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1 Taurine: An Essential Nutrient for the Cat1 KAREN KNOPF, J. A. STURMAN, MARCIA ARMSTRONG ANDK. C. HAYES Department of Nutrition, Harvard School of Public Health, Boston, Massachusetts and Division of Human Development and Genetics, Institute for Basic Research in Mental Retardation, Staten Island, New York and Department of Pediatrics, Mount Sinai School of Medicine of the City University of New York, New York, New York ABSTRACT Cats fed a purified diet containing purified casein as the source of protein develop retinal degeneration due to the lack of taurine in the diet. To test whether cats can synthesize this sulfur amino acid from sulfate or cystine, radioisotopes of these substances were injected into taurine-depleted and control cats. Sulfate did not serve as a precursor for taurine synthesis, whereas cystine underwent only a moderate conversion to taurine. This is in keeping with the low level of cysteinesulfinic acid ( CSA ) decarboxylase activity in cat liver. There was no difference between the activity of CSA decarboxylase in tissues from control cats and that in tissues from taurine-depleted cats. The pattern of tissue accumulation of [35S]taurine and from [35S]cystine also indicated that tissues from taurinedepleted cats do not synthesize [35S]taurine more rapidly than tissues from control cats. The data did not indicate a difference tissues of control and deficient cats, but progressive in taurine uptake by accumulation in de ficient cats suggested that the turnover rate of taurine is decreased by the deficiency. Since supplementation of the purified diet with cysteine has been found previously to be inadequate depletion of the retina and its subsequent to prevent progressive taurine degeneration and since conver sion of sulfur compounds to taurine in vivo is inadequate, taurine can be considered an essential nutrient for the cat. J. Nutr. 108: , INDEXING KEY WORDS taurine â essential nutrients â cats â cysteine It has been demonstrated in a series of studies that cats fed synthetic, diets connot convert to glycine as occurs in other species. Instead, the concentration of free taining purified casein as the source of cholic acid increasesâ an increase that is protein develop retinal degeneration which less marked in adult cats than in kittens results from progressive depletion of retinal taurine. Supplementing this diet with ( 3 ). These observations suggest that the cat, taurine, but not with methionine or with and particularly the kitten, is incapable of cysteine, maintained the retinal taurine synthesizing sufficient taurine to meet the concentration and prevented the degeneration from developing (1, 2). Furthermore, Received for publication December 22, the bile acids of the Cat are Conjugated »Supported in part by grants-in-ald from the exclusively with taurine and, under condã - ^SS^Sf^Sftà Sff^^SIS^ SñSSà tions Of taurine depletion during feeding of Harvardjcnoo^of B^McJBtoatt (Haye^, and^the such a casein diet, this conjugation does (sturman). 773

2 774 KNOPF, STURMAN, ARMSTRONG AND HAYES requirements for maintaining retinal func tion and structure, making taurine an es sential nutrient in this species. All species are thought to convert methionine to tau rine via cysteine and cysteinesulfinic acid (CSA), although considerable variation in the activity of CSA decarboxylase, the en zyme directly responsible for the synthesis of taurine, has been reported in different species (4). Rats (5) and chicks (6) are able to convert inorganic sulfate to taurine in vivo, presumably by activation to 3'- phosphoadenosine-5'-phosphosulfate (PAPS) and transfer of the sulfate to serine by PAPS-sulfotransferase. Other species also may be able to form taurine by this route, since activity of PAPS-sulfotransferase has been found in all species so far investigated including the cat (7). According to these authors the enzymatic mechanism for syn thesizing taurine in vitro from inorganic sulfate is present in the heart and liver of the cat as well as in the heart and liver of the chick, dog, guinea pig, hamster, mon key, mouse, rabbit, rat and sheep. The hy pothesis "regardless is advanced of diet or by anatomical these authors differ that ences, this enzyme appears to be a com ponent of all animal tissues." We have studied the possibility of conversion of in organic sulfate and cystine to taurine in the cat and report the results in this communi cation. MATERIALS AND METHODS Kittens obtained from random sources and of the domestic variety, ranging in weight from 1,100 to 1,600 g, were fed a purified casein diet (3, 8) alone or supple mented with 0.4% taurine or 0.6% sulfate for periods of TA weeks or 15 weeks