Agric.
Biol
Chern., 52 (7),
1765-1769,
1988
1765
Effect of Dietary Protein Levels on the Urinary Excretion of Nicotinamide and Its Metabolites in Rats Katsumi Shibata,
Rika Nomamoto and Kazuo Iwai*
Laboratory of Nutritional Biochemistry, Department of Food Science and Nutrition, Faculty of Domestic Science, Teikoku Women's University, Moriguchi, Osaka 570, Japan * Laboratory of Nutritional Chemistry, Department of Food Science and Technology, Faculty of Agriculture, Kyoto University,
Received
January
Kyoto 606, Japan
28, 1988
Male rats of the Wistar strain (seven weeks old) were fed adlibitum with a 10%, 20% or 40% casein diet (these diets contained sufficient nitocinic acid of 6 mg per 100 g of diet) for 20 days. Urine was collected for the last 2 days, and the urinary excretion of nicotinamide, A^-methylnicotinamide (MNA),
A^-methyl^-pyridone-S-carboxamide
(2-py)
and A^-methyM-pyridone-S-carboxamide
(4-py) was measured using the most reliable determination methods with a high-performance liquid chromatograph. The urinary excretion of nicotinamide and 2-py was about the same in the three
groups. The urinary excretion of 4-py increased with increasing dietary MNA,which decreased with increasing dietary protein levels. The nicotinamide, MNA,2-py and 4-py was about the sameamountin the of total nicotinamide and MNAin the liver decreased with increasing these results being inconsistent with the established theory.
It is known that niacin is de novo biosynthesized from tryptophan in mammals. The conversion ratio of tryptophan to niacin in humanshas been accepted as 60:1 (on a mg basis) based on the studies ofHorwitt et al.l) in 1956 and of Goldsmith et al2) in 1961. With this assumption, the amount of niacin intake wasroughly the same as the amount of intake from tryptophan in Japanese college women.3) Therefore, the tryptophan intake cannot be ignored as the provitamin of niacin, although the proportion of tryptophan converted to niacin is not constant. Nakagawa et al.4) reported that this ratio decreased from about 122 to about 75 as the amounts of added tryptophan were increased, and they concluded that the conversion of tryptophan to niacin depended on the proper dietary conditions, and whether tryptophan was deficient or niacin was saturated in the body.5) Recently, Murata and Motooka6) reported that this ratio was 44.7±17.6 (21.4-60.1) in
protein levels, contrary to total urinary excretion of three groups. The contents dietary protein levels, all
Japanese college women,and Patterson et al.7) reported that the average conversion ratio of tryptophan to niacin was approximately 72 : 1 in American
college
men. In rats,
Singal
et
al.20) and Bell et al.21) showed that an increase
in the casein content of the diet did not increase niacin and A^-methylnicotinamide (MNA) excretions; however, the addition of free tryptophan in an adequate ratio resulted in a marked increase in the excretion of both niacin and MNA. Satyanarayana and Narasinga Rao22) reported that quinolinate phosphoribosyltransferase activity in rat liver was inversely related to dietary protein level, while tryptophan oxygenase was directly related to the protein level. Sanada and Miyazaki16) and Hayakawa and Iwai17) re-
ported
that when rats were fed with a high
protein diet, the liver aminocarboxymuconatesemialdehyde
decarboxylase
activity
greatly
increased compared to that with a normal diet. Consequently, it is nowknownthat the con-
K. Shibata, R. Nomamoto and K. Iwai
1766
version ratio of tryptophan to niacin in humans is changeable, and the control mechanism of tryptophan-niacin metabolism must
accordingly be elucidated.
It has been reported and accepted that the urinary excretion of nicotinamide and its metabolites depends on the amountof niacin and tryptophan ingested.1-2-4~7) However, we have found this to be incosistent, and in this paper wedescribe the relationship between the urinary excretion of niacin and its metabolites
and the dietary protein level in rats. MATERIALSANDMETHODS
Table I. Composition of the Diets
10% casein 20% casein 40% casein (%)
Shokuhin Kako Co., Ltd., respectively. A^!-Methyl-2pyridone-5-carboxamide (2-py) and A^1-methyl-4-pyridone-3-carboxamide (4-py) were respectively synthesized
by the methods of Pullman and Colowick8) and Shibata et al.9) All other chemicals used were of the highest purity obtainable.
Columns.
The columns
for high-performance
liquid
chromatography, a 300-7-SCX (150 x 4.6mm i.d.) and 7ODS-L(250 x 4.6mm i.d.), were purchased from Chemco Scientific Co., Ltd. Animals and diets. Male rats of the Wistar strain (seven weeks old) were purchased from Clea Japan Inc. and used immediately.
