The Prevalence of Cobalamin Deficiency Among Vegetarians

REVIEW The prevalence of cobalamin deﬁciency among vegetarians assessed by serum vitamin B12: a review of literature

R Pawlak, SE Lester and T Babatunde

Individuals following vegetarian diets are at risk for developing vitamin B12 deficiency owing to suboptimal intake. As vitamin B12 is essential for the synthesis of nucleic acids, erythrocytes and in the maintenance of myelin, deficiency may result in a variety of symptoms. Some of these symptoms may be severe while others may be irreversible. The objective of this review was to assess vitamin B12 deficiency, based on reported serum vitamin B12, among individuals adhering to different types of vegetarian diets. A systematic literature search was carried out using multiple search engines including PubMed, Medline, CINAHL plus, ERIC, Nursing and Allied Health Collection and Nursing/Academic Edition. The inclusion criteria consisted of original studies that assessed serum vitamin B12, studies written in English, non-case studies and studies that reported actual percentages of vitamin B12 deficiency. Forty research studies were included. The deficiency prevalence among infants reached 45%. The deficiency among the children and adolescents ranged from 0 to 33.3%. Deficiency among pregnant women ranged from 17 to 39%, dependent on the trimester. Adults and elderly individuals had a deficiency range of 0–86.5%. Higher deficiency prevalence was reported in vegans than in other vegetarians. Thus, with few exceptions, the reviewed studies documented relatively high deficiency prevalence among vegetarians. Vegans who do not ingest vitamin B12 supplements were found to be at especially high risk. Vegetarians, especially vegans, should give strong consideration to the use of vitamin B12 supplements to ensure adequate vitamin B12 intake. Vegetarians, regardless of the type of vegetarian diet they adhere to, should be screened for vitamin B12 deficiency.

European Journal of Clinical Nutrition (2014) 68, 541–548; doi:10.1038/ejcn.2014.46; published online 26 March 2014

INTRODUCTION vitamin B12.1,5,6 Vegans, who do not ingest any foods of animal Vitamin B12 is a water-soluble vitamin that is found naturally in origin, are at the highest risk of developing a deficiency. meat and animal products. It contains a corrin ring with mineral Vegetarians, other than vegans, consume some foods that are a cobalt. Physiologic functions of vitamin B12 include erythro- natural source of vitamin B12 (e.g. milk, yoghurt, cheese, eggs). 2 poiesis, the synthesis and maintenance of myelin sheath and the Still, even they have limited natural sources of this vitamin. Even synthesis of nucleic acid (DNA).1,2 The current Recommended though some items such as cereal or soy products are fortified Dietary Allowance for vitamin B12 has been set by the Institute of with vitamin B12, studies have shown that vegetarians often have 2,7 Medicine at 2.4 μg per day for male and female subjects, aged 14 suboptimal vitamin B12 intake. years and older.2 Recommended intake amounts depend on age The aim of this review was to assess the prevalence of vitamin and physiological development and range from 0.4 μgin B12 deficiency among individuals adhering to vegetarian diets. infants 2.6 and 2.8 μg per day in pregnant and lactating women, The review is based on studies that reported percentage of respectively.2 vitamin B12 deficiency assessed by serum vitamin B12. According to data from 1999 to 2000 National Health and Nutrition Examination Survey, the median daily intake of vitamin B12 in the United States is 3.4 μg.1 Although this amount is higher than the recommended intake, deficiency of vitamin B12 can be MATERIALS AND METHODS 1–6 seen in high-risk individuals. These individuals include older A systematic literature search was carried out using multiple internet- adults and the elderly, individuals with gastrointestinal disorders based search engines including PubMed, Medline, CINAHL plus, ERIC, or those who have undergone gastrointestinal surgery and Health Source, Nursing and Allied Health Collection, Ebscohost and individuals following a vegetarian or vegan diet.1,2,5,6 Clinical Nursing/Academic Edition. To identify relevant literature, a title search symptoms of deficiency include fatigue, weakness, constipation, using combinations of the following key words was used, ‘B12’, loss of appetite, weight loss, numbness and tingling in hands ‘cobalamin’, ‘vegetarian’, ‘vegan’ and ‘deficiency’. Following the literature and feet, difficulty maintaining balance, depression, confusion, search, the titles and abstracts of the manuscripts were screened to assess 1,2,6 the eligibility of identified manuscripts. Inclusion criteria consisted of dementia, poor memory and soreness of the mouth or tongue. original studies that assessed serum vitamin B12 concentration, were Many individuals who adhere to vegetarian diets and do not written in English, were non-case studies and studies that reported an use vitamin B12 supplements can develop vitamin B12 deficiency, actual percentage of B12 deficiency. Studies that reported serum vitamin 7 regardless of the type of vegetarian diet they consume. This is in B12 values such as means or medians but did not report the percentage of part due to the fact that plant products do not naturally contain deficiency in their samples were excluded.

