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Journal of the Formosan Medical Association xxx (xxxx) xxx

Available online at www.sciencedirect.com

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journal homepage: www.jfma-online.com

Original Article

Vitamin B12 deficiency and anemia in 140

Taiwanese female lacto-vegetarians
Yi-Pang Lee a, Ching-Hui Loh b, Ming-Jay Hwang a,c,**,
Chun-Pin Lin a,c,d,*

a
Department of Dentistry, Hualien Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, Hualien,
Taiwan
b
Department of Geriatrics and Gerontology, Hualien Tzu Chi Hospital, Buddhist Tzu Chi Medical
Foundation, Hualien, Taiwan
c
Department of Dentistry, National Taiwan University Hospital, College of Medicine, National Taiwan
University, Taipei, Taiwan
d
School of Dentistry and Graduate Institute of Clinical Dentistry, National Taiwan University, Taipei,
Taiwan

Received 26 March 2021; received in revised form 4 April 2021; accepted 8 April 2021

KEYWORDS Background/Purpose: Lacto-vegetarians (LVs) tend to have vitamin B12 deficiency (B12D). This
Lacto-vegetarians; study assessed whether 140 female LVs, including 16 B12D/LVs and 124 non-B12D/LVs, had
Normocytic anemia; significantly higher frequencies of microcytosis, macrocytosis, and of blood hemoglobin
Microcytic anemia; (Hb), red blood cell (RBC), and serum vitamin B12 deficiencies than 140 healthy control sub-
Vitamin B12 jects (HCSs).
deficiency Methods: The complete blood count and serum vitamin B12 level in 140 female LVs and 140
female HCSs were measured and compared.
Results: We found that 8.6%, 4.3%, 22.9%, 20.0%, and 11.4% of 140 LVs had microcytosis,
macrocytosis, and blood Hb, RBC, and serum vitamin B12 deficiencies, respectively. The 140
LVs, 16 B12D/LVs, and 124 non-B12D/LVs had significantly higher frequencies of microcytosis
as well as blood Hb and RBC deficiencies than 140 HCSs (all P-values < 0.005). Moreover, both
140 LVs and 124 non-B12D/LVs had significantly higher frequencies of macrocytosis than 140
HCSs. In this study, 32 (22.9%) of 140 LVs including 5 B12D/LVs and 27 non-B12D/LVs had ane-
mia. Of the 5 anemic B12D/LVs, three had normocytic anemia, one had iron deficiency anemia
(IDA), and one had thalassemia trait-induced anemia. Moreover, of the 27 anemic non-B12D/
LVs, 18 had normocytic anemia, one had IDA, one had thalassemia trait-induced anemia,
and 7 had microcytic anemia other than IDA and thalassemia trait-induced anemia.
Conclusion: LVs have significantly higher frequencies of microcytosis, macrocytosis, blood Hb,
RBC, and serum vitamin B12 deficiencies than HCSs. Normocytic and microcytic anemias are

** Corresponding author. Department of Dentistry, Hualien Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, No. 707, Section 3,
Chung-Yang Road, Hualien, 970, Taiwan.
* Corresponding author. School of Dentistry and Graduate Institute of Clinical Dentistry, National Taiwan University and National Taiwan
University Hospital, No. 1, Changde Street, Taipei, 10048, Taiwan.
E-mail addresses: mingjays@gmail.com (M.-J. Hwang), chunpinlin@gmail.com (C.-P. Lin).

