Is vitamin B12 deficiency a risk factor for cardiovascular disease in vegetarians?

Is Vitamin B12 Deficiency a Risk Factor for Cardiovascular Disease in Vegetarians? Roman Pawlak, PhD, RD The goal of this paper is to describe the role of vitamin B12 deficiency in cardiovascular disease development among vegetarians. Vegetarians have a high prevalence of vitamin B12 deficiency. Deficiency of this vitamin is associated with a variety of atherogenic processes that are mainly, but not exclusively, due to vitamin B12 deficiency–induced hyperhomocysteinemia. Each 5-μmol/L increase above 10 μmol/L of serum homocysteine is associated with a 20% increased risk of circulatory health problems. Mean homocysteine concentration 410 μmol/L among vegetarians was reported in 32 of 34 reports. Macrocytosis associated with vitamin B12 deficiency is also associated with fatal and non-fatal coronary disease, myocardial infarction, stroke, and other circulatory health problems. Compared with non-vegetarians, vegetarians have an improved profile of the traditional cardiovascular disease risk factors, including serum lipids, blood pressure, serum glucose concentration, and weight status. However, not all studies that assessed cardiovascular disease incidence among vegetarians reported a protective effect. Among studies that did show a lower prevalence of circulatory health problems, the effect was not as pronounced as expected, which may be a result of poor vitamin B12 status due to a vegetarian diet. Vitamin B12 deficiency may negate the cardiovascular disease prevention benefits of vegetarian diets. In order to further reduce the risk of cardiovascular disease, vegetarians should be advised to use vitamin B12 supplements. (Am J Prev Med 2015;48(6):e11–e26) & 2015 American Journal of Preventive Medicine

Introduction

H

eart disease is the leading cause of mortality worldwide, causing more than 17 million deaths annually, which accounts for 31% of all mortalities (32% among women and 27% among men).1,2 Vegetarian diets are protective against some chronic health conditions. Vegetarian diets are devoid of all flesh foods, but may include eggs (ovo-vegetarian); milk and dairy products (lacto-vegetarian [LV]); or eggs, milk, and dairy (lacto-ovo-vegetarian [LOV]). A vegan diet does not contain meats, fish, or poultry and does not contain any products of animal origin (milk, dairy, and eggs). These diets improve many modifiable risk factors for heart disease, including serum lipid profile, serum glucose concentration, and systolic and diastolic blood pressure.3–8 Consequently, fewer vegetarians than nonvegetarians are diagnosed with hyperlipidemia, hyperglycemia, and hypertension. Furthermore, fewer From the Department of Nutrition Science, East Carolina University, Greenville, North Carolina Address correspondence to: Roman Pawlak PhD, RD, Department of Nutrition Science, East Carolina University, Rivers West 337, Greenville NC 27858. E-mail: [email protected]. 0749-3797/$36.00 http://dx.doi.org/10.1016/j.amepre.2015.02.009

& 2015 American Journal of Preventive Medicine

vegetarians than non-vegetarians are overweight or obese.6 In general, fewer vegetarians smoke or abuse alcohol, and they eat more fruits and vegetables compared with non-vegetarians.9–11 Additionally, recent findings have shown that vegetarian diets improve markers of inflammation such as C-reactive protein, reduce oxidative stress, and protect against plaque formation.9 The profile of the traditional heart disease risk factors is especially beneficial among vegans. Based on these factors alone, vegan individuals should exhibit the lowest risk of circulatory health problems. Interestingly, the improved heart disease risk factors in vegetarians compared with non-vegetarians do not always translate into a lower risk of incidence of and mortality from circulatory diseases. Studies that did find a lower prevalence of and mortality from circulatory health conditions among vegetarians did not always report substantial benefits, as should have been expected based on the impact of vegetarian diets on cardiovascular disease (CVD) risk factors. The death rate ratio from ischemic heart disease (IHD) among vegetarians in comparison with non-vegetarians ranged from 0.45 (95% CI¼0.22, 0.95) for German vegetarians included in the Heidelberg Study to 0.97 (95% CI¼0.78, 1.21) among the European Prospective Investigation into

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Pawlak / Am J Prev Med 2015;48(6):e11–e26

Cancer and Nutrition (EPIC)-Oxford participants reported by Key et al.9 When data on all circulatory death rate ratios and cerebrovascular disease death ratios were analyzed, some cohorts actually reported higher rates among vegetarians, including vegans, compared with non-vegetarians.12,13 Although vegetarians seem to have a lower risk of circulatory health problems, it must be concluded that vegetarian diets are not as protective as would be assumed based on the pattern of the CVD traditional risk factors, data were not adjusted for a sufficient number of confounding factors, or that other factors may negate any or some protective effect of these diets on serum lipids, serum glucose, blood pressure, and weight status. Although plant foods provide a variety of nutrients, including antioxidant vitamins and phytochemicals as well as dietary fiber, they provide no (in the case of a vegan diet) or inadequate amounts of vitamin B12. Vitamin B12 deficiency leads to hyperhomocysteinemia. Elevated homocysteine (Hcy) is associated with arterial endothelial dysfunction and is considered an independent risk factor for CVD.14 Although other nutritional deficiencies reported among vegetarians may contribute to the circulatory health problems (e.g., inadequate serum vitamin D, eicosapentaenoic and docosahexaenoic acids), the magnitude of the reported prevalence of vitamin B12 deficiency and the variety of associated pathologic processes may cause this deficiency to be the single most important factor in explaining the disappointing findings of CVD prevalence among vegetarians. The goal of the current paper is to describe the role of vitamin B12 deficiency in CVD development. The objectives are to (1) describe the association between Hcy and heart disease; (2) provide an overview of the prevalence of hyperhomocysteinemia among vegetarians; (3) review vitamin B12 status among vegetarians; (4) discuss the impact of B12 on Hcy in vegetarians; (5) describe the role of Hcy in atherogenesis; and (6) examine the role of red blood cell (RBC) distribution width (RDW) and mean corpuscular volume (MCV) in circulatory health problems.

