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1 From the Course of Food Science and Nutrition, Department of Life Style Studies, School of Human Cultures, The University of Shiga Prefecture, Hikone, Shiga, Japan (HO, AI, TF, and KS), and the Division of Health Promotion, National Institute of Health and Nutrition, Shinjuku-Ku, Tokyo (YY, NK, and MN).
2 Supported by a Grant-in-Aid for Scientific Research, The Ministry of Health, Labor and Welfare, Japan, and a Grant-in-Aid for Scientific Research, The Ministry of Education, Japan. 3 Address reprint requests to H Okamoto, Course of Food Science and Nutrition, Department of Life Style Studies, School of Human Cultures, The University of Shiga Prefecture, 2500 Hassaka, Hikone, Shiga 522-8533, Japan. E-mail: hokamoto{at}shc.usp.ac.jp.
ABSTRACT
Background: More than 500 enzymes need niacin coenzymes. Therefore, elucidation of the control mechanisms of coenzyme metabolism is fundamentally important.
Objective: NAD+ is involved in ATP production. Because energy expenditure is generally higher during the day than at night, we investigated whether the metabolism of nicotinamide changes at various times of day and whether stress affects nicotinamide metabolism.
Design: Twelve women were housed in the same facility and followed the same schedule for activities of daily living for 12 d. Urinary outputs were collected during 5 specific periods to investigate diurnal variations in nicotinamide metabolism. The effects of cold exposure (physical stress), having to perform arithmetic calculations (mental stress), and dark exposure (emotional stress) on nicotinamide metabolism were investigated.
Results: A diurnal variation in the nicotinamide metabolites N1-methylnicotinamide, N1-methyl-2-pyridone-5-carboxamide, and N1-methyl-4-pyridone-3-carboxamide was observed. Of the stresses studied, cold exposure significantly increased the urinary excretory outputs of the nicotinamide metabolites.
Conclusions: Diurnal variations in nicotinamide metabolism were found in these women. The biosynthesis of nicotinamide from tryptophan seemed to be increased by cold exposure.
Key Words: Diurnal variation • nicotinamide • N1-methylnicotinamide • N1-methyl-2-pyridone-5-carboxamide • N1-methyl-4-pyridone-3-carboxamide • stress • human urine • women
INTRODUCTION
Through starvation, eating, exercise, or stress, the human body keeps biological functions fairly constant. However, extremely strong stresses occasionally disturb biological functions. For example, chronic exercise affects vitamin B-6 metabolism (1), blood pantothenic acid concentrations increase in marathon runners (2), and changes in taste perception are reported after mental or physical stress (3). Extra nutrients sometimes overcome such stresses. To determine the adequate amount of nutrients needed in conditions of stress, we must elucidate the basic metabolic changes in the nutrients.
Because >500 enzymes need nicotinamide coenzymes, it is important to determine the control mechanisms of coenzyme metabolism. The metabolism of nicotinamide is changed in some disorders. Barlow et al (4) reported that urinary outputs of the nicotinamide metabolites N1-methylnicotinamide (MNA) and N1-methyl-2-pyridone-5-carboxamide (2-Py) are significantly increased in burned or scalded children. Cazzullo et al (5) found that the frequency of low MNA excretion is significantly higher in patients with primary affective disorders and in healthy first-degree relatives of patients with primary (but not secondary) affective disorders. Lis et al (6) reported that the urinary excretion of 2-Py is low in many children with autistic tendencies. Furthermore, Shibata et al (7) showed that blood NAD concentrations significantly decrease after rats are forced to swim until fatigued.
NAD is involved in ATP production, and NAD turnover can be investigated by the urinary outputs of its major metabolites: MNA, 2-Py, and N1-methyl-4-pyridone-3-carboxamide (4-Py) (8). Because energy expenditure is generally higher during the day than at night, Shibata (9) investigated diurnal variations in the urinary excretion of the catabolites of nicotinamide in female college students and observed some variation in each subject but not a diurnal variation. This seemed to be because the subjects ate self-selected food and daily activity was not controlled. Therefore, we have reinvestigated whether the metabolism of nicotinamide changes during the day when all subjects are housed in the same facility, eat the same food, and have the same type of activities of daily living and whether such diurnal variations in metabolism are affected by stress.
