Glycative Stress Research
Online edition : ISSN 2188-3610
Print edition : ISSN 2188-3602
Received : April 8, 2016
Accepted : June 9, 2016
Published online : June 30, 2016

Original paper

Physical effects of a 12-week protocol of daily gyudon (beef bowl) ingredientsconsumption: Implications for fast food researchMari Ogura 1, 2), Wakako Takabe 1), Yuichi Nagano 3), Ken-ichi Onodera 3), Yoshikazu Yonei 1)
1) Anti-Aging Medical Research Center and Glycative Stress Research Center, Graduate School of Life and Medical Sciences,
Doshisha University, Kyoto, Japan
2) Department of Food Science and Nutrition, Kyoto Bunkyo Junior College, Kyoto, Japan3) Yoshinoya Holdings Ltd., Tokyo, Japan

Abstract

Objectives: Gyudon, a bowl of rice topped with beef and onions, is a fast food item. We evaluated the physical effect andsafety of a 12-week protocol of once daily gyudon ingredients consumption.
Methods: A total of 24 adult volunteers (12 men and 12 women, mean age 44.8 ± 8.5 years) were included and asked
to consume “Frozen Gyudon Ingredients” (heated according to the printed instructions), the test food, once daily for 12
consecutive weeks. The test food, provided by Yoshinoya Holdings Ltd. (Tokyo, Japan), was identical to the regular-size
gyudon provided at Yoshinoya stores. The subjects underwent physical and hematological/biochemical examinations before
and at 4, 8 and 12 weeks after the start of test food consumption and at 4 weeks after the completion of test food consumption.
This study was designed to compare the test food with other food items under development in a double-blind, parallel-groupstudy and was conducted with approval by an ethics committee. The current report however, only presents the results of thegroup of subjects who consumed the test food.
Results: The compliance rate for test food consumption during the observation period was more than 94%, with no subject
withdrawn. No significant change was observed in body weight (63.1 ± 13.9 kg at baseline) or body fat percentage (23.2 ±
6.4% at baseline) during the test food consumption period. As for blood pressure, no significant change was observed insystolic blood pressure while a 4.7% increase in diastolic blood pressure from the baseline value (70.4 ± 9.5 mmHg) was
noted at week 4 (p <0.05), with no significant change from baseline at week 8 or 12. Among lipid metabolism parameters,
the total cholesterol level showed a 4.4% increase from the baseline value (197.3 ± 27.8 mg/dL) at week 8 (p < 0.05), withno significant change from baseline at week 12. The LDL and HDL cholesterol levels showed no significant change during
the consumption period. Although the triglyceride level showed a 30.3% significant increase from the baseline value (76.3± 33.2 mg/dL) at week 8 (p < 0.05), there was no significant increase at week 12. The fasting blood glucose level increased
from baseline (85.4 ± 9.2 mg/dL) by 3.5% at week 4 (p < 0.05), with no significant change from baseline at week 8 or 12. No
adverse event related to the test food was observed during the observation period.
Conclusion: Under the current study conditions, no adverse event and no significant change in glycolipid metabolism
parameters was observed during or after the 12-week protocol of daily test food (gyudon ingredients) consumption. Further
well-designed studies are needed to verify the safety of the test food.

KEY WORDS: Gyudon, continuous intake, fast food, trans fatty acids, glycative stress
Introduction

Gyudon is a bowl of rice topped with thin-sliced (cutor chopped) beef and onion simmered with sugar and soy
sauce. Its sweet soy sauce taste is preferred by the majorityof Japanese people and its attractive flavor of umami-richbeef tallow renders it a famous national dish of Japan.
Japan has recently seen an increasing prevalence of

metabolic syndrome and an associated rapid increase inlifestyle-related diseases. The major causes of these trends
are decreased physical activity and increased energy intake
due to the Westernization of diet. However, there is a strong
trend among people to link the image of a Western diet withmeat consumption. This trend is related to gyudon, a cuisinethat proposes to be a Japanese traditional way of consuming

Corresponding author: Prof. Yoshikazu Yonei
Anti-Aging Medical Research Center and Glycative Stress Research Center,
Graduate School of Life and Medical Sciences, Doshisha University
1-3 Tatara Miyakodani, Kyotanabe City Kyoto 610-0394, Japan
TEL & FAX: +81-774-65-6394 Email: yyonei@mail.doshisha.ac.jp
Contact for Co-authors: Ogura M, m-ogura@po.kbu.ac.jp ;Takabe W, wtakabe@mail.doshisha.ac.jp ; Nagano Y, y.nagano@ysn.yoshinoya.com ;Onodera K, k.onodera@ysn.yoshinoya.com_ 99 _

Glycative Stress Research 2016; 3 (2): 99- 109(c) Society for Glycative Stress ResearchEffect of 12-Week Protocol of Daily Beef Bowl Consumption
due to shift-based or night shift work.
When the subjects were examined for baseline glucose
metabolism parameters, 5 of 24 subjects had high normal
to borderline levels of fasting plasma glucose (FPG) during
the pre-study screening or a 75-g oral glucose tolerance test
(OGTT) 2-hour value of 140-199 mg/dL. With regard todyslipidemia, 8 and 4 of 24 subjects had borderline (123-139
mg/dL) and abnormal (140-187 mg/dL) levels of low density
lipoprotein cholesterol (LDL-C), respectively, while no
subject had a high density lipoprotein cholesterol (HDL-C)
level of < 40 mg/dL or a triglyceride (TG) level of ≥150 mg/
dL. As for lifestyle-related variables, 15 of 24 subjects wereconsuming alcohol (equivalent to ≥2 gou sake/day), 4 were
smokers (≥20 cigarettes/day) and 2 were former smokers.
Exercise habit (≥twice/week) was reported by 7 of 24 subjects.
Subjects were asked to consume one serving of test
food “Frozen Gyudon Ingredients” (heated according to
the printed instructions) daily by adding it to any one of
the three daily meals for 12 consecutive weeks. The test
food, provided by Yoshinoya Holdings Ltd. (Tokyo, Japan),
was identical to the regular-size gyudon (135 g) provided at
Yoshinoya stores.
The study was conducted at Chiyoda Paramedical Care
Clinic (Tokyo, Japan) between April 2015 and October
2015. Subjects were given a full explanation of the study andprovided a written informed consent.