https://doi.org/10.1016/j.jfma.2021.04.007
0929-6646/Copyright ª 2021, Formosan Medical Association. Published by Elsevier Taiwan LLC. This is an open access article under the CC
BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Please cite this article as: Y.-P. Lee, C.-H. Loh, M.-J. Hwang et al., Vitamin B12 deficiency and anemia in 140 Taiwanese female lacto-
vegetarians, Journal of the Formosan Medical Association, https://doi.org/10.1016/j.jfma.2021.04.007
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Y.-P. Lee, C.-H. Loh, M.-J. Hwang et al.
the two most common types of anemia in our LVs.
Copyright ª 2021, Formosan Medical Association. Published by Elsevier Taiwan LLC. This is an
open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
Introduction
Vitamin B12 is known to be predominantly present in animal
tissue and generally absent in plants. Therefore, vegetar-
ians who consume less animal-source foods are considered
to be a risk group for vitamin B12 deficiency. Moreover,
higher frequencies of vitamin B12 deficiency have been
reported in vegans than in vegetarians as well as in in-
dividuals who adhere to a vegetarian diet since birth than in
those who adopt such a diet later in life.1,2 In addition to
vitamin B12 deficiency, it is also well known that elimi-
nating all animal products from the diet increases the risk
of vitamin D, calcium, long-chain n-3 fatty acids, and zinc
deficiencies.3
Vitamin B12 is an essential micronutrient that plays an
important role in cell division, one-carbon metabolism, DNA
synthesis, erythropoiesis, and neurologic function. Vitamin
B12 deficiency can result in increased mean corpuscular
volume (MCV) and anemia through the alteration of eryth-
ropoiesis.4 Vitamin B12 also plays a key role in neuronal
health, and a severe vitamin B12 deficiency inhibits the
physiological formation of the myelin sheath, altering cor-
rect nerve transmission.4 Thus, it is important to measure
the serum vitamin B12 level and complete blood count in
vegetarians to assess who have vitamin B12 deficiency and
anemia and who should take vitamin B12 supplement.
Vegetarians eat mainly plant-based foods and there are
several types of vegetarians. Vegans eat diets that exclude
meat, fish, poultry, eggs, and dairy products. Lacto-
vegetarians (LVs) eat diets that exclude meat, fish,
poultry, and eggs, but allow dairy products, such as milk,
cheese, yogurt and butter. Ovo-vegetarians eat diets that
exclude meat, fish, poultry, and dairy products, but allow
eggs. Lacto-ovo-vegetarians eat diets that exclude meat,
fish and poultry, but allow dairy products and eggs. Pesco-
vegetarians eat diets that exclude meat, poultry, dairy
products, and eggs, but allow fish. Because vegans and LVs
do not eat meat, fish, poultry, and eggs, they are prone to
have deficiencies of vitamin B12, vitamin D, calcium, long-
chain n-3 fatty acids, and zinc.4
In this study, 140 female LVs were recruited from those
who underwent whole body healthy examination in our
hospital. The 140 LVs were further divided into 16 vitamin
B12-deficient LVs (B12D/LVs), and 124 non-B12D/LVs.5,6 We
tried to assess whether the 140 LVs, 16 B12D/LVs, and 124
non-B12D/LVs had significantly higher frequencies of
microcytosis (mean corpuscular volume or MCV < 80 fL),7,8
macrocytosis (MCV  100 fL),9,10 and of blood hemoglobin
(Hb < 12 g/dL),11 red blood cells (RBC < 4  106/mL), mean
corpuscular hemoglobin concentration (MCHC < 31 g/dL),
and serum vitamin B12 (<200 pg/mL) deficiencies,5,6 and of
high RBC distribution width-coefficient of variation (RDW-
CV > 14.5%) than 140 female healthy control subjects. We
2
also evaluated whether 16 B12D/LVs had significantly
higher frequencies of microcytosis, macrocytosis, and of
blood Hb, RBC, MCHC, and serum vitamin B12 deficiencies,
and of high RDW-CV level than 124 non-B12D/LVs. In addi-
tion, we further divided the 140 LVs into 122 LVs with
normocytosis (normocytosis/LVs),12,13 12 LVs with micro-
cytosis (microcytosis/LVs),7,8 6 LVs with macrocytosis
(macrocytosis/LVs)9,10 according to the MCV of RBCs and
subsequently assessed whether 122 normocytosis/LVs, 12
microcytosis/LVs, or 6 macrocytosis/LVs had significantly
higher frequencies of blood Hb, RBC, MCHC, and serum
vitamin B12 deficiencies and of high RDW-CV level than 140
healthy control subjects.
Materials and methods
Subjects
This study consisted of 140 female LVs (140 women, age
range 23e88 years, mean age 49.4  12.3 years) including
16 B12D/LVs (16 women, age range 30e66 years, mean age
45.0  11.6 years) and 124 non-B12D/LVs (124 women, age
range 23e88 years, mean age 49.9  12.4 years). For each
LV, one age- (2 years of each LV’s age) and sex-matched
healthy control subject was selected. Thus, 140 female
age-matched healthy control subjects (age range 24e88
years, mean 49.8  12.4 years) were selected and included
in this study. All the 140 female LVs were selected from
those who underwent whole body health examination in
Hualien Tzu Chi Hospital (HTCH) from January 2016 to
August 2020. The 140 female healthy control subjects had
dental caries, pulpal disease, malocclusion, or missing of
teeth but did not have any oral mucosal or systemic dis-
eases. They were selected from those who were seen
consecutively, diagnosed, and treated in the Department of
Dentistry, National Taiwan University Hospital (NTUH) from
July 2007 to July 2017.14
The blood samples were drawn from 140 LVs to 140
healthy control subjects for the measurement of complete
blood count and serum vitamin B12 level. All LVs and
healthy control subjects signed the informed consents
before entering the study. This study was reviewed and
approved by the Institutional Review Board at the HTCH
(IRB109-258-B).
Determination of complete blood count and serum
vitamin B12 level
The complete blood count and serum vitamin B12 level
were determined by the routine tests performed in the
Department of Laboratory Medicine, HTCH for the 140 LVs
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Journal of the Formosan Medical Association xxx (xxxx) xxx