Homocysteine and Heart Disease Perhaps the most important way that low vitamin B12 status increases risk of CVD is via its role in Hcy metabolism. Hyperhomocysteinemia was hypothesized to play a role in atherogenesis 46 years ago. In 1969, McClully15 described a case of an infant’s death with homocystinuria, cystathioninuria, and methylmalonic aciduria due to an abnormal vitamin B12 status. He noted that the arterial atherosclerotic lesions were similar to those developed by children with progressive

arterial disease that results from the lack of normal activity of cystathionine synthetase. He concluded that the common vascular changes found in these children were a result of elevated concentrations of Hcy or its derivatives.16 A number of publications have supported McClully’s hypothesis. Jacobsen17 proposed that an Hcy concentration of 10–15 mmol/L is a substantial CVD risk factor and that this risk should be considered as a continuum: the higher the concentration, the higher the risk. Jacobson’s conclusion is consistent with findings from the Framingham study, where an increased risk of carotid stenosis was reported at Hcy concentrations of 9.1–11.3 μmol/L among men and 11.4–14.3 μmol/L among women.18 The meta-analysis of the Homocysteine Studies Collaboration group evaluated the impact of Hcy on ischemic heart disease and stroke based on 30 prospective and retrospective studies.14 After adjustment for heart disease risk factors, lowering Hcy by 25% or 3 μmol/L was associated with a statistically significant (11%) lower risk of IHD and a 19% lower risk of stroke. Humphrey and colleagues19 conducted another metaanalysis to assess the effect of Hcy on coronary heart disease risk. They included 31 studies (eight prospective cohort studies and 23 nested case-control studies). The combined effect for each 5-μmol/L increase in Hcy concentration yielded a significantly (18%) higher risk of coronary events, independent of the traditional risk factors. Consequently, these authors suggested that for each 5-μmol/L increase in plasma Hcy, there is a corresponding 20% increase in the risk of coronary heart disease that is independent of traditional heart disease risk factors.19

Prevalence of Hyperhomocysteinemia Among Vegetarians The prevalence of hyperhomocysteinemia depends on several factors, including the serum concentration defined as normal, age, gender, presence/absence of circulatory health conditions, and presence/absence of vegetarianism. In the Third National Health and Nutrition Examination Survey, hyperhomocysteinemia was defined as concentration of at least 11.4 μmol/L for male participants and at least 10.4 μmol/L for female participants.20 In this study, the reported prevalence of high Hcy concentration varied by age and gender, with the lowest prevalence reported for female adolescents (aged 12–19 years, 7.9%) and the highest reported for elderly women (aged Z60 years, 46.5%). In the Framingham cohort, Selhub18 reported hyperhomocysteinemia as a risk factor for extracranial carotid artery stenosis in at least 25% of the sample. Pruefer et al.20 suggested that www.ajpmonline.org


hyperhomocysteinemia is found in 9%–15% of the general population and about 40% of individuals with coronary or cerebrovascular disease.21 Almost without exception, studies that compared Hcy concentrations among vegetarians with non-vegetarians reported a higher serum mean total Hcy (tHcy) concentration among the former, with the highest concentration found among vegans.22 Similarly, vegetarians, especially vegans, consistently show a higher prevalence of hyperhomocysteinemia.22 In the review of Elmadfa and Singer,22 nine of ten comparisons of tHcy concentrations between vegetarians and non-vegetarians showed a higher concentration among vegetarians. All four comparisons between vegans and vegetarians showed higher concentrations among vegans. Obersby and colleagues23 published a meta-analysis of total Hcy concentration among vegetarians (LOVs, LVs, and vegans) in comparison with non-vegetarians. They identified 17 studies (six cohort and 11 case-control) with 3,230 participants. The mean (SD) Hcy concentration among vegetarians based on 15 studies was 13.91 (3.5) μmol/L, that among vegans based on nine studies was 16.41 (4.8) μmol/L. Nonvegetarians had the lowest mean tHcy concentration, at 11.3 (2.89) μmol/L.23 Data included in Table 1 show the prevalence of hyperhomocysteinemia among vegetarians, which ranges from 12% (hyperhomocysteinemia defined as serum tHcy 415 μmol/L) in LV and LOV from Germany to 78% (hyperhomocysteinemia defined as serum tHcy 412 μmol/L) among vegetarians from Slovakia. Of the 20 samples that assessed the prevalence of hyperhomocysteinemia among vegetarians, 13 reported a prevalence higher than 50% of the sample, ranging from 52% to 78%. Furthermore, only two (one of which was for semi-vegetarians) of 34 reports of the mean Hcy concentration were o10 μmol/L (Table 1). Thus, research findings suggest that (1) vegetarians have a higher Hcy concentration compared with non-vegetarians; (2) the prevalence of hyperhomocysteinemia among vegetarians is higher than that among nonvegetarians; and (3) the prevalence of hyperhomocysteinemia among vegetarians may actually be higher than that among non-vegetarians already diagnosed with heart disease.

Vitamin B12 Status Among Vegetarians Almost universally, research findings show a poor vitamin B12 status among vegetarians, regardless of the type of biochemical assessment. The earliest reports of vitamin B12 deficiency among vegetarians from India were published in 1960.24 The earliest reports on a deficiency among vegetarian members of the SeventhDay Adventist Church were published in 1970s.25 The June 2015

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most recent results showed a deficiency among 62% of pregnant women, 25% to almost 86% of children, 21%– 41% of adolescents, and 11%–90% of the elderly.26 Fifteen of 26 reports, among the 18 studies, reported either elevated serum or urinary methylmalonic acid in 50% or more of their respective samples. The prevalence of B12 deficiency is summarized in Tables 1 and 2.

