J. Nutr. Sci. Vitaminol., 38, 93-101, 1992

Note

Accumulation of Hypotaurine in Tissues and Urine of Rats Fed an Excess Methionine Diet

Takanori KASAI, Yutaka OGO, Yuichi OTOBE, and Shuhachi KIRIYAMA

Department of Agricultural Chemistry, Faculty of Agriculture, Hokkaido University, Sapporo 060, Japan (Received November 29, 1991)

Summary Hypotaurine increased in some tissues, especially in muscle, and urine of rats fed methionine excess diet. The significant depression of the body weight and food intake of rats caused by excess methionine was remarkably alleviated as previous reports and hypotaurine content in muscle and urine increased further by supplement with glycine to the excess methionine diet. Key Words hypotaurine, methionine

Methionine, an essential amino acid for the adequate growth and development of mammals, is one of the most highly toxic amino acids and the excess intake of methionine results in growth retardation and tissue damage (1, 2). We found the accumulation of ophthalmic acid and a decrease of glutathione in the livers of rats fed a low (8%) casein diet. On the other hand, glutathione accumulated in the livers of rats fed a low casein diet supplemented with excess (3%) methionine. Hepatic ophthalmic acid content of the excess methionine diet group was signi ficantly lower than that of 8% casein diet group (3, 4). It was also described in the previous report that ƒÁ-glutamylserylglycine, which was a hitherto unknown glu tathione analog, was isolated from rat liver, but the amount of ƒÁ-glutamylserylgly cine could not be determined because of its overlap with another very sharp peak on the amino acid analyzer (4). The unusually sharp peak (named U-14), with nearly the same elution time on the amino acid analyzer as that of ƒÁ-glutamyl serylglycine, was also detected in urine and other tissues such as muscle, kidney and spleen of rats fed excess methionine diets. This paper describes the identification of U-14 as hypotaurine and the increase of the taurine precursor in certain tissues, especially in muscle, of rats fed methionine excess diets. Animals and diets. Male weanling rats of the Sprague-Dawley strain (about

53g, Japan SLC Co., Ltd) were individually housed in metabolic cages to facilitate optimum separation of feces from urine in a room lighted from 0800 to 2000 at 23•Ž. Animals were fed 25% casein diet for a week prior to experimental feeding

93 94 T. KASAI et al.

Table 1. Composition of the experimental diets (in 1kg diet).

1 Vitamin mixture (g/kg): L-ascorbic acid 5, nicotinic acid 3, calcium pantothenate

1.6, pyridoxine hydrochloride 0.7, thiamine hydrochloride 0.6, riboflavin 0.6, folic

acid 0.2, menadione 0.1, biotin 0.02, cyanocobalamin-sucrose mixture (10mg+9.99

g) 1, sucrose 987.18. 2 Chocola A: retinol palmitate 20mg (30,000IU)/ml, Eisai. 3 Chocola D: ergocalcifrerol 0 .4mg (16,000IU)/ml, Eisai. 4 Juvela: tocopherol acetate

200mg/100g, Eisai. 5 Mineral mixture, micro (mg/100g): Na2SeO3 21.90, NH4VO3

28.70, Co(CH3COO)2•E4H2O 42.27, SnSO4 95.02, Na2MoO4•E2H2O 126 .09, CrCl3•E6H2O 128.11, NiCl2•E6H2O 202.50, Na2HAsO4•E7H2O 208.22, Na2B4O7/•E10H2O 220.50, NaF 300.55, Na2SiO3•E9H2O 10,119.10, NaCl 88,507.04. 6 Mineral mixture , macro (g/kg): KIO3 0.014, CuSO4•E5H2O. 0.62, MnSO4•E5H2O 1.15, Zn(NO3)2•E6H2O

4.16, CaHPO4•E2H2O 4.31, Fe(C6H5O7)•E6H2O 16.64, MgSO4•E7H2O 100 .08, CaCO3 292.63, KH2PO4 343.12. NaCl 237.28.

and then divided into 2 groups (6 and 12 rats, respectively) with similar initial body weight. Food and tap water were provided ad libitum. Food consumption and body weight were recorded daily. One group consisting of 6 rats (103 .3•}1.4g) was given 8% casein (8C) for 2 weeks and one group of 12 rats (102.9•}1.9g) was

given 8% casein supplemented with 3% methionine (8C3M) . At the end of the 1st week, the 8C3M group was further divided into 2 groups of 6 rats each. One group

(88.6•}1.8g) was fed the same diet (8C3M) and the other group (88.8•}1.3g) was fed 8C3M diet supplemented with 3% glycine (8C3M3G) for an additional 1 week , because it has been established that methionine toxicity can be alleviated effectively by the addition of glycine to the diet containing excess methionine (2, 6-8). The compositions of the experimental diets are shown in Table 1. The body weight gain , food intake, protein efficiency ratio and weights of the liver , kidney and spleen of rats of each group are given in Table 2. The significant depression of the body weight and food intake of rats of 8C3M group compared to those of 8C group was

J. Nutr. Sci. Vitaminol. ACCUMULATION OF HYPOTAURINE BY EXCESS METHIONINE 95 Table 2. Weight gain, food intake, protein efficiency ratio and weights of the liver, kidney and spleen of rats fed 8% casein (8C) , 8% casein supplemented with 3% methionine 8C3M casein and supplemented 8% with 3% methionine and 3% glycane (8C3M 3G) diets.

