UTILIZATION OF D- AND L-TRYPTOPHAN BY THE GROWING DOG 1'2

Transcription

1 UTILIZATION OF D- AND L-TRYPTOPHAN BY THE GROWING DOG 1'2 Gail L. Czarnecki and David H. Baker University of Illinois, Urbana Summary Two 4 x 4 Latin square experiments were conducted with English Pointer puppies to determine the growth promoting capacity of I> tryptophan (trp) relative to L-trp and to estimate the trp requirement of the growing dog. Based upon a multiple linear regression sloperatio technique, D-trp had an efficacy value of % relative to L-trp. The urine of dogs fed D-trp contained 14.6% of the ingested trp; no measurable trp was recovered in the urine of dogs fed L-trp. The trp requirement for maximal weight gain was at least.16% for 6-to 10-wk-old dogs, between.12 and.16% for 10-to 12-wk-old dogs and -<.12% for 12- to 14-wk-old dogs. The overall requirement for the 6- to 14-wk age period was.15%. Total N, as well as urea-n, in urine decreased and N retention increased with increasing levels of dietary L-trp; NHB-N, creatinine-n and fecal-n were unaffected by treatment. (Key Words: D-Tryptophan, L-Tryptophan Requirement, Dog, Performance, Nitrogen Balance, Urinary Tryptophan.) I ntroduction Increasing feed costs, more economical methods of amino acid manufacture and the use of by-product feeds in animal diets may make crystalline amino acid supplementation more feasible in the future. Accordingly, quantitative estimates of the efficacy of D-amino acids are of value. D-tryptophan (trp) is of t Dept. of Anim. Sci. 2 The authors would like to acknowledge Dr. Katherine P. B0ebel for her help in developing the crystalline amino acid diet used in these experiments and Dr. J. E. Corbin for his help in management of the puppies. 3 Pre-Jel. The Hubinger Co., Keokuk, IA. 4 Young puppies, in contrast to the young of other mammalian and avian species, develop loose stools when fed regular powdered cornstarch. additional interest because of its potential use as a sweetening agent (Berg, 1953). The efficacy of D-trp has been evaluated in the pig (Baker et al., 1971), rat (Oesterling and Rose, 1952; Ohara et al., 1980) and chick (Wilkening and Schweigert, 1947; Morrison et al., 1956; Sugahara et al., 1967; Ohara et al., 1980). Although the metabolism of D-trp has been studied in the dog (Triebwasser et al., 1976), a quantitative estimate of its nutritive value has not been made. Our principal objective, therefore, was to determine the biopotency of D-trp (relative to L-trp) for the growing puppy. A secondary objective was to examine the trp requirement for maximal weight gain. Experimental Procedure The trp-free cystalline amino acid basal diet used in these experiments is presented in table 1. This diet, which was developed in our laboratory, allows excellent rates of growth in the weanling puppy when fully supplemented with L-trp (Czarnecki and Baker, unpublished data). Pregelatinized cornstarch 3 was used to prevent the loose stool problem that occurs when regular cornstarch is fed to young dogs 4. All dietary additions were made at the expense of cornstarch. Weanling English Pointer puppies from two litters were used in the experiments. The first litter consisted of four males with an average weight at the initiation of the experiment of 2,413 g. The second litter contained two males and two females with an average initial weight of 2,640 g. After weaning at 5.5 wk of age, the puppies were placed in individual stainless steel cages that were modified to facilitate quantitative collection of urine and feces. Puppies within each litter were randomly assigned positions in a 4 x 4 Latin square. Hence, two 4 x 4 Latin square experiments were conducted simultaneously, each square containing four puppies from the same litter JOURNAL OF ANIMAL SCIENCE, Vol. 55, No. 6, 1982

