Nutritional Supplementation with Chlorella pyrenoidosa Lowers Serum Methylmalonic Acid in Vegans and Vegetarians with a Suspected Vitamin B 12 Deficiency

Abstract

Since vitamin B₁₂ occurs in substantial amounts only in foods derived from animals, vegetarians and particularly vegans are at risk of developing deficiencies of this essential vitamin. The chlorella used for this study is a commercially available whole-food supplement, which is believed to contain the physiologically active form of the vitamin. This exploratory open-label study was performed to determine if adding 9 g of Chlorella pyrenoidosa daily could help mitigate a vitamin B₁₂ deficiency in vegetarians and vegans. Seventeen vegan or vegetarian adults (26–57 years of age) with a known vitamin B₁₂ deficiency, as evidenced by a baseline serum methylmalonic acid (MMA) level above 270 nmol/L at screening, but who otherwise appeared healthy were enrolled in the study. Each participant added 9 g of C. pyrenoidosa to their daily diet for 60 ± 5 days and their serum MMA, vitamin B₁₂, homocysteine (Hcy) levels as well as mean corpuscular volume (MCV), hemoglobin (Hgb), and hematocrit (Hct) were measured at 30 and 60 days from baseline. After 30 and 60 days, the serum MMA level fell significantly (P < .05) by an average ∼34%. Fifteen of the 17 (88%) subjects showed at least a 10% drop in MMA. At the same time, Hcy trended downward and serum vitamin B₁₂ trended upward, while MCV, Hgb, and Hct appeared unchanged. The results of this work suggest that the vitamin B₁₂ in chlorella is bioavailable and such dietary supplementation is a natural way for vegetarians and vegans to get the vitamin B₁₂ they need.

Introduction

Vitamin B ₁₂, also known as cobalamin, is an essential nutrient. The vitamin is thought by many to be synthesized exclusively from bacterial sources and converted in the body to its physiological forms, methylcobalamin and adenosylcobalamin.¹ The vitamin is water soluble and most of it in the body is stored in the liver. Vitamin B₁₂ is normally involved in the metabolism of virtually every cell of the body, affecting DNA synthesis and regulation, as well as fatty acid synthesis and energy production. There are only two enzymatic reactions in mammalian cells that require the vitamin as a cofactor. Methylcobolamin is a cofactor for methionine synthase and the enzyme methylmalonyl-CoA mutase requires adenosylcobalamin as a cofactor.^1,2 When the availability of B₁₂ is inadequate, the result is an increased concentration of methylmalonic acid (MMA) and homocysteine (Hcy) due to inhibition of methylmalonyl-CoA mutase and methionine synthase, respectively.^1,2 Therefore, serum concentrations of Hcy and MMA can be expected to increase when the intake of vitamin B₁₂ is deficient.

Since humans are unable to synthesize vitamin B₁₂, foods derived from animals and fish are its usual source.² The recommended intake of vitamin B₁₂ per day is 2–3 μg. B₁₂ deficiency is common worldwide and can result from a number of ways. Malabsorption of the vitamin in those over 50 years of age is thought to occur as a result of the slow and steady loss of parietal cells in the stomach with aging. Diseases such as AIDS and Crohn's have also been linked to malabsorption of the vitamin.^1,2 People who ingest low amount of the vitamin in their diet like vegetarians and vegans can also often develop a deficiency.^1,2 Deficiencies in vitamin B₁₂ status have been associated with neurological and cognitive impairment and at its extreme, pernicious anemia.³

Nutraceuticals are a category of natural, bioactive chemical compounds that may have disease-preventing, health-promoting, or medicinal properties. Vitamins, minerals, and herbal supplements fall into this category. Also included are functional foods that are touted to have particular healthful benefits based on their ingredients. Functional foods go beyond providing the basic nutrition and calories of foodstuffs by providing some physiologic benefit such as boosting immune function, improving energy levels and memory, and lowering serum cholesterol and blood pressure. Some examples of these foods are garlic, certain mushrooms, fish oils, green and ginseng teas, ginkgo biloba, and some species of algae such as Chlorella.