beef, thereby alluding that it is not an unhealthy food.
We have measured changes in postprandial glucose
levels after consumption of various food ingredients 1, 2) andfound that consuming rice with gyudon resulted in reduced
postprandial hyperglycemia compared to consuming white
rice alone 3). Meanwhile, no study has examined the physicaleffect of the continuous consumption of gyudon ingredients.
The objective of this study was to evaluate the health effectof the current commercial product by analyzing the physicaleffect of a 12-week protocol of daily gyudon consumption,in particular focusing on changes in lipid metabolism
parameters known to be associated with glycative stress.
Although this study was designed to compare the test foodwith other products under development in a double-blind,parallel-group study, the current report only presents theresults of the group of subjects who consumed the test food(gyudon ingredients).

Methods
Subjects

The subjects consisted of 24 adult volunteers aged ≥20
and <65 years (12 men and 12 women, mean age 44.8 ± 8.5
years). Subjects were excluded from the study if they: 1) wereregularly taking a medicine, health food or food for specified
health use considered to possibly affect study results; 2) had
a previous or current history of a serious disorder of heart,
liver, kidney or gastrointestinal tract; 3) were pregnant,suspected of being pregnant, or breastfeeding; 4) were heavy
alcohol drinkers, defined as those with an average daily
alcohol consumption of > 60 g, according to References on
Promotion of Healthy Japan 21 (Second edition); or 5) had
extremely irregular dietary habits or irregular rhythm of life

Test food

Each bag of Yoshinoya Frozen Gyudon Ingredients(content: 135 g) was defined as 1 serving. The component
composition and nutritional analysis results of the test foodare shown in Tables 1 and 2, respectively.

Table 1. Component of the test sample in 1 package for 1 day.

Item

Unit

Amount

g

65

Beef

Onion

g

Sauce (Tare)

30

g

40

Item

Unit

Amount per 100 g

Energykcal

249

Table 2. Nutrition analysis of the test sample.

Protein
Lipid

Carbohydrate
Sodiumg
g
g

mg※135 g per package_ 10 0 _

9.6
20.8
5.8
696

Glycative Stress Research

Test parameters

Baseline characteristics, anthropometry andphysical examination

The following subject’s baseline characteristics were
evaluated: gender, date of birth, age, alcohol habits, dietary
habits, medical history, comorbidities (subjective and objective
symptoms), allergy ( food and drug ), bowel movement history,
names of drugs taken, names of medicines, health food andfood for specified health use taken regularly, and volume ofblood samples collected within 12 months. Subjects were alsointerviewed regarding their physical condition. Anthropometricparameters included body height and weight as measured
with InBody 3.2 (InBody Japan, Tokyo, Japan), body fat
percentage as measured by the bioelectrical impedance
method on InBody 3.2 (InBody Japan), and body mass index(BMI). On physical examination, systolic and diastolic bloodpressures and pulse rate were measured.
Subjects were asked to record the following information
in a diary during the study period: the details of meals(frequency and amount), alcohol consumption, amount of
exercise, frequency of bowel movement, sleeping time,
subjective symptoms, presentation to a medical institution
and details of treatment received, and use of medicines andhealth food.

Hematology and biochemistry

The following hematological and biochemical parameters
were measured to assess the safety of the test food. The
hematology parameters included white blood cell count(WBC; /μL), red blood cell count (RBC; x10 4/μL), hemoglobin(Hb; g/dL), hematocrit (Hct; %) and platelet count (Plt; x10 4 /μL). The biochemical parameters included total protein (g/dL),
albumin (g/dL), AST (GOT; IU/L), ALT (GPT; IU/L), LDH(IU/L), ALP (IU/L), γ -GTP (IU/L), CPK (IU/L), blood urea
nitrogen (BUN; mg/dL), creatinine (mg/dL), uric acid (mg/dL), serum electrolytes (sodium, potassium and chloride;mEq/L) and calcium (mg/dL). The glycolipid metabolism
parameters included FPG (mg/dL), total cholesterol (TC;mg/dL), HDL-C (mg/dL), LDL-C (mg/dL) and TG (mg/dL).
The hematological and biochemical examinations were
performed at LSI Medience Corporation (Tokyo, Japan).
Urinalysis included a semi-quantitative measurement ofprotein, sugar, urobilinogen and bilirubin.
Subjects were instructed to fast from 21:00 on the day
before testing until the completion of testing, during which
no beverage other than water was allowed. On the day of
testing, subjects were prohibited from smoking from rising
until the completion of testing. Subjects were asked to cometo the clinic in the morning and undergo a series of tests anda medical interview, followed by blood collection. Subjects
were kept in a resting state in the clinic and asked to stay inthe clinic until the completion of testing.
Subjects with normal high to borderline FPG levels at
the pre-study screening were subjected to OGTT, in which
only the 2-hour data were collected.

Study scheduleThis study consisted of the pre-study screening andthe main study. For the pre-study screening, the consenting
subjects were asked to come to the clinic within 4 weeks

before the start of the main study and undergo the necessary
tests. During the main study, each subject made 5 visits tothe clinic (before and at 4, 8 and 12 weeks after the start oftest food consumption, and at 4 weeks after the completionof test food consumption) to undergo the necessary tests.

Statistical Analysis

For each parameter, the number of subjects, mean andstandard deviation (SD) were calculated and tested forstatistical significance as described below. All statistical tests
were performed with a significance level of 5%. For the resultsof anthropometry, physical examination, hematological andbiochemical examinations, the Dunnett’s test was performed
to compare the measured values at each measurement point(at weeks 4, 8 and 12 and 4 -week follow-up) with those atbaseline. The Wilcoxon’s one-sample test was used to compare
urinalysis parameters (protein, sugar, urobilinogen andbilirubin) at each measurement point with those at baseline.

