Journal of the Formosan Medical Association (2019) 118, 1299e1307

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journal homepage: www.jfma-online.com

Original Article

Significantly higher frequencies of hematinic

deficiencies and hyperhomocysteinemia in
oral precancer patients
Yu-Hsueh Wu a,b, Yang-Che Wu c, Fang-Yeh Chu d,e,f,
Shih-Jung Cheng b,g,h, Andy Sun b,g, Hsin-Ming Chen b,g,h,*

a
Department of Dentistry, Far Eastern Memorial Hospital, New Taipei City, Taiwan
b
Graduate Institute of Clinical Dentistry, School of Dentistry, National Taiwan University, Taipei,
Taiwan
c
School of Dentistry, College of Oral Medicine, Taipei Medical University, Taipei, Taiwan
d
Department of Clinical Pathology, Far Eastern Memorial Hospital, New Taipei City, Taiwan
e
Graduate School of Biotechnology and Bioengineering, Yuan Ze University, Hsinchu City, Taiwan
f
Department of Medical Laboratory Science and Biotechnology, Yuanpei University of Medical
Technology, Taoyuan City, Taiwan
g
Department of Dentistry, National Taiwan University Hospital, College of Medicine, National Taiwan
University, Taipei, Taiwan
h
Graduate Institute of Oral Biology, School of Dentistry, National Taiwan University, Taipei, Taiwan

Received 19 April 2019; received in revised form 29 April 2019; accepted 21 May 2019

KEYWORDS Background/Purpose: Our previous studies found relatively higher frequencies of anemia, he-
Oral leukoplakia; matinic deficiencies, and hyperhomocysteinemia in patients with different types of oral
Oral precancer; mucosal diseases. This study evaluated whether patients with oral precancerous lesions (oral
Folic acid deficiency; precancer patients) had significantly higher frequencies of anemia, hematinic deficiencies,
Vitamin B12 and hyperhomocysteinemia than healthy control subjects.
deficiency; Methods: The complete blood count, serum iron, vitamin B12, folic acid, and homocysteine
Hyperhomo levels in 131 oral precancer patients including 96 oral leukoplakia, 26 oral erythroleukoplakia,
cysteinemia and 9 oral verrucous hyperplasia patients and in 131 age- and sex-matched healthy control sub-
jects were measured and compared.
Results: We found significantly lower mean serum iron (for women only), vitamin B12, and folic
acid levels and a significantly higher mean serum homocysteine level in oral precancer patients
than in healthy control subjects (all P-values < 0.05). Moreover, 131 oral precancer patients
had significantly higher frequencies of blood hemoglobin (3.1%), vitamin B12 (43.5%), and folic
acid (46.6%) deficiencies and hyperhomocysteinemia (22.1%) than 131 healthy control subjects
(all P-values < 0.05). Of 131 oral precancer patients, lower mean serum folic acid levels were

* Corresponding author. Department of Dentistry, National Taiwan University Hospital, No. 1, Chang-Te Street, Taipei, 10048, Taiwan. Fax:
þ02 2389 3853.
E-mail address: hmchen51@ntuh.gov.tw (H.-M. Chen).

https://doi.org/10.1016/j.jfma.2019.05.016
0929-6646/Copyright ª 2019, 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/).
1300 Y.-H. Wu et al.

found in 87 cigarette smokers than in 44 non-smokers (P Z 0.002), in 26 smokers

consuming > 20 cigarettes per day than in 61 smokers consuming  20 cigarettes per day
(P Z 0.024), and in 52 betel quid chewers than in 79 non-chewers (P Z 0.051).
Conclusion: There are significantly higher frequencies of anemia, serum vitamin B12, and folic
acid deficiencies, and hyperhomocysteinemia in oral precancer patients than in healthy con-
trol subjects.
Copyright ª 2019, 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 of oral epithelial cells,7,56e61 iron is essential to the normal