Chlorella pyrenoidosa is a unicellular green alga that grows in fresh water. It has the highest content of chlorophyll of any known plant and also contains high concentrations of certain vitamins, minerals, dietary fiber, nucleic acids, amino acids, and other phytochemicals. This alga has a strong cell wall that prevents its native form from being adequately digested so that only after Dyno-Mill processing to break its cell wall can the organism be digested by humans.⁴ A number of scientific reports out of Japan have shown that broken cell wall preparations and extracts of C. pyrenoidosa and other Chlorella species, when either given orally or injected, promote growth and healing, stimulate the immune system such that the host is protected from infection, and exert a significant anticancer activity.^4

–9 Clearly, there remains a need to conduct additional comprehensive studies to identify the macro- and micronutrients in Chlorella and, as well, how these nutrients may act together in concert to promote good health, as well as exert their beneficial effects to mitigate symptoms of conditions such as hypertension and possibly cure chronic diseases.

Although the studies conducted to date suggest that there may be a health benefit from adding C. pyrenoidosa to the diets of healthy people as well as those with a cancer or other disease, there remains a clear need for more scientific research specifically directed at mechanisms of action in restoring health and combating disease. Vitamin B₁₂ is a critical nutrient that is often inadequate in the plant-based, vegan, or vegetarian diet. The inclusion of a reliable source of vitamin B₁₂ in their diet is therefore not only recommended but also essential. Unfortunately, some natural sources of vitamin B₁₂ have been proven to contain biologically inactive vitamin B₁₂ analogues, inadequate for human supplementation. Laboratory testing has indicated that the B₁₂ found in chlorella is the active form of the vitamin, but it has to be shown unequivocally that it is physiologically active in humans. The present exploratory study involved subjects who consumed a vegan or vegetarian diet and had a deficiency in their vitamin B₁₂ activity; that is, a serum MMA level of 270 nmol/L or higher at screening. We hypothesized that adding 9 g of C. pyrenoidosa into their daily diet for 2 months would improve their B₁₂ status, as evidenced by increased serum B₁₂ levels, along with decreased serum MMA and Hcy levels.

Materials and Methods

The protocol and informed consent form (ICF) were reviewed and approved by Copernicus^® Group Institutional Review Board in compliance with relevant federal regulations and guidelines for good clinical practice. Data were collected for this prospective exploratory study between January 2013 and July 2014. All of the screening and follow-up visits were done at a single center (Medicus Research, LLC, Northridge, CA, USA).

Study subjects

This study was open to healthy adults, between the ages of 18 and 60, who had consumed a vegetarian or vegan diet for at least the past year. At screening, all potential subjects received a written copy of the ICF to review and discuss with clinic staff and their families. The ICF provided the subject with information on their role and responsibilities, as well as made them aware of the potential benefits, risks, and discomforts in this study of C. pyrenoidosa. The ICF also described the exploratory nature of the study and reminded them that their participation was totally voluntary. Beside agreeing to add C. pyrenoidosa to their diet, subjects were also agreeing not to initiate any new exercise regimens or change from their current vegetarian or vegan diet during the entire study period. Conventional drugs and other treatments given to treat a preexisting health condition were allowed. However, subjects were cautioned that if any new medication was started after the screening visit and/or during their time in the study, they must notify the investigator. Any dietary supplement that the subject was taking before the screening visit could be continued, but no new supplements/vitamins could be added, while they were enrolled in the study. Before conducting any of the subsequent screening procedures, all participants signed the ICF.

Screening visit

Once a subject agreed to participate and had signed the ICF, they were screened to establish eligibility. A medical history was taken and a routine physical examination was performed. Blood was collected for a complete blood count (CBC) with manual differential and to determine their serum MMA level.