from 6 weeks of age. The casein diet contained (calculated in g/100 cystine, 0.1; taurine, g): methionine, 0.5; 0.0. The cats were killed and blood samples obtained using heparin as anticoagulant. Samples of urine were obtained from the bladder and the following tissues removed: liver, heart, gastrocnemius muscle, retina, occipital lobe and cerebellum. Plasma, urine and tissues were prepared for amino acid analysis and the concentration of taurine determined (9). Seven other cats were fed the casein diet alone or the casein diet supplemented with taurine. Three cats were killed 5 days after the intravenous injection of 1 ml of saline containing 2.87 mci/ml [35S]sulfate2 (disodium salt, specific activity 969 mci/ mmole) (two fed the casein diet, one the casein diet supplemented with taurine). Four cats were killed 24 hours or 14 days after injection of 1 ml of saline containing 2.14 mci/ml [35S]cystine 3 ( specific activity 88.2 mci/mmole) (one fed the casein diet and one the casein diet supplemented with taurine at each time). Tissues were re moved and analyzed for taurine and [36S]- taurine as previously described (9). In addition, bile samples were collected and analyzed for bile acids (3) and the pres ence of 35Sas sulfate or taurine. Bile acids were subjected to solvolysis to remove sul fate (10, 11) and separated into a watersoluble sulfate fraction, a dichloromethane fraction containing the cleaved bile acids, and a diethyl ether fraction containing residual uncleaved bile acids. Radioactivity in each fraction was measured using a liquid scintillation counter.* The activity of CSA decarboxylase in liver, occipital lobe and cerebellum of cats fed the casein diet or the casein diet sup plemented with taurine was determined as previously described (12). RESULTS The addition of sulfate to the casein diet had no effect on the concentration of tau rine in any of the tissues studied. In addi tion, since there was no significant differ ence between the concentration of taurine in the tissues of cats fed the casein diet for 7% weeks and those fed the diet for 15 weeks, all of the data are pooled accord ing to dietâ casein versus casein supple mented with taurine. The concentration of taurine is smaller in all of the tissues studied from the cats fed the casein diet than in the same tissues from the cats fed the casein diet supplemented with taurine (table 1). The greatest depletion occurs in liver (almost 100-fold) and the smallest * New England Nuclear. Cambridge, Massachusetts. 3 Amergham/Searle Corp., Chicago, Illinois. 'Model LS-250, Beck Instruments Company.

3 TAURINE IN CATS 775 occurs in retina (2-fold). Taurine is not detectable in most plasma samples from cats fed the casein diet and the concentra tion of taurine in the urine of these cats is 200-fold lower than in urine of control cats. No conversion of [35S]sulfate to [35S]- taurine in vivo could be detected in any tissue of the control or taurine-depleted cats despite the large amounts of radio active sulfate injected. Even in urine, where samples analyzed contained in excess of IO6 dpm, no trace of radioactive taurine was detected (the analytical system used can detect as little as 200 dpm above back ground). Both plasma and urine contained another 35S-labeled compound, eluted in the same position as sulfate, but not pre cipitated by BaClg. In plasma this com pound comprised 40% of the radioactivity in the samples and in urine 1% of the radioactivity in the samples. No further attempt was made to identify this com pound since it clearly was not taurine (in this analytical system, inorganic sulfate is eluted virtually with the solvent front and a further 20 ml of buffer are needed to elute taurine). Radioactivity was present TABLE 1 Concentration of taurine in various tissues of cats fed the casein diet or the casein diet supplemented with taurine Tissue + Taurine itmole/g wet wt. Liver 0.21± ±5.08 Heart 1.18± ±2.08 Gastrocnemius muscle Retina Occipital lobe ±0.29 ± ± ± ± ±0.20 Cerebellum1.54 ±0.22/ mole/mlplasma 0.45± Urine<0.01 ± ± ±2.48 These values represent the meanâ±sem/â mole taurine/g wet weight of tissue or per ml plasma or urine from 19 cats fed the casein diet, or from eight cats fed the casein diet supplemented with taurine. The values for taurine concentration in tissues and fluids of the cats fed casein diet alone are all signifi cantly different from the values for taurine con centration in the same tissues and fluids of the cats fed the casein diet supplemented with taurine as determined by Students f-test (P < 0.001). TABLE 2 Bile taurine concentration and radioactivity 5 days after injection of cats with [3*S]sulfate Bile taurine1 Diet -f-taurine Concentration (/imole/ml)radioactivity2(io ,0.0, dpm/ml)specific activity(io3 dpm//umole)98.7, 1Derived from taurine conjugated bile acids following solvolysis (10, 11). 