Ethical considerations

Subjects were examined by physicians for any change
in their physical condition caused by test food consumption.
This study was conducted in compliance with the Declarationof Helsinki and the Ethical Guidelines for Medical Researchin Humans and in accordance with the Good Clinical Practice(GCP), with ongoing consideration to the protection ofsubjects’ human rights. This double-blind, parallel-groupstudy was conducted in accordance with the protocol which
had been reviewed for ethical adequacy and appropriatenessof the study plan and approved by Research Ethics Committee
at Chiyoda Paramedical Care Clinic. This report only presents
the results of the group of subjects who consumed the test
food (gyudon ingredients).

Results

Compliance to test food consumption

The mean compliance rate for test food consumption by
the subjects was 99.6 ± 1.1%, ranging from 94.0% to 100%.
Thus, all subjects achieved more than 90% of compliance totest food consumption. There was no significant differencein the compliance rate between men and women. With nosubject withdrawn prematurely, all 24 subjects were includedin the analysis.

Results of anthropometry and physicalexamination (Table 3)

No significant change in body weight was observed at
week 4, 8 or 12 compared to baseline (63.1 ± 13.9 kg), whereas
a significant decrease to 62.3 ± 13.5 kg (-1.3%) was observedat the 4-week follow-up ( p < 0.01). The changes in body weightin individual subjects ranged from -4.5 to 1.6 kg.
For BMI, no significant change was observed at weeks
4, 8 or 12 compared to baseline (22.6 ± 3.6 kg/m 2 ), whereas
a significant decrease to 22.3 ± 3.6 kg/m 2 (-1.3%) was
observed at the 4-week follow-up (p < 0.01). The changes in
BMI in individual subjects ranged from -1.5 to 0.6 kg/m 2.

_ 101 _

Effect of 12-Week Protocol of Daily Beef Bowl Consumption
Table 3. Physical information.

Item

WeightBMI
Body fat
Blood pressure(systolic)(diastolic)Pulse rate

Unit

kg
kg/m2
%
mmHg
mmHg
Bpm

Before4 weeks

8 weeks

12 weeks

4 weeks after the test

63.1 ± 13.9

62.8 ± 13.7

62.9 ± 14.0

63.0 ± 13.5

62.3 ± 13.5 * *22.6 ± 3.6

22.5 ± 3.6

22.5 ± 3.7

115.2 ± 13.7

117.0 ± 14.1

116.9 ± 13.9

70.4 ± 9.5

73.7 ± 9.7 *72.2 ± 10.4

23.2 ± 6.4

73.6 ± 10.3

22.7 ± 6.5

22.9 ± 6.6

75.4 ± 9.2

73.3 ± 9.7

22.6 ± 3.6

22.8 ± 7.0

22.3 ± 3.6

22.5 ± 7.1

115.4 ± 15.7

115.9 ± 13.7

73.3 ± 12.0

72.5 ± 9.7

73.5 ± 9.5***

74.5 ± 9.6

Data are expressed as mean ± standard deviation. *p<0.05, **p<0.01 vs Pre values by Dunnett’ s test, n = 24. BMI, body mass index.

As for body fat percentage, no significant change wasobserved at week 4, 8 or 12 compared to baseline (23.2 ±
6.4%), whereas a significant decrease to 22.5 ± 7.1% (-2.2%)
was observed at the 4-week follow-up (p <0.05). The changes
in body fat percentage in individual subjects ranged from
-4.4 to 2.3%.
In terms of blood pressure, the diastolic blood pressure
significantly increased from 70.4 ± 9.5 mmHg at baseline to73.7 ± 9.7 mmHg (+4.7%) at week 4 (p < 0.05), whereas no
significant difference compared to baseline was observed at
week 8 or 12. No significant change was observed in systolicblood pressure. The changes in systolic and diastolic bloodpressures in individual subjects ranged from -13 to 16 mmHg
and -13 to 17 mmHg, respectively.
No significant change was observed in pulse rate.
While there were significant differences between men
and women in the baseline values of body weight and BMI,
no significant sex difference was observed in percent changes
in the post-baseline values of these variables. No significant
sex difference was observed in any other variable.

Hematological and biochemical examinations(Tables 4 and 5)

When the measured values of biochemical andhematological parameters at each measurement point werecompared to the baseline values, significant changes wereobserved in the following parameters. Since significant sex
differences were observed in the baseline values of RBC,
Hb, Ht, γ - GTP, CPK and Cre, the values of these variables
are presented separately for men and women. No significant
sex difference was observed in any other variable.
Among hematological parameters, Ht in womensignificantly decreased from 40.7 ± 2.9% at baseline to 39.1
± 2.4% (-3.9%) at week 4 ( p <0.05), whereas no significant
difference compared to baseline was observed at week 8. No
significant change was observed in WBC, RBC, Hb or Plt.
Among biochemical parameters, TP showed nosignificant change from baseline (7.08 ± 0.35 g/dL) at week
4, 8 or 12, but showed a significant increase to 7.21 ± 0.30
g/dL (+1.8%) at the 4 -week follow-up ( p < 0.05). LDH
significantly increased from 154.5 ± 21.5 U/L at baseline to164.1 ± 28.2 U/L (+6.2%) at week 12 (p < 0.01), whereas no
significant difference compared to baseline was observedat the 4-week follow-up. γ - GTP in women showed nosignificant change from baseline (14.3 ± 3.5 U/L) at week 4,
8 or 12, but showed a significant increase to 17.3 ± 7.2 U/L(+21.0%) at the 4-week follow-up (p < 0.05).