Department of Nutrition Science, East Carolina University, Greenville, NC, USA. Correspondence: Dr R Pawlak, Department of Nutrition Science, East Carolina University, Rivers West 337, Greenville, NC 27858, USA. E-mail: [email protected] Received 23 August 2013; revised 26 November 2013; accepted 11 February 2014; published online 26 March 2014 B12 deficiency in vegetarians R Pawlak et al 542 RESULTS Outcomes The literature search yielded a total of 1097 manuscripts. One Pregnant women. One study assessed vitamin B12 status in thousand and fifty-seven studies were excluded because they did vegetarian pregnant women.27 This study was conducted in not meet the inclusion criteria. Thus, 40 studies remained and were Germany and included 27 lacto-ovo-vegetarian participants with a included in the review.8–47 The samples included in these studies mean age of 30.8 ± 0.9 years. For the first, second and third consisted of a wide range of individuals at different life stages, trimesters, serum B12 deficiency was defined as o130 pmol/l, including infants,47 children and adolescents,12,14,22,25,31 pregnant o120 pmol/l and o100 pmol/l, respectively. Approximately 33% women,27 adults8–10,12,13,15–24,26,28–30,32–39,41–46 and the elderly.11,12,40 of women in the first trimester of pregnancy were diagnosed as Most studies included both sexes, but 10 studies assessed vitamin deficient. The deficiency rate for women in the second and third 27 B12 status in female subjects only8,17,21,24,25,27,33,40,41,45 and three trimesters was 17% and 39%, respectively. included male subjects only.9,18,42 Individuals in these studies were from several countries, including New Zealand,8,25,39 Jordan,16 12,19 32 10,30,37,43–46 18,34 15 Infants and toddlers. One study evaluated serum vitamin B12 in Israel, Italy, United States, India, Canada, infants and toddlers, aged 10.1–20.4 months (n = 47). These United Kingdom,9 Germany,26–29,35 Netherlands,11,29,47 Austrailia,38 33 21 42 24 17,36,41 31 children were born to women who adhered to a macrobiotic Taiwan, Korea, Chad, Turkey, Slovak Republic, Sweden, – 14,22,40 20 23 43 13 diet. Most were exclusively breastfed for 4 8 months. The rate of China, Poland, Austria, Finland and Thailand. Study vitamin B12 deficiency, defined as serum vitamin B12 o136 participants were from various ethnic, religious and cultural back- pmol/l, was reported in 45% of these children.47 grounds, including Asian Indians or South Asians living in the United States,30 New Zealand8,25 or Canada;15 Buddhists;21,33 and members of the Seventh-day Adventist Church.38 Children and adolescents. Five of the reviewed studies examined Participants in each study adhered to a variety of vegetarian diet vitamin B12 status in children and adolescents (aged 4–20 types. Table 1 summarizes specific diet characteristics of the studies. years).12,14,22,25,31 Two of these studies included individuals who 12,31 Vegetarian categories consisted of semivegetarians (including ‘low adhered to a vegan diet. One of the two vegan studies meat eaters’ and ‘plant-based diet’),17,24,35,42 vegetarians,8,9,19,23,30,34,39 indicated that there was no incidence of vitamin B12 deficiency ‘non-meat eaters’,25 lacto-vegetarians,11,13,15,18,21,22,24,29,30,35,36 lacto- among children 8–19 years of age (n = 5) at serum vitamin B12 12 ovo-vegetarians,11,13–15,22,24,27,29,30,32,33,35,36,38,40,41 ovo-vegetarians,13,22 o96 pmol/l. Among the vegan adolescents (aged 16–20 years, vegans,8,12,15,16,20,22,23,26,28–33,35–40,43,46 individuals adhering to a n = 30) included in the other study, 10% had a deficiency, defined 31 22 14 macrobiotic diet44,45,47 and those adhering to raw food diets.10,43 as vitamin B12 o95 pmol/l. Deficiency rates of 0% and 3.9% The semivegetarians were defined as individuals consuming no red were reported in two studies that included Chinese children (mean – meat products, but occasionally ingesting white meat (poultry) age 5.2 ± 1.5 years, n = 21) and adolescents (aged 4 14 years, fi 17,24 fi n = 51), respectively. These two studies used similar serum vitamin and sh. A similar de nition was used in a study that included 22 ‘ ’ 35 ‘ B12 values to define deficiency, o156 pmol/l and o160 pmol/l, low meat eaters , and among those described as eating a plant- 14 based diet’.42 Two additional