2 1406 CZARNECKI AND BAKER Squares consisted of four 10-d periods of four dietary treatments:.08,.12 or.16% L-trp and.16% D-trp. A 4-d adjustment period (during which the.16% L-trp diet was fed) preceded each 10-d assay period. The Latin square design allowed estimation of the L-tryptophan requirement over time. It also minimized error variance due to dog-to-dog variation such that treatment differences could be assessed more accurately. To minimize feed wastage and consequent contamination of the urine and feces, the puppies were meal-fed three times daily (0730, 1200, 1700 h) and orts were removed and weighed after each meal. Feed intake was assessed daily. Water was provided ad libitum, and the dogs were weighed after an overnight fast at the end of each period. Urine (under H2 SO4) and feces were collected daily on d 5 s Beckman (Model 119CL) Amino Acid Autoanalyzer. Beckman Instruments, Palo Alto, CA. through 10 of each assay period for N balance determination. Nitrogen was determined by the Kjeldahl procedure. Urinary urea-n and NH3-N were determined according to procedures outlined by Fawcett and Scott (1960) as modified by Chancy and Marback (1962); urinary creatinine-n was measured by the method of Oser (1965). A 24-h urine sample, representative of the last day of each period, was collected under toluene and analyzed for free trp by ion-exchange chromatography using an automated amino acid analyzer s. Fivemilliliter samples of urine were adjusted to ph 11.5 to 12.0 with 4 N NaOH. The beakers of urine, together with a beaker of concentrated H2 SO4, were then placed in a vacuum desiccator and continuously evacuated with a water aspirator for 7 h. After removing the beakers containing urine samples from the desiccator and adjusting the ph to 1.8 to 2.5 with 6 N HC1, samples were transferred to volumetric flasks and adjusted to a volume of 10 ml with lithium citrate buffer (ph 2.2). This TABLE 1. COMPOSITION OF PURIFIED BASAL DIET AND CRYSTALLINE AMINO ACID MIXTURE a Ingredient % Amino acid mixture % Pregelatinized cornstarch to L-Arginine, HCI 1.15 Sucrose L-Histidine.HCl.H Amino acid mixture L-Ly'~ine.HC Corn oil L-Tyrosine.45 Mineral mixture b 5.37 L-Tryp tophan Solka floc 3.00 L-Phenylalanine.50 NaI-ICO DL-Methionine.40 Choline 9 CI.30 L-Cystine.40 Vitamin mixture c.30 L-Threonine.65 MgSO 4 9 7H a O.06 L-Leucine 1.00 ZnCO 3.02 L-Isoleucine.60 DL-~-tocopheryl acetate (50 mgkg) + L-Valine.69 Ethoxyquin (125 mgkg) + Glycine 1.60 L-Proline.80 L-Glutamic acid 2.40 L-Alanine 1.60 L-Asparagine. H L-Serine.80 adiet contains 3,482 kcal MEkg. Total bmineral mix provided per kg of diet: CaCO3, 3.0 g; Ca 3 (PO 4 )2, 28.0 g; K~ HPO4, 9.0 g; NaCI, 8.8 g; MgSO 4 9 7H20, 3.5 g; MnSO 4.H20,.65 g; ferric citrate,.50 g; ZnCOa,.10 g; CuSO 4.5H20, mg; H3BO3, 90.2 mg; Naa MoO 4-2H20, 90.2 mg; KI, mg; COSO4.7H20, 1.0 mg; Na2 SeO 3,.215 mg. cvitamin mix provided per kg of diet: thiamin.hcl, 150 rag; niacin, 150 mg; riboflavin, 24 mg; Ca-pantothenate, 30 mg; vitamin B12,.03 mg; pyridoxine-hc1, 9 mg; biotin,.9 mg; folic acid, 6 mg; inositol, 150 mg; paraaminobenzoic acid, 3 rag; menadione, 7.5 rag; ascorbic acid, 375 mg; retinyl acetate (250,000 IUg), 15,000 IU; cholecalciferol (200,000 IUg), 900 IU.