2The samples con taining taurine did not have any radioactivity above that of background. Each value was derived from one cat. in bile acids, but was essentially completely removed by solvolysis indicating that it represented direct sulfation of the bile acid sterol nucleus and not incorporation of label into taurine (10) (table 2). [35S]Cystine was converted to [35S]taurine in vivo and found in easily measured quantities in liver, heart and retina (table 3). Trace amounts of radioactive taurine were detected in urine, plasma and brain from the cats fed the casein diet supple mented with taurine, but not from the cats fed the casein diet alone. There were some differences between the amounts of 35S taurine formed in the cats fed the casein diet and those fed the casein diet supple mented with taurine. After injection of TABLE 3 Radioactivity in taurine after injecting [3SiS]ct/s<ine into four cats fed the casein diet or the casein diet supplemented with taurine Tissue and time afterinjectionradioactivity+ Taurine10' gliver24 hours14 daysheart24 hours14 daysretina24 dpm/ Activity Taurinedpm + limole hours days<0.50< specific 77 Each value was derived from one cat.

4 776 KNOPF, STURMAN, ARMSTRONG AND HAYES TABLE 4 Bile taurine concentration and radioactivity after injecting [3SS~]cystineinto cats fed the casein diet or the casein diet supplemented with taurine Bile taurine1 and time after injection Concentration Gimole/ml) 24 hours 14 days Radioactivity (IO3dpm/ml) 24 hours 14 days Specific activity (IO3dpm//imole) + Taurine hours days See footnote in table 2. rived from one cat. Each value was de- [35S]cystine, [35S]taurine could not be de tected in liver from the cats fed the casein diet, whereas it was readily measured in the liver of cats fed the casein diet sup plemented with taurine. There was little difference in total radioactivity in taurine in heart and retina after 24 hours between the cats fed the casein diet or the casein diet supplemented with taurine, and only a 2-fold difference in radioactivity in bile (table 4). The radioactive taurine in heart and retina of the cats fed the casein diet supplemented with taurine was unchanged after 14 days but accumulated in heart and retina of the cats fed the casein diet. Thus, after 14 days the heart and retina of these cats contained more than 3 times the amount of [35S]taurine than was present in these same tissues of cats fed the casein diet supplemented with taurine (table 3). After 14 days the bulk of the radioactivity in extracts of liver and heart was present as taurine and all of the radioactivity in extracts of retina was present as taurine. The radioactivity in bile acids from cats fed the casein diet supplemented with taurine decreased 7-fold from 24 hours to 14 days after injection of [35S]cystine, whereas it increased 3-fold in the cats fed the casein diet over this period. Thus, 14 days after injection of [35S]cystine, there was approximately 50-fold more radio active taurine conjugated to bile acids in TABLE 5 Activity of cysfeine sulfinic acid decarboxytase in liver and brain of cats fed the casein diet or the casein diet supplemented with taurine Tissue + Taurine limole COà /mgprotein/hr Liver 4.4 ± ±0.4 Occipital lobe 58.8± ±5.5 Cerebellum 55.1± ±2.8 Each value represents the meanâ±sem from three cats fed the casein diet or the casein diet supplemented with taurine. The values for tissues from cats fed the casein diet alone are not signifi cantly different from the values for the same tissues from cats fed the casein diet supplemented with taurine as determined by Student's i-test. cats fed the casein diet alone than to bile acids in those cats fed the casein diet sup plemented with taurine (table 4). Solvolysis and separation and counting of fractions indicated that the 35S cystine was exclu sively incorporated as taurine conjugated bile acids. There was no difference in CSA decarboxylase activity in liver, occipital lobe and cerebellum from cats fed the casein diet alone or supplemented with taurine (table 5). Activity in liver was low compared to that in brain and the values obtained are similar to those previously reported ( 4 ). DISCUSSION These results provide evidence that cats fed a synthetic diet containing purified casein as the source of protein have a de creased concentration of taurine in a wide variety of tissues. They show further that the addition of sulfate to such a diet has no effect on the concentration of taurine in any of the tissues, and that sulfate is not converted to taurine in the cat. Taurine was formed in limited amounts from cystine; its formation presumably being limited by the low activity of CSA decarboxylase present. The formation of taurine from cystine did not appear to be enhanced by taurine depletion since the activity of CSA decarboxylase was similar for both dietary groups. Furthermore, since the amount of radioactive taurine present in heart and retina after 24 hours was similar, the forma tion and/or uptake of [35S]taurine by these