studies described their participants respectively. Finally, researchers reported a 33.3% of vitamin B12 fi o as ‘vegetarian’, but included in their sample individuals with de ciency (serum vitamin B12 170 pmol/l) in a small sample of – 25 consumption of poultry or fish at a frequency of no more than one migrant Indian girls (n = 6), aged 9 12 years living in New Zealand. time per week39 or between one and four times per month.30 The macrobiotic diets were described as one consisting of whole-grain Adults and elderly. The majority of the reviewed studies assessed cereal (50–60%), soups (5%), vegetables (20–25%) and beans and B12 deficiency in non-pregnant adults, with ages ranging from 18 44,45,47 sea vegetables (5–10%). Individuals included in one of these to 97 years.8–13,15–24,26,28–30,32–46 Participants in these studies diets were reported to consume occasionally small amounts of adhered to a variety of different vegetarian diets. seafood and little or no dairy or eggs.44 Participants of another Vegans: Thirteen studies specifically reported deficiency rates study were described as using a very limited amount of products of for vegan subjects.9,12,16,20,23,26,28,29,32,36,37,43,46 The age of animal origin.47 Individuals included in another of these studies vegan subjects varied from 18 to 84 years. Vitamin B12 avoided intake of all animal products including dairy and eggs.45 In deficiency in vegan subjects ranged from 0% to addition, one of the studies was based on results obtained from 86.5%.9,12,16,20,23,26,28,29,32,36,37,43,46 Adeficiency rate of 0% (serum individuals who adhered to the ‘Hallelujah diet’. This diet was vitamin B12 o142 pmol/l) was reported in one study that described as a pure vegetarian diet using raw fruits and vegetables, included vegan subjects whose diet consisted of vitamin B12- nuts, seeds, limited cooked tubers and whole-grain products.10 fortified foods. The same study reported vitamin B12 deficiency in All studies reviewed included the assessment of vitamin B12 20% of vegan subgroup that did not consume fortified foods.20 status using serum measurements. Various specific methods of More detailed results of these studies are presented in Table 1. serum vitamin B12 assessment were used, including radio- Raw food vegan diets: Donaldson10 reported 12.2% of the immunoassays, immunochemiluminometric methods, microparti- sample having serum value o147 pmol/l and 76% of the sample cle assays, chemiluminescence immunoassays and microbiologial with serum vitamin B12 o221 pmol/l. Participants of this study assays. Five studies either did not report their specific lab methods adhered to a Hallelujah diet, of which 85% of ingested foods were or only reported that their serum B12 results were measured using raw foods. Rauma et al.43 reported a 57% deficiency rate among ‘standard’ or ‘validated’ laboratory procedures.12,13,20,26,41 individuals consuming a ‘living raw food diet’. As with a diverse sample of laboratory methods used to Vegetarians (lacto-, ovo- and lacto-ovo-) and vegans combined: evaluate serum B12, the included studies also contained a diverse Twenty studies reported the prevalence of vitamin B12 deficiency researcher-defined serum limit that was used to categorize for lacto-vegetarians, ovo-vegetarians, lacto-ovo-vegetarians and their subjects as vitamin B12 deficient. Most studies defined vegans as combined samples.8,9,11–13,15,18,19,21–23,29,30,32–34,36,38,40,41 vitamin B12 deficiency as serum concentration of o130–150 Prevalence of deficiency ranged from 0 to 81%. This prevalence was pmol/l.9,11,12,16,17,19–22,25,27,28,30,34,35,38,40,41,45–48 The lowest defined highly dependent on the definition used by researchers with deficiency was serum concentration below 95 pmol/l.13,32 The respect to what value constituted a deficiency. highest defined deficiency was defined as serum vitamin B12 Macrobiotic diet: Two studies investigated vitamin B12 status in o250 pmol/l.27 In addition, two different units of measurement adults adhering to a macrobiotic diet. Both studies were were used in most studies with regard to serum vitamin B12. conducted with subjects from the United States and both used Serum concentrations were reported either as pmol/l or pg/ml. a serum vitamin B12 value o148 pmol/l to define deficiency.

