the consumer. The observation that some mushrooms when exposed to UVB radiation also produce vitamin D₃ and vitamin D₄ can also provide the consumer with at least two additional vitamin Ds.

Glossary

Abbreviations:

25(OH)D

25-hydroxyvitamin D

7-DHC

7-dehydrocholesterol

cZc

5,6-s-cis-s-cis previtamin D

E. huxleyi

Emiliania huxleyi

HPLC

high performance liquid chromatograph

IU

international units

LCMS/MS

liquid chromatography tandem mass spectroscopy

S. cerevisiae

Saccharomyces cerevisiae

tZc

5,6-s-trans-s-cis previtamin D

UV

ultraviolet

UVB

ultraviolet B

Disclosure of Potential Conflicts of Interest

This work was supported by The Mushroom Council and from the National Institutes of Health Clinical Translational Science Institute Grant UL1-TR000157. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript; ClinicalTrials.gov Identifier: NCT01815437.