Homocysteine in Vegetarians Is Affected Mainly by Vitamin B12 Status Homocysteine is a sulfur-containing amino acid that is synthesized in the metabolism of the dietary amino acid methionine. Hcy undergoes metabolism via one of two pathways: remethylation to methionine by either methionine synthetase or methylene tetrahydrofolate reductase enzymes, or trans-sulfuration via cystathionine βsynthase enzyme. Vitamins B12 and folate are required for the first pathway and vitamin B6 is needed for the latter (Figure 1). Consequently, a high intake of methionine and low folate, vitamin B12, or vitamin B6 status may result in hyperhomocysteinemia. In the U.S. and a number of other countries, following studies that documented the effectiveness of folic acid in preventing neural tube defects, some grain and other products have been fortified with folic acid. This public health measure increased the overall intake of folic acid and improved RBC folate concentration. The higher folate status in the post-fortification era has also been associated with a reduction in Hcy concentration. Studies have also shown that in the post-fortification era, vitamin B12 is the predominant cause of hyperhomocysteinemia. Green and Miller,27 based on a study with 1,096 participants aged Z60 years, showed that when three different criteria of vitamin B12 deficiency were taken into account (o148 pmol/L, holotranscobalamin o35 pmol/L, creatinine 4115 mmol/L), 50.8% of those with hyperhomocysteinemia were diagnosed with B12 deficiency. By contrast, only 1.1% of hyperhomocysteinemia participants had an RBC folate concentration o365 nmol/L (indicative of folate deficiency). Only 0.3% of the risk for hyperhomocysteinemia was attributed to RBC folate o365 nmol/L, whereas 29.7%, 36.4%, and 41.5% of the attributable risk was related to serum vitamin B12 o148 pmol/L, holotranscobalamin o35 pmol/L, and creatinine 4115 mmol/L, respectively. They concluded that in the post-fortification period, vitamin B12 is the dominant risk factor for hyperhomocysteinemia.27 The hypothesis that the Hcy concentration is affected mainly by vitamin B12 status in groups of high folate consumers is supported by findings from other studies. Waldmann et al.28 assessed cardiovascular risk factors

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Table 1. Homocysteine Concentration and Prevalence of Hyperhomocysteinemia Among Vegetarian Adults

Study

Country

Sample (number and age)

Diet length of adherence

Vitamin B12 deficiency criteria

Prevalence of B12 deficiency

Vitamin B12 status

22

Hyperhomocysteinemia defined as Z or 4 15 lmol/L India

N¼78 (LV: n¼77; vegans: n¼1); age range 27–55 years

Not reported

415

76%

Herrmann et al. (2001)30

Germany

N¼41 (LV/LOV: n¼34, M age¼22 years [range 19.5–49.3]; vegans: n¼7, M age¼22 years [range 19–30])

Criteria of inclusion: 41 year of constant dietary pattern

415

LV/LOV: 12% Vegans: 57%

Bissoli et al. (2002)31

Italy

N¼45 (vegans: n¼31, M age¼45.8⫾15.8 years, LV: n¼14, M age¼48.5⫾14.5 years)

5 years

415

53.3%

All vegetarians: 23.9⫾21.3 Vegans: 26.9⫾24.1 LV: 17.4⫾11.1

Karabudak et al. (2008)32

Turkey

N¼26 Turkish women (SV: n¼7; LOV: n¼9; LV: n¼10); M age 29.0⫾8.84 years (range 20–50)

Diet duration 10.5⫾6.77

Z15

34.6%

12.6⫾5.97

Naik et al. (2013)33

India

N¼51 LV (men: n¼15; women: n¼36); M age¼27.6 years (range 26– 30)

Not reported

Z15

65%

21.1

Huang et al. (2003)34

Taiwan

N¼37 (vegans: n¼3; nLV: n¼18, LOV: n¼16) M age¼28.9 years (range 27.1– 30.7)

M¼5.6 years (from 1.3 to 19 years)

Z15.0

21.6%

13.2

Z12.0

78%

16.5⫾5.6 LV/LOV: 10.6 (6.4, 27.7) Vegans: 12.8 (5.9, 57.1)

Refsum et al. (2001)

29

LV/LOV: 11.0 Vegans: 15.2

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Krivosikova et al. (2010)35

Slovakia

N¼141 LOV; M age¼41.9 years (range 20–70)

“Long-term”—no details/definition


Germany Netherlands

N¼95 LV/LOV: n¼66; M age¼48 years (range 24–75), and vegans: n¼29; M age¼37 years (range 15–64)


412

LV-LOV: 38% Vegans: 67%

Elmadfa et al. (2009)22

Austria

N¼78 (vegans: n¼42 and LOV: n¼36; age not reported

Not reported

412

Vegans: 66% Vegetarians: 52%

Not reported


Germany

N¼164 LV/LOV: n¼114, M age¼50 years (range 35–71) and vegans: n¼50, M age¼44 years (range 20–66)

Not reported

412

Vegans: 62% LV/LOV: not reported

LV/LOV: 10.9 Vegans: 13.0

Majchrzak et al. (2006)38

Austria

N¼78; vegan: n¼42, M age¼30.7⫾9.9 and LOV: n¼36, M age¼34.2⫾13.6

67% of vegetarians and vegans followed their own diet for at least 5

Z12

Vegan: 65.9% LOV: 52.8%

Vegan men: 15.72⫾5.94; vegan women: 17.25⫾10.06 LOV men: ¼17.01⫾6.47; LOV women: 12.80⫾4.56 (continued on next page)


Hyperhomocysteinemia defined as Z or 412 lmol/L

June 2015

Table 1. Homocysteine Concentration and Prevalence of Hyperhomocysteinemia Among Vegetarian Adults (continued)

Study

Country


Sample (number and age)



Vitamin B12 status

Geisel et al. (2005)39

Germany

N¼71 LV/LOV: n¼48 M age¼53 years (range 21–75) and vegans: n¼23, M age¼51 years (range 28–76)


412

45%

Obeid et al. (2002)40

Germany Netherlands

N¼113 LV/LOV: n¼64, Vegan: n¼29, SV: n¼20), M (SD) age¼46 (15)

Not reported

412

36% for the entire sample


Germany, Netherlands

N¼111; M age¼46 (18–73)