Head and neck cancer, including oral cancer, is the sixth
most common cancer in males worldwide.1 In Taiwan, oral
cancer remains the fifth leading cause of cancer death in
the whole population and the fourth leading cause of can-
cer death in males.2 Oral leukoplakia (OL), oral eryth-
roleukoplakia (OEL), and oral verrucous hyperplasia (OVH)
are the three common oral precancerous lesions that may
transform into an oral cancer.3e6 The malignant trans-
formation rate is reported to be 1e7% for homogenous thick
OL, 4e15% for granular or verruciform OL, and 18e47% (28%
in average) for OEL.7 Cohort study found that the average
dwelling times are 24 years for OL and 7 years for OEL.
Furthermore, the risks of developing oral cancer after 20
years of follow-up are 42.2% for OL and 95.0% for OEL.8
These findings suggest a higher malignant transformation
potential for OEL lesions than for OL lesions. A retrospec-
tive clinical study showed a malignant transformation rate
of 3.1% and a mean malignant transformation duration of
54.6 months for 324 OVH lesions arising from Taiwanese
patients.9 Our previous study also demonstrated a 5-year
malignant transformation rate of 3% for 30 plaque-typed
and of 17% for 30 mass-typed OVH lesions.10 The high ma-
lignant transformation rates of OL, OEL and OVH lesions
highlight the importance of early detection and treatment
of these three types of oral precancerous lesions and indi-
cate the urgent need of finding any factors that can delay
the progression and malignant transformation of these
three oral precancerous lesions.
In our oral mucosal disease clinic, patients with atrophic
glossitis, burning mouth syndrome, oral lichen planus,
recurrent aphthous stomatitis, and oral submucous fibrosis
(OSF) are frequently encountered and patients with Beh-
cet’s disease are less commonly seen.11e30,31-55 For patients
with one of these six specific diseases, complete blood
count, serum iron, vitamin B12, folic acid, and homocys-
teine levels are frequently examined to assess whether
these patients have anemia, hematinic deficiencies, and
hyperhomocysteinemia.11e30.31-55 Indeed, our previous
studies found relatively higher frequencies of blood he-
moglobin (Hb), iron, vitamin B12, and folic acid de-
ficiencies, and hyperhomocysteinemia in patients with
aforementioned six oral mucosal diseases.11,16,18,30,36,43
In Taiwan, betel quid chewing, cigarette smoking, and
alcohol consumption are involved in multistate progression
of OL, OEL and OVH lesions.3e10 Because folic acid and
vitamin B12 are involved in DNA synthesis and cell division
functioning of oral epithelial cells,7 iron deficiency anemia
(e.g., Plummer-Vinson syndrome) is associated with upper
aerodigestive tract malignancy,7 and carcinogens in to-
bacco smoke and betel quid can damage the DNA of oral
epithelial cells,62e65 the rapid DNA repair and proliferation
of oral epithelial cells may consume a large amount of folic
acid, vitamin B12, and iron in patients with oral precan-
cerous lesions (so-called oral precancer patients in this
study).66 Thus, it was interesting to know whether the
above-mentioned three high-risk oral habits could result in
anemia, hematinic deficiencies, and hyper-
homocysteinemia in oral precancer patients.
In this study, the complete blood count, serum iron,
vitamin B12, folic acid, and homocysteine levels in 131 oral
precancer patients and 131 healthy control subjects were
measured and compared. The main purpose of this study
was to evaluate whether there were significantly higher
frequencies of anemia, serum iron, vitamin B12, and folic
acid deficiencies, and hyperhomocysteinemia in these 131
oral precancer patients than in 131 healthy control sub-
jects. In addition, we also assessed whether oral precancer
patients with betel quid chewing, cigarette smoking, or
alcohol drinking habit were prone to have anemia, serum
iron, vitamin B12, and folic acid deficiencies, and
hyperhomocysteinemia.
Materials and methods
Study participants
The study group consisted of 131 oral precancer patients
(122 men and 9 women; age range, 25e78 years; mean,
48.6  11.6 years) including 96 (89 men and 7 women; age
range, 27e73 years; mean, 48.4  10.7 years) OL, 26 (24
men and 2 women; age range, 25e78 years; mean,
49.3  15.0 years) OEL, and 9 (9 men; age range, 33e69
years; mean, 47.9  11.0 years) OVH patients. The normal
control group consisted of 131 age- (3 years of each pa-
tient’s age) and sex-matched healthy control subjects. All
the patients were seen consecutively, diagnosed, and
treated in the Department of Dentistry, Far Eastern Me-
morial Hospital, New Taipei City, Taiwan from 2017 to 2019.
They were selected according to the following criteria: 1) a
clinical presentation of oral mucosal white patches or pla-
ques (OL) that could not be identified clinically or patho-
logically as any other oral disease,3,4 2) combined oral
Hyperhomocysteinemia in oral precancer patientsArticle type: original article
mucosal red and white lesions (OEL) that could not be
diagnosed clinically or pathologically as any other oral
disease,5,6 and 3) single or multiple elevated oral mucosal
verrucous lesions without marginal induration (OVH) that
could not be recognized clinically or pathologically as any
other oral disease.5,6 All of these clinically-diagnosed OL,
OEL, and OVH lesions were confirmed by histopathological
examination of the biopsy specimens taken from the char-
acteristic part of the OL, OEL, and OVH lesions at the pa-
tient’s first visits, respectively. Histologically, OL lesions
were diagnosed as epithelial hyperplasia with either hy-
perkeratosis or parakeratosis, when no dysplastic cell was
found in the hyperplastic epithelium. Furthermore, OL le-