The screening data were reviewed to ensure patient eligibility, whether the ICF was properly executed and if all screening laboratory procedures and examinations had been performed. If a subject had a serum MMA level of 270 nmol/L or more and met all other eligibility requirements, they were contacted and instructed as to when to return to the study site for their baseline visit and begin consuming the nutritional supplement according to the protocol.

Baseline and follow-up visit assessments

Subjects returned to the study center for their baseline visit, within 14 days of their screening visit for further tests and to begin their consumption of the dietary supplement. At the baseline visit and again after consuming the supplement for 30 ± 5 and 60 ± 5 days, the subjects were interviewed regarding their current status of health, asked about any adverse events (AEs), and if any changes were made in any medications they currently were taking. They also underwent a general physical examination, and had their body temperature, heart rate, respiratory rate, blood pressure, and weight recorded. Blood was collected on each occasion for a CBC with manual differential and determination of mean corpuscular volume (MCV), hemoglobin (Hgb), and hematocrit (Hct) along with MMA, Hcy, and vitamin B₁₂ levels. At the baseline visit only, urine was collected from women of childbearing potential to rule out that they were pregnant, as pregnancy would have made them ineligible to participate in the study.

Materials

Sun Chlorella^® A, which is produced and marketed internationally by the Sun Chlorella Corporation (Kyoto, Japan), has been studied in various preclinical and human trials. Sun Chlorella A tablets are formed from a pulverized cell wall preparation of C. pyrenoidosa (95% by weight) and lecithin (5% by weight). Each tablet is composed of 60% protein, 20% carbohydrate, 11% unsaturated fats, and 3% chlorophyll. The proteins contain all the amino acids known to be essential for the nutrition of animals and human beings. Vitamins found in Sun Chlorella A tablets include vitamin C, provitamin A (ß-carotene), thiamine (B₁), riboflavin (B₂), pyridoxine (B₆), niacin, pantothenic acid, folic acid, vitamin B₁₂, biotin, choline, vitamin K, lipoic acid, leutin, and inositol. Minerals include phosphorus, calcium, zinc, iodine, magnesium, iron, and copper. The daily maintenance dosage recommended by the manufacturer is 3–6 g C. pyrenoidosa tablets. Subjects ate 9 g of Chlorella per day and began their consumption of C. pyrenoidosa tablets according to the dose escalation schedule shown in Table 1. They were instructed that the dietary supplement should be taken with meals. The amount of vitamin B₁₂ in 9 g of C. pyrenoidosa is estimated to be 21 μg.^10,11

Table 1.

Schedule of Daily Dosing of Chlorella pyrenoidosa

Days	Dose (g)	Daily number of Sun Chlorella^® A tablets
1–2	3	15 Tablets total: 5 tablets with morning, midday, and evening meals
3–4	6	30 Tablets total: 10 tablets with morning, midday, and evening meals
5–60	9	45 Tablets total: 15 tablets with morning, midday, and evening meals

Subjects were told to increase their daily dosage according to the above schedule, unless they experienced an AE. If a subject experienced an AE during this escalation period or anytime thereafter while in the study, they were instructed to notify the investigator who would determine if they should continue in the study or not.

Participants were given a log book and instructed to record every day the total number of C. pyrenoidosa tablets they consumed, the name and total dose of any concomitant drugs (if any), and to describe any AEs or symptoms they experienced. Subjects were asked to bring their study diary along with them to each follow-up visit, so that it could be reviewed by the study coordinator.

Statistical analysis

All the data collected were summarized and tabulated using Excel^™ spreadsheets. Appropriate descriptive statistics were presented for subject demographics, laboratory parameters under study, as well as any other variables that were collected. For every subject, the incidence and relative severity of any AEs were assessed by body system.

For all subjects, laboratory measures were evaluated for trends over time and shifts from normal range. Student's t-tests were used to analyze changes from baseline in serum MMA and the other laboratory values under study. All statistical tests were conducted at the 5% level of significance.