Not reported

412

36%

10.4

410

Strict vegans: 71.1%; 57.1%

Strict vegans: 13.3 (range 5.97–82.0) Moderate vegans: 11.1 (range 3.60–25.7)

LV/LOV: from Germany: 14.4 Vegans from Germany: 15.9 Indian LOV: 14.0

LV/LOV: 11.1 (range 6.3–21.8) Vegans ¼ 13.0 (range 5.5–38.0) LV/LOV: 10.5; Vegans¼12.8 SV¼8.7

Waldmann et al. (2005)

Germany

N¼154 vegans (strict vegans: n¼98, M age 43.4⫾15.4; and moderate vegans: n¼56), M age 45.7⫾14.2 years

M diet duration 7.70⫾6.40 – strict 5.06⫾4.03 – moderate

No specified definition of hyperhomocysteinemia 42

Germany, Oman

n¼54 LV/LOV from Germany M age 50 years, n¼23 Vegans from Germany M age¼45 years, and n¼19, Indian LOV M age¼45 years

Criteria of inclusion: Z2 year of constant dietary pattern

Not reported

Not reported

Mezzano et al. (1999)43

Chile

N¼26 LV/LOV: n¼23 and vegans: n¼3; women: n¼14 and men: n¼12) M age¼39⫾12.7 years

41 year

Not reported

Not reported

13.5

Hung et al. (2002)44

Taiwan, Buddhist

N¼45 LV women; age range 31–45 years

Not reported

Not reported

Not reported

11.20⫾4.27

Su et al. (2006)45

Taiwan

N¼57 healthy postmenopausal women (vegan: n¼51 and vegetarian: n¼6); M age¼59.2⫾6.4 years

M diet duration¼ 10.4⫾4.2

Not reported

Not reported

11.0⫾3.3

Chen et al. (2008)46

Taiwan

N¼99; M age 51.24⫾8.88 years

Not reported

Not reported

Not reported

10.97⫾6.69

M diet duration¼ 10.8⫾6.0 years

Not reported

Not reported

10.8⫾3.2

4.2 years

Not reported

Not reported

7.9

Herrmann et al. (2009)

Su et al. (2011)

47

Haddad et al. (1999)48

N¼49 healthy postmenopausal LOV women; M age¼58.6⫾6.0 years U.S.

b

N¼25 vegans; age range 20–60 years; 9 used B12 supplements þ 4 used multivitamin supplements

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LOV, lacto-ovo-vegetarians; LV, lacto-vegetarians; SV, semi-vegetarians.


Hyperhomocysteinemia defined as 410 lmol/L 28

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Table 2. Vitamin B12 Status Among Vegetarian Adults

Study

Country

Sample (Number, and age)




75%

Vitamin B12 status

Methylmalonic acid M MMA¼0.53 μmol/L

India

N¼78 (LV: n¼77; vegans: n¼1), age range 27–55 years

Not reported

MMA4260 nmol/L


Germany



MMA4271 nmol/L


LV/LOV: M MMA¼205 Vegans: M MMA¼246



N¼95 LV/LOV: n¼66, M age¼48 years (range 24–75), and vegans: n¼29, M age¼37 years (range 15–64)


MMA4271 nmol/L

LV/LOV: 68% Vegan: 83%

LV/LOV: M serum B12¼192 (range 127–450) Vegans: M serum B12¼148 (range 99–314) pmol/L

Obeid et al. (2002)40


N¼113 LV/LOV: n¼64, vegan: n¼29, SV: n¼20), M (SD) age¼46 (15)

Not reported

MMA4271

58% for the entire sample

LV/LOV: M serum B12¼192 pmol/L, MMA¼355 nmol/L Vegans: M serum B12¼148 pmol/L, MMA¼708 nmol/L SV: M serum B12¼218 pmol/ L, MMA¼206 nmol/L



N¼111; M age¼46 (18–73)

Not reported

MMA4271 nmol/L

60%

M MMA¼356 nmol/L


U.S.

N¼25 vegans, age range 20–60 years; 9 used B12 supplements þ 4 used multivitamin supplements

4.2 years

MMA4376 nmol/L

20%

M MMA¼316 nmol/L


Germany

N¼164 LV/LOV: n¼114, M age¼50 years (range 35–71) and vegans: n¼50, M age¼44 years (range 20–66)

Not reported

MMA (value not specified)

LV/LOV:57% Vegans:74%

LV/LOV: M MMA¼308 pmol/L Vegans: M MMA¼698

HoloTC

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Refsum et al. (2001)29

India

N¼78 (LV: n¼77; vegans: n¼1), age range 27–55 years

Not reported

HoloTC IIo35 pmol/L

76%



N¼95 LV/LOV: n¼66, M age¼48 years (range 24– 75), and vegans: n¼29; M age¼37 years (range 15–64)


HoloTC IIo35 pmol/L

LV/LOV: 77%, vegan: 92%

M holoTC II¼19.0 pmol/L

LV/LOV: M serum B12¼192 (range 127–450) Vegans: M serum B12¼148 (range 99–314) pmol/L (continued on next page)


Refsum et al. (2001)29

June 2015

Table 2. Vitamin B12 Status Among Vegetarian Adults (continued)

Study

Country





Germany

N¼164 LV/LOV: n¼114, M age¼50 years (range 35–71); and vegans: n¼50, M age¼44 years (range 20–66)

Not reported

HoloTC IIo35 pmol/L


Germany Netherlands

N¼111; M age¼46 (18–73)

Not reported

HoloTC IIo35 pmol/L

Prevalence of B12 deficiency LV/LOV: 61% Vegans: 76%

Vitamin B12 status LV/LOV: M holoTC II¼31 pmol/L Vegans: M holoTC II¼13 pmol/L

72%

M holoTC II¼23 pmol/L

LV/LOV: M serum B12¼180 pmol/L; HoloTC II¼40 pmol/ L; M MMA¼273 Vegans: M serum B12¼145 pmol/L; HoloTC II¼22 pmol/ L; M MMA¼695