sions were diagnosed as mild, moderate or severe
dysplasia, when enough dysplastic cells were present in the
basal one third, in the basal two thirds, or in more than the
basal two thirds but not the complete layer of the oral
epithelium, respectively.3,4 Histologically, OEL lesions
usually showed mild, moderate, or severe epithelial
dysplasia.5,6 The histological criteria for a diagnosis of OVH
were: 1) epithelial hyperplasia with parakeratosis or hy-
perkeratosis and verrucous surface, and 2) no invasion of
the hyperplastic epithelium into the lamina propria as
compared to adjacent normal oral mucosal epithelium.
Mild, moderate or severe dysplasia detected in OVH lesions
was also recorded.5,6 However, patients with autoimmune
diseases (such as systemic lupus erythematosus, rheuma-
toid arthritis, Sjogren’s syndrome, pemphigus vulgaris, and
cicatricial pemphigoid), inflammatory diseases, malig-
nancy, or recent surgery were excluded. In addition, all
patients with serum creatinine concentrations indicative of
renal dysfunction (men, > 131 mM; women, > 115 mM) and
who reported a history of stroke, heavy alcohol use, or
diseases of the liver, kidney, or coronary arteries were also
excluded. Healthy control subjects were those who came to
Far Eastern Memorial Hospital for general health examina-
tion from 2017 to 2019 and were found to have none of oral
mucosal or systemic diseases. In addition, none of our oral
precancer patients had taken any prescription medication
for their oral precancerous lesions at least 3 months before
entering the study.
Details of patients’ oral habits, including daily/weekly
consumption of betel quid, cigarette, and alcohol as well as
the duration of these habits, were recorded. Oral pre-
cancer patients were defined as betel quid chewers when
they chew 2 or more betel quids daily for at least one year,
as cigarette smokers when they smoked every day for at
least one year and consumed more than 50 packs of ciga-
rettes per year, and as alcohol drinkers when they drank
more than three days and consumed more than 20 g of pure
alcohol per week for at least one year. The betel quid
chewing and cigarette smoking habits were available for all
131 oral precancer patients, but the alcohol drinking habit
was available for only 77 oral precancer patients. According
to above-mentioned definitions, 52 (39.7%) patients were
betel quid chewers, 87 (66.4%) were smokers, and 42
(54.5%) were drinkers. The oral habit data for the 131
healthy control subjects were not available.
The blood samples were drawn from 131 oral precancer
patients and 131 healthy control subjects for the mea-
surement of blood Hb, iron, vitamin B12, folic acid, and
homocysteine concentrations. This study was reviewed and
1301
approved by the Institutional Review Board at the Far
Eastern Memorial Hospital (FEMH No.: 107116-E).
Determination of blood Hb, iron, vitamin B12, folic
acid, and homocysteine concentrations
The blood Hb, iron, vitamin B12, folic acid, and homocys-
teine concentrations were determined by the routine tests
performed in the Department of Laboratory Medicine, Far
Eastern Memorial Hospital.11,16,18,30,36,43
Statistical analysis
Comparisons of the mean corpuscular volume (MCV) and
mean blood levels of Hb, iron, vitamin B12, folic acid, and
homocysteine between 131 oral precancer, 96 OL, 26 OEL,
or 9 OVH patients and 131 age- and sex-matched healthy
control subjects were performed by Student’s t-test. The
differences in frequencies of microcytosis (MCV < 80 fL),
macrocytosis (MCV  100 fL), deficiencies of blood Hb, iron,
vitamin B12, and folic acid, and hyperhomocysteinemia
between 131 oral precancer, 96 OL, 26 OEL, or 9 OVH pa-
tients and healthy control subjects were compared by chi-
square test. The mean blood levels of Hb, iron, vitamin B12,
folic acid, and homocysteine between two groups of oral
precancer patients with or without alcohol drinking, betel
quid chewing, or cigarette smoking habits as well as be-
tween two groups of chewers or smokers consuming
different amount or duration of betel quids or cigarettes
respectively were compared by Student’s t-test. The result
was considered to be significant if the P-value was less than
0.05.
Results
The MCV and mean blood concentrations of Hb, iron,
vitamin B12, folic acid, and homocysteine in 131 oral pre-
cancer, 96 OL, 26 OEL, and 9 OVH patients and in 131
healthy control subjects are presented in Table 1. Because
men usually had higher blood levels of Hb and iron than
women, these two mean levels were calculated separately
for men and women. We found that 131 oral precancer and
96 OL patients had the significantly lower mean serum iron
(for women only), vitamin B12, and folic acid levels and the
significantly higher MCV and mean serum homocysteine
level than healthy control subjects (all P-values < 0.05,
Table 1). The 26 OEL patients had significantly lower mean
serum iron (for men only), vitamin B12, and folic acid levels
than healthy control subjects (all P-values < 0.05, Table 1).
In addition, 9 OVH patients had the significantly lower mean
serum vitamin B12 and folic acid levels and the significantly
higher MCV than healthy control subjects (all P-
values < 0.05, Table 1).
According to the World Health Organization (WHO)
criteria, men with Hb < 13 g/dL and women with Hb < 12 g/
dL were defined as having Hb deficiency or anemia.67
Furthermore, by our previous studies for OSF patients, pa-
tients with serum iron level  70 mg/dL, vitamin B12
level  450 pg/mL, or folic acid level  6 ng/mL were
defined as having iron, vitamin B12, or folic acid deficiency,
respectively.43 In addition, patients with the serum
1302 Y.-H. Wu et al.