Results

Over the course of the study, a total of 223 potential subjects were screened and all but 23 failed to meet eligibility requirements and were enrolled. The far majority were not eligible because their serum MMA levels were within the normal range; 70–270 nmol/L. Of those enrolled, 17 completed the entire study (through day 60). All six of the participants who did not finish, voluntarily withdrew; two completed 30 days and four withdrew between screening and the baseline visits. For this report, the analysis of data was limited to 17 subjects (9 females and 8 males) who completed the entire 60 days of dietary supplementation with C. pyrenoidosa.

For the 17 subjects who consumed the supplement daily for 60 ± 5 days, clinic visits were conducted at baseline (day 0) and after 30 ± 5 and 60 ± 5 days. At each clinic visit, subjects had a general physical examination and their body temperature, heart rate, respiratory rate, blood pressure, and weight were also recorded. Subjects also were asked about any AEs and if any changes were made in any medications they currently were taking. Samples of their blood were taken and analyzed for cell counts with differential, Hct, Hgb, and MCV according to standard clinical laboratory methods. Serum concentrations of Hcy and MMA were measured by gas chromatography–mass spectrometry and vitamin B₁₂ by chemiluminescence immunoassay.¹²

None of the subjects experienced any appreciable changes in their body temperature, heart rate, respiratory rate, blood pressure, or weight between baseline and day 60. No AEs or dose-limiting toxicity to C. pyrenoidosa, as defined using the NCI Common Toxicity Criteria (Version 2.0), were observed among the participants.

For the 17 subjects who consumed Chlorella for 60 days, the average serum MMA levels fell by 32% after 30 ± 5 days and 33% after 60 ± 5 days compared to baseline (Table 2). After 30 and 60 days, 15 (88.23%) showed a >10% decrease in their serum MMA levels from baseline. The range of the drop was 11–65%. For the two who did not show at least a 10% drop after 60 days, one subject showed a 1% decrease and the other showed an increase of 21%. Taken together, the average drop in the serum MMA level for all 17 subjects after 30 and 60 days was statistically significant; 142 and 145 nmol/L, respectively.

Table 2.

Serum MMA Levels (nmol/L) Over the Course of Study

Subject	Baseline	30 Day	60 Day	Difference ^a	% Change ^a
1	424	287	215	−209	49
2	245^b	181	218	−27	11
3	774	505	596	−178	23
4	343	180	177	−166	48
5	397	373	334	−63	16
6	312	254	240	−72	23
7	239^b	171	187	−52	22
8	337	266	212	−125	37
9	346	352	342	−4	1
10	840	668	1021	181	−21
11	527	348	291	−236	45
12	631	357	239	−392	62
13	285	164	145	−140	49
14	381	219	167	−214	56
15	567	457	275	−292	51
16	445	167	157	−288	65
17	410	172	225	−185	45
Mean	441	301	297	−145	34
SD	174	142	214

Change from baseline visit.

MMA level was above 270 nmol/L at screening visit.

MMA, methylmalonic acid; SD, standard deviation.

With regards to Hcy, 12 of 17 (71%) subjects showed a decrease in Hcy levels (range of 2–42%) and 5 showed increases ranging from 1% to 19% at the end of the study period (Table 3). The overall Hcy level trended downward from baseline to day 60 by 10% on an average. The serum levels of vitamin B₁₂ on the other hand, trended upward rising by an average of 21% after 60 days (Table 4). Twelve (76%) subjects showed an increase in serum B₁₂ levels between baseline and day 60 (range of 6–89% increase), while five showed modest decreases ranging from 5% to 11%. The average increase in the serum B₁₂ level was 76 mg/dL (a 27% increase). With regards to MCV, Hgb, and Hct, there were no appreciable changes from baseline, and all three remained in their normal ranges of variation at all time points (Table 5).

Table 3.