5 TAURINE IN CATS 777 tissues in the control and taurine-dã ficient cats was similar. The difference in specific activity of [35S]taurine between the tissues of the taurine-dã ficientand control cats is the result of the differences in concentra tion of taurine between those same tissues. Thus, taurine deficiency does not appear to influence the formation of taurine from cystine. However, the low activity of CSA decarboxylase in liver of the cats makes it likely that because synthesis of taurine is limited, any effect on uptake of taurine is obscured. The inability to detect radio active taurine in liver of cats fed the casein diet alone may have been due to the result either of the small amount of endogenous taurine present in liver to trap any labeled taurine or of the immediate utilization of any taurine (labeled or not) for conjuga tion with bile acids. By 14 days after the injection of [35S]- cystine, there were other differences found between cats fed the casein diet alone and the casein diet supplemented with taurine in the behavior of [35S]taurine. Thus [35S]taurine continued to accumulate in heart, retina and bile of the taurine-dã fi cient cats but not in those tissues from the control animals. This accumulation may have resulted from the remaining taurine having a slower rate of turnover in the tis sues of the deficient cats than it did in the tissues of the control cats. It is possible also that the amounts of [35S]taurine accumulated by the various tissues in the taurine-dã ficient cats reflect the importance of taurine for the function of that tissue. On this basis bile acids would have the most important functional need for taurine, followed by the retina and heart. Interestingly, [35S]taurine was not detected in the occipital lobe or cerebellum from either control or taurine-dã ficientcats. Apparently the remaining 10% of taurine in brain tissue is adequate for vital control of central nervous system functions. Other studies have suggested also that only a small fraction of the total amount of taurine in brain is associated with synaptic ves icles (13). These results support the concept that taurine is an essential nutrient for the cat. Further evidence has recently been re ported from two independent laboratories. The first demonstrated that the isolated perfused cat liver, unlike the rat liver, was incapable of maintaining taurine synthesis for bile acid conjugation (14). The second found that the retinal dysfunction was still apparent when a purified amino acid mix ture without taurine was substituted for the dietary casein ( 15). In essence, taurine must be supplied by the diet to prevent depletion of retinal taurine and the im paired visual function and structure that results from its depletion. Under normal circumstances cats do consume foods which are rich sources of taurine, such as fish and meat (16). The present data indicate that the cat can convert a limited amount of cystine to taurine; however, it was demon strated previously that replacing dietary taurine with equimolar amounts (0.8%) of cystine (or methionine) failed to maintain normal body concentrations of taurine, par ticularly the plasma and retinal pools. This occurred despite elevated plasma levels of methionine eventually and cysteine. Those kittens developed retinal degeneration (2), clearly indicating that the ability to convert cysteine (or rine was not adequate methionine) to tau to meet at least one important physiological need, i.e., that re quired for vision. The fact that the cat is unable to utilize sulfate as a source of taurine further indicates this species de pendence on a dietary supply of taurine and the essentiality of this compound for the cat. These dietary studies in cats have as sumed added significance in light of re ports that human infants fed a mã¼kformula based on casein have appreciably lower serum and urinary taurine concentrations than breast-fed infants (17, 18). Whether the human neonate has a poorly developed enzyme capability for synthesis of taurine remains to be determined. ACKNOWLE DOME NT The authors thank Ms. Judith Fagan and Mr. Barry Rabin for expert technical as sistance. LITERATURE CITED 1. Hayes, K. C. (1976) A review of the bio logical function of taurine. Nutr. Rev. 34,

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