or in the Department of Laboratory Medicine, NTUH for the
140 healthy control subjects.14
Statistical analysis
Comparisons of the mean age, MCV, mean blood Hb, mean
RBC number, MCHC, mean RDW-CV level, and mean serum
vitamin B12 level between 140 LVs, 16 B12D/LVs, or 124
non-B12D/LVs and 140 healthy control subjects as well as
between 16 B12D/LVs and 124 non-B12D/LVs were per-
formed by Student’s t-test. Moreover, comparisons of fre-
quencies of microcytosis,7,8 normocytosis (MCV between
80 fL and 99.9 fL),12,13 macrocytosis,9,10 and of blood Hb,
RBC, MCHC, and serum vitamin B12 deficiencies,5,6 and of
high RDW-CV level between 140 LVs, 16 B12D/LVs, or 124
non-B12D/LVs and 140 healthy control subjects as well as
between 16 B12D/LVs and 124 non-B12D/LVs were per-
formed by chi-square or Fisher’s exact test, where appro-
priate. In addition, comparisons of frequencies of blood Hb,
RBC, MCHC, and serum vitamin B12 deficiencies, and of high
RDW-CV between any two of the four groups, including 122
normocytosis/LVs,12,13 12 microcytosis/LVs,7,8 6 macrocy-
tosis/LVs,9,10 and 140 healthy control subjects were per-
formed by chi-square or Fisher’s exact test, where
appropriate. The result was considered to be significant if
the P-value was less than 0.05.
Results
Comparisons of the mean age, MCV, mean blood Hb, RBC
number, MCHC, RDW-CV, and serum vitamin B12 levels be-
tween 140 LVs, 16 B12D/LVs, or 124 non-B12D/LVs and 140
healthy control subjects as well as between 16 B12D/LVs
and 124 non-B12D/LVs are shown in Table 1. We found
significantly lower mean blood Hb, MCHC, and serum
vitamin B12 levels in 140 LVs, 16 B12D/LVs, and 124 non-
B12D/LVs than in 140 healthy control subjects (all P-
values < 0.001, Table 1). Moreover, 140 LVs and 124 non-
B12D/LVs had significantly lower mean RBC numbers than
140 healthy control subjects (both P-values < 0.001, Table
1). In addition, 16 B12D/LVs had significantly lower mean
serum vitamin B12 level than 124 non-B12D/LVs (P < 0.001,
Table 1).
According to the World Health Organization (WHO)
criteria, microcytosis of RBC was defined as having
MCV < 80 fL,7,8 normocytosis of RBC was defined as having
MCV between 80 fL and 99.9 fL,12,13 macrocytosis of RBC
was defined as having MCV  100 fL.9,10 Women with
Hb < 12 g/dL were defined as having Hb deficiency or
anemia.11 Patients with RBC count < 4  106/mL,
MCHC < 31 g/dL, serum vitamin B12 level < 200 pg/mL5,6
were defined as having RBC, MCHC, and serum vitamin
B12 deficiencies, respectively. Furthermore, patients with
the RDW-CV > 14.5% were defined as having high RDW-CV.
By the above-mentioned definitions, 8.6%, 87.1%, 4.3%,
22.9%, 20.0%, 7.1%, 11.4%, and 8.6% of 140 LVs were diag-
nosed as having microcytosis, normocytosis, macrocytosis,
and blood Hb, RBC, MCHC, and serum vitamin B12 de-
ficiencies, and high RDW-CV, respectively (Table 2). We
found that 140 LVs, 16 B12D/LVs, and 124 non-B12D/LVs had
significantly higher frequencies of microcytosis as well as
blood Hb and RBC deficiencies than 140 healthy control
subjects (all P-values < 0.005, Table 2). Moreover, both 140
LVs and 124 non-B12D/LVs had significantly higher fre-
quencies of macrocytosis than 140 healthy control subjects
(both P-values < 0.05, Table 2). Both 140 LVs and 16 B12D/
LVs had significantly higher frequencies of serum vitamin
B12 deficiency than 140 healthy control subjects (both P-
values < 0.001, Table 2). In addition, 140 LVs, 16 B12D/LVs,
and 124 non-B12D/LVs had significantly lower frequencies
of normocytosis than 140 healthy control subjects (all P-
values < 0.005, Table 2).
In this study, 32 (22.9%) of 140 LVs including 5 (31.3%) of
16 B12D/LVs and 27 (21.8%) of 124 non-B12D/LVs had anemia
(Hb concentration < 12 g/dL for women).11 Of the 5 anemic

