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Plasma riboflavin is a useful marker for studying riboflavin requirement in Chinese male adults
N U TR I TION RE S E ARCH 3 6 ( 2 0 16 ) 5 3 4–5 40
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Original Research
Plasma riboflavin is a useful marker for studying riboflavin requirement in Chinese male adults Changjiang Guo⁎, 1 , Jingyu Wei 1 , Weina Gao, Lingling Pu, Zhenchuang Tang, Lingyan Li Department of Nutrition, Tianjin Institute of Health and Environmental Medicine, No 1, Dali Dao, Tianjin 300050, PR China
ARTI CLE I NFO
A BS TRACT
Article history:
Urinary riboflavin excretion and erythrocyte glutathione reductase activation coefficient are
Received 18 November 2015
frequently applied in determining riboflavin requirement. Previously, we found that plasma
Revised 12 February 2016
riboflavin is a sensitive marker in the assessment of riboflavin status in rat models. Here, we
Accepted 16 February 2016
hypothesize that plasma riboflavin is a useful maker in studying riboflavin requirement. This study examines the changes of fasting plasma riboflavin and urinary riboflavin excretion in response to different riboflavin intake levels in Chinese male adults. The estimated average
Keywords:
requirement (EAR) of riboflavin was extrapolated. Seventy-eight participants were randomly
Riboflavin
divided into the control and 5 riboflavin-supplemented groups. A 6-week riboflavin
Estimated average requirement
supplementation was performed at the doses of 0, 0.2, 0.4, 0.6, 0.8, or 1.0 mg daily. The
Plasma
energy expenditure was 15.4 ± 1.9 MJ/d, as estimated by the 24-hour physical activity recording
Urinary excretion
method. Dietary riboflavin intake was 1.0 ± 0.2 mg/d, based on chemical analysis. The fasting plasma riboflavin was increased significantly in a dose-dependent manner when the supplemented riboflavin exceeded 0.4 mg/d and the EAR of riboflavin was suggested to be between 1.3 and 1.5 mg/d. In addition, we found a significant increase in fasting urinary riboflavin excretion when the supplemented riboflavin exceeded 0.6 mg/d. The critical point was calculated as 1.4 mg/d, based on the intersecting point of the 2 regression lines at lower and higher riboflavin intakes. These findings demonstrate that plasma riboflavin is a sensitive marker for riboflavin status, and the EAR of riboflavin for Chinese male adults is 1.4 mg. © 2016 Elsevier Inc. All rights reserved.
1.
Introduction
Riboflavin acts as coenzymes in the forms of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) for numerous oxidases, reductases, and dehydrogenases in metabolic processes. Riboflavin deficiency results in low FMN and FAD contents in the cell and reduced activity of a series of flavin-containing enzymes [1,2]. Mitochondrial β-oxidation was
impaired remarkably by riboflavin deficiency because several flavin-containing enzymes, such as acyl-CoA dehydrogenase, are involved in mitochondrial β-oxidation [3]. Riboflavin also plays a role in glutathione synthesis, in which FAD is a cofactor for glutathione reductase. The erythrocyte glutathione reductase activation coefficient (EGRAC) is frequently used as a marker in the assessment of riboflavin status [4]. Moreover, riboflavin is required for methylenetetrahydrofolate reductase and plays an
Abbreviations: CNS, Chinese Nutrition Society; EAR, estimated average requirement; EGRAC, erythrocyte glutathione reductase activation coefficient; FAD, flavin adenine dinucleotide; FMN, flavin mononucleotide. ⁎ Corresponding author. Tel.: +86 22 84655429; fax: +86 22 84655020. E-mail address: [email protected] (C. Guo). 1 The first 2 authors contributed equally. http://dx.doi.org/10.1016/j.nutres.2016.02.003 0271-5317/© 2016 Elsevier Inc. All rights reserved.
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Table 1 – Baseline characteristics of the study participants Parameter
Group 1
Group 2
Group 3
Group 4
Group 5
Group 6
Participants (n) Age (y) Height (cm) Body weight (kg) Body mass index (kg/m2)
13 21 ± 2 175.2 ± 3.9 62.9 ± 6.5 20.5 ± 1.6
13 20 ± 1 174.8 ± 7.0 68.8 ± 13.8 22.5 ± 3.1
13 20 ± 2 174.9 ± 5.9 67.5 ± 6.4 22.1 ± 1.9
13 21 ± 2 170.0 ± 5.2 66.1 ± 6.0 22.9 ± 1.4
13 21 ± 2 176.8 ± 6.1 67.9 ± 8.7 22.0 ± 2.8
13 20 ± 1 172.5 ± 5.8 65.4 ± 5.8 22.0 ± 2.4
Values are expressed as means ± SD.
important role in homocysteine metabolism, especially in individuals with a methylenetetrahydrofolate reductase 677 TT genotype [5]. The present estimated average requirement (EAR) of riboflavin in the United States is based mainly on studies conducted more than 50 years ago, in which 24-hour urinary riboflavin excretion was used as an indicator [6]. The critical point of urinary riboflavin excretion, a point of intersection of the regression lines fitted to the excretion at higher and lower riboflavin intakes, is considered the level of intake at which organ or tissue riboflavin saturation occurs [7]. Later, the EGRAC method was developed and applied in determining riboflavin requirement in children, women, and the elderly [8–10]. However, the acceptable range of activity coefficient values is too small in magnitude (from 1.0 to 1.2), as recommended by Sauberlich et al [11]. Boisvert et al [10] found that the changes of the EGRAC value were without a clear delineation between inadequate and adequate riboflavin status, although a decreasing trend was recorded with increasing riboflavin intake in aged participants. In 2005, Xu et al [12] reported that fasting plasma riboflavin was decreased rapidly in response to a riboflavin-deficient diet and recovered soon after a riboflavin-containing diet was provided in rats. In riboflavin malnourished humans, it was confirmed that fasting plasma riboflavin was increased significantly after 1 week of riboflavin supplementation [13]. A consistent result was also reported by Hustad et al [14] in a human study, in which plasma riboflavin was demonstrated to be useful in the assessment of riboflavin status. Therefore, it appears that plasma riboflavin is a sensitive index for riboflavin status. The purpose of this study is to validate the effectiveness of plasma riboflavin in assessing riboflavin status and further to determine the EAR of riboflavin for Chinese male adults. We hypothesize that plasma riboflavin is a useful maker in studying riboflavin requirement. To test this hypothesis, the changes of fasting plasma riboflavin in response to different riboflavin intake levels were determined in Chinese male adults after 6 weeks of intervention. In addition, fasting urinary riboflavin excretion was simultaneously measured.