Status assessed by both MMA and holoTC Germany

N¼71 LV/LOV: n¼48 M age¼53 years (range 21–75) and vegans: n¼23, M age¼51 years (range 28–76)


Both present: HoloTC IIo35 pmol/L and MMA4271 nmol/L

58%


Germany Oman

n¼54 LV/LOV from Germany, M age 50 years; n¼23 vegans from Germany, M age¼45 years; and n¼19 LOV from India, M age¼45 years

Criteria of inclusion: Z2 year of constant dietary pattern

Both present: holoTCo35 pmol/ L and MMA4271 nmol/L

German vegetarians¼66% Indian vegetarians¼69%

LV/LOV: from Germany: HoloTC II¼22; MMA¼424 n/ mol/L; M serum B12¼163 pmol/L Vegans: HoloTC II¼20; MMA¼727 n/mol/L; M serum B12¼138 pmol/L Indian LOV: HoloTC II¼18; MMA¼723 n/mol/L; M serum B12¼107 pmol/L


Geisel et al. (2005)39

Serum Vitamin B12 Refsum et al. (2001)29

India

N¼78 (LV: n¼77; vegans: n¼1), age range 27– 55 years

Not reported

Serum B12o150 pmol/L

60%

Naik et al. (2013)33

India

N¼51 LV (men: n¼15 women: n¼36); M age¼27.6 years (range 26–30)

Not reported


56.9%

Waldmann et al. (2005)28

Germany

N¼154 vegans (strict vegans: n¼98, M age 43.4⫾15.4 and moderate vegans: n¼56), M age 45.7⫾14.2 years

M diet duration 7.70⫾6.40 – strict 5.06⫾4.03 – moderate


Strict vegan¼86.5% Moderate vegans¼69.1%

M serum B12¼124 pmol/L

M serum B12¼130 pmol/L; M holoTC II¼19.6 Strict vegan: M serum B12¼130 pmol/L Moderate vegans: M serum B12¼187 pmol/L (continued on next page)

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e18


Study

Country





Vitamin B12 status

Mezzano et al. (1999)43

Chile

N¼26 (LV/LOV: n¼23, and vegans: n¼3; women: n¼14 and men: n¼12) M age¼39⫾12.7 years

41 year

First criteria: serum B12o100 pg/mL Second criteria: serum B12o200 pg/mL


U.S.

N¼25 vegans; age range 20–60 years; 9 used B12 supplements þ 4 used multivitamin supplements

4.2 years



Germany





LV/LOV: M serum B12¼253pmol/L Vegans: M serum B12¼217pmol/L

Bissoli et al. (2002)31

Italy

N¼45 (vegans: n¼31, M age¼45.8⫾15.8 years; LV: n¼14, M age¼48.5⫾14.5 years)

5 years


Vegans: 47.8% LV: 33.3%

M serum B12¼171.2⫾73.6 pmol/L

Karabudak et al. (2008)32

Turkey

N¼26 Turkish women (SV: n¼7, LOV: n¼9, LV: n¼10), M age 29.0⫾8.84 years (range 20–50)

Diet duration 10.5⫾6.77


26.9%

M serum B12¼200.5⫾137.28 pmol/L

Krivosikova et al. (2010)35

Slovakia

N¼141 LOV; M age¼41.9 years (range 20–70)

“Long-term”—no details/ definition

Serum B12r220 pmol/L

47%


Elmadfa et al. (2009)22

Austria

N¼78 (vegans: n¼42 and LOV: n¼36); age not reported

Not reported

Serumo110 pmol/L

Majchrzak et al. (2006)38

Austria

N¼78 (vegan: n¼42, M age¼30.7⫾9.9; and LOV: n¼36, M age¼34.2⫾13.6)

67% of vegetarians and vegans followed their own diet for at least 5 years




N¼111; M age¼46 (18–73)

Not reported


First criteria¼26.9% Second criteria¼80.8% 12%

LOV¼0% Vegan¼2.4%


Not reported

LOV: M serum B12¼238.54⫾99.1 pmol/L Vegans: M B12¼203.17⫾101.5 pmol/L

www.ajpmonline.org

30%


Not reported


B12 status without reported deficiency criteria or deficiency rate Hung et al. (2002)44

Taiwan, Buddhist

N¼45 LV women Age range 31–45 years

Not reported

Not reported

(continued on next page)


Vegans¼2.4%

M serum B12¼166⫾89 pg/ mL

June 2015

M serum B12¼191.8 pmol/L range 164.0–220.0 HoloTC II, holotranscobalamin; LOV, lacto-ovo-vegetarians; LV, lacto-vegetarians; MMA, methylmalonic acid; SV, semi-vegetarians.

Not reported M¼5.6 years (from 1.3 to 19 years) N¼37 (vegans: n¼3, LV: n¼18, LOV: n¼16) M age¼28.9 years (range 27.1–30.7) Taiwan Huang et al. (2003)34

Not reported

M serum B12¼273.4⫾184.8 pmol/L Not reported M diet duration¼10.8⫾6.0 years N¼49 healthy postmenopausal LOV women, M age¼58.6⫾6.0 years Su et al. (2011)47

Not reported

M serum B12¼265.2⫾179.3 pmol/L Not reported Not reported M diet duration¼10.4⫾4.2 N¼57 healthy postmenopausal women (vegan: n¼51 and vegetarian: n¼6); M age¼59.2⫾6.4 years Taiwan Su et al. (2006)45