Serum Homocysteine Levels (μ mol/L) Over the Course of Study

Subject	Baseline	30 Day	60 Day	Difference ^a	% Change ^a
1	10.5	10	9.3	−1.2	−11
2	8.7	11.3	10.7	2	19
3	13.9	12.2	14.9	1	7
4	8.1	7.3	6.7	−1.4	−20
5	6.6	5.6	5.1	−1.5	−29
6	7.1	4.6	6.7	−0.4	−6
7	7.6	9.1	7.4	−0.2	−29
8	11.3	9.5	9.6	−1.7	−15
9	6.9	8.5	7	0.1	1
10	17	18.5	16.6	−0.4	−2
11	11.6	11.7	11.7	0.1	1
12	9.9	7.3	7.6	−2.3	−23
13	9.6	8.3	9.1	−0.5	−5
14	12.6	11.6	8.2	−4.4	−35
15	9.0	8.9	9.1	0.1	1
16	8.8	7.6	5.1	−3.7	−42
17	11.2	9.4	7.7	−3.5	−31
Mean	10.02	9.49	8.97	−1.05	−13
SD	2.74	3.13	3.11

Change from baseline to day 60 visit.

Table 4.

Serum Vitamin B₁₂ Levels (mg/dL) Over the Course of Study

Subject	Baseline	30 Day	60 Day	Difference ^a	% Change ^a
1	248	326	362	114	45
2	475	491	435	−40	−8
3	339	356	415	76	22
4	242	408	458	216	89
5	534	441	476	−58	−11
6	444	507	508	64	14
7	247	317	341	94	38
8	517	623	615	98	19
9	1004	978	954	−50	−5
10	148	189	198	50	34
11	325	303	346	21	6
12	320	414	502	182	57
13	274	277	323	49	18
14	491	768	765	274	56
15	234	217	222	−12	−5
16	277	528	447	170	61
17	165	194	216	51	30
Mean	369	432	446	76	27
SD	203	209	195

Change from baseline to day 60 visit.

Table 5.

Summary of Laboratory Results Over the Course of the Study

Visit	MMA (nmol/L)	Hcy (μmol/L)	B₁₂ (mg/dL)	MCV	Hgb	Hct
Baseline	441 ± 174	10.0 ± 2.7	370 ± 203	89.3 ± 4.8	13.8 ± 1.2	41.0 ± 2.9
Day 30	301 ± 142	9.5 ± 3.1	432 ± 209	89.8 ± 4.8	14.0 ± 1.4	41.5 ± 3.8
Change^a (%)	−32^*	−5	+17	+0.6	+1.4	+1.2
Day 60	297 ± 214	9.0 ± 3.5	446 ± 195	90.2 ± 4.4	13.9 ± 1.5	41.3 ± 3.7
Change^a (%)	−33^*	−10	+21	+0.08	+0.7	+0.7

Change from baseline visit.

Statistically significant decrease, P < .05.

Hct, hematocrit; Hcy, homocysteine; Hgb, hemoglobin; MCV, mean corpuscular volume.

Discussion

The principal focus of this open-label exploratory study was to provide solid evidence that dietary supplementation with C. pyrenoidosa is a natural way for vegetarians and vegans to get the vitamin B₁₂ needed for optimal health. The study recruited vegetarians or vegans who showed an abnormally high level of MMA at screening; indicative of a vitamin B₁₂ deficiency. To determine if adding the dietary supplement helped improve values of parameters associated with a vitamin B₁₂ deficiency, serum was tested for MMA, Hcy, and B₁₂ at baseline and then after 30 ± 5 and 60 ± 5 days of supplementation. Whole blood was also assessed at these same times for MCV, Hgb, and Hct.

A low vitamin B₁₂ status is defined as a plasma level below 148 pmol/L.¹³ Total serum cobalamin, however, is neither sensitive nor specific for vitamin deficiency, and this might explain why many deficient subjects are overlooked by utilizing this test alone.^2,12 Since it is known that concentrations of both MMA and Hcy increase in the blood of B₁₂-deficient subjects, these now appear to be complementary and, together, the most useful functional markers.^13
–15 Serum Hcy levels >12 μmol/L combined with an MMA value above 270 nmol/L suggest that a functional B₁₂ deficiency exits.¹³ One of the first studies to demonstrate this was performed by Savage et al. who showed that measuring serum metabolite concentrations provided a highly sensitive test of deficiency.¹⁶ Of the 434 people with a vitamin B₁₂ level below the normal range, 98.4% of their serum MMA levels and 95.9% of their serum Hcy levels were elevated (>3 standard deviations above the mean in normal subjects).

