The Journal of Nutrition, Health & Aging©

Volume 17, Number 4, 2013

Ankle-brAchiAl pressure index And mini nutritionAl

Assessment in community-dwelling elderly people
B.A. MUZEMBO1, Y. NAgANO2, N. DUMAVIBHAT1, N.R. NgATU1, T. MATSUI1, S.A. BHATTI1,
M. EITOKU1, R. HIROTA1, K. ISHIDA2, N. SUgANUMA1
1. Division of Social Medicine, Department of Environmental Medicine, Kochi Medical School, Kochi University, Kochi, Japan; 2. Department of Orthopaedics, Kochi Medical School,
Kochi University, Kochi, Japan. Corresponding author: Muzembo B. Andre, Division of Social Medicine, Department of Environmental Medicine, Kochi Medical School, Kochi
University, Kohasu, Oko-cho, Nangoku-shi, Kochi 783-8505, Japan (e-mail: andersonbasilua@yahoo.fr), Telephone number: 088-880-2407, Fax number: 088-880-2407

Abstract: Background: A low ankle-brachial pressure index (an ABPI value <0.90) is considered predictive of
cardiovascular disease, and is widely thought to increase morbidity and mortality in the elderly. However, good
nutrition is beneficial both for the health and the ability to resist and recover from the disease. Objective: The aim
of the present study was to evaluate the relationship between the ankle-brachial pressure index and the nutritional
status of the elderly in a city of Kochi prefecture, Japan. Methods: This was a cross-sectional study in which a
total of 100 elderly people, both males and females, were screened for ankle-brachial pressure index (ABPI),
nutritional status (through the use of the short form of the mini nutritional assessment), activities of daily living,
lifestyle, gait speed (10MWT), postural stability (OLST), and functional mobility (TUg). Results: About 67% of
the participants were found to have a normal nutritional status, 27% were at risk of malnutrition, and six percent
were classified as malnourished. The mean ABPI of the participants was 1.08±0.10, and three participants had an
ABPI lower than 0.90. The ABPI was statistically higher in well nourished participants compared with those at
risk of malnutrition or the malnourished. The mean ABPI was significantly higher in non-smokers compared with
former smokers. The ABPI was found to correlate negatively with gait speed and with TUg score. Conclusion:
Well-nourished elderly had a higher normal ankle-brachial pressure index as compared with the malnourished
elderly. This study provides supportive evidence for the necessity of adequate nutrition for elderly people.

key words: ABPI, MNA, functional mobility, elderly.

introduction changes in nutritional condition (14). Vitamin D deficiency

Peripheral arterial disease (PAD) is common among elderly
people; it is a strong independent predictor of mortality,
morbidity and functional disability in the elderly (1). Its
prevalence is estimated to be between 12% and 14% and
increases with age (1-3). PAD is an indicator of atherosclerosis
and life expectancy in patients with PAD is lower than that in
patients without PAD (4). The ankle-brachial pressure index
(ABPI) is a marker of subclinical PAD; it is the ratio of ankle to
brachial systolic blood pressure. It is a non-invasive,
reproducible and reliable diagnostic tool in primary care and in
the diagnosis of individuals with subclinical atherosclerosis (5-
7). Low ABPI ( an ABPI <0.9) has been associated with an
increased risk of cardiovascular mortality independent of
metabolic syndrome and traditional risk factors (8). In the
general population, smoking, hypertension,
hypercholesterolemia and diabetes are all believed to play a
crucial role in the reduction of ABPI, in addition to age (9).
Malnutrition is both a common condition and an important
health concern among the elderly. It increases morbidity (10)
and mortality (11) among older persons and it is also a risk
factor for osteoporotic fracture (12). Previous studies had
suggested that there might be a relationship between
malnutrition and PAD. A low body mass index (BMI) caused
by malnutrition was found to contribute to death in patients
with PAD (13). Early life malnutrition may contribute to the
possibility of developing PAD at older ages, possibly due to
through lower dietary intake contributed to PAD in the National
Health and Nutrition Examination Survey (NANHES 2001-
2004) (15), probably because vitamin D deficiency leads to
high blood pressure by inappropriate stimulation of the renin-
angiotensin system (16).
ABPI measurement can help in identifying asymptomatic
individuals at risk of cardiovascular disease (6). In addition,
occlusive PAD and malnutrition in elderly people are common
and remain underdiagnosed and undertreated. Since keeping a
good quality of life in the elderly is one of the primary public
health goals, assessment of the health situation of the elderly
was an important public health objective and scientifically
relevant as a cardiovascular disease prevention goal.
The aim of this study was to evaluate the relationship
between the ankle-brachial pressure index and the nutritional
status in community-dwelling rural elderly people.
methods and materials
Study design and recruitment
This study was a cross-sectional study using data from 100
elderly, both males and females, living in one of the cities in
Kochi prefecture, on Shikoku island, Japan. Selection was on a
voluntary basis through the local welfare committee, and 1103
postal invitations explaining the aim and the schedule of the
health check were sent to eligible participants. The inclusion
criteria were as follow: age 65 years and above, and the ability