SUBJECTS AND METHODS
Subjects
The 12 female subjects were 20.9 ± 2.1 y of age (
All subjects were housed in the same facility for 12 d (24 July to 4 August 1999). The lights were turned off at 2200 and turned on at 0600. We did not check when the subjects indeed slept and awakened. The first 4 d were used for environmental adaptation. Days 5, 6, 7, 9, 10, and 11 were the experimental days, and day 8 was an intermission. Four daily menus were devised and were fed in rotation. Intakes of niacin, tryptophan, and energy were calculated on the basis of Japanese food-composition tables (10) (Table 1). The niacin intake from tryptophan was calculated by assuming that 60 mg tryptophan is converted to 1 mg niacin (10). The sum of the amount of niacin in the diet and the niacin converted from dietary tryptophan was the niacin equivalent intake.
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TABLE 1 . Composition of the experimental diet1
Urine samples were collected and pooled during 5 specific periods (0630 to 0830, 0830 to 1300, 1300 to 1830, 1830 to 2130, and 2130 to 0630 of the next day). Although the subjects were allowed to urinate whenever they wished, they were obligated to urinate just before 0630, 0830, 1300, 1830, and 2130. The volume of each urine sample was measured, and the sample was placed in a bottle containing 1 mL of 1 mol HCl/L and then stored at -25°C until needed.
Stresses
A complete crossover design in the sequence of the stresses was used to avoid the influence of diet and stress.
Cold exposure
Cold exposure is a typical physical stress (11). The area of the cold room was 5.4 m x 4.5 m x 2.4 m (height), and a temperature of from 2 to 6°C was maintained. The subjects wore ski suits, including ski gloves, wool caps, and double socks, over their normal winter clothes. The cold exposure stress was carried out from 0930 to 1230 and from 1400 to 1700. The subjects sat on chairs in the cold room, read books, and played crossword games. The control subjects wore self-selected clothes, sat on chairs in front of the cold room, read books, played crossword games, and monitored whether the subjects for cold exposure were all right.
Calculation exercises
This treatment is a typical mental stress (11). The area of the calculation room was 7.3 m x 7.3 m x 2.2 m (height). The room temperature was maintained at 25°C. The subjects (in both the treatment and control groups) wore self-selected clothes and sat on chairs in the room. The arithmetic problems for the calculation exercises were of the third-grade level (in Japanese elementary schools), for example, addition (double figures + double figures), subtraction (3 figures - double figures), multiplication (double figures x single figure), and division (4 figures/single figure). The calculation exercise stress was carried out from 0930 to 1230 and from 1400 to 1700. The control subjects checked every page completed by the subjects in the treatment group. When the answers were not correct, the mistakes were indicated and the pages were immediately corrected by the subjects in the treatment group.
Dark exposure
This exposure is a typical emotional stress (11). The dark exposure stress was carried out from 0930 to 1230 and from 1400 to 1700. The area of the dark room and the room temperature were exactly as in the calculation room. The subjects wore self-selected clothes and sat on stools in the room. The subjects in the treatment group wore eye masks and were restricted in movement and were prohibited from talking with one another. The control subjects sat on chairs and read books and monitored whether the stress subjects were sleeping.
Chemicals
MNA chloride was purchased from Tokyo Kasei Kogyo Co (Tokyo). 2-Py and 4-Py were synthesized by the methods of Pullman and Colowick (12) and Shibata et al (13). Other chemicals used were of the highest purity available from commercial sources.
Analyses
Urinary MNA was measured by the HPLC method of Shibata (14). The 2-Py and 4-Py contents in urine were measured simultaneously by the HPLC method of Shibata et al (13). Urinary creatinine was measured by the HPLC method of Shibata and Matsuo (8).
Statistics
Variables between groups in terms of total daily urine were compared by using paired Student’s t tests. Diurnal variations in the urinary excretion of MNA, 2-Py, 4-Py, the sum of the metabolites, and the ratio (2-Py + 4-Py)/MNA were determined by one-factor repeated-measures analysis of variance; the individual time points were compared by using Tukey’s test for multiple comparisons. STATVIEW software (version 5.0; Abacus Concepts, Berkeley, CA) was used for all analyses.