2.
Methods and materials
2.1.
Participants and study design
in this study. No classic signs of riboflavin deficiency, such as cheilosis, angular stomatitis, and glossitis, were found in a routine physical examination. A written informed consent was signed by all participants. Approval for this study was granted by the ethical committee of the Institute of Health and Environmental Medicine. The participants were randomly assigned to the control and 5 riboflavin-supplemented groups by selection of a random number. The general characteristics of the study participants are presented in Table 1. During the 6week experimental period, all participants led a similar lifestyle and were engaged in routine physical training. They were asked to abstain from any riboflavin-containing supplements and were group fed in a dining room on a menu designed by a dietitian. Riboflavin supplementation was performed via the drinks containing different amounts of riboflavin for the different groups. The drinks were prepared freshly by dissolving riboflavin (≥98% in purity, provided by Tianjin Feiying Pharmaceutical Co, Ltd, Tianjin, PR China) in 200 mL of water and consumed by the participants every morning under the supervision of the field staff. The doses of riboflavin for different riboflavin-supplemented groups were 0.2, 0.4, 0.6, 0.8, and 1.0 mg daily, respectively. At these low concentrations, riboflavin was completely soluble in the water. During the experimental period, 5 participants withdrew from groups 1 (control), 2 (0.2 mg), 3 (0.4 mg), 5 (0.8 mg), and 6 (1.0 mg), respectively, and their data were excluded from the final statistical analyses (Fig. 1).
2.2.
A 4-day dietary survey was performed in the last week, using a food-weighting method. Dietary intakes of energy and nutrients (including riboflavin) were calculated based on food intakes and Chinese Food Composition, which was compiled by the Institute of Nutrition and Food Safety, Chinese Center for Disease Control and Prevention [15]. From the second week to the last week, daily duplicate food samples consumed by the participants were selected randomly from each group, collected, and stored at −80°C before being analyzed fluorophotometrically for riboflavin content by a standardized procedure approved by the Ministry of Public Health, PR China [16].
2.3.
Seventy-eight male adults aged 18 to 22 years from an army unit in Tianjin, PR China, were recruited. They were nonsmokers and physically healthy and volunteered to participate
Assessment of dietary intakes
Estimation of daily energy expenditure
From the third week to the fifth week, 3 to 4 participants were selected randomly from each group for estimation of daily energy expenditure by the 24-hour physical activity recording method [17]. The field staff followed the participants from the
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Fig. 1 – Illustration of the experimental process.
wake-up time in the morning to the sleep time at night. All the activities the participants performed, as well as how long they lasted, were recorded accordingly. Daily energy expenditure was summed up based on the energy expenditure by individual activities, in addition to the basic metabolic expenditure.
2.4.
Measurement of fasting plasma riboflavin
Fasting blood samples from the antecubital vein were collected at the beginning and end of the experiment. Vacuum tubes containing heparin were used to obtain blood samples. Plasma was separated by centrifugation and stored at −80°C before the content of free riboflavin was determined by a high-performance liquid chromatography procedure described by Wei et al [13].
2.5.
Measurement of fasting urinary riboflavin
Fasting urine samples were collected at the beginning and end of the experiment. The pH of the urine samples was adjusted to 4.0 by addition of oxalate and stored at −80°C before the content of riboflavin was assayed fluorophotometrically [18]. The content of urinary creatinine was measured by the reaction of creatinine with picrate in alkaline medium, using a commercial assay kit purchased from Jiancheng Bioengineering Institute (Nanjing, China).
2.6.
Statistical analyses
Data are expressed as means ± SD and checked for normality using the Kolmogorov-Smirnov test before subjected to further
analysis. We found that the data of plasma riboflavin and urinary riboflavin excretion were not in a normal distribution. Thereby, Mann-Whitney rank sum test was performed to analyze the differences among different groups. The level of significance was set at P < .05. Linear regression was used to investigate the relationship between riboflavin intake and plasma riboflavin or urinary riboflavin excretion. The critical point was calculated based on 2 regression lines fitted to the urinary riboflavin excretion at lower and higher riboflavin intakes. Power analyses were conducted to predict the sample size to achieve 80% power for plasma riboflavin (minimum n = 8) and urinary riboflavin excretion (minimum n = 10) with statistical significance at .05.
3.
Results
3.1.
Dietary intake of energy and nutrients
As shown in Table 2, the mean dietary intake of energy and protein reached the EAR recommended by the Chinese Nutrition Society (CNS) [19]. However, 33.8% energy was contributed by lipids, which is higher than the level recommended by the CNS. The mean dietary intake of selenium and zinc was adequate, whereas calcium was insufficient, based on the EAR. The mean dietary intake of vitamin A, vitamin C, vitamin E, thiamine; riboflavin, and niacin reached the EAR. However, the mean dietary intake of iron exceeded the EAR by 380%.