Table 1 Comparisons of mean corpuscular volume (MCV) and mean blood concentrations of hemoglobin (Hb), iron, vitamin
B12, folic acid, and homocysteine between 131 patients with oral precancerous lesions (oral precancer patients), 96 patients
with oral leukoplakia (OL), 26 patients with oral erythroleukoplakia (OEL), or 9 patients with oral verrucous hyperplasia (OVH)
and 131 healthy control subjects.
Group MCV (fL) Hb (g/dL) Iron (mg/dL) Vitamin B12 Folic acid Homocysteine
Men Women Men Women (pg/mL) (ng/mL) (mM)

Oral precancer 90.4  6.2 15.4  1.4 13.7  1.4 96.9  34.7 84.0  20.4 531.1  253.6 8.2  7.2 11.8  5.1
patients (n Z 122) (n Z 9) (n Z 122) (n Z 9)
(n Z 131)
a
P-value 0.035 0.231 0.699 0.160 0.033 0.001 <0.001 <0.001
OL patients 90.6  5.6 15.4  1.3 14.1  0.8 96.6  36.6 85.9  21.3 560.5  264.0 8.6  8.0 11.7  4.5
(n Z 96) (n Z 89) (n Z 7) (n Z 89) (n Z 7)
a
P-value 0.020 0.431 0.658 0.199 0.046 0.048 <0.001 <0.001
OEL patients 88.7  8.5 15.0  1.4 12.3  2.6 90.3  26.1 91  28.3 467.3  218.0 7.6  4.9 12.3  7.3
(n Z 26) (n Z 24) (n Z 2) (n Z 24) (n Z 2)
a
P-value 0.850 0.101 0.528 0.044 0.373 0.002 <0.001 0.125
OVH patients 93.1  3.7 15.7  1.5 0 (n Z 0) 117.9  30.3 0 (n Z 0) 401.4  163.3 5.9  2.3 11.2  2.9
(n Z 9) (n Z 9) (n Z 9)
a
P-value 0.010 0.698 NA 0.172 NA 0.003 <0.001 0.243
Healthy control 89.0  3.8 15.5  0.9 13.9  0.9 102.5  26.2 118.2  36.9 622.7  181.4 12.3  5.1 10.0  1.7
subjects (n Z 122) (n Z 9) (n Z 122) (n Z 9)
(n Z 131)
NA Z not assessed.
a
Comparisons of means of parameters between patients and healthy control subjects by Student’s t-test.