The rats were kept individually
in wire-
bottomedcages, witha06:00~18:00 (light) and 18:00~ 06 : 00 (dark) schedule. The room temperature was kept at 22±2°C and the humidity
20 23.0 46.0 5 5 1
40
26.3 52.7 5
Mineral mixture" Vitamin mixture^
5 1
16.3 32.7 5 5 1
a Oriental's ratio (14.56g of CaHPO4-2H2O, 25.72g of KH2PO4, 9.35g of NaH2PO4, 4.66g of NaCl, 35.09g of Ca-lactate, 3.18g of Fe-citrate, 7.17g of MgSO4, 0.1 1 g ofZnCO3, 0.12g ofMnSO4à"4-6H2O, 0.03g of CuSO4-5H2O and 0.01g of KI) was used and obtained from Oriental Yeast Co., Ltd. Oriental's
ratio
(50,000IU
of vitamin
A-acetate,
10,000IU of vitamin D3, 500mg of vitamin Eacetate, 520mg of vitamin K3, 120mg of vitamin B^HCl, 400mg of vitamin B2, 80mg of vitamin B6HC1, 0.05mg of vitamin B12, 3000mg of vitamin C, 2mg of D-biotin, 20mg of folate, 500mg of calcium panthotenate, 500 mg of /rara-aminobenzoic acid,
600mg of nicotinic acid, 600mg of inositol, 20,000mg ofcholine chloride and made up to lOOg by cellulose powder) was used and obtained from
Oriental
Yeast Co., Ltd.
of Shibata.10) Determination of creatinine. The creatinine urine was measured by the Jaffe reaction.ll'
content in
Statistical analysis. The significance of differences between means was evaluated by Bartlett's test and by Duncan's new multiple range test.12) RESULTS
was about 60%. The 10%, 20%
or 40%casein diet (Table I) and water were fed ad libitum
(%)
10
Casein Sucrose a-Cornstarch Corn oil
b
Chemicals. MNA,creatinine, vitamin-free milk casein, sucrose and nicotinamide were obtained from WakoPure Chemical Industries Ltd. a-Cornstarch and corn oil were purchased from Nichiden Kagaku Co., Ltd. and Nippon
(%)
to the rats for 20 days, these diets containing sufficient nicotinic acid of 6mg per 100g of diet. Body weight and food intake were measured daily at 9 :00~ 10 :00a.m. Urine was collected for the last 2 days in a flask
Body weight gain, food intake and food efficiency ratio
containing 0.5 ml oftoluene and 1.0ml of 1 mhydrochloric acid, using a metabolic cage, and was stored at -25°C until used. The rats were killed by decapitation at around
the 10%, 20% and 40% casein diets. These values are significantly higher in the groups
9:00a.m. and the liver was removed.
Determination ofnicotinamide, 2-py and 4-py in liver and urine. The nicotinamide, 2-py and 4-py contents in liver and urine were simultaneously measured by the method of Shibata
et al9)
Determination of MNAin liver and urine. The MNA contents in liver and urine were measured by the method
Table II shows the body weight gain, food intake and food efficiency ratio in rats fed with
with the 20 and 40%casein diets than in the group with the 10% casein diet. Urinary excretion of nicotinamide, and 4-py Table III
shows the urinary
MNA,2-py excretion
of
nicotinamide, MNA,2-py and 4-py when the 10%, 20% and 40% casein diets were fed to the
1767
Effect of Protein Levels on Niacin Formation in Rats Table II. Effect of Dietary Protein Levels on the Gain in Body Weight, Food Intake and Food Efficiency Ratio of Rats
10% casein 55.0±2.2a
Gain in body weight (g)
88.6±3.3b 311.1+4.5*
289.2±6Aa 0.189+0.005°
Food intake (g) Food efficiency ratio
0.284±
Values are means± S.E. for 10 rats; means having a different significant difference at p < 0.05. Table III.
AND ITS
10% casein
Creatinine Omol/daily urine) Nicotinamide (nmol/daily urine) (nmol/^mol
urine)
4-py (nmol/daily
urine)
(nmol/yumol
± 26.00
3492.84± 244.31° 107.00±5.91a 298.81 ± 17.22* 9.21±0.53
creatinine)
2097.19± 64.61
(nmbl/^mol creatinine) Nicotinamide + MNA+ 2-py+4-py (nmol/daily urine) (nmol/^mol
1.88*
290.52 8.82±0.60°
creatinine)
2-py (nmol/daily
32.94±
creatinine)
urine)
(nmol//imol
0.009b
40% casein 96.0±3.6b 312.7+5.7* 0.305±0.008*
superscript letter in the same line mean a statistically
Effect of Dietary Protein Level on the Urinary Excretion of NlCOTINAMIDE
MNA(nmol/daily
20% casein
135.16° +4.24°
6179.36±388.95 189.64± 10.05
creatinine)
2-py + 4-py/MNA
0.696±0.036°
Values are means± S.E. for 10 rats; means having a different significant difference at p < 0.05.
METABOLITES
20% casein 36.27±
1.92a'fc
330.20± 8.93±0.61fl
37.80
40°/ casein 40.15±1.96* 272.38± 13.74 5.39±0.31ft
819.25± 121.43* 21.98±2.34b
600.00 14.90±
± 65.79" 1.33c
358.73
385.41 9.65±0.68
± 30.57b
± 21.46fl'ft
10.35±0.62 4740.82± 313.63" 137.52±9.99b
5766.52 ± 376.60c 145.40+9.81*
6137.24± 347.52 179.38+ll.13
6971.99±454.81 175.34+ ll-.09
7.827+
1.060b
10.792+0.836c
superscript letter in the same line mean a statistically
Table IV. Effect of Dietary Protein Level on the Contents of Total Nicotinamide and MNAin Liver
10% casein Total nicotinamide (nmol/g wet weight) 1616.21
MNA(nmol/g wet weight)
±72.95°
58.19±3.78"
20% casein
40% casein
1408.20±
1236.72+28.34b
38.47±3.16fe
73.79*
34.46+2.83*
Values are means+S.E. for 10 rats; means having a different superscript letter in the same line mean a statistically significant difference at p