Table 1 Comparisons of the mean age, mean corpuscular volume (MCV), mean blood hemoglobin (Hb), mean red blood cell
(RBC) number, mean corpuscular hemoglobin concentration (MCHC), mean red blood cell distribution width-coefficient of
variation (RDW-CV), and mean serum vitamin B12 level between 140 lacto-vegetarians (LVs), 16 vitamin B12-deficient LVs (B12D/
LVs), or 124 non-B12D/LVs and 140 healthy control subjects as well as between 16 B12D/LVs and 124 non-B12D/LVs.
Group Age (year) MCV (fL) Hb (g/dL) RBC (106/mL) MCHC (g/dL) RDW-CV (%) Vitamin B12 (pg/mL)
LVs (n Z 140) 49.4  12.3 90.0  6.9 12.6  1.2 4.3  0.4 32.6  1.0 13.2  1.0 484.4  274.0
a
P-value 0.787 >0.999 <0.001 <0.001 <0.001 0.311 <0.001
B12D/LVs 45.0  11.6 88.8  8.3 12.6  1.7 4.4  0.5 32.5  1.3 13.2  1.1 172.1  29.5
(n Z 16)
a
P-value 0.142 0.282 <0.001 0.245 <0.001 0.570 <0.001
b
P-value 0.136 0.484 >0.999 0.363 0.717 >0.999 <0.001
Non-B12D/LVs 49.9  12.4 90.1  6.8 12.6  1.1 4.3  0.4 32.6  1.0 13.2  1.0 524.7  265.3
(n Z 124)
a
P-value 0.984 0.879 <0.001 <0.001 <0.001 0.311 <0.001
Healthy control 49.8  12.4 90.0  3.5 13.6  0.7 4.5  0.3 33.4  0.9 13.1  0.6 685.7  207.7
subjects
(n Z 140)
a
Comparisons of means of parameters between 140 LVs, 16 B12D/LVs, or 124 non-B12D/LVs and 140 healthy control subjects by
Student’s t-test.
b
Comparisons of means of parameters between 16 B12D/LVs and 124 non-B12D/LVs by Student’s t-test.

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Y.-P. Lee, C.-H. Loh, M.-J. Hwang et al.

B12D/LVs, three had normocytic anemia (defined as having

Comparisons of frequencies of parameters between 140 LVs, 16 B12D/LVs, or 124 non-B12D/LVs and 140 healthy control subjects by chi-square or Fisher’s exact test, where
Table 2 Comparisons of frequencies of microcytosis (mean corpuscular volume or MCV < 80 fL), normocytosis (MCV between 80 fL and 99.9 fL), macrocytosis (MCV  100 fL),
as well as of blood hemoglobin (Hb < 12 g/dL), red blood cells (RBC < 4  106/mL), mean corpuscular hemoglobin concentration (MCHC < 31 g/dL), and serum vitamin B12
(<200 pg/mL) deficiencies, and of high RBC distribution width-coefficient of variation (RDW-CV > 14.5%) between 140 lacto-vegetarians (LVs), 16 vitamin B12-deficient lacto-