Table 1. Detailed information of reviewed studies

Reference Country/ethnicity Participants Diet characteristics Assessment criteria used Rate of deﬁciency

o150 pg/ml or o200 pmol/l Majchrzak et al.23 Austria N = 36 vegetarians (10 Vegetarian—no meat, fish, poultry Serum B12 reference value 0% Vegetarian males and 26 females) Vegan avoided all products of animal defined according to 2.4% Vegans Mean age 34.2 ± 13.6 years origin Sauberlich, normal >147 N = 42 vegans (21 males Diet duration: pmol/l, deficiency o110 and 21 females) 67% at least 5 years pmol/l Mean age 30.7 ± 9.9 years 25–31% 1–5 years Radioimmunoassay 2–8% o1year Lee and Korea, Buddhists N = 54 Buddhist nuns Ages Lacto-vegetarian Serum B12 deficiency o150 1.9% Krawinkel21 21–44 years Diet duration >3years pmol/l Electrochemiluminescence immunoassay Leung et al.14 Chinese N = 51 children (24 boys Lacto-ovo-vegetarian diet (includes Serum B12 normal range 3.9% (N = 2 ‘below and 27 girls) dairy, eggs) 160–625 pmol/l normal’) Ages 4–14 years Diet duration >2years Lab method not reported Yen et al.22 Chinese N = 21 parents (4 males and Vegetarian status included lacto-, Serum B12 deficiency o156 4.8% Parents 17 females) ovo-, lacto-ovo- and vegan pmol/l 0% Children Mean age 34.8 ± 4.5 years Diet duration: Standard competitive N = 21 children (7 boys and Parents—mean 8.8 years immunochemiluminometric 14 girls) Mean age 5.2 ± 1.5 Children—mean 4.5 years methods years Gilsing et al.9 United Kingdom N = 231 vegetarians Vegetarians defined as eating dairy Serum B12 biochemical Vegetarian: 7% deficient; 96% White N = 232 vegans but no meat or fish deficiency o118 pmol/l 17% depleted 4% Bangladeshi or (all males) Diet duration median 11 years depletion 118–149 pmol/l Vegan: 52% deficient; Chinese Mean age = 47 years Vegans ate no foods of animal origin Quantaphase II radioassay 21% depleted Diet duration median 7 years Herrmann et al.35 Germany N = 60 (14 males and 46 Lacto-ovo ate no meat, poultry, fish Serum B12 reference interval Lacto-ovo/lacto-vegan: females) Lacto—also excluded eggs (lacto- 156–674 pmol/l 6% Median age 22 years ovo/lacto-vegan N = 34) Chemiluminescence Vegan: 14% Vegan exclude all foods of animal immunoassay Low meat eaters: 11% origin (N = 7) Low meat eaters ate no red meat, but white meat or fish 1–2 × per week (N = 19) Diet duration defined as consistent habits for ⩾ 1 year Larsson and Sweden N = 30 (15 males and 15 Vegan diet defined as eating food of Adequate blood 10% (N = 3) Johansson31 females), plan origin concentrations of B12 mean age 17.5 ± 1.0 years Diet duration for least 6 months (and defined as 95–568 pmol/l planning to continue) Quantaphase II B12/folate radioassay Haddad et al.37 United States N = 25 (10 males and 15 Vegans defined as excluding meat, Serum B12 deficiency o150 12% (N = 3) females) fish, poultry, dairy products, eggs pmol/l Mean age 20.5 ± 2.5 years Diet duration mean 4.2 years Quantaphase II radioassay Donaldson10 USA N = 49 (17 males and 32 Adherence to Hallelujah diet defined Serum B12 deficiency o147 12.2% females) as pure vegetarian, with raw veg, fruit, pmol/l 2 Men (11.7%) Average age 55 ± 9 years raw nuts and seeds, limited cooked Immunochemiluminometric 4 Women(12.5%) tubers and whole-grain products method Diet duration mean 39 months Bar-Sella et al.12 Israel N = 36 (males and females) Vegan diet Serum B12 deficiency o130 13.9% Deficient (N = 5) Age 8–19 years: N = 5 Diet duration 5–35 years pg/ml Age 20–39 years: N = 5 Borderline 130–200 pg/ml Age 40–59 years: N = 13 Radioimmunoassay Age 60–79 years: N = 13 30.5% Borderline (N = 11) Ingenbleek and McCully42 Africa (Chad) N = 24 (rural males) Ages 18–30 Vegetarian diet Serum B12 lower 16.7% (N = 4) described as limit of normal plant-based, eat defined as ⩽ 140 very few dairy pmol/l foods, rarely eat Chemiluminescence meat, occasionally consume poultry and eggs Diet duration not reported Lowik et al.11 Netherlands; N = 40 (17 males and 23 Lacto-ovo vegetarian ate no meat, Serum B12 deficiency o138 22.5% Caucasian females) fish, poulty pmol/l 6 Men (35.3%) Mean age: Lacto-vegetarians also excluded eggs Lab method not available 3 Women (13.0%) Diet duration described as ‘32

Table. 1. (Continued )

Reference Country/ethnicity Participants Diet characteristics Assessment criteria used Rate of deﬁciency