RESULTS
Diurnal variations in the urinary excretion of nicotinamide metabolites
The urinary excretory patterns of MNA, 2-Py, 4-Py, the sum of the nicotinamide metabolites, and the ratio (2-Py + 4-Py)/MNA in control subjects are shown in Figure 1. MNA output (Figure 1A) was highest in the urine samples collected from 0630 to 0830, gradually decreased during the day, and was lowest in the urine sample collected from 1830 to 2130. The decreased output seemed to increase slightly during sleep.
FIGURE 1. . Mean (±SD) diurnal variations in the urinary excretion of N1-methylnicotinamide (MNA; A), N1-methyl-2-pyridone-5-carboxamide (2-Py; B), N1-methyl-4-pyridone-3-carboxamide (4-Py; C), the sum of the metabolites (D), and the ratio (2-Py + 4-Py)/MNA (E). The mean obtained from 3 control days for each person was combined, and each time point is the mean for 12 persons. The dots were plotted on 0730, 1045, 1545, 2000, and 2600. The P values for the main effect of time were significant: 0.0002 (MNA), 0.0001 (2-Py), 0.0000 (4-Py), 0.0006 (sum), and 0.00000 (the excretory ratio). Means with different letters are significantly different, P < 0.05.
The amount of 2-Py excreted (Figure 1B) was not significantly different in the urine samples collected from 0630 to 0830, from 0830 to 1300, and from 1300 to 1830, but dropped significantly in the evening and night samples collected from 1830 to 2130 and from 2130 to 0630 the next day. The diurnal pattern of 4-Py output was similar to that of 2-Py (Figure 1C).
The sum of urinary outputs (Figure 1D) was significantly higher in samples collected from 0630 to 0830, from 0830 to 1300, and from 1300 to 1830 than in the samples collected from 1830 to 2130 and from 2130 to 0630 the next day.
Diurnal variation of the ratio (2-Py + 4-Py)/MNA was found (Figure 1E). The ratio was the highest in the urine sample collected from 1300 to 1830, decreased in the urine samples collected from 1830 to 2130, and was lowest in the urine samples collected from 0630 to 0830 and from 2130 to 0630 the next day.
Effect of stress on the excretory pattern of nicotinamide metabolites
The effect of cold exposure on the urinary excretion of nicotinamide metabolites is shown in Table 2 in terms of total daily urine output. MNA excretion and the excretion of the sum of the metabolites were higher during cold exposure than during the control period. No significant effect of the calculation exercises (mental stress; Table 3) or of dark treatment (emotional stress; Table 4) was seen in the urinary excretion of nicotinamide metabolites in term of total daily urine.
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TABLE 2 . Effects of cold exposure on the urinary excretion of nicotinamide metabolites1
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TABLE 3 . Effects of performing arithmetic calculation exercises on the urinary excretion of nicotinamide metabolites1
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TABLE 4 . Effects of dark exposure on the urinary excretion of nicotinamide metabolites1
DISCUSSION
The urinary excretion of MNA and 2-Py has been used as an index of niacin nutrition (15). However, Shibata and Matsuo (8) reported that a better method is to measure the daily urinary excretion of the pyridones 2-Py and 4-Py (end metabolites of niacin) rather than of MNA. Shibata (9) then determined that there is no fixed diurnal variation in the urinary excretion of MNA, 2-Py, and 4-Py or in the resulting excretion ratio, (2-Py + 4-Py)/MNA, in young women. In that study, however, the subjects ate self-selected food and daily activity was not controlled. In the present experiment, all subjects were housed together for 12 d. Environmental adaptation occurred in the first 4 d, the daytime schedule was very regulated, and the subjects ate a cycle of 4 menus, which were accepted by the subjects, who said that they felt no stress. The daily niacin equivalent intake was 25 mg in all of the diets. This value is more than the RDA in Japan (15 mg/d). The subjects slept at 2200, awoke at 0600, and ate breakfast at 0830. Under these conditions, a diurnal variation in the urinary excretion of niacin metabolites was found. The urinary excretion of MNA was lowest before the hour of sleeping and highest between the hours of rising and of breakfast. The urinary outputs of 2-Py, 4-Py, and the sum were higher during the day than in the evening and at night, dropping sharply after supper and sharply increasing after the hour of rising. The excretory ratio, (2-Py + 4-Py)/MNA, gradually increased during the day and peaked in the afternoon sample (collected from 1300 to 1830).