Vitamin B12 deficiency

anemia and an MCV between 80.0 fL and 99.9 fL),12,13 one
had iron deficiency anemia (IDA, defined as having anemia,
an MCV < 80 fL, and a Mentzer index (MCV/RBC) > 16),15e20

(<200 pg/mL)
and one had thalassemia trait-induced anemia (TTIA,
defined as having anemia, a RBC count > 5.0  106/mL, an

16 (100.0)
16 (11.4)
MCV < 74 fL, and a Mentzer index < 13)21

<0.001

<0.001
<0.001
0 (0.0)

0 (0.0)
(Table 3). Moreover, of the 27 anemic non-B12D/LVs, 18

NA
had normocytic anemia,12,13 one had IDA,15e20 one had
TTIA,21 and 7 had microcytic anemia (defined as having
High RDW-CV

anemia, an MCV < 80 fL, and a Mentzer index between 13

and 16)7,8 other than IDA and TTIA (Table 3).
(>14.5%)

12 (8.6)

11 (8.9)
1 (6.3)

4 (2.9)
If 140 LVs were divided into three groups including 122

Comparisons of frequencies of parameters between 16 B12D/LVs and 124 non-B12D/LVs by chi-square or Fisher’s exact test, where appropriate.
0.072

0.985
0.903

0.066
LVs with normocytosis (normocytosis/LVs),12,13 12 LVs with
vegetarians (B12D/LVs), or 124 non-B12D/LVs and 140 healthy control subjects as well as between 16 B12D/LVs and 124 non-B12D/LVs.

microcytosis (microcytosis/LVs),7,8 and 6 LVs with macro-

cytosis (macrocytosis/LVs)9,10 according to the MCV of
(<31 g/dL)
deficiency

RBCs. We found that 122 normocytosis/LVs had significantly

10 (7.1)

1 (6.3)

9 (7.3)

3 (2.1)

higher frequencies of blood Hb, RBC, and vitamin B12 de-

MCHC

0.088

0.881
0.713

0.090

ficiencies than 140 healthy control subjects, and had

significantly lower frequencies of blood Hb and MCHC de-
ficiencies and of high RDW-CV than 12 microcytosis/LVs (all
RBC deficiency
(<4  106/mL)

P-values < 0.001, Table 4). Moreover, 12 microcytosis/LVs

had significantly higher frequencies of blood Hb, MCHC, and
28 (20.0)

23 (18.5)
Subject number (%)

5 (31.3)
<0.001

<0.001

<0.001

vitamin B12 deficiencies and of high RDW-CV than 140

3 (2.1)
0.388

healthy control subjects, and significantly higher fre-

quencies of blood Hb deficiency and of high RDW-CV than 6
macrocytosis/LVs (all P-values < 0.05, Table 4). In addition,
(Hb < 12 g/dL)
Hb deficiency

6 macrocytosis/LVs had significantly higher frequencies of

blood RBC deficiency than 140 healthy control subjects, 122
32 (22.9)

27 (21.8)

normocytosis/LVs, or 12 microcytosis/LVs (all P-

5 (31.3)
<0.001

<0.001

<0.001
0 (0.0)

values < 0.005, Table 4).

0.594

Discussion
(MCV  100 fL)
Macrocytosis

The major finding of this study was that 12 (8.6%), 6 (4.3%),

6 (4.3)

0 (0.0)

6 (4.8)

0 (0.0)

32 (22.9%), 28 (20.0%), and 16 (11.4%) of 140 LVs had

0.039

0.808

0.026

microcytosis, macrocytosis, and blood Hb, RBC, and serum

NA

vitamin B12 deficiencies, respectively. Moreover, the 140

LVs had significantly higher frequencies of microcytosis,
80 fL and 99.9 fL)

macrocytosis, and blood Hb, RBC, and serum vitamin B12

(MCV between
Normocytosis

deficiencies than 140 healthy control subjects (all P-

140 (100.0)

values < 0.05). Thus, our findings indicate that vitamin B12
122 (87.1)

108 (87.1)
14 (87.5)
<0.001

<0.001

deficiency and anemia are commonly observed in LVs.