Men—82.8 years (subjects) had been vegetarian for Women—81.1 years more than 45 years’ Gammon et al.8 South Asians living N = 34 females Vegetarian defined as absence of Serum B12 deficiency o150 23.5% Deficient in New Zealand Mean age 36 ± 10.1 years meat in diet pmol/l 35% Marginal status (India, Pakistan, Sri Diet duration not reported Marginal B12 status 150–221 Lanka, Fiji, other) pmol/l Radioassay Herrmann et al.29 Germany and N = 66 vegetarians Lacto-ovo—excluded meat, poultry, Low serum B12 26% Vegetarians Netherlands Median age 48 years fish concentration o156 pmol/l 52% Vegans N = 29 vegans Lacto—excluded exclude meat, Chemiluminescence Median age 37 years poltry, fish, egg immunoassay Males and females Vegan excluded all foods of animal origin Diet duration ⩾ 1 year Krajcovicova- Slovak Republic N = 62 vegetarians (24 Vegetarian not defined but included Serum B12 deficient 26% Vegetarians Kudlackova et al.36 males and 38 females) lacto- and lacto-ovo-vegetarians o179.0 pmol/l 78% Vegans Mean age 35.1 ± 0.9 years Vegan not defined Elecsys 2010 immunoassay N = 32 vegans (10 males Diet duration: and 22 females) Vegetarian mean 8.85 ± 0.33 years Mean age 41.5 ± 1.8 years Vegan mean 7.87 ± 0.41 years Karabudak et al.24 Turkey N = 26 (all females) Vegetarian duration ⩾ 2 years, Serum B12 deficiency ⩽ 150 26.9%* Mean age 29.0 ± 8.84 years included semiveg (no red meat, pmol/l *results combined all occasional poultry, fish), lacto-ovo- Solid-phase, vegetarian types veg and lacto-veg chemiluminescent Diet duration mean 10.5 ± 6.77 years competitive binding assay Waldmann et al.28 Germany N = 86 strict vegans (48.8% Strict vegan defined as ‘pure vegan Serum B12 deficiency o110 28.2% All vegans males and 51.2% females) diet’ pmol/l (52% ⩽ 156 pmol/l) Mean age 43.8 ± 15.6 years Moderate vegan—ate maximum of (also assessed at ⩽ 156 39.5% Strict vegans N = 45 moderate vegans 5% of total energy from eggs and/or pmol/l) (65.5% ⩽ 156 pmol/l) (35.6% males and 64.4% dairy products Chemiluminescence 6.67% Moderate vegans females) Diet duration: (37.8% ⩽ 156 pmol/l) Mean age 44.6 ± 15.0 years Strict vegan mean 7.14 ± 6.69 years Moderate vegan mean 4.69 ± 2.97 years Krajcovicova- Slovak Republic N = 52 (all females) Vegetarian defined as diet of Serum B12 deficiency o179 31% Kudlackova et al.17 Mean age 25.2 ± 0.4 years predominantly plant food, dairy, eggs pmol/l and ⩽ 2 times per months white meat Elecsys immunoassay (Roche (chicken, fish) test) Diet duration 9.7 ± 0.5 years Hung et al.33 Taiwan, Buddhists N = 45 (39 lacto-vegetarians Lacto-vegetarian consumed o240 ml Serum B12 deficiency 31% (N = 14) and 6 vegans) milk per day o147.8 pmol/l (all females) Vegan not defined Microparticle enzyme Ages 31–45 years Diet duration avg. 7.9 years immunoassay Koebnick et al.27 Germany N = 27 (pregnant women) Ovo-lacto-vegetarian defined as Serum B12 o130 pmol/l (1st 33% First tri Mean age 30.8 ± 0.9 years completely omit meat and meat tri); o120 pmol/l (2nd tri); 17% Second tri products o100 pmol/l (3rd tri) 39% Third tri Diet duration at least 3 years IMx cobalamin assay (all % extrapolated from (microparticle) graph—Figure 1) Alexander et al.39 New Zealand N = 18 (13 vegetarians and Vegetarian defined as never Serum B12 reference range 33.3% (N = 6) 5 vegans; males and consuming red meat and consume 150–680 pmol/l females) ckn or fish no more than once per SimulTRAC radioassay kit Females—Vegetarian Mean week (Becton Dickson) age 26 years Vegan consume no meat, eggs, dairy Vegan mean age 30y Diet duration vegetarians 5 years; not Males —Vegetarian Mean specified for vegans age 28 years Rush et al.25 Migrant Indian girls, N = 6 (all girls) Only defined as ‘non-meat eaters’ Serum B12 deficiency o170 33.3% living in Auckland, Ages 9–11 years Diet duration described by mothers pmol/l New Zealand as ‘practiced for many generations in Lab methods not available their family’ Carmel et al.30 Asian-Indian N = 8 (6 males and 2 Not defined, but group consisted of Serum B12 normal range 37.5% (N = 3) individuals living in females) ovo-lacto-vegetarians, lacto- 180–600 pmol/l United States Ages 26–36 years vegetarian and vegans Quantaphase II radioisotope Some vegetarians reported eating dilution poultry and fish 1–4 × per month Diet duration not reported Dagnelie et al.47 Netherlands N = 47 (males and females) Diet described by researchers as Serum B12 o136 pmol/l 45% Mean age 15.2 months consisting of grain cereals (mainly Competitive protein-binding (10.1–20.4 months) rice), vegetables, pulses (mainly soy), assay and sea vegetables and only very

Table. 1. (Continued )