Sample (Number, and age) Country Study





Vitamin B12 status


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among 154 German vegans. The group was divided into strict and moderate (a maximum of 5% of the ingested energy was derived from eggs, milk, or dairy products) vegans. Elevated Hcy concentrations (410 μmol/L) were present in 71.1% of strict vegans and 57.1% of moderate vegans. Neither RBC folate nor vitamin B6 predicted Hcy concentration. Vitamin B12 was the only predictor of Hcy concentration among these participants (r’s¼–0.684, po0.001, vs r’s¼0.033, po0.689, for RBC folate; r’s¼ –0.08, po0.342 for vitamin B6). Hcy concentration 410 μmol/L was found among 66% of the study participants (combined strict and moderate vegans), of which 67% had a serum B12 concentration of o150 pmol/L and 93% had a concentration o250 pmol/L. Similar findings were reported by Herrmann and colleagues36: Folate deficiency, defined as serum folate o7 nmol/L, was not found among any of the 66 LVs or LOVs or among the 29 vegans. They did, however, find vitamin B12 to be the strongest predictor of Hcy (β coefficient for holotranscobalamin II¼–0.237, po0.001). Karabudak et al.32 found a similar association between vitamin B12 (regression correlation¼–0.969, po0.001) but not folate (regression correlation¼0.004, po0.488) and serum Hcy among 26 Turkish women (mean [SD] age¼29.0 [8.84] years). Serum vitamin B12 among 26 vegetarians from Chile was associated with Hcy in a study conducted by Mezzano and colleagues43 (Spearman correlation for serum B12¼–0.46, p¼0.017). They did not find statistically significant correlations between Hcy and serum folate (r¼–0.03, p¼not significant [NS]); pyridoxal-5-phosphate (B6) (r¼0.27, p¼NS); or creatinine (r¼0.27, p¼NS). Serum vitamin B12 concentration was the most significant predictor of Hcy among the 31 vegans and 14 vegetarians (r¼–0.776, po0.001) included in a study of Bissoli et al.31 The correlation between serum folate was much weaker (r¼–0.34, po0.05). These authors also concluded that their results supported the hypothesis that poor vitamin B12 status appears to explain hyperhomocysteinemia among vegetarians.

Homocysteine and Atherogenesis Studies have documented several mechanisms for the role of hyperhomocysteinemia in atherogenesis, including auto-oxidation of Hcy, which leads to synthesis of compounds associated with the initiation of atherogenic processes, such as superoxide, hydrogen peroxide, superoxide anion, and hydroxyl radicals.49 These compounds have been implicated in atherogenesis via oxidation of low-density lipoprotein, suppression of nitric oxide synthesis, arterial stiffness, endothelial inflammation, and foam cell formation.49,50 Chronic hyperhomocysteinemia

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Figure 1. Pathway of homocysteine metabolism.

reduces the activity of superoxide dismutase, an enzyme that is important in the dismutation of superoxide radicals.51 Elevated serum Hcy concentrations result in suppression of endothelium-derived nitric oxide synthesis. Nitric oxide plays a critical role in CVD development by several mechanisms, including vasoanticonstricting and vaso-antiaggregating effects.52 Van Campenhout and colleagues53 found that Hcy is present in advanced atheromas. They further documented a positive association between total Hcy concentration and severity of aortic calcification. Mahalle et al.54 reported a negative association between vitamin B12 and a positive association between Hcy and triglycerides, very low-density lipoprotein, and C-reactive protein, respectively. They also found a positive association of B12 and an inverse association of total Hcy with high-density lipoprotein. The link between hyperhomocysteinemia and CVD is supported by six of seven recent meta-analyses.18,19,55–59 These meta-analyses included data from prospective and case-control studies.18,19 They also included data on the impact of Hcy lowering on intima-media thickness and flow-mediated vasodilation.55,56,59

Vitamin B12, Red Blood Cell Distribution Width, Mean Corpuscular Volume, and Circulatory Health Problems In addition to vitamin B12 deficiency–induced hyperhomocysteinemia, deficiency of this nutrient may increase the risk of circulatory health problems via its role in macrocytosis. Elevated MCV and RDW are symptoms of vitamin B12 deficiency. RDW has been associated with several circulatory problems. Tonelli and colleagues60 reported a significantly increased risk of fatal coronary disease and nonfatal myocardial infarction (8% increase in risk for each 1% increase in RDW); stroke (20% increase for each 1% increase in RDW); and symptomatic heart failure (15% increase for each 1%

increase in RDW). Dabbah et al.61 found a progressively increased risk of death among patients with acute myocardial infarction with each of the higher quintiles of RDW compared to the lowest quintile. The risk among the extreme quintiles was 2.8 (95% CI¼1.6, 4.7, p for trendo0.0001). RDW has been associated with morbidity and mortality among heart failure patients from the North American Candesartan in Heart Failure: Assessment of Reduction in Mortality and Morbidity (CHARM) Program (hazard ratio¼1.17 per 1-SD increase, po0.001) and the Duke Databank (hazard ratio¼1.29 per 1-SD increase, po0.001) cohorts.62 Petal and colleagues63 reported an increased death from CVD for each quintile of RDW among individuals aged Z45 years who participated in the 1988–1994 National Health and Nutrition Examination Survey. The risk for the fifth quintile compared to the first quintile was 2.9, 2.5, and 2.1, respectively, for each of the three reported statistical models (Model 1 adjusted for age, sex, and ethnicity; Model 2 additionally adjusted for education, BMI, smoking status, hospitalizations, renal function, hemoglobin concentration, MCV, and Creactive protein; and Model 3 additionally adjusted for iron, folate, and vitamin B12 deficiency risk factors). Mueller et al.64 showed an increased risk of peripheral artery disease among patients with elevated MCV.