In electrolyte analysis, the potassium level showed asignificant increase from 4.13 ± 0.25 mEq/L at baseline to4.30 ± 0.41 mEq/L (+4.1%) at week 8 (p <0.05), whereas no
significant difference compared to baseline was observed at
week 12.
Among glycolipid metabolism-related parameters
known to be associated with glycative stress, the TC level
showed a significant increase from 197.3 ± 27.8 mg/dL atbaseline to 206.0 ± 31.3 mg/dL (+4.4%) at week 8 (p <0.05),
whereas no significant difference compared to baseline wasobserved at week 12. TC also significantly increased to205.2 ± 28.4 mg/dL (+ 4.0%) at the 4-week follow-up (p< 0.05). No significant change was observed in LDL-C or
HDL-C. TG significantly increased from 76.3 ± 33.2 mg/
dL at baseline to 99.4 ± 71.1 mg/dL (+30.3%) at week 8(p < 0.05), whereas no significant difference compared
to baseline was observed at week 12. FPG significantly
increased from 85.4 ± 9.2 mg/dL at baseline to 88.4 ± 8.2
mg/dL (+ 3.5%) at week 4 (p < 0.05), whereas no significant
difference compared to baseline was observed at or after
week 8.

Qualitative urinalysis (Table 6)

Qualitative urinalysis showed a change to (±) (from (-)
at baseline) in 3 subjects at week 8, changes to (±) in 3 subjects
and to (+) (from (-) at baseline) in 1 subject at week 12, andchanges to (±) in 4 subjects and to (+) (from (-) at baseline)in 1 subject at the 4 -week follow-up. When the semiquantitsative values of urinalysis parameters at eachmeasurement point were compared to the baseline values,
no significant change was observed in any of the parameters
tested. No abnormal changes in qualitative sugar, urobilinogenor bilirubin levels were observed in any of the subjects.

Adverse events

The number of adverse events observed during the study
period is shown in Tables 7 and 8. The incidence of adverse
events was 16.7% (4/24). No serious adverse event wasobserved in this study. The subjective symptoms reported
as adverse events included acute pharyngitis in 1 man andcold symptoms (cough, malaise and anorexia) in 1 man and1 woman. These events were considered unrelated to the test
food consumption. Abnormal laboratory changes reported asadverse events included a change in urine protein in a woman
and an increase in γ -GTP in a man. These events were

_ 10 2 _Glycative Stress Research

Table 4. Peripheral blood examination.

Item

Unit

Sex

Before

WBC

/μL

-

5741.7 ± 1657.1

5729.2 ± 1503.5 5208.3 ± 1468.8 5358.3 ± 1472.6

5391.7 ± 1410.5

RBC

104/μL

Male

493.7 ± 27.0

499.3 ± 34.6

498.5 ± 27.1

489.7 ± 25.7

492.8 ± 28.7

Female428.2 ± 38.7

428.2 ± 35.3

430.3 ± 40.7

421.0 ± 40.8

435.8 ± 27.9

Hb

g/dL

Male

15.1 ± 1.0

15.1 ± 1.0

15.2 ± 0.9

15.0 ± 0.8

14.9 ± 1.0

Female12.6 ± 0.9

12.6 ± 0.9

12.7 ± 1.1

12.5 ± 1.1

12.7 ± 0.9

Ht

%

Male

47.3 ± 2.2

46.3 ± 2.8

46.5 ± 2.0

47.0 ± 2.2

46.4 ± 2.7

Female40.7 ± 2.9

39.1 ± 2.4 * *39.8 ± 3.1

39.8 ± 3.0

40.6 ± 2.6

Plt

10 /μL

-25.1 ± 3.4

25.8 ± 3.8

25.4 ± 3.4

24.6 ± 3.4

26.1 ± 4.3

12 weeks

4 weeks after the test4

4 weeks

8 weeks

12 weeks

4 weeks after the test

Data are expressed as mean ± standard deviation. **p<0.01 vs Pre values by Dunnett’ s test, n = 24.

Table 5. Peripheral blood examination.

Item

Unit

Sex

Before4 weeks

8 weeks

TP

g/dL

-

7.08 ± 0.35

7.06 ± 0.31

7.18 ± 0.36

7.18 ± 0.41

7.21 ± 0.30 *

ALB

g/dL

-

4.36 ± 0.24

4.38 ± 0.18

4.40 ± 0.23

4.42 ± 0.24

4.43 ± 0.20

AST (GOT)

U/L-

17.5 ± 3.6

17.3 ± 4.1

18.0 ± 4.4

17.9 ± 4.3

18.4 ± 5.2

ALT (GPT)

U/L-

16.7 ± 8.7

16.5 ± 8.5

17.5 ± 8.9

16.3 ± 8.3

LDH (LD)