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Table 2 – Daily dietary intake of energy and nutrients Energy and nutrients
Daily dietary intake
Energy (MJ) Protein (g) Calcium (mg) Iron (mg) Zinc (mg) Selenium (μg) Vitamin A (μg RE) Vitamin E (mg) Thiamine (mg) Riboflavin (mg) Niacin (mg) Vitamin C (mg)
13.9 89.6 615.7 43.2 13.2 53.7 801.3 25.5 1.3 1.2 18.2 127.9
± ± ± ± ± ± ± ± ± ± ± ±
0.6 10.1 62.8 1.1 1.0 8.4 90.4 4.8 0.2 0.2 8.5 32.4
Values are expressed as means ± SD. The dietary survey was performed in the last week using a foodweighting method. Dietary intakes of energy and nutrients were calculated based on food intakes and Chinese Food Composition.
3.2.
Energy expenditure
No significant difference was found in daily energy expenditure among different groups (Table 3). The average energy expenditure was 15.4 ± 1.9 MJ/d, which is 10.9% higher than the energy intake as estimated by the dietary survey.
3.3.
Dietary riboflavin intake by chemical analysis
Based on chemical analysis, no significant difference was found for dietary riboflavin intake among different groups (Table 4). The average dietary riboflavin intake was 1.0 ± 0.2 mg/d, which is 16.7% lower than that estimated by the dietary survey and reflects a possibility of marginal riboflavin status. This is due to the losses caused by the cooking process, as demonstrated by Xu et al [20]. The total riboflavin intake should be the combination of the dietary riboflavin and the supplemented riboflavin from the drinks. Therefore, the actual daily riboflavin intakes are 1.0, 1.3, 1.5, 1.6, 1.9, and 2.0 mg for groups 1, 2, 3, 4, 5, and 6, respectively.
3.4.
Fasting plasma riboflavin
As indicated in Table 5, there was no remarkable difference in fasting plasma riboflavin among the different groups at the beginning. At the end of the experiment, fasting plasma riboflavin was increased significantly in a dose-dependent manner when the supplemented riboflavin exceeded 0.4 mg/d (P < .05 or .01). A positive correlation was found between riboflavin intake and fasting plasma riboflavin among the
groups 2, 3, 4, 5, and 6 (R2 = 0.9231, P < .01). It is not logical to run a linear regression based on 2 points at the lower riboflavin intakes (groups 1 and 2). Therefore, the critical point was not calculated from the fasting plasma riboflavin. Based on the changes of fasting plasma riboflavin, the EAR of riboflavin is between 1.3 and 1.5 mg/d.
3.5.
Fasting urinary riboflavin excretion
It was noted that fasting urinary riboflavin excretion was relatively lower at the beginning, without significant differences among different groups. At the end of the experiment, fasting urinary riboflavin excretion was increased significantly compared with the beginning. Moreover, a sharp increase in fasting urinary riboflavin excretion was found when the supplemented riboflavin exceeded 0.6 mg/d (Table 6). Correlation analysis showed that a positive linear correlation was found between riboflavin intake and fasting urinary riboflavin excretion among groups 3, 4, 5, and 6 (R2 = 0.9667, P < .01). Based on the changes of fasting urinary riboflavin excretion, 2 linear regression lines were constructed between riboflavin intake and fasting urinary riboflavin excretion (Fig. 2). One line was developed among groups 1, 2, and 3 and another among groups 3, 4, 5, and 6. The critical point (the intersecting point of the 2 linear regression lines) is calculated as 1.4 mg/d.
4.
Discussion
Low riboflavin intake is common in many parts of the world, particularly in developing countries [21,22]. In China, inadequate riboflavin status is believed to be widespread. It was estimated that the average daily riboflavin intake was approximately 0.8 mg per capita; this is remarkably lower than the current EAR recommended by CNS (1.2 mg for males and 1.0 mg for females), which is based on urinary excretion data obtained more than 50 years ago [23]. However, Campbell et al [24] thought that the riboflavin requirement might be set too high for the Chinese population if based on Western experimental data because they found that no clear evidence of riboflavin deficiency symptoms was observed in their field surveys. Hence, it is important to determine riboflavin EAR for Chinese populations and lay a solid basis for the revision of Chinese Dietary Reference Intakes. Previously, it was demonstrated that plasma riboflavin levels tended to be variable and reflected recent intake of riboflavin [11]. However, a rat study conducted in our laboratory indicated that fasting plasma riboflavin responded sensitively to riboflavin depletion and repletion. Comparatively, urinary riboflavin excretion was also a reliable
Table 3 – Estimated daily energy expenditure Parameter
Group 1
Group 2
Group 3
Group 4
Group 5
Group 6
Participants (n) Energy expenditure (MJ/d)
4 15.9 ± 3.1
4 16.0 ± 1.2
3 15.7 ± 2.2
3 14.3 ± 1.4
3 14.5 ± 2.8
3 15.6 ± 2.2
Values are expressed as means ± SD. The 24-hour physical activity recording method was used to estimate daily energy expenditure.
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Table 4 – Dietary riboflavin intake by chemical analysis Parameter
Group 1
Group 2
Group 3
Group 4
Group 5
Group 6
Duplicate foods (n) Riboflavin intake (mg/d)
16 1.0 ± 0.2
17 1.1 ± 0.3
15 1.1 ± 0.3
16 1.0 ± 0.2
16 1.1 ± 0.3
15 1.0 ± 0.2
Values are expressed as means ± SD. The riboflavin content of duplicate foods was analyzed by a standardized procedure (GB/T 5009.85-2003) approved by the Ministry of Public Health, PR China.