370
Received January 10, 2012
Accepted for publication August 20, 2012
The Journal of Nutrition, Health & Aging©
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to answer a questionnaire. A total of 180 elderly people agreed
to participate in the study, however, 80 of them (44.4%) were
not available during our three day fieldwork period. The
primary reason for non-participation was due to the
participants’ work schedules. Another reason for non-
participation was probably the absence of reminder invitation.
The elderly interested in the study were instructed to visit the
local community centers once for a health check-up.
Questionnaire
The participants were instructed to fill out a questionnaire
(which took about 30 minutes), with visually impaired
participants being assisted by our research team. The research
team included five medical doctors and two nurses. All the
participants visited the local community center once during the
three days field work period. Information collected on their
lifestyle included food intake frequency, smoking habits, and
alcohol consumption. In addition, participants were screened
for nutritional status, activities of daily living (ADL) (17),
instrumental activities of daily living (IADL) (18), medications,
current and past diseases, and living area. Nutritional status was
evaluated using the mini nutritional assessment short form
(MNA-SF) and scores were classified as “normal” (12-14), “at
risk of malnutrition” (8-11) or as exhibiting “malnutrition” (0-
7) (19).
Measurements
The participants’ ankle-brachial pressure index, gait speed,
postural stability, basic functional mobility, and blood pressure,
height, body weight measurements were assessed in the local
health centers. The ABPI of both brachial arteries (arms) and
both dorsalis pedis and posterior tibial arteries (ankles) was
measured by a trained orthopedist doctor according to
American College of Cardiology Foundation/American Heart
Association (ACCF/AHA) guidelines (20). A device (VP-1000;
Colin Co., Komaki, Japan) that automatically measures ABPI
and pulse wave velocity was used for both validity and
reproducibility of results (21). ABPI measurement was then
classified as follows: “low” (ABPI ≤0.90), “normal” (0.91-
1.30), and “high” (ABPI >1.30) indicating the presence of
medial arterial calcification (MAC). For each participant, the
lowest ABPI was used for data analysis.
gait speed was assessed by the 10-meter walk test (10MWT)
(22), postural stability by the one leg standing test (OLST) (23),
and functional mobility by the Timed Up and go test (TUg).
The TUg was expressed in seconds, and was defined as the
time required for a participant to rise from an arm chair, walk
three meters, return to the chair and sit down again (24).
Blood pressure (BP) was measured with a standard mercury
sphygmomanometer after the participants had had a ten minute
rest. BP was classified in the following four categories
according to the Seventh Report of the Joint National
Committee on Prevention, Detection, Evaluation, and
Treatment of High Blood pressure (25). Normal (systolic
371
BP<120 mm Hg and diastolic BP<80mm Hg), prehypertension
(systolic BP=120-139 mm Hg, or diastolic BP=80-89 mm Hg),
hypertension stage one (systolic BP= 140-159 mm Hg, or
diastolic BP=90-99 mm Hg), and hypertension stage two
(systolic BP≥160 mm Hg or diastolic BP≥100 mm Hg).
The participants’ height and body weight were measured
using a stadiometer, and a mechanical medical scale (subjects
wore normal indoor clothing without shoes), respectively.
Body mass index (BMI) was calculated by dividing weight in
kg by height in meters squared, and was categorized based on
guidelines of the National Heart, Lung, and Blood Institute
(26).
This study was approved by the Research Ethics Committee
of Kochi Medical School, and all subjects were registered after
giving their informed consent to participate in this study.
Statistical analysis
Data was analyzed using Stata software package version 10
(STATACORP LP, 4905 Lakeway Drive, College Station, TX
77845, USA), and p<0.05 was considered to be statistically
significant. The Kolmogorov-Smirnov test was used to test for
the normality of data distribution. The correlation between
ABPI and health parameters was conducted using Pearson’s
correlation. The Student’s t-test and the Chi-squared test were
used respectively to evaluate differences in ABPI between
different groups and categorical variables. Multiple regression
analysis was performed to evaluate the relationship between
ABPI and studied parameters. The results of quantitative
variables are presented both as the mean ± standard deviation of
the mean.
results
General characteristics of the participants
As shown in Table 1, the study included more females (79%)
than males (21%) and the mean age of the participants was
76.7±6 years. Fourteen participants (14%) consumed alcohol
everyday while twelve (12%) reported as consuming alcohol
occasionally. One participant (one percent) was a tobacco
smoker, and eleven participants (11%) were former smokers.
Fifty-four participants (54%) reported having regular physical
activity. The mean BMI of participants was 23.5±3 kg/m2
(range, 12.3-30) and five percent of the participants were
classified as underweight (BMI<18.5 kg/m2). The mean
systolic blood pressure was 139±22 mmHg while the mean
diastolic blood pressure was 71±11 mmHg. Pre-hypertension
was noted in forty-three participants (43%), hypertension stage
one in thirty participants (30%), hypertension stage two in
sixteen participants (16%), while eleven participants (11%)
were normotensive. A small number of participants (seven
percent) had a diastolic blood pressure greater than 90 mmHg.
Ten percent of participants had a limitation in at least one item
of ADL. There were no statistically significant differences
between men and women in terms of age, BMI, blood pressure
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ANKLE-BRACHIAL PRESSURE INDEX AND MINI NUTRITIONAL ASSESSMENT