Reference Country/ethnicity Participants Diet characteristics Assessment criteria used Rate of deﬁciency

small amounts of fruits and products of animal origin Majority of infants exclusively breastfed by macrobiotic mothers for 4–8 months and then introduced solids; breastfeeding continued for most until 10–18 months (median 13 months) Miller et al.44 United States N = 110 (46 males and 64 Macrobiotic diet defined as typically Low vitamin B12 51% females) consisting of 50–60% whole cereal concentrations o148 pmol/l Median age 33 years grains, 5% soups, 20–25% vegetables Radioimmunoassay (21–70) and 5–10% beans and sea vegetables; occasional small amounts seafood eaten, but no meat and little or no dairy or eggs Woo et al.40 Chinese (elderly) N = 106 (all females) Vegetarian not specifically defined Serum B12 deficiency o150 53.8% (N = 57) Mean age 81.3 ± 7.6 years but included both vegans and lacto- pmol/l ovo-veg Measured ‘using standard Diet duration median 25 years laboratory methods’ Specker et al.45 United States N = 17 (all females) Vegetarians described as practicing a Low serum B12 o148 56% Mean age 31 years (22–40) macrobiotic diet—50–60% whole pmol/l cereal grains, 5% soups, 20–25% Radioimmunoassay vegetables, 5–10% beans and sea vegetables; avoid animal products; no dairy products or eggs Diet duration median 72 months (14–137 months) Rauma et al.43 Finland N = 21 (20 females and 1 Uncooked vegan diet (‘living food Serum B12 lower reference 57% male) diet’) with most food items fermented limit 200 pmol/l Females mean age 47 years or sprouted, thought to enhance Dual radioassay (27–69) growth of vitamin-B12-producing Male age 40 years bacteria Diet duration average 5.2 years (0.7–14) Waldmann et al.26 Germany N = 98 strict vegans (49% Strict vegan not further defined Serum B12 deficiency 58.3% Strict vegans males and 51% females) Moderate vegan—ate o5% of defined as o150 pmol/l (86.5% o250 pmol/l) Mean age 43.4 ± 15.4 years ingested energy from eggs, milk and (also assessed deficiency at 34.5% Moderate vegans N = 56 moderate vegans dairy o250 pmol/l) (69.1% o250 pmol/l) (33.9% males and 66.1% Diet duration: Chemiluminescence females) Strict vegan mean 7.70 ± 6.40 years Mean age 45.7 ± 14.2 years Moderate vegan mean 5.06 ± 4.03 years Refsum et al.34 Asian Indians N = 78 (males and females) Subjects considered vegetarian if Serum B12 deficiency o150 60% Ages 27–55 years never ate mutton, poultry, fish or pmol/l eggs Microbiological assay Diet duration not reported Gupta et al.15 South Asians (India, N = 172 (70 males and 102 Vegetarian classification included Serum B12 deficiency ⩽ 132 66.3% Pakistan) living in females) vegans, lacto- and lacto-ovo- pmol/l 51 Men (72.8%) Toronto Ages 18–84 years (avg age vegetarians Microparticle intrinsic factor 63 Women (61.7%) 42 years) Diet duration not reported assays (three types) Yajnik et al.18 India N = 137 males Lacto-vegetarian Serum B12 low 68% Rural (41% rural, 11% slum, 44% Diet duration not reported concentration o150 pmol/l 51% Slum urban) Radioimmunoassay 81% Urban middle-class

Between 150 and 221 pg/ml or 200 –300 pmol/l Madry et al.20 Poland N = 20 (6 males and 14 Subjects agreed to switch to vegan Serum B12 considered 20% Strict vegans females) diet for 5 years abnormal o193 pg/ml 0% Vegan with B12- Median age = 23 years N = 10 strict vegan Chemiluminescence fortified foods N = 10 vegan with B12-fortified foods immunoassay Diet duration 5 years Morad et al.19 Israel, individuals N = 47 (25 males and 22 ‘Vegetarian’ not defined Serum B12 deficiency o157 25.5% with intellectual females) Diet duration not reported pg/ml disability Ages 19–61+ years Lab method not reported, used Clalit Health Service Laboratory Bissoli et al.32 Italy N = 31 vegan (19 males and Vegan avoided all animal products Normal range defined as Very low serum B12: 12 females) Lacto-ovo-vegetarian consumed milk 220–740 pmol/l 47.8% vegan Mean age 45.8 ± 15.8 years and eggs, but not meat Very low serum levels o127 33.3% Lacto-ovo- N = 14 lacto-ovo- Diet duration at least 5 years pmol/l vegetarians

Table. 1. (Continued )

Reference Country/ethnicity Participants Diet characteristics Assessment criteria used Rate of deﬁciency