Table 5 – Changes of fasting plasma riboflavin Parameter
Group 1
Group 2
Group 3
Group 4
Group 5
Group 6
Participants (n) Initial (nmol/L) Final (nmol/L)
12 12.7 ± 4.8 11.6 ± 4.8
12 10.0 ± 4.1 10.9 ± 4.8
12 12.4 ± 3.6 19.2 ± 9.4 ⁎
13 11.5 ± 5.6 20.77 ± 11.15 ⁎
12 11.3 ± 4.9 24.7 ± 12.9 ⁎⁎
12 12.5 ± 4.2 27.0 ± 17.3 ⁎⁎
Values are expressed as means ± SD. The total daily riboflavin intakes are 1.0, 1.3, 1.5, 1.6, 1.9, and 2.0 mg for groups 1, 2, 3, 4, 5, and 6, respectively. Plasma riboflavin was determined by a high-performance liquid chromatography procedure. Mann-Whitney rank sum test was used for statistical analyses. ⁎ Significance at P < .05 as compared with group 1. ⁎⁎ Significance at P < .01 as compared with group 1.
indicator in reflecting the short-term changes of riboflavin intake. On the other hand, EGRAC and the contents of erythrocyte riboflavin, FMN, and FAD were relatively insensitive to riboflavin depletion and repletion [12]. It is explainable because the cells are capable of regulating riboflavin transport actively to maintain cellular riboflavin homeostasis, as demonstrated previously by Werner et al [25]. Consistent data were also obtained from riboflavin supplementation trials in humans, in which fasting plasma riboflavin was used as a marker in assessing riboflavin status [13,14]. In the present study, the changes of fasting plasma riboflavin were determined in participants with different riboflavin intakes. It was found that the fasting plasma riboflavin responded sensitively to the riboflavin supplementation in a dosedependent manner when the supplemented dose was greater than 0.2 mg. The EAR of riboflavin is suggested to be between 1.3 and 1.5 mg/d. The 24-hour urinary riboflavin excretion was used frequently by many investigators as an index of riboflavin status and proved to be valuable in determining riboflavin requirement [11]. For example, Horwitt et al [26] reported that the 24-hour
urinary riboflavin excretion increased markedly when the riboflavin intake exceeded 1.1 mg/d in adult men. However, it is often difficult practically to collect 24-hour urine samples in the field study. It was demonstrated that the riboflavin content of fasting urine samples was also useful in the assessment of riboflavin status after corrected by creatinine, because it was not affected by dietary and nondietary factors in the daytime [27,28]. In the present study, the changes of fasting urinary riboflavin excretion were monitored simultaneously. The results showed that the critical point was located at the riboflavin intake level of 1.4 mg/d, which is close to the EAR extrapolated from the changes in fasting plasma riboflavin. Therefore, the EAR of riboflavin is suggested to be 1.4 mg/d for Chinese male adults. In conclusion, the results of this study suggest that plasma riboflavin is a reliable marker for riboflavin status. It is also indicated, for the first time, that the EAR of riboflavin for Chinese male adults is 1.4 mg, based on the changes of fasting plasma riboflavin and urinary riboflavin excretion. However, we are aware of the limitation of our study because it has been known that riboflavin requirement is affected by several
Table 6 – Changes of fasting urinary riboflavin excretion Parameter
Group 1
Group 2
Group 3
Group 4
Group 5
Group 6
Participants (n) Initial (μg/g Cr) Final (μg/g Cr)
12 248.2 ± 138.8 538.4 ± 325.5
12 171.0 ± 76.9 507.3 ± 222.8
12 194.4 ± 92.5 542.6 ± 276.0
13 238.1 ± 107.1 741.6 ± 306.4
12 223.1 ± 108.0 963.3 ± 300.9 ⁎⁎
12 204.4 ± 90.9 1066.0 ± 475.7 ⁎⁎
Values are expressed as means ± standard deviation. The total daily riboflavin intakes are 1.0, 1.3, 1.5, 1.6, 1.9, and 2.0 mg for groups 1, 2, 3, 4, 5, and 6, respectively. The urinary riboflavin was assayed fluorophotometrically. Mann-Whitney rank sum test was used for statistical analyses. ⁎⁎ Significance at P < .01 as compared with group 1.
Fasting urinary riboflavin excretion (µg/gCr)
N U T RI TI O N RE S E ARCH 3 6 ( 2 0 16 ) 5 3 4–5 40
1,300 1,200
y = 965.34x - 860.96 R² = 0.9667
1,100
[6]
1,000 900
[7]
800 700
y = -0.4132x + 529.93 R² = 3E-05
[8]
600 500 400 0.8
1.1
1.4
1.7
2
2.3
Riboflavin intake (mg)
Fig. 2 – Critical point of fasting urinary riboflavin excretion at the end of the experiment. Two linear regression lines were constructed between riboflavin intake and mean fasting urinary riboflavin excretion. One line was developed among groups 1, 2, and 3 and another among groups 3, 4, 5, and 6. The total daily riboflavin intakes are 1.0, 1.3, 1.5, 1.6, 1.9, and 2.0 mg for groups 1, 2, 3, 4, 5, and 6, respectively. The critical point of fasting urinary riboflavin excretion was 1.4 mg/d, based on the intersecting point of the 2 regression lines at lower and higher riboflavin intakes.
factors, including age, energy intake, physical activity, and dietary composition. Several countries and the Food and Agriculture Organization/World Health Organization express riboflavin requirements in relation to energy intake, although it still remains controversial [1,6,10,23]. The mean energy intake of the participants in this study was estimated to be 13.9 MJ/d, or 3322 kcal/d. If expressed in terms of energy intake, the EAR of riboflavin is 0.1 mg/MJ, or 0.4 mg/1000 kcal, for Chinese male adults. Further studies should be undertaken to investigate riboflavin requirements in other age groups or conditions. In addition, possible impacts of excess iron intake on riboflavin requirement need to be further explored.
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16] [17]
Acknowledgment This work is supported financially by a grant (DIC2012-02) from Danone Institute of China. We acknowledge the cooperation of the volunteers participating in this study.