homocysteine level > 13.4 mM (which was the mean serum
homocysteine level of healthy control participants plus two
standard deviations) were defined as having hyper-
homocysteinemia. Based on the aforementioned defini-
tions, 4 (3.1%), 28 (21.4%), 57 (43.5%), 61 (46.6%), and 29
(22.1%) oral precancer patients were diagnosed as having
blood Hb, iron, vitamin B12, and folic acid deficiencies and
hyperhomocysteinemia, respectively (Table 2). We found
that 131 oral precancer patients had significantly higher
frequencies of blood Hb, vitamin B12, and folic acid de-
ficiencies and hyperhomocysteinemia than healthy control
subjects (all P < 0.05, Table 2). The 96 OL patients had
significantly higher frequencies of serum iron, vitamin B12,
and folic acid deficiencies and hyperhomocysteinemia than
healthy control subjects (all P < 0.05, Table 2). Moreover,
26 OEL patients had significantly higher frequencies of
microcytosis, macrocytosis, blood Hb, vitamin B12, and
folic acid deficiencies, and hyperhomocysteinemia than
healthy control subjects (all P < 0.05, Table 2). The 9 OVH
patients had significantly higher frequencies of serum
vitamin B12 and folic acid deficiencies and hyper-
homocysteinemia than healthy control subjects (all
P < 0.005, Table 2).
In this study, in addition to anemia, normocytic anemia
was defined as having the MCV between 80 and 99.9 fL,32e34
iron deficiency anemia as having the MCV <80 fL and serum
iron <60 mg/dL,49,67,68 and thalassemia trait-induced ane-
mia as having the red blood cell count >5.0 M/mL, the MCV
<74 fL, and a Mentzer index (MCV/RBC) < 13.50 By these
definitions, we discovered that of 4 anemic oral precancer
patients, 3 had normocytic anemia, one had thalassemia
trait-induced anemia, and none had iron deficiency
anemia.
We further investigated whether the oral habits might
influence the serum hematinic levels in 131 oral precancer
patients. The relations between alcohol drinking, betel
quid chewing, or cigarette smoking habit and the blood
level of Hb, iron, vitamin B12, folic acid, or homocysteine
are shown in Table 3. Of 131 oral precancer patients,
significantly lower mean serum folic acid levels were found
in 87 cigarette smokers than in 44 non-smokers (P Z 0.002)
and in 26 smokers consuming > 20 cigarettes per day than
in 61 smokers consuming  20 cigarettes per day
(P Z 0.024). The mean serum folic acid level was also lower
in 52 betel quid chewers than in 79 non-chewers
(P Z 0.051, marginal significance, Table 3). Moreover, the
87 cigarette smokers had significantly higher mean serum
homocysteine level than the 44 non-smokers (P Z 0.034).
Furthermore, the mean serum vitamin B12 level was also
lower in 26 smokers consuming >20 cigarettes per day than
in 61 smokers consuming 20 cigarettes per day
(P Z 0.059, marginal significance, Table 3).
Discussion
This study found a significantly higher frequency of Hb
deficiency (3.1%) and a higher frequency of serum iron
deficiency (21.4%, marginal significance) in 131 oral pre-
cancer patients and a significantly higher frequency of
serum iron deficiency (24.0%) in 96 OL patients than in 131
healthy control participants (0% and 13.0% for Hb and iron
deficiencies, respectively). Moreover, we discovered
significantly lower mean serum iron levels in female oral
precancer and OL patients (84.0 and 85.9 mg/dL, respec-
tively) than in female healthy control subjects (118.2 mg/
dL). Our male oral precancer and OL patients also had
lower mean serum iron levels (96.9 and 96.6 mg/dL,
respectively) than that (102.5 mg/dL) in male healthy con-
trol subjects, although the differences were not significant.
Hyperhomocysteinemia in oral precancer patientsArticle type: original article 1303