vegetarians (6 males and 8 Automated (% deficient after females) chemiluminescence exclusion for B vitamin Mean age 48.5 ± 14.5 years supplementation—7 vegans, 2 lacto-ovo- vegetarians) Krivosikova et al.41 Slovak Republic N = 141 (all females) Vegetarian not specifically defined Serum B12 normal range 48% Mean age 41.9 ± 19.7 years but described subjects as on ‘long- defined as >220 pmol/l term ovo-lacto-veg diet’ Elecsys 2010 system, inter- Diet duration ‘long-term’ and intra-assay Hokin and Butler38 Australia (Seventh- N = 245 N = 234 lacto-ovo-vegetarians Serum B12 laboratory 53% day Adventist (predominantly males) N = 11 vegans reference range 171–850 (73% o221 pmol/l) ministers) Ages 22–80 years Diet duration mean 32 years pmol/l (also assessed at o221 pmol/l as recommended by cited author) Abbott IMx analyzer, reagents and calibrators Crane and United States N = 78 (males and females) Vegetarians described as consuming Serum B12 deficiency o200 60% Sample46 Mean age 41 years (18–84) a ‘total vegetarian diet’; selected food pg/ml ad lib from fruit, whole grains, (defined normal values for legumes, tubers, green leafy serum B12 >200 pg/ml) vegetables and nuts Radioassay Diet duration: 12 months or longer Barghouti et al.16 Jordan N = 53 (males and females) ‘strict vegetarians’ loosely defined as Serum B12 deficiency o180 62.3% Ages 18–78 years ‘vegan, eating a diet of exclusively of pg/ml vegetable origin’ Chemiluminescent Diet duration not reported microparticle immunoassay

No speciﬁcally deﬁned levels Pongstaporn and Thailand N = 68 (35 males and 33 Vegetarian included lacto-, ovo-, Pg/ml 39.7% Bunyaratavej13 females) Ages 18–40 year lacto-ovo Diet duration 1–40 years Microparticle enzyme immunoassay

Fifty-one percent (n = 110) and 56% (n = 17) of subjects, respec- authors as indicative of vitamin B12 deficiency. According to the tively, were found to be deficient.44,45 Institute of Medicine, a serum of 120–180 pmol/l is indicative of Semivegetarians: Five studies reported deficiency rates for a deficiency. Most of the studies in this review defined vitamin B12 sample that included participants who consumed vegetarian and deficiency in this range.2 semivegetarian diets.17,24,35,39,42 Deficiency rates in these five Of the four studies that assessed vitamin B12 deficiency at studies ranged from 8%35 to 33.3%.39 Study-specificdeficiency higher serum values, two reported results specifically for adult criteria ranged from ⩽ 140 pmol/l42 to o179 pmol/l.17 More vegans. Reported results found 47.8% deficiency among vegans details are provided in Table 1. (o220 pmol/l)32 and 86.5 and 69.1% (o250 pmol/l) in ‘strict’ and ‘moderate’ vegans, respectively.26 These results suggest that the prevalence of vitamin B12 deficiency among vegans is especially Gender-specificdeficiency rates (non-pregnant adults) high even when their vitamin B12 status is assessed using a Some of the authors specifically looked at subjects of one 8,9,17,18,21,24,33,40–42,45 relatively high serum vitamin B12 value. The fact that a higher sex or reported their results by separating prevalence of deficiency is found among ‘strict’ vegans clearly fi 10,11,15 de ciency rates for male and female subjects. Some of the indicates the importance of consuming foods containing vitamin fi highest de ciency rates across all studies reviewed were reported B12 or using vitamin B12 supplements. in lacto-vegetarian, urban, middle-class Indian male subjects fi 18 De ciency prevalence of 0% was reported among vegans who (81%) and in adult American female subjects (56%) who consumed vitamin B12-fortified foods.20 Although these findings 45 fi consumed a macrobiotic diet. Overall, de ciency rates for can be in part explained by a relatively lenient deficiency fi fi female subjects ranged from 1.9 to 56% with de ciency de nitions definition (o142 pmol/l), vitamin B12 provided by fortification o o fi ranging from 147.8 to 220 pmol/l. De ciency rates for male undoubtedly was a factor in preventing the occurrence of a fi fi subjects ranged from 7 to 81% using de ciency de nition from deficiency. It needs to be pointed out, however, that even among o o 118 to 150 pmol/l. In the studies that reported results participants consuming vitamin B12-fortified foods, the research- comparing male and female subjects, no considerable differences 10,11,15 ers documented a steady decline in serum vitamin B12 over a were noted between the two sexes. 5-year period of study duration. This finding indicates that in long- term prevention of vitamin B12 deficiency, higher doses of vitamin B12 than the dose currently used in some fortified foods may be DISCUSSION needed. This may especially be true for older adults and the This review assessed vitamin B12 deficiency prevalence among elderly owing to age-related decline in absorption rate. Additional individuals adhering to vegetarian diets. A comparison of studies are needed to support or refute this conclusion. These deficiency prevalence between studies is not easy considering findings indicate that including vitamin B12 supplements even the wide range of serum vitamin B12 values defined by study when foods fortified with vitamin B12 are ingested is the most