[18]
[19]
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Available online at www.sciencedirect.com
ScienceDirect www.nrjournal.com
Original Research
Plasma riboflavin is a useful marker for studying riboflavin requirement in Chinese male adults Changjiang Guo⁎, 1 , Jingyu Wei 1 , Weina Gao, Lingling Pu, Zhenchuang Tang, Lingyan Li Department of Nutrition, Tianjin Institute of Health and Environmental Medicine, No 1, Dali Dao, Tianjin 300050, PR China
ARTI CLE I NFO
A BS TRACT
Article history:
Urinary riboflavin excretion and erythrocyte glutathione reductase activation coefficient are
Received 18 November 2015
frequently applied in determining riboflavin requirement. Previously, we found that plasma
Revised 12 February 2016
riboflavin is a sensitive marker in the assessment of riboflavin status in rat models. Here, we
Accepted 16 February 2016
hypothesize that plasma riboflavin is a useful maker in studying riboflavin requirement. This study examines the changes of fasting plasma riboflavin and urinary riboflavin excretion in response to different riboflavin intake levels in Chinese male adults. The estimated average
Keywords:
requirement (EAR) of riboflavin was extrapolated. Seventy-eight participants were randomly
Riboflavin
divided into the control and 5 riboflavin-supplemented groups. A 6-week riboflavin
Estimated average requirement
supplementation was performed at the doses of 0, 0.2, 0.4, 0.6, 0.8, or 1.0 mg daily. The
Plasma
energy expenditure was 15.4 ± 1.9 MJ/d, as estimated by the 24-hour physical activity recording
Urinary excretion
method. Dietary riboflavin intake was 1.0 ± 0.2 mg/d, based on chemical analysis. The fasting plasma riboflavin was increased significantly in a dose-dependent manner when the supplemented riboflavin exceeded 0.4 mg/d and the EAR of riboflavin was suggested to be between 1.3 and 1.5 mg/d. In addition, we found a significant increase in fasting urinary riboflavin excretion when the supplemented riboflavin exceeded 0.6 mg/d. The critical point was calculated as 1.4 mg/d, based on the intersecting point of the 2 regression lines at lower and higher riboflavin intakes. These findings demonstrate that plasma riboflavin is a sensitive marker for riboflavin status, and the EAR of riboflavin for Chinese male adults is 1.4 mg. © 2016 Elsevier Inc. All rights reserved.
1.
Introduction
Riboflavin acts as coenzymes in the forms of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) for numerous oxidases, reductases, and dehydrogenases in metabolic processes. Riboflavin deficiency results in low FMN and FAD contents in the cell and reduced activity of a series of flavin-containing enzymes [1,2]. Mitochondrial β-oxidation was
impaired remarkably by riboflavin deficiency because several flavin-containing enzymes, such as acyl-CoA dehydrogenase, are involved in mitochondrial β-oxidation [3]. Riboflavin also plays a role in glutathione synthesis, in which FAD is a cofactor for glutathione reductase. The erythrocyte glutathione reductase activation coefficient (EGRAC) is frequently used as a marker in the assessment of riboflavin status [4]. Moreover, riboflavin is required for methylenetetrahydrofolate reductase and plays an
Abbreviations: CNS, Chinese Nutrition Society; EAR, estimated average requirement; EGRAC, erythrocyte glutathione reductase activation coefficient; FAD, flavin adenine dinucleotide; FMN, flavin mononucleotide. ⁎ Corresponding author. Tel.: +86 22 84655429; fax: +86 22 84655020. E-mail address: [email protected] (C. Guo). 1 The first 2 authors contributed equally. http://dx.doi.org/10.1016/j.nutres.2016.02.003 0271-5317/© 2016 Elsevier Inc. All rights reserved.
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N U T RI TI O N RE S E ARCH 3 6 ( 2 0 16 ) 5 3 4–5 40
Table 1 – Baseline characteristics of the study participants Parameter
Group 1
Group 2
Group 3
Group 4
Group 5
Group 6
Participants (n) Age (y) Height (cm) Body weight (kg) Body mass index (kg/m2)
13 21 ± 2 175.2 ± 3.9 62.9 ± 6.5 20.5 ± 1.6
13 20 ± 1 174.8 ± 7.0 68.8 ± 13.8 22.5 ± 3.1
13 20 ± 2 174.9 ± 5.9 67.5 ± 6.4 22.1 ± 1.9
13 21 ± 2 170.0 ± 5.2 66.1 ± 6.0 22.9 ± 1.4
13 21 ± 2 176.8 ± 6.1 67.9 ± 8.7 22.0 ± 2.8
13 20 ± 1 172.5 ± 5.8 65.4 ± 5.8 22.0 ± 2.4
Values are expressed as means ± SD.
important role in homocysteine metabolism, especially in individuals with a methylenetetrahydrofolate reductase 677 TT genotype [5]. The present estimated average requirement (EAR) of riboflavin in the United States is based mainly on studies conducted more than 50 years ago, in which 24-hour urinary riboflavin excretion was used as an indicator [6]. The critical point of urinary riboflavin excretion, a point of intersection of the regression lines fitted to the excretion at higher and lower riboflavin intakes, is considered the level of intake at which organ or tissue riboflavin saturation occurs [7]. Later, the EGRAC method was developed and applied in determining riboflavin requirement in children, women, and the elderly [8–10]. However, the acceptable range of activity coefficient values is too small in magnitude (from 1.0 to 1.2), as recommended by Sauberlich et al [11]. Boisvert et al [10] found that the changes of the EGRAC value were without a clear delineation between inadequate and adequate riboflavin status, although a decreasing trend was recorded with increasing riboflavin intake in aged participants. In 2005, Xu et al [12] reported that fasting plasma riboflavin was decreased rapidly in response to a riboflavin-deficient diet and recovered soon after a riboflavin-containing diet was provided in rats. In riboflavin malnourished humans, it was confirmed that fasting plasma riboflavin was increased significantly after 1 week of riboflavin supplementation [13]. A consistent result was also reported by Hustad et al [14] in a human study, in which plasma riboflavin was demonstrated to be useful in the assessment of riboflavin status. Therefore, it appears that plasma riboflavin is a sensitive index for riboflavin status. The purpose of this study is to validate the effectiveness of plasma riboflavin in assessing riboflavin status and further to determine the EAR of riboflavin for Chinese male adults. We hypothesize that plasma riboflavin is a useful maker in studying riboflavin requirement. To test this hypothesis, the changes of fasting plasma riboflavin in response to different riboflavin intake levels were determined in Chinese male adults after 6 weeks of intervention. In addition, fasting urinary riboflavin excretion was simultaneously measured.