Discussion The available data strongly support the hypothesis that hyperhomocysteinemia is associated with atherogenesis. Although Hcy concentration is a function of several nutrients, the data equally strongly support that among individuals with low methionine, adequate vitamin B6, and high folate intake, low vitamin B12 status is the main predictor of hyperhomocysteinemia.28 Furthermore, low vitamin B12 status may increase the mortality and morbidity from CVD due to factors other than hyperhomocysteinemia, such as elevated RDW and MCV. www.ajpmonline.org


Consequently, vegetarians with low vitamin B12 status are predisposed to developing circulatory health problems regardless of their favorable profile of traditional heart disease risk factors. In fact, because of their low risk of CVD mediated by traditional risk factors, it may be correct to suggest that low vitamin B12 status and hyperhomocysteinemia may play an even bigger role in developing circulatory problems among vegetarians compared with their non-vegetarian counterparts. Although vegetarians do show a modest reduction in CVD (Table 3), the described research findings support the hypothesis that the compromised vitamin B12 status that leads to hyperhomocysteinemia, elevated RDW, and MCV largely negates these benefits.23 The hypothesis that low vitamin B12 and hyperhomocysteinemia are risk factors for CVD is supported by data from vegan individuals.12,28 Vegans consistently have been shown to have the lowest vitamin B12 status and highest Hcy concentration. At the same time, data show that vegans have the best profile of all traditional risk factors (serum glucose concentration, blood pressure, serum lipids, and weight status). Yet, though some studies have shown a reduced risk of circulatory health problems among vegans compared with non-vegetarians, other studies have reported a higher risk of CVD among vegans compared with LOV and even non-vegetarians (Table 3).12,28 Results of six of seven recent meta-analyses support the association between hyperhomocysteinemia and increased risk of CVD.18,19,55–59 However, only one of two meta-analyses of randomized trials that assessed the impact of Hcy lowering on CVD events supports the association.58,59 Several explanations have been suggested for the lack of reduction in CVD outcomes in interventional studies with reduced Hcy concentration. Is it possible that folic acid, B12, and vitamin B6 supplements carry an undetermined adverse effect that negates any benefits associated with Hcy reduction?65 Is it possible that folic acid supplements stimulate cell proliferation and plaque formation? Would the results of interventional studies be different if the intervention to lower Hcy concentration with folic acid would consist of adequate food folate intake rather than synthetic folic acid? Is it possible that Hcy concentration is a surrogate for another causative agent?65 Taking a closer look at the individual studies included in the meta-analysis of Clarke and colleagues66–72 may also, at least in part, explain the lack of reduction in CVD events. Folic acid supplementation was included in each of the studies as the intervention agent. Although reduction in Hcy concentration was reported among the intervention group(s) in each of the groups that used folic acid and B12 supplements, in only two of these June 2015

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studies the post-intervention mean Hcy concentration was substantially lower than 10 μmol/L; in two of them it was about 10 μmol/L, and in two it was higher than 10 μmol/L. As stated previously, Jacobsen17 proposed that an Hcy concentration of 10–15 mmol/L constitutes a substantial CVD risk factor and that this risk should be considered as a continuum: the higher the concentration, the higher the risk. If Jacobsen were correct, the postintervention mean Hcy concentration in a bulk of the participants of these studies would have been too high to expect any reduction in CVD outcomes. Thus, results of the trials that did not show improvements/reduction in CVD outcomes in secondary prevention among highrisk individuals may at least in part be attributed to inadequate intervention results in terms of postintervention Hcy concentration. Although the metaanalysis of Clarke et al.59 did not find a statistically significant improvement in cardiovascular event occurrence or mortality within the median of 5 years of using folic acid supplements to lower Hcy concentration (these studies also used B12 supplements), ample evidence exists to support a conclusion that hyperhomocysteinemia promotes oxidative stress, inflammation, thrombosis, endothelial dysfunction, and cell proliferation and thus should be considered an atherogenic agent.59,60 Herrmann and colleagues36 suggested that their findings, which showed the association between vitamin B12 and hyperhomocysteinemia, support the following recommendations: (1) to screen all vegetarians for vitamin B12 deficiency and (2) that all vegetarians, regardless of the type of vegetarian diet they adhere to, use vitamin B12 supplements. Taking an adequate dose of supplemental vitamin B12 would virtually eliminate a deficiency among vegetarians. Furthermore, findings reported by Kwok et al.73 showed that 500 μg/day B12 supplementation for 12 weeks used by asymptomatic vegetarians not only statistically significantly increased serum B12 (mean baseline serum B12¼134.0 pmol/L vs 379.6 pmol/L at 12 weeks, p¼0.0001) and reduced Hcy (mean baseline Hcy¼16.7 μmol/L vs 11.3 μmol/L at 12 weeks, p¼0.01) concentrations but also improved flow-mediated dilation of the brachial artery and intima–media thickness of the carotid artery. Unfortunately, recommending taking B12 supplements may meet opposition among vegetarians because misconceptions regarding this nutrient are prevalent. Many individuals still hold on to the old myth that deficiency of this vitamin is rare and occurs only in a small proportion of vegans. This myth is accepted not only by some vegetarians but also by some researchers.74 Future studies with vegetarians should focus on identifying ways of convincing vegetarians to routinely take vitamin B12 supplements in order to prevent a deficiency.

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Table 3. Incidence and Mortality of Cardiovascular Disease, Ischemic Heart Disease, and Stroke Among Vegetarians and Non-Vegetarians

Reference

Country

Incidence/prevalence vegetarians and/or vegan versus non-vegetarians

Study design Circulatory/cardiovascular disease

United Kingdom

EPIC-Oxford Study; N¼55,041 (32% vegetarians); age range¼20–89 years

DRR for all circulatory deaths¼0.93 (95% CI¼0.65, 1.32)

Key et al. (2009)75

United Kingdom

EPIC-Oxford Study; N¼47,254; age range¼20–89 years

DRR for circulatory diseases¼0.97 (95% CI¼0.78, 1.21)

Orlich et al. (2013)12

North America

Adventist Health Study-2; N¼73,308; vegans: n¼5,548 and LOV: n¼21,177; M age: vegans¼57.9 years; LOV¼57.5 years; non-vegetarians¼55.9 years

DHR for CVD: Vegans¼0.91 (95% CI¼0.71, 1.16); vegan men¼0.58 (95% CI¼0.38, 0.89), vegan women¼1.18 (95% CI¼0.88, 1.60) LOV¼0.90 (95% CI¼0.76, 1.06); LOV men¼0.77 (95% CI¼0.59, 0.99), LOV women¼0.99 (95% CI¼0.81, 1.22)

IHD

www.ajpmonline.org

Key et al. (2003)13

United Kingdom

Health Food Shoppers Study N¼10,736 (43% vegetarians)

DRR for IHD mortality¼0.85 (95% CI¼0.71, 1.01)

Key et al. (2003)13

United Kingdom

Oxford Vegetarian Study; N¼11,045 (42% vegetarians)


Key et al. (2003)13

United Kingdom

EPIC-Oxford Study; N¼55,041 (32% vegetarians); participants’ age range: 20–89 years


Crowe et al. (2013)76

United Kingdom

EPIC-Oxford Study; N¼44,561 (34% vegetarians), age Z20 years

Hazard ratio¼0.68 (95% CI¼0.58, 0.81); the cumulative probability of IHD between ages 50 and 70 years was 6.8% for non-vegetarians compared with 4.6% for vegetarians

Key et al. (2009)75

United Kingdom

EPIC-Oxford Study; N¼47,254; age range: 20–89 years

DRR¼0.83 (95% CI¼0.59, 1.18)

Key et al. (1999)9

U.S.