The excretion of the sum of metabolites could be subject to 3 kinds of enzymatic activity: NAD-degrading enzymes, nicotinamide phosphoribosyltransferase (EC 2.4.2.12), and nicotinamide methyltransferase (16; EC 2.1.1.1), as shown in Figure 2. The excretion of MNA and its pyridones is controlled by enzymes involved in the reactions of nicotinamideMNA (catalyzed by nicotinamide methyltransferase), MNA2-Py (2-Py-forming MNA oxidase; EC 1.2.3.1), and MNA4-Py (4-Py-forming MNA oxidase). Formation of 2-Py and 4-Py reflects activities of the respective enzymes (17, 18), whereas the ratio (2-Py + 4-Py)/MNA reflects the ratio of 2 kinds of MNA oxidase activities and nicotinamide methyltransferase activity (17, 18). The different diurnal excretory patterns of MNA, its pyridones, and the sum suggest different control mechanisms for these enzymes. Nicotinamide methyltransferase activity increases in poor nutritional conditions such as thiamine deficiency and fat deficiency (19). The activities of 2-Py-forming MNA oxidase and 4-Py-forming MNA oxidase are subject to nutritional conditions; enzymes increase in good conditions and decrease in poor conditions (16). Increased urinary outputs of nicotinamide metabolites indicate improved niacin nutrition because these urinary outputs increase after the pool of pyridine nucleotide coenzymes is filled (20).
FIGURE 2. . Niacin metabolism. MNA, N1-methylnicotinamide; 2-Py, N1-methyl-2-pyridone-5-carboxamide; 4-Py, N1-methyl-4-pyridone-3-carboxamide; NMN, nicotinamide methyltransferase, EC 2.1.1.1 (after the pool is filled with nicotinamide, nicotinamide is mainly metabolized to MNA); nicotinamide phosphoribosyltransferase, EC 2.4.2.12 (after the NAD pool is filled with NAD, this enzyme activity is inhibited by NAD); NMN adenyltransferase, EC 2.7.7.18; 2-Py-forming MNA oxidase, EC 1.2.3.1.
Energy expenditure and use of NAD are generally higher in the daytime than in the early morning, the evening, and at night. In mammals including humans, nicotinamide is biosynthesized from tryptophan (21). Thus, the conversion of tryptophan to nicotinamide may increase to complement the need for NAD in the daytime. NAD is degraded after being utilized 200000 times (22), and urinary outputs of nicotinamide metabolites increase in the daytime. Higher urinary outputs of nicotinamide metabolites in the daytime seem to denote increased NAD utilization, whereas lower urinary outputs in the evening and night might reflect decreased NAD utilization.
After the NAD pool is filled during the night, NAD inhibits nicotinamide phosphoribosyltransferase activity (23), and as a result, nicotinamide is metabolized to MNA. Higher MNA excretion in the early morning might indicate high nicotinamide methyltransferase activity and low MNA oxidase activities. Furthermore, the higher excretory ratio (2-Py + 4-Py)/MNA at noon indicates that MNA oxidase activity increases gradually from early morning to noon. The diurnal variation studies showed that nicotinamide was synthesized from tryptophan more during the day than at night.
Total daily MNA excretion and excretion of the sum of metabolites were significantly higher during cold exposure than during the control condition (Table 2). These findings suggest that cold exposure caused an increase in the metabolic rate and energy expenditure for thermogenesis, and that, as a result, the turnover ratio of the NAD cycle was immediately activated. High urinary outputs of MNA and 2-Py also occur in burned or scalded patients (4), indicating a high need for NAD in skin repair. Stress may also be a factor.
The calculation exercises (mental stress) had no significant effect on the metabolism of niacin in terms of total daily urine output (Table 3). Dark treatment (emotional stress) also had no significant effect on the metabolism of niacin in term of total daily urine output (Table 4). However, in patients with primary affective disorders (5) and autism (6), disturbances of nicotinamide metabolism have been reported.
In summary, diurnal variations in the nicotinamide metabolites MNA, 2-Py, and 4-Py were observed, and nicotinamide metabolism was affected by only the physical stress of cold exposure. Cold exposure raised energy expenditure (24), resulting in activated biosynthesis of nicotinamide from tryptophan and increased nicotinamide metabolism.
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