Table 2 Comparisons of frequencies of microcytosis (mean corpuscular volume or MCV < 80 fL), macrocytosis (MCV  100 fL),
blood hemoglobin (Hb), iron, vitamin B12, and folic acid deficiencies, and hyperhomocysteinemia between 131 patients with
oral precancerous lesions (oral precancer patients), 96 patients with oral leukoplakia (OL), 26 patients with oral eryth-
roleukoplakia (OEL), or 9 patients with oral verrucous hyperplasia (OVH) and 131 healthy control subjects.
Group Patient number (%)
Microcytosis Macrocytosis Hb deficiency Iron Vitamin B12 Folic acid Hyperhomo
(MCV < 80 fL) (MCV  100 fL) (Men < 13 g/dL, deficiency deficiency deficiency cysteinemia
women < 12 g/dL) (70 mg/ (450 pg/mL) (6 ng/mL) (>13.4 mM)
dL)
Oral precancer 6 (4.6) 2 (1.5) 4 (3.1) 28 (21.4) 57 (43.5) 61 (46.6) 29 (22.1)
patients
(n Z 131)
a
P-value 0.055 0.156 0.044 0.072 <0.001 <0.001 <0.001
OL patients 3 (3.1) 1 (1.0) 2 (2) 23 (24.0) 35 (36.5) 44 (45.8) 21 (21.9)
(n Z 96)
a
P-value 0.182 0.242 0.097 0.032 <0.001 <0.001 <0.001
OEL patients 3 (11.5) 1 (3.8) 2 (2.2) 4 (15.4) 16 (61.5) 12 (46.2) 6 (23.1)
(n Z 26)
a
P-value 0.001 0.024 0.001 0.742 <0.001 <0.001 <0.001
OVH patients 0 (0) 0 (0) 0 (0) 1 (11.1) 6 (66.7) 5 (55.6) 2 (22.2)
(n Z 9)
a
P-value 0.793 NA NA 0.872 <0.001 <0.001 0.002
Healthy 1 (0.8) 0 (0.0) 0 (0.0) 17 (13.0) 17 (13.0) 4 (3.1) 3 (2.3)
control
subjects
(n Z 131)
NA Z not assessed.
a
Comparisons of frequencies of parameters between patients and healthy control subjects by chi-square test.

Similar findings of blood Hb or serum iron deficiency in oral
precancer patients have also been reported by oth-
ers.43,69e73 Rajendran et al.69 found a significant decrease
in the Hb and serum iron levels in 50 OSF patients and in 50
OL patients than in 50 healthy control subjects. Tiwari
et al.70 estimated the serum iron level in 40 patients with
potentially malignant disorders (PMD, including 14 OL, 7
erosive oral lichen planus, and 19 OSF patients), 40 oral
cancer patients, and 30 healthy control subjects. They
demonstrated that the mean serum iron level is signifi-
cantly lower in the PMD group (111 mg/dL) or in the oral
cancer group (114 mg/dL) than in the healthy control group
(137 mg/100 mL). Khanna and Karjodkar71 discovered that
the mean serum iron level is reduced significantly in 30
OSF/OL patients (101 mg/dL) and in 30 oral squamous cell
carcinoma patients (105 mg/dL) compared to that (138 mg/
dL) in 30 healthy control subjects. Saurabh et al.72 found a
significantly lower serum iron level in 30 OSF patients than
in 30 normal control subjects. Karthik et al.73 showed
significantly lower blood Hb and serum iron levels in 30 OSF
patients than in 15 healthy control subjects. Wang et al.43
also demonstrated a significantly greater frequency of Hb
deficiency (7.4%) in 68 OSF patients than in 136 healthy
control subjects (0%).
On the contrary, Challacombe74 found the low serum
iron level in 3 (10%) of 30 OL patients and 5 (5%) of 100
healthy control subjects; the difference is not significant.
Jaber75 demonstrated no significant difference in the mean
serum iron level between 120 oral epithelial dysplasia
patients (89 smokers and 31 non-smokers) and 120 age- and
gender-matched healthy control subjects (59 smokers and
61 non-smokers). Wang et al.43 also did not find a significant
difference in the frequency of serum iron deficiency be-
tween 68 OSF patients (20.6%) and 136 healthy control
subjects (14.7%).
This study found significantly lower mean serum folic
acid and vitamin B12 levels in 131 oral precancer, 96 OL, 26
OEL, and 9 OVH patients and significantly higher mean
serum homocysteine levels in 131 oral precancer and in 96
OL patients than in 131 healthy control participants.
Furthermore, we also showed significantly greater fre-
quencies of folic acid and vitamin B12 deficiencies and
hyperhomocysteinemia in 131 oral precancer, 96 OL, 26
OEL, and 9 OVH patients than in 131 healthy control sub-
jects. Hematological alterations of the serum folic acid,
vitamin B12, and homocysteine levels in the head and neck
cancer and precancer patients have also been reported.
Ramaswamy et al.76 found significantly lower mean serum
folic acid and vitamin B12 levels in 50 OL patients than in 50
age- and sex-matched normal controls including 25 tobacco
chewers and 25 non-chewers. Almadori et al.77 discovered
significantly lower serum folic acid levels in 144 head and
neck squamous cell carcinoma (HNSCC) patients and in 40
laryngeal leukoplakia patients than in 90 control smokers
and 120 control nonsmokers. Serum homocysteine levels
are significantly higher in HNSCC patients than in both
control smokers and nonsmokers and in laryngeal leuko-
plakia patients. However, there are no significant
1304 Y.-H. Wu et al.