3 TRYPTOPHAN UTILIZATION BY THE DOG L-Trp o v ~ " " "" " ~c Z < <3 ~ O D-Trp x! I i I i x 2 3 4! 5 TRYPTOPHAN INTAKE (g10-d) Figure 1. Multiple linear regression of weight gain on D- (x) and L- (.) trp intake. material was then placed on the amino acid analyzer. Data were subjected to analysis of variance using procedures appropriate for Latin square experiments (Steel and Torrie, 1980). The efficacy of D-trp was determined by multiple linear regression slope-ratio techniques (Boebel and Baker, 1982). The regression model consisted of one dependent variable (weight gain) and two independent variables (D- and L-trp intake). The model thus estimated two straight lines which were fitted to a common intercept. Because gain was regressed on trp intake r~ther than on dietary trp concentration, there were eight observations (two squares, four observationssquare) for D-trp and 24 observations for L-trp (figure 1). An estimate of the L-trp requirement for maximal weight gain was determined by fitting the gain data to a sigmoid curve (Robbins et al., 1979) and determining the point on the curve where 95% of the upper asymptote was reached. Results and Discussion The performance and N balance data are summarized in tables 2 and 3, respectively. For each of the measurements, a treatment square mean square was calculated and tested against the error term to determine whether the treatment response was different in the two squares. Because no differences (P>.05) were detectable, the treatment square mean square was incorporated into the error term, and the data from the two squares pooled. The three dietary treatments involving graded increments of L-trp were then subjected to linear regression analysis, and an apriori comparison of.16% L-trp vs.16% D-trp was also made. Puppies fed.16% D-trp gained less (P<.001)

4 1408 CZARNECKI AND BAKER TABLE 2. PERFORMANCE OF PUPPIES FED D-TRYPTOPHAN OR GRADED LEVELS OF L-TRYPTOPHAN a Dietary addition Gain, glo d be Gainfeed cd.08% L-trp % L-trp 1, % L-trp 1, % D-trp Pooled SE aresuits represent mean values of eight observations in two 4 X 4 Latin square designs; puppies wer~ fed the experimental diets from 6 to 14 wk of age. Average initial weight at the start of the first period was 2,526 g. bquadratic (P<.04) response to L-trp. e.16% L-trp different (P<.001) from.16% D-trp. dlinear (P<.O01) response to L-trp. weight, had lower (P<.001) gain:feed ratios (table 2) and ate less feed (P<.003) than those fed an equivalent level of L-trp. As evidenced by increased (P<.005) concentrations of urea-n and total-n in urine and by decreased (P<.005) N-balance, D-trp was less efficiently utilized than L-trp (table 3). The multiple linear regression analysis of weight gain on D- or L-trp intake (figure 1) resulted in a regression equation of Y = Xl X2, where Y = gain (g10-d period), XI = g L-trp consumed10-d period and X2 = g D-trp consumed10-d period (r =.91). Only points on the linear portion of the curve, i.e., <5 g trp intake10-d period, were used in the slope-ratio analysis. The ratio of slopes of the two lines indicated that D-trp had % of the growth-promoting ability of L-trp. If one assumes that D-trp must be inverted to L-trp before it can be utilized by the tissues, our estimate of 35.5% agrees closely with the conclusions of Triebwasser et al. (1976) who estimated that at least 30% of an oral dose of D-[14CJ-trp was inverted by the dog. It should be noted, however, that with only one dietary level of D-trp (.16%), we were unable to determine whether higher or lower levels of D-trp would be utilized with the same efficiency. The rat can utilize D-trp with at least 75% efficiency (Oesterling and Rose, 1952). More recent evidence, in fact, indicates close to 100% utilization (Ohara et al., 1980). The efficacy of D-trp for weight gain in the pig has been estimated at 60% (Baker et al., 1971), and recent research with the chick indicates a bioactivity of 20% (Oharaet al., 1980), although other researchers have reported values ranging from 7 to 40% (Wilkening and Schweigert, 1947; Morrison et al., 1956; Sugahara et al., 1967). The human (Langner and Berg, 1955; Berg, 1959; Hankes et al., 1972), rabbit (Schayer, 1950; Loh and Berg, 1971) and mouse (Celander and Berg, 1953) have little or no capacity to utilize TABLE 3. NITROGEN METABOLISM OF PUPPIES FED D-TRYPTOPHAN OR GRADED LEVELS OF L-TRYPTOPHAN a Daily N Urine Dietary addition Urea-N bd NH s -hi Creatinine-N Total ed Feces Balance cd.08% L-trp % L-trp % L-trp % D-~p Pooled SE aresults represent mean values of eight observations in two 4 4 Latin square designs; puppies were fed the experimental diets from 6 to 14 wk of age. Urine and feces were collected on d 5 through 10 of the lo-d assay periods. blinear (P<.002) response to L-trp. CQuadratie (P<.01) response to L-trp. d.16% L-trp different (P<.O05) from. 16% D-trp.