2.
Methods and materials
2.1.
Participants and study design
in this study. No classic signs of riboflavin deficiency, such as cheilosis, angular stomatitis, and glossitis, were found in a routine physical examination. A written informed consent was signed by all participants. Approval for this study was granted by the ethical committee of the Institute of Health and Environmental Medicine. The participants were randomly assigned to the control and 5 riboflavin-supplemented groups by selection of a random number. The general characteristics of the study participants are presented in Table 1. During the 6week experimental period, all participants led a similar lifestyle and were engaged in routine physical training. They were asked to abstain from any riboflavin-containing supplements and were group fed in a dining room on a menu designed by a dietitian. Riboflavin supplementation was performed via the drinks containing different amounts of riboflavin for the different groups. The drinks were prepared freshly by dissolving riboflavin (≥98% in purity, provided by Tianjin Feiying Pharmaceutical Co, Ltd, Tianjin, PR China) in 200 mL of water and consumed by the participants every morning under the supervision of the field staff. The doses of riboflavin for different riboflavin-supplemented groups were 0.2, 0.4, 0.6, 0.8, and 1.0 mg daily, respectively. At these low concentrations, riboflavin was completely soluble in the water. During the experimental period, 5 participants withdrew from groups 1 (control), 2 (0.2 mg), 3 (0.4 mg), 5 (0.8 mg), and 6 (1.0 mg), respectively, and their data were excluded from the final statistical analyses (Fig. 1).
2.2.
A 4-day dietary survey was performed in the last week, using a food-weighting method. Dietary intakes of energy and nutrients (including riboflavin) were calculated based on food intakes and Chinese Food Composition, which was compiled by the Institute of Nutrition and Food Safety, Chinese Center for Disease Control and Prevention [15]. From the second week to the last week, daily duplicate food samples consumed by the participants were selected randomly from each group, collected, and stored at −80°C before being analyzed fluorophotometrically for riboflavin content by a standardized procedure approved by the Ministry of Public Health, PR China [16].
2.3.
Seventy-eight male adults aged 18 to 22 years from an army unit in Tianjin, PR China, were recruited. They were nonsmokers and physically healthy and volunteered to participate
Assessment of dietary intakes
Estimation of daily energy expenditure
From the third week to the fifth week, 3 to 4 participants were selected randomly from each group for estimation of daily energy expenditure by the 24-hour physical activity recording method [17]. The field staff followed the participants from the
536
N U TR I TION RE S E ARCH 3 6 ( 2 0 16 ) 5 3 4–5 40
Fig. 1 – Illustration of the experimental process.
wake-up time in the morning to the sleep time at night. All the activities the participants performed, as well as how long they lasted, were recorded accordingly. Daily energy expenditure was summed up based on the energy expenditure by individual activities, in addition to the basic metabolic expenditure.
2.4.
Measurement of fasting plasma riboflavin
Fasting blood samples from the antecubital vein were collected at the beginning and end of the experiment. Vacuum tubes containing heparin were used to obtain blood samples. Plasma was separated by centrifugation and stored at −80°C before the content of free riboflavin was determined by a high-performance liquid chromatography procedure described by Wei et al [13].
2.5.
Measurement of fasting urinary riboflavin
Fasting urine samples were collected at the beginning and end of the experiment. The pH of the urine samples was adjusted to 4.0 by addition of oxalate and stored at −80°C before the content of riboflavin was assayed fluorophotometrically [18]. The content of urinary creatinine was measured by the reaction of creatinine with picrate in alkaline medium, using a commercial assay kit purchased from Jiancheng Bioengineering Institute (Nanjing, China).
2.6.
Statistical analyses
Data are expressed as means ± SD and checked for normality using the Kolmogorov-Smirnov test before subjected to further
analysis. We found that the data of plasma riboflavin and urinary riboflavin excretion were not in a normal distribution. Thereby, Mann-Whitney rank sum test was performed to analyze the differences among different groups. The level of significance was set at P < .05. Linear regression was used to investigate the relationship between riboflavin intake and plasma riboflavin or urinary riboflavin excretion. The critical point was calculated based on 2 regression lines fitted to the urinary riboflavin excretion at lower and higher riboflavin intakes. Power analyses were conducted to predict the sample size to achieve 80% power for plasma riboflavin (minimum n = 8) and urinary riboflavin excretion (minimum n = 10) with statistical significance at .05.
3.
Results
3.1.
Dietary intake of energy and nutrients
As shown in Table 2, the mean dietary intake of energy and protein reached the EAR recommended by the Chinese Nutrition Society (CNS) [19]. However, 33.8% energy was contributed by lipids, which is higher than the level recommended by the CNS. The mean dietary intake of selenium and zinc was adequate, whereas calcium was insufficient, based on the EAR. The mean dietary intake of vitamin A, vitamin C, vitamin E, thiamine; riboflavin, and niacin reached the EAR. However, the mean dietary intake of iron exceeded the EAR by 380%.