U/L-

154.5 ± 21.5

155.5 ± 24.5

155.3 ± 24.5

164. ± 28.2

T-BIL

mg/dL

-

0.76 ± 0.25

0.78 ± 0.24

0.80 ± 0.29

10.78 ± 0.28

0.83 ± 0.26

ALP

U/L

-

166.8 ± 45.2

171.4 ± 46.8

173.7 ± 49.6

173.4 ± 54.4

173.9 ± 49.9

γ -GTP

U/L

Male

31.7 ± 18.2

32.6 ± 19.8

34.5 ± 22.4

36.6 ± 32.2

37.2 ± 25.8

Female14.3 ± 3.5

14.9 ± 5.3

15.4 ± 5.4

15.0 ± 6.0

17.3 ± 7.2 *

CPK

U/L

Male

138.3 ± 53.1

123.3 ± 33.3

110.0 ± 32.0

180.0 ± 144.9

151.6 ± 67.2

Female74.2 ± 30.6

76.8 ± 30.6

93.5 ± 56.0

82.6 ± 42.2

74.2 ± 31.1

BUN

mg/dL

-

11.4 ± 3.1

11.6 ± 2.8

11.1 ± 2.8

11.6 ± 3.0

12.2 ± 4.4

Cre

mg/dL

Male

0.862 ± 0.103

0.833 ± 0.104

0.859 ± 0.128

0.858 ± 0.127

0.848 ± 0.142

Female0.592 ± 0.068

0.566 ± 0.049

0.600 ± 0.062

0.601 ± 0.075

0.614 ± 0.055

Na

mEq/L

-

140.3 ± 1.8

139.8 ± 1.5

140.5 ± 1.6

140.3 ± 1.7

140.3 ± 1.3

Cl

mEq/L

-

104.4 ± 1.8

104.0 ± 1.8

104.5 ± 1.9

103.7 ± 2.0

104.0 ± 1.4

K

mEq/L

-

4.13 ± 0.25

4.27 ± 0.37

4.30 ± 0.41 *4.30 ± 0.34

4.24 ± 0.32

Ca

mg/dL

-

9.42 ± 0.38

9.45 ± 0.38

9.48 ± 0.35

9.47 ± 0.37

9.53 ± 0.30

TC

mg/dL

-

197.3 ± 27.8

202.5 ± 34.4

206.0 ± 31.3 *199.0 ± 29.9

205.2 ± 28.4 *

LDL-C

mg/dL

-

116.8 ± 26.9

120.8 ± 28.7

120.8 ± 30.2

117.1 ± 27.2

121.0 ± 22.5

HDL-C

mg/dL

Male

59.8 ± 8.7

59.1 ± 10.8

60.0 ± 8.9

61.3 ± 7.4

59.7 ± 8.3

Female67.0 ± 19.5

67.1 ± 18.5

70.4 ± 21.8

64.7 ± 20.0

65.8 ± 23.3

TG

mg/dL

76.3 ± 33.2

77.9 ± 38.1

99.4 ± 71.1 *84.5 ± 37.6

92.5 ± 56.0

85.4 ± 9.2

88.4 ± 8.2 *86.1 ± 7.5

86.6 ± 8.1

85.0 ± 9.8

FPG

mg/dL

Data are expressed as mean ± standard deviation. *p< 0.05, **p<0.01 vs Pre values by Dunnett’ s test, n = 24.

_ 10 3 _**18.1 ± 9.3
159.0 ± 24.0

Effect of 12-Week Protocol of Daily Beef Bowl Consumption
Table 6. Physical information.

Item

Judgement

Before4 weeks

8 weeks

12 weeks

4 weeks after the test

Protein

(+ -)

1

2

4

6

6

21

(-)

(1+)

2

0

17

1

1

24

24

24

24

(1+)

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

(+ -)

24

24

24

24

24

(-)

24

24

24

24

24

(1+)

0

0

0

0

0

(1+)

0

(+ -)

Bilirubin

0

17

24

(-)

Urobilinogen

19

(-)

(+ -)

Sugar

22

0

0

0

0

0

0

0

0

0

No significant difference from pre-values “Before” is analyzed by Wilcoxon test.

Table 7. Adverse event in subjective symptoms.
Sex

Manifestation
Intakedate tostart
disappearance
date
date

Event

Treatment

Treatment
content

DegreeFollow-up

Outcome

Seriousness

Acutepharyngitis

Doctorconsultation
andmedication

PL 3tablet/day
Flomox3 tablet/day
Transamine
3 tablet /dayModerate

No

RecoveredNot
serious

Cold-like
symptoms
July 14 to 22 (cough, fatigue Medication
andappetite loss)

PabronGold A
3 tablet/day

Mild

No

Recovered

July 31 toAugust 7

Female May 13

Male

May 14

Not
serious

Test
Causalcontinuation relationship

Yes

No

Yes

No

Degree is divided into “mild”, “moderate” and “severe”, and seriousness into “not serious” and “serious”.

Table 8. Adverse event in examination data.
Time course
Sex

Event

CausalTest
4 weeks Treatment Degree Outcome Follow- Seriouscontinuation relationship
up
ness
after
the test

Before4 weeks

Female Proteinuria
(+)

(−)

(−)

(±)

(±)

(+)

No

Mild

UnknownNo

Not
seriousYesNo

γ - GTPelevation

72

75

83

125

101

No

Mild

UnknownNo

Not
serious

Yes

No

Female8 weeks 12 weeks

Degree is divided into “mild”, “ moderate” and “severe”, and seriousness into “not serious” and “serious”.

_ 10 4 _Glycative Stress Research

considered unrelated to the test food consumption.
In the first case, urine protein (+) was observed at the
4-week follow-up and reported as an adverse event. Because
this change was observed at the 4-week follow-up in a subject
during her menstrual period, the event was considered to beunrelated to the test food consumption. Since this was amenstruation-related change, no further follow-up wasconsidered necessary.
In the second case, the γ -GTP value, with a baseline
value of 72 U/L, started to gradually increase from week 4
and reached 125 U/L at week 12, the highest value during
the study period, followed by a slight decrease at the 4-weekfollow-up. Thus, this change was originally considered as
an adverse event. A detailed interview with this subject
revealed that the subject tends to drink more alcohol at anincreased frequency during the summer season. Thus, theobserved increase in γ -GTP was considered to be due tochanges in the subject’s drinking habit and was consideredunrelated to the test food consumption. Since a decrease in
the value was observed at the 4-week follow-up, no further
follow-up was considered necessary for this variable.

Discussion
The accumulation of AGEs and carbonyl stress have
beenThe objective of this study was to evaluate the healtheffect of gyudon, a representative national dish of Japan,
using the current commercial product as the first step tofurther refine the dish as a healthier food choice. Since it isdifficult to conduct a study using gyudon bowls served atstores due to subject management issues, we conducted thestudy using frozen gyudon ingredients, an equivalent productin terms of nutritional composition. We evaluated the safety
and health effect of gyudon ingredients by measuring
anthropometric, hematological/biochemical and urine
parameters and abnormal changes in these parameters
exceeding the range of physiological variations before, after
and at 4 weeks after the completion of a 12-week protocol ofdaily gyudon consumption. The results showed no significant
change in body weight or body fat percentage and only slightchanges in some physical parameters, including a 4.7 %increase in diastolic blood pressure. With regard to glycolipid
metabolism parameters, no significant change was observedin LDL-C and, with the exception of TG which exhibited a
30.3% increase, only transient, slight changes were observedin TC (+4.4%) and FPG (+3.5%). No serious adverse event
related to the test food was observed during the observation
period.