Table 3 Comparisons of mean blood levels of hemoglobin (Hb), iron, vitamin B12, folic acid, and homocysteine between two
groups of oral precancer patients with or without alcohol drinking, betel quid chewing, or cigarette smoking habits as well as
between two groups of chewers or smokers consuming different amount or duration of betel quids or cigarettes, respectively.
Group Mean  SD
Hb (g/dL) Iron (mg/dL) Vitamin B12 (pg/mL) Folic acid (ng/mL) Homocysteine (mM)
Alcohol drinking (n [ 77)
Drinker (n Z 42) 15.0  1.3 94.5  36.9 521  294 7.2  6.3 12.5  5.7
Non-drinker (n Z 35) 15.4  1.4 96.5  27.2 579  235 8.2  6.5 11.1  2.9
a
P-value 0.177 0.784 0.342 0.480 0.181
Betel quid chewing (n [ 131)
Chewer (n Z 52) 15.3  1.3 96.7  4.0 522  284 6.8  5.4 11.3  3.8
Non-chewer (n Z 79) 15.2  1.5 95.9  29.4 537  233 9.2  8.1 12.1  5.8
a
P-value 0.743 0.908 0.752 0.051 0.339
Daily consumption (n Z 52)
>20 quids (n Z 24) 15.3  1.2 103.5  49.9 462  185 5.8  2.2 12.2  3.8
20 quids (n Z 28) 15.3  1.4 90.9  29.6 574  343 7.7  7.0 10.5  3.7
a
P-value 0.886 0.287 0.142 0.174 0.102
Duration (n Z 52)
>10 years (n Z 25) 15.3  1.3 100.3  48.6 512  218 6.3  2.5 10.8  3.4
10 years (n Z 27) 15.3  1.3 93.3  31.4 531  338 7.3  7.2 11.8  4.2
a
P-value 0.868 0.545 0.813 0.514 0.374
Cigarette smoking (n [ 131)
Smoker (n Z 87) 15.4  1.4 98.2  34.5 507  261 6.6  5.1 12.4  5.7
Non-smoker (n Z 44) 15.0  1.4 92.3  33.0 578  235 11.5  9.4 10.7  3.4
a
P-value 0.101 0.339 0.117 0.002* 0.034*
Daily consumption (n Z 87)
>20 cigarettes (n Z 26) 15.7  1.2 103.4  37.9 436  196 5.2  2.2 12.3  4.9
20 cigarettes (n Z 61) 15.3  1.5 96.0  33.0 537  280 7.2  6.0 12.4  6.1
a
P-value 0.199 0.396 0.059 0.024* 0.970
Duration (n Z 87)
>10 years (n Z 80) 15.3  1.5 98.3  34.2 512  271 6.6  5.4 12.2  5.5
10 years (n Z 7) 15.8  0.8 97.9  40.5 448.7  49.5 6.0  1.4 13.7  0.7.9
a
P-value 0.158 0.980 0.080 0.416 0.658
a
Comparisons of means of parameters between two groups of patients by Student’s t-test.