table 1
general characteristics of the study population

Variables normal nutrition risk of malnutrition malnutrition (n=6) total (n=100) p value (normal p value (normal
status (n=67) (n=27) nutrition vs risk of nutrition vs
malnutrition) malnutrition)

Sex (%) 0.887 0.806

Males 14 6 1 21
Females 53 21 5 79
Age group (%)
65-69 10 4 1 15 - -
70-79 36 12 3 51 - -
80-89 20 11 2 33 - -
≥ 90 1 0 0 1 - -
Mean age (y) 76.7±6 76.9±6 76.8±6 76.7±6 0.442 0.484
Smoking (%)
Non-smokers 59 24 5 88
Former smokers 7 3 1 11 0.943 0.651
Current smoker 1 0 0 1 0.525 0.443
Alcohol consumption (%)
No alcohol 48 22 4 74
Everyday 12 2 0 14 0.195 0.321
Occasionaly 8 3 1 12 0.781 0.730
Systolic blood pressure 136±24 140±20 150±27 139±22 0.223 0.089
(mmHg )
Diastolic blood pressure 69±10 67±11 72±10 71±10 0.198 0.241
(mmHg )
Activities of daily living
Without limitation 61 25 4 90
With limitation 6 2 2 10 0.808 0.067
Mean Timed Up and 8.2±3 10.6±7 9.2±2 8.9±5 0.010* 0.218
go score (sec)
Mean gait speed (sec) 6.9±2.2 7.5±1.8 7.9±2.2 7.2±2.2 0.106 0.144

*p<0.05 (by Student's t-test or Chi-squared test))