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N U T RI TI O N RE S E ARCH 3 6 ( 2 0 16 ) 5 3 4–5 40
Table 2 – Daily dietary intake of energy and nutrients Energy and nutrients
Daily dietary intake
Energy (MJ) Protein (g) Calcium (mg) Iron (mg) Zinc (mg) Selenium (μg) Vitamin A (μg RE) Vitamin E (mg) Thiamine (mg) Riboflavin (mg) Niacin (mg) Vitamin C (mg)
13.9 89.6 615.7 43.2 13.2 53.7 801.3 25.5 1.3 1.2 18.2 127.9
± ± ± ± ± ± ± ± ± ± ± ±
0.6 10.1 62.8 1.1 1.0 8.4 90.4 4.8 0.2 0.2 8.5 32.4
Values are expressed as means ± SD. The dietary survey was performed in the last week using a foodweighting method. Dietary intakes of energy and nutrients were calculated based on food intakes and Chinese Food Composition.
3.2.
Energy expenditure
No significant difference was found in daily energy expenditure among different groups (Table 3). The average energy expenditure was 15.4 ± 1.9 MJ/d, which is 10.9% higher than the energy intake as estimated by the dietary survey.
3.3.
Dietary riboflavin intake by chemical analysis
Based on chemical analysis, no significant difference was found for dietary riboflavin intake among different groups (Table 4). The average dietary riboflavin intake was 1.0 ± 0.2 mg/d, which is 16.7% lower than that estimated by the dietary survey and reflects a possibility of marginal riboflavin status. This is due to the losses caused by the cooking process, as demonstrated by Xu et al [20]. The total riboflavin intake should be the combination of the dietary riboflavin and the supplemented riboflavin from the drinks. Therefore, the actual daily riboflavin intakes are 1.0, 1.3, 1.5, 1.6, 1.9, and 2.0 mg for groups 1, 2, 3, 4, 5, and 6, respectively.
3.4.
Fasting plasma riboflavin
As indicated in Table 5, there was no remarkable difference in fasting plasma riboflavin among the different groups at the beginning. At the end of the experiment, fasting plasma riboflavin was increased significantly in a dose-dependent manner when the supplemented riboflavin exceeded 0.4 mg/d (P < .05 or .01). A positive correlation was found between riboflavin intake and fasting plasma riboflavin among the
groups 2, 3, 4, 5, and 6 (R2 = 0.9231, P < .01). It is not logical to run a linear regression based on 2 points at the lower riboflavin intakes (groups 1 and 2). Therefore, the critical point was not calculated from the fasting plasma riboflavin. Based on the changes of fasting plasma riboflavin, the EAR of riboflavin is between 1.3 and 1.5 mg/d.
3.5.
Fasting urinary riboflavin excretion
It was noted that fasting urinary riboflavin excretion was relatively lower at the beginning, without significant differences among different groups. At the end of the experiment, fasting urinary riboflavin excretion was increased significantly compared with the beginning. Moreover, a sharp increase in fasting urinary riboflavin excretion was found when the supplemented riboflavin exceeded 0.6 mg/d (Table 6). Correlation analysis showed that a positive linear correlation was found between riboflavin intake and fasting urinary riboflavin excretion among groups 3, 4, 5, and 6 (R2 = 0.9667, P < .01). Based on the changes of fasting urinary riboflavin excretion, 2 linear regression lines were constructed between riboflavin intake and fasting urinary riboflavin excretion (Fig. 2). One line was developed among groups 1, 2, and 3 and another among groups 3, 4, 5, and 6. The critical point (the intersecting point of the 2 linear regression lines) is calculated as 1.4 mg/d.
4.
Discussion
Low riboflavin intake is common in many parts of the world, particularly in developing countries [21,22]. In China, inadequate riboflavin status is believed to be widespread. It was estimated that the average daily riboflavin intake was approximately 0.8 mg per capita; this is remarkably lower than the current EAR recommended by CNS (1.2 mg for males and 1.0 mg for females), which is based on urinary excretion data obtained more than 50 years ago [23]. However, Campbell et al [24] thought that the riboflavin requirement might be set too high for the Chinese population if based on Western experimental data because they found that no clear evidence of riboflavin deficiency symptoms was observed in their field surveys. Hence, it is important to determine riboflavin EAR for Chinese populations and lay a solid basis for the revision of Chinese Dietary Reference Intakes. Previously, it was demonstrated that plasma riboflavin levels tended to be variable and reflected recent intake of riboflavin [11]. However, a rat study conducted in our laboratory indicated that fasting plasma riboflavin responded sensitively to riboflavin depletion and repletion. Comparatively, urinary riboflavin excretion was also a reliable
Table 3 – Estimated daily energy expenditure Parameter
Group 1
Group 2
Group 3
Group 4
Group 5
Group 6
Participants (n) Energy expenditure (MJ/d)
4 15.9 ± 3.1
4 16.0 ± 1.2
3 15.7 ± 2.2
3 14.3 ± 1.4
3 14.5 ± 2.8
3 15.6 ± 2.2
Values are expressed as means ± SD. The 24-hour physical activity recording method was used to estimate daily energy expenditure.
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N U TR I TION RE S E ARCH 3 6 ( 2 0 16 ) 5 3 4–5 40
Table 4 – Dietary riboflavin intake by chemical analysis Parameter
Group 1
Group 2
Group 3
Group 4
Group 5
Group 6
Duplicate foods (n) Riboflavin intake (mg/d)
16 1.0 ± 0.2
17 1.1 ± 0.3
15 1.1 ± 0.3
16 1.0 ± 0.2
16 1.1 ± 0.3
15 1.0 ± 0.2
Values are expressed as means ± SD. The riboflavin content of duplicate foods was analyzed by a standardized procedure (GB/T 5009.85-2003) approved by the Ministry of Public Health, PR China.