differences in serum vitamin B12 levels among the above 4
groups.77 Jaber75 discovered a significant decrease in the
mean serum folate level and red blood cell (RBC) folate
level in 120 oral epithelial dysplasia patients (89 smokers
and 31 non-smokers) compared to 59 healthy control to-
bacco smokers (P < 0.05). However, no significant differ-
ence in the mean serum vitamin B12 level is found between
120 oral epithelial dysplasia patients and 120 healthy con-
trol subjects (59 smokers and 61 non-smokers). The above
findings consistently support the folic acid deficiency in oral
precancer and oral cancer patients. Because the folic acid
and vitamin B12 deficiencies were the main factors causing
hyperhomocysteinemia,11,16,18,30,36,43 it was easy to explain
why parts of our oral precancer patients with folic acid and
vitamin B12 deficiencies might have hyperhomocysteinemia
or great frequencies of hyperhomocysteinemia.
Previous studies supported the findings of significantly
lower mean folate levels in buccal mucosal cells and sera of
smokers than in those of non-smokers.62,63 Piyathilake
et al.62 found significantly lower mean buccal mucosal cell
folate levels in 25 smokers than in 34 non-smokers. The
mean plasma folate level of 25 smokers, although within
normal limits, is also significantly lower than that of 34 non-
smokers. These results indicate that cigarette smoking may
cause a localized folate deficiency in buccal mucosal cells,
independent of the plasma folate levels.62 Pivathilake
et al.63 further studied the local and systemic effects of
cigarette smoking on plasma and RBC folate and vitamin
B12 levels in 39 current smokers and 60 noncurrent
smokers. They found significantly lower plasma and RBC
folate concentrations in subjects who have the last smoking
event less than one hour before collecting the blood sam-
ples than in subjects who have smoked earlier. They also
demonstrated lower buccal mucosal cell folate and vitamin
Bl2 concentrations in current smokers than in noncurrent
smokers.63 This study also found significantly lower mean
serum folic acid levels in 87 cigarette smokers than in 44
non-smokers and in 26 smokers consuming >20 cigarettes
per day than in 61 smokers consuming 20 cigarettes per
day. The mean serum folic acid level was also lower in 52
betel quid chewers than in 79 non-chewers. Heimburger66
reviewed the possible existence of localized folate de-
ficiencies in the aerodigestive tract of the smoker and
collected the evidences including lower serum folate levels
in smokers than in nonsmokers, lower serum folate levels in
smokers with bronchial metaplasia than in those without
Hyperhomocysteinemia in oral precancer patientsArticle type: original article
bronchial metaplasia, lower folate levels in scrapings of the
buccal mucosal cells of smokers than in those of non-
smokers, apparent improvement in bronchial atypical
metaplasia in smokers supplemented with folic acid, lower
erythrocyte folate levels and higher prevalence of cellular
features compatible with folate deficiency in geographic
areas and individuals in South Africa at high risk for
esophageal cancer, and a trend toward a lower prevalence
of colonic dysplasia in ulcerative colitis patients who use
folic acid supplements. He finally suggested that folate
deficiency may be co-carcinogenic and the mechanisms of
folate deficiencies may arise from elevated folate turnover
in response to rapid tissue proliferation or repair, inacti-
vation or alteration of its function by external agents such
as tobacco, alcohol or drugs, or altered metabolism or tis-
sue uptake caused by an inborn error.66 The findings of our
study also add the evidence that oral precancer patients
with cigarette smoking or betel quid chewing habit are
prone to have lower serum folic acid levels than oral pre-
cancer patients without cigarette smoking or betel quid
chewing habit, respectively.
This study found significantly lower mean serum levels of
iron (for women only), folic acid, and vitamin B12 and a
significantly higher mean serum homocysteine level in our
oral precancer and OL patients than in healthy control
subjects. Moreover, we also demonstrated greater fre-
quencies of blood Hb, iron, vitamin B12, and folic acid
deficiencies and hyperhomocysteinemia in our oral pre-
cancer and OL patients than in healthy control subjects.
The significantly greater frequencies of serum iron, vitamin
B12, and folic acid deficiencies in our oral precancer pa-
tients may be partially due to the tobacco, betel quid, and
alcohol consumption in a relatively high proportion of our
oral precancer patients. Further studies should be carried
out to explore the other possible mechanisms that cause
serum iron, vitamin B12, and folic acid deficiencies in our
oral precancer patients and to clarify whether supple-
mentations of these hematinics (iron, vitamin B12, and folic
acid) may delay the progression and malignant trans-
formation of OL, OEL, and OVH lesions.
Conflicts of interest
The authors have no conflicts of interest relevant to this
article.
Acknowledgements
This study was partially supported by the grant (FEMH-2019-
C-059) of Far Eastern Memorial Hospital, New Taipei City,
Taiwan.
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