and ADL, though more women were found with impaired
IADL.
Forty-seven participants (47%) were free of chronic
diseases. Five participants (five percent) reported a past history
of atherosclerosis, one participant (one percent) a past history
of asthma, one participant (one percent) a chronic obstructive
pulmonary disease, while seven participants reported kyphosis.
Other chronic diseases such as diabetes, arthritis, anaemia, and
peptic ulcers were also reported. Forty participants (40%)
declared using more than three medications per day.
The mean gait speed, OLST, and TUg expressed in seconds
was the following, respectively: 7.2±2.2 , 36.8±21.6, and
8.9±5.0. gait speed and TUg were found to correlate
significantly (r= 0.470; P<0.001), while a negative correlation
was found between gait speed and OLST (r= -0.480; P<0.001).
Mini nutritional assessment
The mean of the MNA score was 12.2±1.8. About 67% of
the participants were found to have a normal nutritional status
(MNA score, 13.2±0.8), 27% were at risk of malnutrition
(MNA score, 10.5±0.6), and six percent were classified as
malnourished (MNA score, 5.7±2.3). For all participants, the
MNA were found to correlate positively with BMI and gait
speed, while the MNA was negatively correlated with impaired
ADL. In regard to the relationship between the MNA score and
co-morbidities, among the six participants who were classified
as malnourished, five (83.3%) suffered from at least one
chronic disease. Fourteen participants (51.9%) who were at risk
of malnutrition reported having at least one chronic disease and
thirty-one participants (46.3%) with normal nutritional status
had at least one chronic disease.
Ankle-brachial pressure index
The mean ABPI of the participants was 1.08±0.10 (range,
0.35-1.27), and it did not differ between right ABPI and left
ABPI (1.11±0.10 vs 1.10±0.10; P=0.251). An ABPI greater
than 1.30 that could have suggested the presence of MAC was
not observed. Three participants had an ABPI lower than 0.90,
and these three participants were asymptomatic with past
medical conditions of hypertension, diabetes and smoking. The
ABPI was slightly higher in females than in males, but the
difference was not statistically significant (P=0.070).
Participants who lived along the coast had a higher ABPI than
those living in the mountainous areas, and the differences were
statistically significant (0.004). The mean ABPI in non-
smokers was significantly higher than in former smokers
(p<0.001).

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Correlation between ABPI and health assessment
parameters
Figure 1 shows the ABPI of the participants according to
their nutritional status rated by MNA score. The ABPI was
statistically higher in well nourished participants compared
with those at risk of malnutrition or malnourished.
Figure 1
Ankle-brachial index of the participants according to their
nutritional status rated by mini nutritional assessment (MNA)
score (n= 100)
and age (r= -0.232; P=0.020). Postural stability was also
positively correlated with ABPI (r=0.347; P=0.002). The ABPI
had a negative correlation with gait speed (r= -0.355; P<0.001)
and TUg test (r= -0.237; P=0.010). A significant negative
correlation was found between systolic blood pressure and
ABPI (r= -0.240; P=0.010) in the group of participants with an
ABPI lower than 1:00.
Multiple regression analysis for ABPI
Table 3 shows the results of multiple regression analysis
with the explanatory set for ABPI. The model was adjusted for
the following parameters as predictors: age, sex, systolic blood
pressure, diastolic blood pressure, tobacco smoking, nutritional
status, ADL, past chronic diseases, the TUg score, the OLST
score, and gait speed. Among the predictors, smoking,
nutritional status and the TUg test were significantly associated
with ABPI; they accounted for 28% of the variation in ABPI.
After removing the TUg and the OLST in the model to avoid
multicollinearity, however, ABPI was negatively associated
with gait speed (beta= -0.260; p=0.023; adjusted R2=0.218).

table 3
Multiple regression analysis for ABPI

predictor standardized p value Adjusted r2

coefficient (β)

0.28
MNA scores 0.230 0.010
* ABPI in elderly at risk of malnutrition vs ABPI in elderly with normal nutrition status, Smoking - 0.380 0.007
p=0.005; ** ABPI in malnourished elderly vs ABPI in elderly with normal nutrition status,
Timed Up and - 0.420 0.001
p=0.020
go scores

The results of Pearson’s correlation analysis between ABPI

MNA: Mini nutritional assessment. The model was adjusted for age, sex, systolic blood
pressure, diastolic blood pressure, tobacco smoking, nutritional status, activities of daily
and health parameters are shown in Table 2. living, past chronic diseases, the Timed Up and go score, the one leg standing test score,

ABPI and nutritional status had a positive correlation

and gait speed.

(r=0.279; P=0.005), while a negative correlation was found discussion

between ABPI and cigarette smoking. In all the participants, a
positive correlation was found between ABPI and ADL In the present study, we investigated the relationship
(r=0.253; P=0.010), and a negative correlation between ABPI between ABPI and the nutritional status in the elderly of a
table 2
Pearson’s correlation of ankle brachial pressure index and health parameters

total (n=100) males (n=21) Females (n=79)

health parameter r p r p r p

MNA scores 0.279 0.005* 0.448 0.041* 0.249 0.030*

Timed Up and go scores (sec) - 0.237 0.010* - 0.264 0.246 - 0.170 0.136
Smoking (No/day/year) - 0.303 0.002* - 0.323 0.152 - 0.103 0.369
Age (y) - 0. 232 0.020* - 0.278 0.221 - 0.195 0.080
gait speed (sec) - 0.355 <0.001* - 0.679 <0.001* - 0.277 0.010*
Activities of daily living 0.253 0.010* 0.500 0.020* 0.040 0.699
Postural stability 0.347 0.002* 0.570 0.010* 0.220 0.080