European Journal of Clinical Nutrition (2014) 541 – 548 © 2014 Macmillan Publishers Limited B12 deficiency in vegetarians R Pawlak et al 547 reliable way of preventing vitamin B12 deficiency among biomarkers are believed to be more reliable indicators of vitamin vegetarians, especially vegans. B12 deficiency.52 Although well-planned vegetarian diets can The study led by Madry et al.20 is of particular importance for provide adequate nutrition for individuals across all life stages, another reason. In the past, it was widely believed that it took a special consideration must be made to ensure reliable sources of very long time for vitamin B12 deficiency to develop when foods vitamin B12 are included to avoid a deficiency.4,52,53 that are a natural source of this vitamin are not ingested. In their 20 study, Madry et al. included participants who switched from Limitations consuming an omnivorous to a vegan diet for a period of 5 years. This review was based on studies that used serum vitamin B12 The results showed a progressive decline in the mean serum fi vitamin B12, with two participants (20%) among those who did de ciency as a marker of vitamin B12 status. It is generally fi accepted that serum vitamin B12 alone may not be the best not consume vitamin B12-forti ed foods being diagnosed fi with vitamin B12 deficiency (o193 pg/ml) within the study marker of de ciency. Serum vitamin B12 in combination with period. This prevalence would likely be higher if in addition to other biomarkers such as methylmalonic acid or homocysteine generally give a more reliable picture of the prevalence of serum vitamin B12 other assessment methods believed to be fi more accurate, such as methylmalonic acid, were used. de ciency. Also, a number of the studies reviewed used relatively fi low serum vitamin B12 as a criteria for what constituted a Vitamin B12 de ciency seems to be higher among vegetarians fi from India and Pakistan compared with those living in other de ciency. Some patients with a higher serum vitamin B12 than 15 fi those used as deficiency cutoffs, in some of the reviewed studies, regions. Gupta et al. reported a de ciency prevalence among fi 52 individuals of Indian and Pakistani origin living in Canada. In this already showed symptoms of vitamin B12 de ciency. Using 15 fi fi higher serum values as indicative of a deficiency, such as that retrospective study, Gupta et al. found 66.3% (de ciency de ned 54 o as serum vitamin B12 ⩽ 132 pmol/l) of their sample having vitamin suggested by Herbert ( 300 or 222 pmol/l), would identify B12 deficiency. Similarly, Yajnik et al.18 reported deficiency rates of more individuals with compromised vitamin B12 status. 81, 68, and 51%, among Indian men with different socioeconomic 18 status. Men from urban, middle-class had notably higher rates of CONCLUSION deficiency compared with both men from rural and slum fi socioeconomic class. Yajnik et al.18 also suggested that the higher The ndings of this review show that individuals who follow a deficiency prevalence among male subjects from the middle-class vegan diet, and do not take vitamin B12 supplements nor eat fi may be reflective of ingestion of less microbial B12 from foods forti ed with this vitamin, have higher prevalence of a fi contaminated food and water sources. Although other studies de ciency compared with those adhering to other types of ‘ ’ confirmed that the contamination of water and unwashed vegetarian diets. Similarly, strict vegans were found to have a fi ‘ ’ products may contain B12, the high deficiency prevalence among higher prevalence of de ciency when compared with moderate individuals included in this study indicates that vegetarians cannot vegans. In fact, every study that reported the prevalence of fi rely on them as means of vitamin B12 deficiency prevention.49 de ciency for vegans separately from other vegetarians showed fi The deficiency prevalence among pregnant women differed higher prevalence of de ciency among vegans. Nevertheless, the fi depending on which trimester the women were in. Even though ndings also suggest that, with few exceptions, there is also a fi serum vitamin B12 indicative of a deficiency among women in the relatively high prevalence of de ciency among individuals who last trimester was lower compared with the criteria in the first two follow all other types of vegetarian diets. Thus, vegetarians, and trimesters (o100 pmol/l, compared with o120 and o130 pmol/l, especially vegans, should strongly consider using vitamin B12 in the first and the second trimesters, respectively), women in the supplements to ensure adequate vitamin B12 intake, and to help fi third trimester were found to have a higher prevalence of prevent de ciency. deficiency (39% vs 33% and 17% for the first and second trimesters, respectively). Although the lower serum vitamin B12 during CONFLICT OF INTEREST pregnancy may be in part explained by expanded blood volume, this dilution effect is unlikely the reason for the higher prevalence The authors declare no conflict of interest. of deficiency among women in the last trimester. Blood volume peaks at about the 34th to 36th week of gestation and remains ACKNOWLEDGEMENTS unchanged until term.50 It is more likely that the higher deficiency prevalence among pregnant vegetarian women in the third The authors did not receive any financial compensation for creating this manuscript. trimester is indicative of depleted vitamin B12 stores rather than expanded blood volume. Pregnant women have higher needs for REFERENCES vitamin B12. The low vitamin B12 status of pregnant vegetarian/ vegan women may result in diagnoses of vitamin B12 deficiency in 1 National Institute of Health NIH Office of Dietary Supplements. Dietary Supple- infants. Such deficiency is often associated with severe symptoms, ment Fact Sheet: Vitamin B12. Available at: http://ods.od.nih.gov/factsheets/ including failure to thrive, inability to accept solid foods, stunting VitaminB12-HealthProfessional/. Accessed on 15 November 2013. and developmental delays. 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