Fast foods and health

There has been a gradual increase in fat intake among
Japanese since around 1990. The mean total cholesterollevel in Japanese has become comparable to that in
Americans, with recent data showing a higher mean totalcholesterol level in Japanese women than in American men
and women 4). Factors contributing to this trend include thewesternization of diet and popularization of fast food chains,family restaurants and convenience stores.
Geographically easy access to supermarkets, fast foodrestaurants, food outlets or convenience stores is known
to influence people’s dietary habits 5). The availability ofsupermarkets has been positively correlated with vegetable/
fruit intake while the availability of fast food restaurants
and convenience stores has been positively correlated with
the intake of ice cream, salty snacks, meat, confectionery
and sugar-sweetened beverages and negatively correlatedwith the intake of vegetables/fruits and low-fat food. The
availability of fast food restaurants also affects vegetableintake. A study on the correlation between the number offast food restaurants and vegetable intake in each prefecture
revealed a significant negative correlation between the twovariables 6).
How often do typical Japanese people use fast foodrestaurants? Although information is limited regarding thefrequency of use of fast food restaurants, data have suggested
that “junk food” intake accounts for about one-third of thestandard calorie intake and that this percentage is higher inyounger age groups. A particularly high frequency of theuse of fast food restaurants and other dine-out restaurants
was reported by children, being about 8 times a month 7).
The frequency of fast food intake among junior high school
students was distributed as follows: “almost every day”
accounting for 1.0%, “3-4 times a week” for 2.3%, “once or
twice a week” for 17.3%, “a few times a month” for 70.0%
and “none” for 9.3% 8). Another survey, conducted with 45
non-dialysis patients with chronic kidney disease (CKD),
showed that 26.7% of the patients used fast food restaurants
once a month and 8.9% used them once a week 9). People
who have started living apart from their family for work are
known to become more dependent on fast food restaurants
or other dine-out places and take-out foods. In a study
comparing those living apart from their family for workand those living with their family, no significant difference
was observed in the prevalence of obesity, dyslipidemia or
diabetes. However, the prevalence of hypertension symptoms
in each group was 0% and 1.6% in those in their 20s, 14.9%
and 4.1% in those in their 30s, 21.4% and 13.8% in thosein their 40s, and 27.6% and 23.2% in those in their 50s,
respectively, with significant differences observed between
groups in those in their 30s and 40s 10).
Several reports are also available regarding the association
between the frequency of use of fast food restaurants andlifestyle-related diseases. A study examining the effect offast foods on pregnancy showed no significant correlation
between the frequency of use of fast food restaurants during
before-pregnancy and children’s birthweight, but it did show
a significant correlation in the preference for fast foods
between before-pregnancy and children’s birthweight 11).
More specifically, those mothers who delivered children witha birthweight of less than 2500 g were less likely to answer
that they liked fast foods and tended to be aware of the highcalorie content of fast foods and thus avoid them.
Regarding the effect of fast foods on younger children,
there has been a report on the association between fast foodintake and prosocial behavior of children. A logistic regression
analysis of the association between factors known to affectchildren’s prosocial behavior and fast food intake wasconducted in a total of 246 kindergarten pupils (age 5-6 years)
in a Japanese kindergarten in Shanghai, China, and inkindergartens in Osaka and Wakayama Prefectures in Japan.
A significant correlation was observed between the factor“getting anxious or reluctant in interacting with children when
they are defiant” and fast food intake only in Shanghai 12). In
Wakayama, a significant correlation was found between TV
viewing time and fast food intake. In Osaka, the amount of_ 10 5 _Effect of 12-Week Protocol of Daily Beef Bowl Consumption
outdoor play time was identified as a significant factor. Fast
food is only one of the many factors that constitute the living
environment. Therefore, the association between people’s
health status or lifestyle-related disease and fast food intake
should be analyzed from a broader point of view.
The percentage of obese children in Japan reached apeak in 2006 and slightly declined thereafter. However, thepercentage remained at a high level of about 8%, which issuggested to be due to junk food/fast food intake. Nowadays,
children are able to buy anything they want at a reasonable
price, anytime and anywhere 7). Since children’s lifestyle islargely influenced by their parents’ and other family members’
lifestyle, it is necessary to provide these children withappropriate advice and support taking into account thesituation of each family.
A survey has been conducted in junior high schools tocollect information about their desire for slenderness, accessto information on diet, dietary habits, bone mass, exercise andlifestyle. The survey revealed a stronger desire for slenderness
among girls than boys and the fact that more than half of thegirls of standard weight have a desire to be slender. In addition,
a comparison of the girls of standard weight with and without
a desire for slenderness identified fast food intake, as wellas information on diet, daily exercise hours and having
breakfast or not, as a factor closely related to the desire
for slenderness 13). These results suggest the importance ofthe proper understanding of health information for healthpromotion and maintenance.
A correlation between oral hygienic status and fast foodintake has been suggested in a survey of 997 female juniorhigh and high school students. In this survey, a questionnaire
was administered to assess associations of subjective oral
symptoms, including “dryness of the mouth and throat”,“sticky feeling in the mouth” and “a sense of swallowing
difficulty in the absence of sufficient moisture”, with risk
factors related to these symptoms, including 1) intake ofcoffee, tea or green tea, 2) skipping a meal, 3) eating fast
foods, 4) having a meal while drinking water or tea, 5)
finishing a meal within 10 minutes, 6) nasal congestion,7) mouth breathing and 8) stress 14). A logistic regression
analysis identified “nasal congestion”, “mouth breathing”
and “stress” as significant factors associated with “sticky
feeling in the mouth”, while “eating fast foods”, as well as“junior-high or high school”, “skipping a meal”, “having ameal while drinking water or tea”, “nasal congestion” and“stress”, were identified as factors significantly associatedwith “a sense of swallowing difficulty in the absence ofsufficient moisture”. No reference was made to the frequency
of use of fast food restaurants.
With the changing social environment, the dietary habitsof pupils and students have also changed, generating problems
such as eating alone and skipping meals. Psychophysiologicalinvestigations have also been conducted to assess theassociation between fast food intake and lifestyle of children.
Dietary habits such as eating fast foods and high saturated fat
diets have been shown to increase the risk of depression 15).
A survey on the association between stress responses(psychological/physical stress) and lifestyle factors in 114
children of grades 4 to 12 showed that children consuming
instant foods at least once a week had significantly higherscores for physical stress response, bad mood/anger, lack ofmotivation and depression/anxiety, as compared to those
consuming instant foods less than once a week 16). In addition,
children consuming fast foods at least once a week hadsignificantly higher scores for lack of motivation anddepression/anxiety, as compared to those consuming fast
foods less than once a week.
In a survey to assess the physical and mental health statusof 244 male high school students (89 grade-10, 81 grade-11
and 74 grade-12 students) using the Todai Health Index 17),
the frequent users of convenience stores or fast food restaurants
had significantly higher scores for “irregularity of life” among
grade-10, “multi-complaints” among grade-11, and “mouth
and anus” and “irregularity of life” among grade-12 students.
This result suggests that the frequency of use of fast foodrestaurants affects the mental status of male high school
students.
A logistic regression analysis of the association between
dietary habits and depression symptoms in 331 hospital nurses(108 males and 223 females) showed an increasing ageadjusted odds ratio with an increasing frequency of eating
instant foods and late evening meals 18). In contrast, an
increasing frequency of consuming dark-colored vegetables,
seaweed and mushrooms was associated with a decrease inage-adjusted odds ratio.
Other reported associations between fast food intake
and diseases include an association between lower fast foodintake and reduced prostate cancer risk 19) and an association
between higher fast food intake and higher likelihood of
increased prostate specific antigen (PSA) levels above thereference range 20).
There are some positive aspects of fast food restaurants.
In Aichi Prefecture, there is a polarization of attitude topassive smoking prevention at eating places dependingon the type of business; smoking bans have been actively
implemented at fast food and curry restaurants, but seldom
implemented at bars, barbecue restaurants, pubs andokonomiyaki restaurants 21). Efforts should be made to findbetter ways to use fast food restaurants by improving ourunderstanding of the nutritional value of foods provided atthese restaurants 22).
The “fast food restaurants” that have been discussed inthis report actually include various types of restaurants thatprovide various types of foods, including hamburgers, friedchicken, kaitenzushi (conveyer belt sushi), udon, soba, curryrice and gyudon. Therefore, future studies should be designed
to address the safety and health management aspects ofindividual food ingredients.