*p<0.05; MNA: Mini nutritional assessment

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The Journal of Nutrition, Health & Aging©
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ANKLE-BRACHIAL PRESSURE INDEX AND MINI NUTRITIONAL ASSESSMENT
community. The major findings of this study were that
nutritional status evaluated by MNA-SF, along with cigarette
smoking, gait speed, and the TUg scores were significantly
correlated with ABPI, and the correlation remained even after
adjusting for confounding factors. Although, impaired ADL,
age, and postural stability were correlated with ABPI; after
controlling for confounding factors, however, correlations were
not statistically significant.
Mini nutritional assessment and ABPI
Twenty-seven participants (27%) were at risk of
malnutrition and six participants (6%) were classified as
malnourished, being of particular concern because malnutrition
increases morbidity and mortality among elderly people. These
results highlight the importance of early detection of
malnutrition or risk of malnutrition in the elderly, so that early
nutritional intervention can occur.
The prevalence of the risk of malnutrition varies
considerably depending on the population studied and the
criteria used for the diagnosis (27). Available data for the risk
of malnutrition worldwide indicates that the prevalence of the
risk of malnutrition as rated by the MNA-SF in community-
dwelling elderly is 24 percent (19). In this study, 27% of the
participants were at risk of malnutrition and 53% of the
participants reported as suffering from at least one disease. This
is in agreement with the report by the Japanese Ministry of
Health, Labor and Welfare, which reported that 30% of
Japanese community-dwelling elderly are at risk of
malnutrition, and 50% are suffering from at least one health
problem requiring medical attention (28).
The findings in this study show that well-nourished
participants had significantly higher normal ABPI than other
participants. ABPI has traditionally been used as a non-invasive
marker of generalized atherosclerosis, since it may help to
detect the individuals in the general population who are at risk
of cardiovascular disease (6). Therefore, ABPI measurement
had been recommended by the AHA (29) as it is highly
efficient for cardiovascular disease prevention programs. To
interpret the relationship between ABPI and MNA, it is
important to consider the mechanism by which good nutritional
status may protect against atherosclerosis. A study by
Chakrabarty et al. (30) reported that malnutrition exposes the
endothelium to oxidative stress injury, while good nutritional
status leads to a better control of abnormal platelet
accumulation, helping to prevent or reduce endothelial injury,
improve the ability to recover in case of endothelial injury, and
stop inflammatory response that may generate atheromatous
plaque. In the present study, the ABPI of most of the
participants was within the normal range (1.08±0.10); however,
three participants had an ABPI lower than 0.90, the three
participants being advised to consult a vascular specialist for
additional monitoring.
The fact of the number of tobacco smoker among the
participants being very small (only one), and the good blood
374
pressure control of the participants (average of 139/71mmHg)
may have contributed to the normal mean ABPI found in this
study. This is in accord with the Framingham Offspring study,
which found that smoking and hypertension were significant
correlates of lower ABPI (31).
ABPI, cigarette smoking and alcohol consumption
Non-smokers had a significantly higher mean ABPI
compared with former smokers and the difference remains
statistically significant even after controlling for confounding
factors; this means that the threat of low ABPI remains years
after cessation of tobacco smoking. This fact shows the need
for programs that prevent people from starting tobacco
smoking. These findings concur with previous reports
regarding ABPI and smoking (9, 32, 33). The study by Fowler
et al. (33) found that previous smoking and current smoking
were responsible for low ABPI in one-third of the 4470
participants aged between 65 and 83 years. The Quebec
vascular study (32) found an inverse association between
cigarette smoking and intermittent claudication (IC) among
4570 participants aged 47 to 76 years at the end of the study,
while the risk of IC in participants who cease tobacco smoking
one year before had a similar risk compared to those who never
smoked. However, another study by Hooi et al. (9) observed
that former smokers had an increased risk of PAD but the
difference was not statistically significant with non-smokers.
In this study, about 14% of the participants reported as
consuming alcohol everyday. However, their mean ABPI did
not differ from both those who consumed alcohol occasionally
and those who never drank alcohol. These findings are
consistent with a previous report which reported no significant
association between low ABPI and alcohol consumption (33),
but are inconsistent with the Edinburgh Artery Study (34), a