Table 5 – Changes of fasting plasma riboflavin Parameter
Group 1
Group 2
Group 3
Group 4
Group 5
Group 6
Participants (n) Initial (nmol/L) Final (nmol/L)
12 12.7 ± 4.8 11.6 ± 4.8
12 10.0 ± 4.1 10.9 ± 4.8
12 12.4 ± 3.6 19.2 ± 9.4 ⁎
13 11.5 ± 5.6 20.77 ± 11.15 ⁎
12 11.3 ± 4.9 24.7 ± 12.9 ⁎⁎
12 12.5 ± 4.2 27.0 ± 17.3 ⁎⁎
Values are expressed as means ± SD. The total daily riboflavin intakes are 1.0, 1.3, 1.5, 1.6, 1.9, and 2.0 mg for groups 1, 2, 3, 4, 5, and 6, respectively. Plasma riboflavin was determined by a high-performance liquid chromatography procedure. Mann-Whitney rank sum test was used for statistical analyses. ⁎ Significance at P < .05 as compared with group 1. ⁎⁎ Significance at P < .01 as compared with group 1.
indicator in reflecting the short-term changes of riboflavin intake. On the other hand, EGRAC and the contents of erythrocyte riboflavin, FMN, and FAD were relatively insensitive to riboflavin depletion and repletion [12]. It is explainable because the cells are capable of regulating riboflavin transport actively to maintain cellular riboflavin homeostasis, as demonstrated previously by Werner et al [25]. Consistent data were also obtained from riboflavin supplementation trials in humans, in which fasting plasma riboflavin was used as a marker in assessing riboflavin status [13,14]. In the present study, the changes of fasting plasma riboflavin were determined in participants with different riboflavin intakes. It was found that the fasting plasma riboflavin responded sensitively to the riboflavin supplementation in a dosedependent manner when the supplemented dose was greater than 0.2 mg. The EAR of riboflavin is suggested to be between 1.3 and 1.5 mg/d. The 24-hour urinary riboflavin excretion was used frequently by many investigators as an index of riboflavin status and proved to be valuable in determining riboflavin requirement [11]. For example, Horwitt et al [26] reported that the 24-hour
urinary riboflavin excretion increased markedly when the riboflavin intake exceeded 1.1 mg/d in adult men. However, it is often difficult practically to collect 24-hour urine samples in the field study. It was demonstrated that the riboflavin content of fasting urine samples was also useful in the assessment of riboflavin status after corrected by creatinine, because it was not affected by dietary and nondietary factors in the daytime [27,28]. In the present study, the changes of fasting urinary riboflavin excretion were monitored simultaneously. The results showed that the critical point was located at the riboflavin intake level of 1.4 mg/d, which is close to the EAR extrapolated from the changes in fasting plasma riboflavin. Therefore, the EAR of riboflavin is suggested to be 1.4 mg/d for Chinese male adults. In conclusion, the results of this study suggest that plasma riboflavin is a reliable marker for riboflavin status. It is also indicated, for the first time, that the EAR of riboflavin for Chinese male adults is 1.4 mg, based on the changes of fasting plasma riboflavin and urinary riboflavin excretion. However, we are aware of the limitation of our study because it has been known that riboflavin requirement is affected by several
Table 6 – Changes of fasting urinary riboflavin excretion Parameter
Group 1
Group 2
Group 3
Group 4
Group 5
Group 6
Participants (n) Initial (μg/g Cr) Final (μg/g Cr)
12 248.2 ± 138.8 538.4 ± 325.5
12 171.0 ± 76.9 507.3 ± 222.8
12 194.4 ± 92.5 542.6 ± 276.0
13 238.1 ± 107.1 741.6 ± 306.4
12 223.1 ± 108.0 963.3 ± 300.9 ⁎⁎
12 204.4 ± 90.9 1066.0 ± 475.7 ⁎⁎
Values are expressed as means ± standard deviation. The total daily riboflavin intakes are 1.0, 1.3, 1.5, 1.6, 1.9, and 2.0 mg for groups 1, 2, 3, 4, 5, and 6, respectively. The urinary riboflavin was assayed fluorophotometrically. Mann-Whitney rank sum test was used for statistical analyses. ⁎⁎ Significance at P < .01 as compared with group 1.
Fasting urinary riboflavin excretion (µg/gCr)
N U T RI TI O N RE S E ARCH 3 6 ( 2 0 16 ) 5 3 4–5 40
1,300 1,200
y = 965.34x - 860.96 R² = 0.9667
1,100
[6]
1,000 900
[7]
800 700
y = -0.4132x + 529.93 R² = 3E-05
[8]
600 500 400 0.8
1.1
1.4
1.7
2
2.3
Riboflavin intake (mg)
Fig. 2 – Critical point of fasting urinary riboflavin excretion at the end of the experiment. Two linear regression lines were constructed between riboflavin intake and mean fasting urinary riboflavin excretion. One line was developed among groups 1, 2, and 3 and another among groups 3, 4, 5, and 6. The total daily riboflavin intakes are 1.0, 1.3, 1.5, 1.6, 1.9, and 2.0 mg for groups 1, 2, 3, 4, 5, and 6, respectively. The critical point of fasting urinary riboflavin excretion was 1.4 mg/d, based on the intersecting point of the 2 regression lines at lower and higher riboflavin intakes.
factors, including age, energy intake, physical activity, and dietary composition. Several countries and the Food and Agriculture Organization/World Health Organization express riboflavin requirements in relation to energy intake, although it still remains controversial [1,6,10,23]. The mean energy intake of the participants in this study was estimated to be 13.9 MJ/d, or 3322 kcal/d. If expressed in terms of energy intake, the EAR of riboflavin is 0.1 mg/MJ, or 0.4 mg/1000 kcal, for Chinese male adults. Further studies should be undertaken to investigate riboflavin requirements in other age groups or conditions. In addition, possible impacts of excess iron intake on riboflavin requirement need to be further explored.
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16] [17]
Acknowledgment This work is supported financially by a grant (DIC2012-02) from Danone Institute of China. We acknowledge the cooperation of the volunteers participating in this study.
[18]
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