Values are means•}SEM for 12 rats 1st week of 8C3M group) or 6 rats all other groups). Means in the same column with a different superscript are significantly different (p<0.05). 1 See Table 1 for the composition of diets and grouping . 2 Weight gain, food intake and protein efficiency ratio of 8C3M3G group are the data obtained from during the second week , for the reason that this group was fed 8C3M diet for the first week and changed to 8C3M3G diet from day 8.Vol. 38, No. 1, 1992 96 T. KASAI et al. remarkably improved by the addition of 3% glycine to the 8C3M diet as in previous reports (2, 6-8). Rats were anesthetized with Nembutal (5mg/100g body wt) on day 14 to obtain various tissues and blood through the abdominal aorta. As shown in Table 2, the enlargement of kidney and spleen caused by the methionine excess diet was also alleviated significantly by the addition of glycine to the diet (8C3M

3G). Assay procedure. The amino acid analysis was carried out with a Hitachi 835 high-speed amino acid analyzer equipped with Hitachi custom ion-exchange resin

#2619 (2.6mm i.d.•~250mm). MCI_??_ PF-Kit for analysis of physiological fluid was used. Urea was determined by biacetylmonooxime method (5). Unless other wise stated, the significance of difference was assessed by Duncan's multiple range test.

Identification of hypotaurine and its concentration in some tissues. As de scribed above, the amount of ƒÁ-glutamylserylglycine in liver could not be determin ed because of its overlap with another peak on the amino acid analyzer. The peak

(named U-14) was unusually sharp compared with other peaks and was detected not only in liver, but in other tissues such as muscle, kidney and spleen and also in the urine or rats fed methionine excess diet (Fig. 1). Urine of 8C3M group was collected in 0.1N H2SO4 and used to identify U-14. U-14 was unstable and decreased during storage of urine sample in a cold room and disappeared entirely by the addition of H2O2 solution. Since U-14 increased by feeding methionine excess diet and was easily oxidized, it was suggested that U-14 was one of the sulfur containing metabolites of methionine. It was shown that hypotaurine had the same

Fig. 1. A part of the amino acid chromatograms of urine of rats fed the following diets. (a) 8% casein (8C). (b) 8% casein supplemented with 3% methionine (8C3M). (c) chromatogram of urine of rats fed 8C3M diet co-chromato graphed with an authentic hypotaurine. See the text for the conditions of the amine acid analysis. U-14, hypotaurine.

J. Nutr. Sci. Vitaminol. ACCUMULATION OF HYPOTAURINE BY EXCESS METHIONINE 97 Table 3. Free amino acids in the liver and muscle musculus gastrocnemius that are the products of the methionine metabolic pathway or showed a considerable difference among the 8% casein (8C), 8% casein supplemented with 3% methionine 8C3M and 8% casein supplemented with 3% methionine and 3% g1ycine 8C3M3G diets.

Values are means•}SEM for 6 rats. Means in the same line in each tissue with a different superscript are significantly different (p< 0.05. 1 Not determined because of its overlap with another peak on the analyzer. 2 Calculated by assuming that ophthalmic acid gives the same color constant as glutathione. 3 tr, trace amount; n.d., not detected.Vol. 38, No. 1, 1992 98 T. KASAI et al.

Table 4. Hypotaurine in kidney and spleen of rats fed 8% casein (8C) and 8% casein supplemented with 3% methionine (8C3M) diets.#

Values are means•}SEM for 6 rats. * Significantly different from control (8C) by

Student's t-test (p<0.001). # Values in this table were obtained in the previous

experiment (3, 4).