Interpretation of data

In the present study, in blood testing, a transient decreasein Ht (-3.9% at week 4 in women) was observed. Based on
the data from the 14 clinical studies we have conducted 23-36),
2-4% decreases in RBC, Hb or Ht were observed at week
4 in 3 23, 27, 30) of the 6 studies 23, 25, 27, 30, 32, 33) in which bloodcollection was done at week 4. In one study, although not atweek 4, a significant decrease in Ht was observed at week8 33). In terms of sex difference, all 4 studies included only
women, with 1 study involving post-menopausal women 30)
and 3 studies involving both pre- and post-menopausal
women 23, 27, 33). The 2 studies showing no significant
difference at week 4 included both men and women 25, 32).
Of the 8 studies in which blood collection was done atweek 8 and not at week 4 24, 26, 28, 29, 31, 34-36), only 1 study(involving only women) showed a significant decrease in Ht
at week 8 31), whereas no significant difference was observedin the remaining 7 studies 24, 26, 28, 29, 34-36). Of these 7 studies,_ 10 6 _Glycative Stress Research

6 included both men and women 24, 26, 28, 29, 34, 35) and 1 included
only women (both pre- and post-menopausal) 36).
Mild variations due to a small blood sampling volumeof 10 -20 mL may be interpreted as significant differencesby a paired t-test if the variations are in the same direction.
These observations are common in studies involving only
women and are probably influenced by such factors as age,presence/absence and volume of menstruation, and potential
iron deficiency. In the present study, a mild decrease in Ht
was observed only at week 4. The reason for the absenceof significant differences at weeks 8 and 12 could not be
identified.
In biochemistry, an increased LDH (+6.2% at week12) was observed. This change was within the range ofphysiological variation, although its cause could not be
identified. Increases in TP and γ -GTP (women) were observed
only after the completion of test food consumption and thus
were considered unrelated to the test food.
As for electrolytes, a transient increase in the potassium
level (+4.1% at week 8) was observed. The transient and mild
increase in the potassium level was within the range ofphysiological variation and was considered negligible. The
potassium source in the test food was onion (containing 150
mg of potassium per 100 g edible part), with each serving of
the test food containing about 30 mg of potassium. The test
food contains about 940 mg of sodium; therefore, gyudon
has a relatively low sodium/potassium ratio compared to otherJapanese dishes. Typical Japanese people are taking too muchsodium, with a mean daily sodium intake of about 10.0 g(Ministry of Health, Labour and Welfare) 37), which is way
over the levels recommended by the Japanese Society ofHypertension (< 6 g/day) and the WHO (<5 g/day). Given thissituation, foods with a low sodium/potassium ratio should be
preferred.
The PFC balance is a measure of dietary nutritionalbalance in terms of the three major nutrients found in food,i.e. protein, fat and carbohydrate. In our previous analysis,
the PFC balance of gyudon was P = 12.45%, F = 31.74% andC=55.82% 3). The ideal PFC balance is said to be 20% protein(P), <25% fat (F) and 50-60% carbohydrate (C), relative tothe total energy intake from a meal 38-40). The PFC balanceshould be used as a measure for assessing the nutritionalbalance of a meal as a whole and may not be pertinent in theassessment of the nutritional balance of individual food items.
The nutritional balance of a meal can be regarded as the sumof individual food ingredients. Therefore, maintaining
the PFC balance of individual food ingredients within areasonable range may lead to a better PFC balance of a meal
as a whole. The PFC balance of gyudon, although with slightly
lower protein and higher fat ratios compared to the idealbalance, is within the reasonable range compared to that of
the staple food alone.
Glycolipid parameters are strongly influenced by dietary
habits. In this study, abnormal changes in glycolipid parameters
included transient increases in TC (+4.4%), TG (+30.3% atweek 8) and FPG (+3.5% at week 4). Based on the PFC balanceof gyudon, a slightly higher fat ratio than the ideal balancemight have contributed to the observed increases in TC and
TG. The change in TC was transient and mild in nature andwas within the range of physiological variation. In addition,
no significant change was observed in LDL-C. Therefore,
given that the usual frequency of gyudon consumption isestimated to be 2-8 times a month, which is less than thefrequency of test food consumption in the present study, theimpact of these changes is considered to be limited.