cross sectional survey which found higher levels of ABPI in the
men with the highest alcohol consumption among 1592
participants aged 55 to 74 years.
ABPI, TUG score, and gait speed
The TUg test and gait speed are used to evaluate a basic
functional mobility that may help to detect the elderly at risk of
health-related events (24, 35). The mean TUg score and gait
speed observed in this study were 8.9±5 seconds and 7.2±2.2
seconds respectively, supporting the classification of
participants as faster, thus confirming that participants in this
study were rather active. The mean TUg score found in this
study is in line with the value reported in the literature
describing healthy Japanese subjects (8.86 seconds) (36);
Steffen et al. also reported a mean TUg score of 9.1 seconds in
96 community dwelling elderly aged between 61 and 89 years
(37). However, a study by Newton (38) reported a mean TUg
score of 15 seconds, ranged between 5.4 and 40.8. The reason
for this discrepancy is that Newton included participants who
were using ambulatory devices but in our study none of the
participants used an ambulatory device.
The Journal of Nutrition, Health & Aging©
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Several studies have evaluated whether ABPI has an
association with functional mobility, but controversy still
exists. In this study, the result of Pearson’s correlation analysis
showed that ABPI was negatively associated with TUg and
gait speed. These associations remained significant even after
adjusting for the age, sex, blood pressure, smoking, nutritional
status, ADL, and history of chronic diseases. These findings
provide support that reduced functional mobility may be a
marker of poor health status. These findings comply with those
of McDermott et al., suggesting that ABPI may be used to
assess patients at risk of mobility loss (39).
Study limitations
The limitation of the study included the following: a
questionnaire was used to assess lifestyle habits and nutritional
status which may contain some recall bias and details may not
be taken into account by participants; this is a common
limitation when using questionnaires. To minimize those
potential errors, the participants were assisted by our research
team in filling out the questionnaire, which was written in
Japanese. Second, the causal relationships cannot be affirmed
between associations observed in this study as it is a cross-
sectional study. Third, the participants who attended the health
check-up were likely to be health cautious elderly in relatively
good health, in spite of the history of some chronic diseases.
The sample size in this study may not be completely
representative of the elderly of the town since the elderly living
alone with IADL dependency such as transportation, and frail
elderly people did not attend the health check-up. It is possible
that if frail elderly people had participated in this study, we
may have seen more malnourished elderly people or those with
a lower ABPI.
Although data reported in this study shows that the
nutritional status evaluated by MNA-SF was significantly
correlated with ABPI, we cannot conclude that MNA-SF has
the potential to predict the risk of atherosclerosis because of the
methodology used. The impact of potential determinants of
health (such as living standards, health care insurance and
educational status) was not analyzed.
In conclusion, the present study enabled us to evaluate the
ankle-brachial pressure index and its correlations with gait
speed, the TUg and the nutritional status in a group of elderly
participants. Results showed that a small number of participants
had lower ankle-brachial pressure index, and that the well-
nourished elderly had a higher normal ankle-brachial pressure
index compared with the malnourished or those at risk of
malnutrition. These findings provided supportive evidence for
the necessity of adequate nutrition for the elderly people.
Acknowledgments: This work was financially supported by Kochi University and city
office of Kuroshio town, Kochi prefecture, Japan. We thank all the participants of this
study. We are also grateful to Mr. Daniel Ribble, Mr Andrew Mugo, Mr Matthew Rowan,
and Ms Mansongi Biyela Carine for their advice and contributions to the manuscript.
Disclosure of Potential Conflicts of Interest: There are no potential conflicts of interest
regarding the content of this article.
375
Author’s contribution: 1. Yasunori NAgANO: data collection, discussion of the study
design, research protocol conception and approval of the manuscript to be published; 2.
Narongpon DUMAVIBHAT: data collection; 3. Nlandu Roger NgATU: data collection; 4.
Tomomi MATSUI: data collection; 5. Sabah Asif BHATTI: data collection; 6. Masamitsu
EITOKU: data collection and discussion of the research protocol; 7. Ryoji HIROTA: data
collection; 8. Kenji ISHIDA: discussion of the study design and research protocol
conception; 9. Narufumi SUgANUMA: study design, research protocol conception and
approval of the manuscript to be published
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