2.2.1. Vitamins

As shown in Table 3, commercially available *Chlorella* products contain all the vitamins required by humans, i.e., B1, B2, B6, B12, niacin, folate, biotin, pantothenic acid, C, D2, E, and K, and α- and β-carotenes. *Chlorella* products contain substantial amounts of vitamins D2 and B12, both of which are well known to be absent in plants. Commercially available *Chlorella* (*C. vulgaris*) products contain higher amounts of folate (approximately 2.5 mg/100 g dry weight) than spinach [31]. Vitamin B12 and

folate deficiencies induce the accumulation of serum homocysteine, which is involved in cardiovascular diseases. In this section, we discuss vitamin D2, vitamin B12, and folate.


**Table 3.** Content of vitamins and related compounds in 13 commercially available *Chlorella* products.

\*1 Vitamin K1 (mg), \*2 β-Carotene (mg), \*3 α-Carotene + β-Carotene (mg).

Vitamin D, a major regulator of calcium absorption, reduces the risk of osteomalacia in adults and rickets in children [32]. The two main dietary forms of vitamin D are vitamin D2 and D3, which are found in fungi such as mushrooms [33,34] and animal-derived foods such as fish and fish products [35], respectively. Mushrooms have the ability to synthesize ergosterol (known as provitamin D2), which is converted into ergocalciferol as vitamin D2 upon ultraviolet irradiation [34,36]. Thus, ultraviolet-irradiated mushrooms are suitable for use as vitamin D2 sources in strict vegetarians [36]. Cell walls of mushrooms contain high concentrations of ergosterol, which plays a physiological role in modulating cell membrane strength and fluidity similar to cholesterol in animals [37]. Sun-dried, commercially available mushrooms reportedly contain approximately 17 μg of vitamin D2 per g dry weight [38]. The bioavailability of vitamin D2from mushrooms has been studied in humans [39,40].

Ergosterol was first reported as the main sterol compound in *C. pyrenoidosa* in the early 1950S [41]. *C. vulgaris* also contains a substantial amount of ergosterol [42,43]. Our unpublished data show that one commercially available *Chlorella* product contains both ergosterol (1.68 mg/g dry weight) and vitamin D2 (15.2 μg/g dry weight), similar in amounts to those in sun-dried mushrooms. The vitamin D2 in this *Chlorella* product is synthesized from ergosterol upon exposure to sunlight during cultivation (Figure 1). Although it has been reported that vitamin D3 is more e ffective than vitamin D2 at increasing the concentration of circulating 25-hydroxyvitamin D [44], *Chlorella* products and sun-dried mushrooms could become sources of vitamin D for vegetarians.

**Figure 1.** Structures of provitamin D2 and vitamin D2 found in commercially available*Chlorella* products.

Serum homocysteine (Hcy) is an established biomarker of cardiovascular disease in humans [45,46]. Hcy is a non-protein forming amino acid (Figure 2) produced as an intermediate compound of methionine metabolism and is further metabolized to cystathionine via cystathionine β-synthetase, a vitamin B6-dependent enzyme [46]. Alternatively, Hcy can be remethylated back to methionine by methionine synthase, a vitamin B12-dependent enzyme. Folate is also required for the remethylation of Hcy by providing 5-methyltetrahydrofolate. Deficiencies in vitamin B12 [47], vitamin B6 [48], and folate [49] cause hyper-homocysteinemia. Several clinical studies report a correlation between atherosclerosis and deficiencies in vitamin B12 and folate [50,51]. Folate deficiency in women before and during pregnancy is associated with neural tube defects in newborns [52]. Plants can synthesize folate compounds de novo, but animals cannot [53]. Thus, plant-derived foods are sources of dietary folates for humans. High concentrations of folate (approximately 1.69–2.45 mg/100 g dry weight) are reported in commercially available *Chlorella* (*C. vulgaris*) products [31], with concentrations similar to those of the products shown in Table 3 (0.3–3.6 mg/100 g dry weight). The main folate compounds identified in *Chlorella* products are 5-CHO-H4 folate (60–62%) and 5-CH3-H4 folate (24–26%) and the minor folate compounds are 10-CHO-folate (5–7%), H4 folate (4%), and fully oxidized folate (3–6%) [31]. The chemical structures of the *Chlorella* folate compounds are shown in Figure 3. The main dietary sources of folates are vegetables (25%), bread and cereal products (22%), dairy products (10%), fruit (10%), and oils and fats (5%) [31]. Spinach has high folate content (165 μg/100 g fresh weight; 1.7 mg/100 g dry weight) [31,54], which is similar to that of *Chlorella* products. Thus, *Chlorella* products are an excellent source of folate for humans.

**Figure 2.** Homocysteine metabolic pathway in mammals. Abbreviations: B6, vitamin B6; B12, vitamin B12; CBS, cystathionine β-synthetase; DHF, dihydrofolate; MS, cobalamin-dependent methionine synthase; SAM, S-adenosyl methionine; SAH, S-adenosyl homocysteine; THF, tetrahydrofolate.

**Figure 3.** Chemical structures of folate compounds found in commercially available *Chlorella* products.

Vitamin B12 (B12) is synthesized by certain bacteria and archaea but not by plants [55]. Animal-derived foods, such as meats, milk, fish, and shellfish, are the major dietary sources of B12 for humans [56]. B12 content is high in beef, pork, and chicken livers (approximately 25–53 μg/100 g fresh weight) [56] and in edible bivalves such as clams (approximately 60 μg/100 g fresh weight) [57]. The reported B12 content of *Chlorella* products varies from <0.1 to 400 μg per 100 g of dry weight [58,59], consistent with that of the products shown in Table 3 (6–500 μg/100 g dry weight). Among *Chlorella* species, the B12 content is much higher in *C. pyrenoidosa* than in *C. vulgaris* when grown under open culture conditions [59]. B12 is not essential for the growth of these *Chlorella* species [59,60], suggesting that *Chlorella* cells absorb and accumulate large amounts of exogenous B12. Some of the high B12-containing *Chlorella* products contain inactive corrinoid compounds such as 5-methoxybenzimidazolylcobamide and cobalt-free corrinoid (Figure 4). Thus, if *Chlorella* products with high B12 are consumed as a sole B12 source, accurate content estimation requires the identification of B12 compounds using liquid chromatography–tandem mass spectrometry [59].

**Figure 4.** Chemical structures of vitamin B12 and related compounds found in commercially available *Chlorella* products. Abbreviations: Factor IIIm, 5-methoxybenzimidazolylcobamide.

Rauma et al. [61] demonstrated that substantial consumption of *Chlorella* products can supply adequate amounts of B12. Another study of strict vegetarians (vegans) with an elevated baseline of serum methylmalonic acid (as an index of B12 deficiency) showed that ingestion of 9 g of *C. pyrenoidosa* daily for 60 days resulted in significant decreases in serum methylmalonic acid in 88% of the subjects [62]; serum Hcy decreased and serum B12 tended to increase, although the mean corpuscular volume, hemoglobin, and hematocrit levels were unchanged. These results sugges<sup>t</sup> that *Chlorella* products with high B12 and without inactive corrinoid compounds are suitable for use as B12 sources in humans, particularly vegans.