A vegetarian/vegan diet provides for the low intake of saturated fat and cholesterol and the consumption of high amounts of dietary fiber along with many health-promoting phytochemicals derived from fruits, vegetables, whole grains, legumes, nuts, and soy products. Vegetarians and vegans typically have lower body–mass index, serum total, low-density lipoprotein cholesterol levels, and blood pressure. They also have reduced rates of death from ischemic heart disease and stroke, as well as lower risks for hypertension, type 2 diabetes, and certain cancers.¹⁷ However since vitamin B₁₂ occurs in substantial amounts only in foods derived from animals, vegetarians and particularly vegans are at a risk to develop deficiencies of this essential vitamin as evidenced by increased levels of Hcy and MMA, elevated MCV of erythrocytes, and lowered Hct and Hgb.^18,19 Therefore, optimal monitoring of cobalamin status in vegetarians should include the measurement of MMA and Hcy.^18,19 Herrmann and Geisel found elevated MMA and Hcy in over 60% of vegetarians they examined.¹⁸ Hyperhomocysteinemia is associated with an increased risk of atherosclerosis and cardiovascular disease, which may partly counteract the beneficial lifestyle of vegetarians.^20,21

While the usual dietary sources of vitamin B₁₂ come from meat, milk, eggs, and fish, substantial amounts of vitamin B₁₂ have been found in some edible algae, including chlorella and the blue-green algae spirulina.^{10,11,20
–22} The amount of vitamin B₁₂ in 9 g of C. pyrenoidosa is estimated to be 21 μg.^10,11 There has, however, been some controversy surrounding the B₁₂ found in algae because it has been claimed that this is pseudo-B₁₂, meaning that, while in tests that measure B₁₂ levels in blood show a presence of the vitamin, these substances do not have a B₁₂ biological activity for humans. To better understand this, Watanabe et al. purified and characterized corrinoid compounds from chlorella and spirulina to clarify the chemical properties and bioavailability of the algal vitamin B₁₂. They found that the true vitamin B₁₂ was the predominant cobamide of chlorella tablets, while the pseudovitamin B₁₂ predominated in the spirulina.¹⁰ For vegetarians and vegans, therefore, chlorella appears to be an edible algae with a bioavailable vitamin B₁₂, while edible cyanobacteria like spirulina are not suitable for use as vitamin B₁₂ sources.^11,22

The results of the present study support our hypothesis that adding 9 g per day of C. pyrenoidosa can help overcome a vitamin B₁₂ deficiency and the B₁₂ in C. pyrenoidosa is the biologically active form of the vitamin. We have observed a significant reduction in serum MMA levels after just 1 month with 15 of the 17 (88%) subjects showing a greater than 10% reduction in serum MMA. Furthermore, 13 subjects showed increases in their serum B₁₂ and 12 showed decreases in their serum Hcy levels over the same time period. Ten (59%) subjects showed the ideal response; that is, reductions in serum MMA and Hcy accompanied by an increase in serum B₁₂. Although the sample size was small and the study lasted only 60 days, the results of this exploratory study supported our hypothesis that dietary supplementation with Sun Chlorella A is a natural way for vegetarians and vegans to get vitamin B₁₂ in their diet.

Footnotes

Acknowledgment

The study was funded by the Sun Chlorella Corporation, 369 Osaka-cho, Karasuma-dori Gojo-sagaru, Shimogyo-ku, Kyoto 600–8177, Japan.

Author Disclosure Statement

Dr. Merchant serves on the scientific advisory board and is a paid consultant for the Sun Chlorella Corporation. Dr. Udani is the Founder, CEO, and Medical Director of Medicus Research, LLC.

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