0.002
0.725

The etiologies of vitamin B12 deficiency include inade-

quate intake (alcohol abuse, patients older than 75 years,
vegans or strict vegetarians), decreased ileal absorption
(MCV < 80 fL)

(Crohn disease, ileal resection, or tapeworm infection),

Microcytosis

decreased intrinsic factor (atrophic gastritis, pernicious

12 (8.6)

10 (8.1)
2 (12.5)
<0.001

anemia, post-gastrectomy syndrome), genetic disorder

0 (0.0)
0.002
0.903

0.002

(transcobalamin II deficiency), and prolonged medication

use (histamine H2 blocker or proton pump inhibitor use for
more than 12 months or metformin use for more than four
Non-B12D/LVs (n Z 124)

Healthy control subjects

months).22 For LVs, it is obvious that the vitamin B12 defi-

ciency is caused mainly by inadequate intake.
B12D/LVs (n Z 16)

NA Z not assessed.

This study detected serum vitamin B12 deficiency and

anemia in 16 (11.4%) and 32 (22.9%) of 140 LVs, respectively.
LVs (n Z 140)

(n Z 140)

Hung et al.23 also demonstrated a significantly lower plasma

appropriate.

vitamin B12 level in 45 female Buddhist LVs

P-value
P-value

P-value

P-value

(207.7  127.1 pmol/L) than in 45 matched omnivores

Group

(403.5  138.9 pmol/L) recruited in Hualien, Taiwan. Kapoor

a

b
b
a

et al.3 found that mean serum vitamin B12 level and mean

4
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Journal of the Formosan Medical Association xxx (xxxx) xxx

Table 3 Anemia types, mean corpuscular volume (MCV), serum vitamin B12 deficiency, and Mentzer index (MCV/RBC) for 5
anemic vitamin B12-deficient lacto-vegetarians (B12D/LVs) and 27 anemic non-B12D/LVs.
Anemia type Patient number (%)
Patient Mean corpuscular Vitamin B12 Mentzer Mentzer
number (%) volume (fL) deficiency index (>16) index (<13)
(<200 pg/mL)
Anemic B12D/LV (n Z 5)
Normocytic anemia 3 (9.4) 80e99.9 3 (100.0) 3 (100.0) 0 (0.0)
Iron deficiency anemia 1 (3.1) <80 1 (100.0) 1 (100.0) 0 (0.0)
Thalassemia trait-induced anemia 1 (3.1) <74 1 (100.0) 0 (0.0) 1 (100.0)
Total 5 (15.6) 5 (15.6) 4 (12.5) 1 (3.1)
Anemic non-B12D/LV(n Z 27)
Normocytic anemia 18 (56.3) 80e99.9 0 (0.0) 18 (100.0) 0 (0.0)
Iron deficiency anemia 1 (3.1) <80 0 (0.0) 1 (100.0) 0 (0.0)
Thalassemia trait-induced anemia 1 (3.1) <74 0 (0.0) 0 (0.0) 1 (100.0)
Other microcytic anemia 7 (21.9) <80 0 (0.0) 0 (0.0) 0 (0.0)
Total 27 (84.4) 0 (0.0) 19 (59.4) 1 (3.1)

blood Hb concentration were significantly lower (P < 0.001)
in 100 vegetarians as compared to 100 non-vegetarians.
Moreover, significantly higher frequency of vitamin B12
deficiency (51%, defined as having vitamin B12 < 200 pg/mL)
are discovered in 100 vegetarians than in 100 non-
vegetarians (21%). Tong et al.24 discovered anemia in 12.8%
of vegetarians and 8.7% of regular meat eaters in white
premenopausal women and in 5.8% of vegetarians and 3.4%
of regular meat eaters in white postmenopausal women.
Chai et al.25 showed anemia in 28.2% of the 177 female adult