Adventist Mortality Study N¼24,538; vegetarians: n¼10,258

DRR¼0.748 (95% CI¼0.63, 0.88)

Key et al. (1999)9

United Kingdom

Health Food Shoppers Study N¼9,878; vegetarians: n¼3,790

DRR¼0.97 (95% CI¼0.81, 1.16)

Key et al. (1999)9

U.S.

Adventist Health Study; N¼28,952; vegetarians: n¼8,003

DRR in AHS¼0.62 (95% CI¼0.53, 0.73)

Key et al. (1999)9

Germany

Heidelberg Study N¼1,757; vegetarians: n¼1,083

DRR¼0.45 (95% CI¼0.22, 0.95)

Key et al. (1999)9

United Kingdom

Oxford Vegetarian Study; N¼11,047; vegetarians: n¼4,674

DRR¼0.90 (95% CI¼0.68, 1.20) (continued on next page)


Key et al. (2003)13

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Table 3. Incidence and Mortality of Cardiovascular Disease, Ischemic Heart Disease, and Stroke Among Vegetarians and Non-Vegetarians (continued)

Reference

Country

Study design

Incidence/prevalence vegetarians and/or vegan versus non-vegetarians

U.S., United Kingdom, Germany

Combined effect of five studies Adventist Mortality Study, Health Food Shoppers Study, Adventist Health Study, Heidelberg Study, and Oxford Vegetarian Study; N¼76,172; vegetarians: n¼27,808

DRR¼0.93 (95% CI¼0.62, 0.94)

Orlich et al. (2013)12

North America

Adventist Health Study-2; N¼73,308; vegans: n¼5,548 and LOV: n¼21,177 M age; vegans¼57.9 years; vegetarians¼57.5 years; nonvegetarians¼55.9 years

Hazard ratio: Vegans¼0.90 (95% CI¼0.60, 1.33); vegan men¼0.45 (95% CI¼0.21, 0.94), vegan women¼1.39 (95% CI¼0.87, 2.24) LOV¼0.82 (95% CI¼0.62, 1.06); LOV men¼0.76 (95% CI¼0.52, 1.12) Vegan woman¼0.85 (95% CI¼0.59, 1.22)

Cerebrovascular disease Key et al. (2003)13

United Kingdom

EPIC-Oxford Study; N¼55,041 (32% vegetarians); participants’ age range: 20–89 years

DRR¼1.13 (95% CI¼0.65, 1.96)

Key et al. (2009)75

United Kingdom

EPIC-Oxford Study N¼47,254

DRR¼1.10 (95% CI¼0.77, 1.58)

Key et al. (1999)9

U.S.

Adventist Mortality Study; N¼24,538; vegetarians: n¼10,258

DRR¼0.65 (95% CI¼ 0.48, 0.87)

Key et al. (1999)9

United Kingdom

Health Food Shoppers Study N¼9,878; vegetarians: n¼3,790

DRR¼0.99 (95% CI¼0.78, 1.26)

Key et al. (1999)9

U.S.

Adventist Health Study; N¼28,952; vegetarians: n¼8,003

DRR¼0.93 (95% CI¼0.73, 1.19)

Key et al. (1999)9

Germany

Heidelberg Study; N¼1,757; vegetarians: n¼1,083

DRR¼1.69 (95% CI¼0.69, 4.15)

Key et al. (1999)9

United Kingdom

Oxford Vegetarian Study; N¼11,047; vegetarians: n¼4,674

DRR¼1.17 (95% CI¼0.76, 1.80)

Key et al. (1999)9

U.S., United Kingdom, Germany

Combined effect of five studies Adventist Mortality Study, Health Food Shoppers Study, Adventist Health Study, Heidelberg Study, and Oxford Vegetarian Study; N¼76,172; vegetarians: n¼27,808

DRR¼0.93 (95% CI¼0.74, 1.17)


Key et al. (1999)9

CVD, cardiovascular disease; DHR, death hazard ratio; DRR, death rate ratio; EPIC, European Prospective Investigation into Cancer and Nutrition; IHD, ischemic heart disease; LOV, lacto-ovo-vegetarian.

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e24


Limitations This study represents a non-systematic review of the literature and, therefore, may not have included all pertinent studies. To the author’s knowledge, no study to date has assessed the risk of CVD incidence or mortality among vegetarians based on vitamin B12 status. Only one published study assessed the impact of improving vitamin B12 status of vegetarians via the use of vitamin B12 supplements on CVD indices.73 Based on available studies, it is not possible to assess how much of the potential risk for CVD in vegetarians may be accounted for by inadequate vitamin B12 status. Any potential cardio-protective benefits of improved vitamin B12 status needs to be confirmed in additional studies.

Conclusions Although vegetarians have an improved profile of traditional heart disease risk factors in comparison to nonvegetarians, available findings show only a modest decrease in CVD prevalence. The low status of vitamin B12 among vegetarians that results in hyperhomocysteinemia, elevated RDW, and MCV predisposes them to CVD development and may negate the benefits associated with their traditional risk factor profile. Health professionals should find ways to convince vegetarians of the importance of improving their vitamin B12 concentration.11,26 No financial disclosures were reported by the author of this paper.

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49.

50.

51.

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