The observed change in TG, although appearing to be
somewhat large, was transient in nature and within the rangeof physiological variation. A large standard deviation alsosuggests that the change was likely due to other foods than
the test food consumed by some subjects. In daily dietary
life, gyudon is often consumed with various other types offoods. Balanced dietary habits should therefore be encouraged.
The observed increase in FPG was also transient and mild
in nature and within the range of physiological variation.
The results of these glycolipid parameters suggest that
12 weeks of daily gyudon consumption does not increase thelevels of glycative stress-related factors. Rather, gyudon has
been associated with a lower postprandial peak plasma glucose
level compared to white rice alone, which is composed mainlyof carbohydrates, and thus gyudon is considered to cause lessglycative stress than meals containing a disproportionately
high percentage of the carbohydrate-rich staple food, suchas white rice alone 3).

Issues to be addressed in future studies:
Trans fatty acids

Trans fatty acids (TFAs) are generated as a result of thepartial hydrogenation of double bonds in vegetable fats
(unsaturated fatty acids). Unlike natural polyunsaturated fatty
acids, which are liquid at room temperature, these artificiallysynthesized polyunsaturated fatty acids, i.e. TFAs, are semisolid, keep for a long time and improve the flavor and tasteof foods, especially fried foods 41). Most TFAs are generated
as a result of hydrogenation during the manufacturing processof hardened oil. An excessive intake of TFAs has been shownto increase the risk of heart diseases. The World HealthOrganization (WHO) recommends the intake of TFAs to beless than 1% of total energy intake.
In 2015, the previous three legal provisions relating tofood labeling were integrated into the Food Labeling Act.
This new food labeling system still has several issues to beaddressed, such as the handling of cholesterol, TFAs andpotassium, and the appropriateness of mandatory nutrition
labeling for liquor products. Since the labeling of TFA content
is not mandatory in Japan, many Japanese, both children andadults, may be consuming TFAs without knowing it 42, 43).
The mean daily intake of TFAs from industrial products
is estimated to be 0.82 g. The mean daily intake of TFAs
estimated from the data from a 6-day daily diet survey in
118 Japanese college students in the Kanto region and Okinawa
prefecture (57 males and 61 females) was 0.43 g for males in
Kanto, and 0.30 g for males in Okinawa, 0.49 g for femalesin Kanto and 0.73 g for females in Okinawa 44).
The intake of TFAs has been shown to correlate positively
with the intake of non-essential food items, such as cookies,
cakes and pastries. As most Japanese foods contain a small
amount of TFAs, the daily intake of TFAs by Japanese does
not substantially exceed the US regulation level of 500 mg
per serving 45). However, care should be taken as some typesof foods, such as fast foods, may contain an amount of TFAs
exceeding the acceptable level. In the younger population,high serum TFA levels have also been reported in patientswith non-alcoholic steatohepatitis (NASH) 46) and coronary
artery disease 47).
A field study of TFA intake in 467 young Japanese
subjects ranging from infants to high school students, where
the mean daily TFA intake was estimated to be 0.63 ± 0.37 g,suggested that infants have a greater intake (relative to body_ 10 7 _Effect of 12-Week Protocol of Daily Beef Bowl Consumptionsize) of TFAs from tasty food items, as compared to children
in other age groups 48). The estimated source of TFAs was
fast foods. When the frequency of use of fast food restaurants
was analyzed, while Mos Burger, Lotteria, Mister Donuts
and Kentucky Fried Chicken were used at an almost
comparable frequency, McDonald's was more frequently
used than the other 4 fast food chains.
TFA-rich fast food items include donuts, karaage, Frenchfries, pies, fried chicken, pizza, pork cutlet, mince cutlet,
shrimp cutlet, tempura and deep-fried food in general. By
comparison, the fat component of gyudon is solely derived
from beef, with about half of the fatty acid content accounted
for by saturated fatty acids and the remaining half by oleic
acid (an omega-9 fatty acid abundant in olive oil), and anegligible part by omega-6 fatty acids (about 240 mg per 65
g beef in a regular-size gyudon; in-house data). It contains
no TFAs derived from harmful additives. Thus, gyudon canbe said to be a fast food item with a low TFA content. Thisshould be further clarified in future studies.

Limitations of study

The current study did not cover all glycolipid metabolismrelated parameters (e.g. HbA1c). Oxidized and glycated formsof LDL, as well as advanced glycation end products (AGEs)
as a measure of glycative stress, should have also been
analyzed.
The subjects were not adequately managed or evaluatedfor dietary intake and exercise amount during the intervention
period. Dietary details were recorded in a diary by the subjects,
but this could not be analyzed quantitatively. The same applies

References

to the evaluation of exercise amount. Future studies should
be designed to allow for the proper evaluation of dietaryintake and exercise amount.
In this study, the subjects were asked to consume thetest food, which was identical to gyudon served at stores,
once daily and achieved a 99% compliance rate for test
food consumption, where even the least compliant subject
achieved 94%. However, we did not specifically instructthem to eat gyudon for breakfast, lunch or dinner, as it has arelatively high fat content. It was also impossible to collect
all the details of meals consumed by the subjects. Therefore,
we could not perform chrono-nutritional analysis.

Conclusion

In this study, a 12-week protocol of daily test food(gyudon ingredients) consumption resulted in no significant
change in parameters related to metabolic syndrome orother lifestyle-related diseases, such as body weight, bodyfat percentage, blood pressure, TG, LDL-C and FPG, or
the development of adverse events. While the present study
has a significance in the pursuit of food safety, further
investigations are needed to verify the safety of gyudon.

Conflict of Interest Statement

This study was mainly supported by Yoshinoya Holdings.

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