Table 4 Comparisons of frequencies of blood hemoglobin (Hb < 12 g/dL), red blood cells (RBC < 4  106/mL), mean
corpuscular hemoglobin concentration (MCHC < 31 g/dL), and serum vitamin B12 (<200 pg/mL) deficiencies, and of high RBC
distribution width-coefficient of variation value (RDW-CV > 14.5%) between any two of the four groups, including 122 lacto-
vegetarians with normocytosis (normocytosis/LVs), 12 LVs with microcytosis (microcytosis/LVs), 6 LVs with macrocytosis
(macrocytosis/LVs), and 140 healthy control subjects.
Group Subject number (%)
Hb deficiency RBC deficiency MCHC deficiency High RDW-CV Vitamin B12
(Hb < 12 g/dL) (<4  106/mL) (<31 g/dL) (>14.5%) deficiency
(<200 pg/mL)
Normocytosis/LVs 21 (17.2) 20 (16.4) 3 (2.5) 3 (2.5) 14 (11.5)
(n Z 122)
a
P-value <0.001 <0.001 0.808 0.853 <0.001
b
P-value 0.584 <0.001 0.321 0.321 0.834
c
P-value <0.001 0.701 <0.001 <0.001 0.950
Microcytosis/LVs 11 (91.7) 2 (16.7) 7 (58.3) 9 (75.0) 2 (16.7)
(n Z 12)
a
P-value <0.001 0.062 <0.001 <0.001 <0.001
b
P-value 0.001 0.004 0.060 0.012 0.791
Macrocytosis/LVs 0 (0.0) 6 (100.0) 0 (0.0) 0 (0.0) 0 (0.0)
(n Z 6)
a
P-value NS <0.001 0.268 0.391 NA
Healthy control 0 (0.0) 3 (2.1) 3 (2.1) 4 (2.9) 0 (0.0)
subjects (n Z 140)
NA Z not assessed.
a
Comparisons of frequencies of parameters between 122 normocytosis/LVs, 12 microcytosis/LVs, or 6 macrocytosis/LVs and 140
healthy control subjects by chi-square or Fisher’s exact test, where appropriate.
b
Comparisons of frequencies of parameters between 122 normocytosis/LVs or 12 microcytosis/LVs and 6 macrocytosis/LVs by chi-
square or Fisher’s exact test, where appropriate.
c
Comparisons of frequencies of parameters between 122 normocytosis/LVs and 12 microcytosis/LVs by chi-square or Fisher’s exact
test, where appropriate.