characteristic sharp peak with the same elution time as that of U-14 on the amino acid analyzer. The peak of U-14 in urine sample increased by co-chromatography with an authentic hypotaurine (Sigma) (Fig. 1). U-14 in the muscle extract, prepared as described below, showed the same chromatographic behavior and lability to oxidation by H2O2 as urinary U-14. U-14 was, therefore, identified as hypotaurine. It has been reported that hypotaurine is unstable in the presence of an oxidizing agent (9) and a photosensitive agent such as flavins (10). Hypotaurine content in muscle increased significantly by the feeding of excess methionine diets and further increased by the addition of glycine to the methionine excess diet (Table 3). Although the same tendency was observed in urine, the accurate estimation of hypotaurine in urine was difficult because of its partial overlap with the peak of urea on the amino acid chromatogram (Fig. 1). Hypo taurine content in liver also could not be determined because of its overlap with ƒÁ- glutamylserylglycine as described above. Amino acid analysis of other tissues was not carried out in this experiment, but hypotaurine contents in kidney and spleen of rats fed 8C and 8C3M diets in the previous experiment (3, 4) could be determined from the amino acid chromatograms of those tissues (Table 4). Hypotaurine content of kidney and spleen increased significantly by excess methionine feeding. No distinct peak of hypotaurine could be detected in any blood sample of rats fed

8C, 8C3M and 8C3M3G diets. This is the first report describing the significant increase of hypotaurine in certain tissues of rats by the feeding of an excess methionine diet, although the accumulation of hypotaurine has been demonstrated in the regenerating rat liver shortly after partial hepatectomy (9).

Amino acid composition of some tissues and urine. Since amino acid patterns of blood, kidney, spleen and liver of rats fed 8C and 8C3M diets had already been reported in previous papers (3, 4), amino acid in muscle tissue (musculus gastroc nemius) was determined in this experiment. The amino acid of liver was analyzed once again in this experiment to confirm the change especially in ophthalmic acid, glutathione and ƒ¿-aminobutyric acid by feeding of an excess methionine diet found in the previous experiment. The liver (1g) and a whole musculus gastrocnemius

J. Nutr. Sci. Vitaminol. ACCUMULATION OF HYPOTAURINE BY EXCESS METHIONINE 99

from one hind leg were homogenized in 10ml and 5ml of 3% sulfosalicylic acid , respectively. The filtrate of each homogenate was analyzed with the amino acid

analyzer. Amino acid composition of rat livers fed 8C and 8C3M diets were nearly

the same as those of the previous experiment (4). The amino acids which were the metabolites of methionine and of which concentrations were significantly different among the 3 diet groups are listed in

Table 3. Metabolites of methionine such as taurine, glutathione and cystathionine. which increased by excess methionine feeding either did not change or decreased by

addition of 3% glycine to the methionine excess diet. Although the increase of hepatic sarcosine after feeding of excess methionine diet was reported (8) , sarcosine was not detected in liver in this experiment. However, sarcosine was detected in

muscle of 8C3M group and increased by the addition of glycine to the diet . It has been reported that the increase of methionine in the diet causes the increases of the transsulfuration enzymes such as methionine adenosyltransferase , glycine methyltransferase, cystathionine ƒÀ-synthetase and cystathionine ƒÁ-lyase (8, 11-15) and the methionine metabolites such as cysteine and taurine (16 , 17). The increase in hepatic cysteine dioxygenase activity and urinary taurine in rats fed cysteine (18-20) and excess methionine (21) suggests that methionine is metabo lized mainly through cysteine sulfinate and that the cysteine sulfinate pathway plays a major role in the regulation of methionine and cysteine degradation (21 , 22). Significant activity of cysteine sulfinate decarboxylase was detected in liver and nearly 70% of cysteine is metabolized via the taurine pathway (23) . The high concentration of cysteine also favors glutathione synthesis (24) . A large portion of sulfur-containing amino acids is used for glutathione synthesis when a large amount of methionine is given orally (16, 25, 26).

Aspartic acid, lysine, histidine, ornithine and arginine decreased significantly in liver of excess methionine feeding groups as previously reported (4) and the decrease of these acidic and basic amino acids could not be alleviated by the supplementation of glycine with the exception of arginine , which was partially recovered by addition of glycine. Lysine and histidine also decreased in muscle of 8C3M group without statistical significance and were further reduced by addition of glycine. Arginine, on the other hand, increased significantly by excess methi onine diet and was restored to the control level by addition of glycine . Urinary urea of excess methionine diet group was significantly lower than that of control group

(Fig. 1), but addition of glycine to the excess methionine diet caused a remarkable increase of urea in urine. The amount of urinary urea in 8C , 8C3M and 8C3M3G groups was 0.96•}0.04bmmol/day/100g body wt, 0.40•}0.02c (8C3M) and 1.16•} 0.04a (8C3M3G), respectively. The cause of the remarkable changes in those acidic and basic amino acids in tissues and urinary ureas of rats fed an excess Met diet is still obscure at the present stage.

Financial support by a Grant-in-Aid for Scientific Research (No . 02660073) from the Ministry of Education, Science and Culture of Japan is gratefully acknowledged .

Vol. 38, No. 1, 1992 100 T. KASAI et al.

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