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Y.-P. Lee, C.-H. Loh, M.-J. Hwang et al.
vegetarians in Malaysia. Moreover, Kwok et al.26 found that
the prevalence of definite vitamin B12 deficiency (vitamin
B12 level < 150 pmol/L and methylmalonic acid or
MMA  0.4 mmol/L) is 42% in 119 Chinese vegetarian women
older than 55 years who have been vegetarian for more than
3 years. Pawlak et al.1 reviewed the prevalence of vitamin
B12 deficiency among vegetarians in 18 different studies and
concluded that vegetarians develop B12 depletion or defi-
ciency regardless of demographic characteristics, place of
residency, age, or type of vegetarian diet. One year later,
Pawlak et al.2 reviewed 40 studies on the prevalence of
vitamin B12 deficiency among vegetarians again. They also
discovered a relatively high prevalence of vitamin B12 defi-
ciency among vegetarians and there is a higher prevalence of
vitamin B12 deficiency in vegans than in other vegetarians.
The aforementioned studies confirm that serum vitamin B12
deficiency and anemia are frequently observed in vegetar-
ians, especially in vegans.
Because vitamin B12 is an important micronutrient that
is related to cell division, DNA synthesis, and erythropoi-
esis, severe vitamin B12 deficiency usually results in anemia
and macrocytosis of erythrocytes.3e6 This study discovered
that 16 (11.4%) of the 140 LVs had severe vitamin B12
deficiency (vitamin B12 < 200 pg/mL). Of these 16 B12D/
LVs, 14 had normocytosis, two had microcytosis, and none
had macrocytosis (Table 4). In addition, anemia was
observed in only 5 (three had normocytic anemia, one had
IDA, and one had TTIA) of 16 B12D/LVs (Table 3), suggesting
that normocytic anemia and microcytic anemia are two
major types of anemia in our 16 B12D/LVs. Severe vitamin
B12 and folic acid deficiencies can impair DNA replication
and retard cell division and S-phase progression of eryth-
roblasts, but it does not affect protein (including Hb) syn-
thesis rates. Therefore, overproduction of protein in
erythroblasts results in macrocytosis in patients with severe
vitamin B12 and folic acid deficiencies.27 LVs usually eat a
lot of plant-based foods and thus they usually have a high
serum level of folate that may in turn mask the signs and
symptoms of severe vitamin B12 deficiency,23 finally leading
to normocytosis rather than macrocytosis in LVs. Kwok
et al.26 also reported that vitamin B12 deficiency is asso-
ciated with a significant decrease in Hb concentration, but
anemia associated with vitamin B12 deficiency is often
normocytic rather than macrocytic in vegetarians. In
addition, although this study did not measure the serum
iron and ferritin levels in LVs, LVs might be prone to have
iron deficiency due to suboptimal intakes of animal-source
foods.26 If anemic patients have concomitant deficiencies
of iron (which causes microcytosis in deficient patients) and
vitamin B12 (which causes macrocytosis in deficient pa-
tients), they may have anemia with normal-sized RBCs
(normocytic anemia).28
This study also analyzed the 6 macrocytosis/LVs and
discovered that all of them had RBC deficiency (<4  106/
mL; mean, 3.7  106/mL), low-normal levels of Hb (mean,
12.4 g/dL), and lower serum vitamin B12 level (mean,
368 pg/mL), although none of them had anemia (Hb < 12 g/
dL for women) and serum vitamin B12 deficiency (vitamin
B12 < 200 pg/mL) by WHO definitions.5,6,11 This finding
indicates that LVs with relatively low-normal serum vitamin
6
B12 level can also have macrocytosis. A previous study
suggested asymptomatic high-risk patients (such as LVs)
with low-normal serum vitamin B12 levels (vitamin B12
level between 200 and 399 pg/mL) may still have vitamin
B12 deficiency.22 In this situation, measurement of serum
MMA can be used to confirm vitamin B12 deficiency. If the
patient’s serum MMA level is elevated (>0.40 mmol/L), it
means that the patient does have vitamin B12 deficiency,22
even though the serum vitamin B12 level is of low-normal
value.
In this study, 12 LVs were discovered to have microcytosis
and 11 of the 12 microcytosis/LVs had anemia, suggesting a
high rate of anemia (91.7%) in our 12 microcytosis/LVs. A
detailed analysis of 11 anemic microcytosis/LVs further
found that two had vitamin B12 deficiency and 9 did not have
vitamin B12 deficiency. In the former two anemic B12D/LVs,
one had IDA and the other had TTIA. Of the latter 9 anemic
non-B12D/LVs, one had IDA, one had TTIA, and the remaining
7 had microcytic anemia other than IDA and TTIA. The only
one non-anemic microcytosis/LV had thalassemia trait
(blood Hb, 12.2 g/dL; MCV, 65.0 fL; RBC number, 6.03  106/
mL; and Mentzer index, 10.8).21 These findings indicate that
the microcytosis of the 12 microcytosis/LVs is majorly due to
iron deficiency (iron is a structure component of heme and
iron deficiency results in microcytosis)15e20 and minorly due
to thalassemia trait (gene mutation resulting in insufficient
synthesis of either the a-globin or b-globin chains of HbA
molecule),21 although other conditions (such as defects in
the synthesis of heme or protoporphyrine IX) may also cause
microcytosis of RBCs.15,16
This study discovered that 32 (22.9%) and 16 (11.4%) of
the 140 LVs had anemia and vitamin B12 deficiency,
respectively. Of 32 anemic LVs, 5 (including three with nor-
mocytic anemia, one with IDA, and one with TTIA) had
vitamin B12 deficiency and 27 (including 18 with normocytic
anemia, one with IDA, one with TTIA, and 7 with microcytic
anemia other than IDA and TTIA) did not have vitamin B12
deficiency. Therefore, normocytic anemia (21/32) and
microcytic anemia (11/32) are the two most common types
of anemia in our 140 LVs. LVs seem to be more prone to
develop vitamin B12 and/or iron deficiency. For those LVs
with vitamin B12 deficiency, oral administration of high-dose
vitamin B12 (1e2 mg daily) is as effective as intramuscular
administration of vitamin B12 (1 mg of hydroxycobalamin
once a week for 2 months and 1 mg of hydroxycobalamin
once a month thereafter) for correcting vitamin B12 defi-
ciency.22,29,30 For those LVs with iron deficiency, oral
administration of iron tablet (one tablet of 100 mg of
Fe(OH)3 polymaltose complex per day) for at least 6 months
is also effective for correcting iron deficiency.29,30 Further-
more, dried purple laver (nori) contains not only high levels
of vitamin B12 but also high levels of iron and n-3 poly-
unsaturated fatty acids.31 Thus, it is the most suitable plant-
derived food sources of vitamin B12, iron, and n-3 poly-
unsaturated fatty acids for vegetarians.31
Declaration of competing interest
The authors have no conflicts of interest relevant to this
article.
 + MODEL
Journal of the Formosan Medical Association xxx (xxxx) xxx

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