5 TRYPTOPHAN UTILIZATION BY THE DOG to I0 wk of age I0 to 12 wk of age 12 to i@ wk of age. I00 o o Ii0 9O z8o I00 I i [ I I i 9 o DIETARY TRYPTOPHAN (%) L I ~,,,.o8.1s.16 Figure 2. Weight gain over time of puppies fed graded levels of L-tryptophan. D-trp. The dog, therefore, appears to be less efficient in utilizing D-trp than the rat or pig, but perhaps slightly more efficient than the chick, human, mouse and rabbit. An examination of the trp content of urine (table 4) offers a partial explanation for the inefficient utilization of D-trp by the puppies in our study. No trp was detected in the urine of dogs fed L-trp, whereas the dogs fed D-trp excreted 227 gtmold, or 14.6% of the total D-trp ingested. These data agree well with 17% of ingested D-trp excreted in dog urine in the study of Triebwasser et al. (1976) and provide evidence that renal tubular reabs0rption of D-trp is inefficient. In addition to poor renal tubular reabsorption of D-trp, Triebwasser et al. (1976) indicated that slow uptake of D-trp by the tissues and(or) slow inversion of D- to L-trp probably contributed to the relatively poor utilization of D-trp by the dog. Intestinal absorption of D-trp appeared to be relatively efficient. The data in table 2 indicate that gain increased quadratically (P<.04) in response to increasing increments of dietary L-trp. The intake of both feed and L-trp required to maximize gain were computed as 95% of the upper asymptote of the corresponding sigmoid curves (Robbins et al., 1979). The percentage dietary trp required for maximal gain was then computed by dividing the grams of trl0 required to maximize gain by the grams of feed required to maximize gain. Accordingly, a trp requirement for the 6- to 14-wk-old English Pointer of.15% was determined. An examination of figure 2 shows that the trp requirement for maximal weight gain decreased with age. The requirement was at least.16% for the 6- to 10-wk-old dog (periods 1 and 2 of the Latin square), between. 12% and. 16% for the 10- to 12-wk-old dog (period 3) and <.12% for the 12- to 14-wkold dog (period 4). Our overall requirement estimate of. 15% L-trp was slightly lower than the estimate recently reported by Burns and Milner (1982) for the 6- to 8-wk-old Beagle. This discrepancy can be accounted for by the TABLE 4. EFFECT OF DIETARY D- OR L-TRYPTOPHAN ON TRYPTOPHAN OUTPUT IN DoG URINE a Urinary tryptophan Dietary addition tzmol24 h % of trp intake.08% L-trp % L-trp % L-trp % D-trp aresults represent mean values of eight dogs in two 4 4 Latin square designs; puppies were fed the experimental diets from 6 to 14 wk of age. Urine samples represent the last 24 h of each 10-d period. The procedure used for detection of trp was sensitive to I>20 nmolml of urine.

6 1410 CZARNECKI AND BAKER differences in the age of the dogs used. When comparing dogs of similar ages our requirement estimate of >.16% trp for the 6- to 10-wk-old dog agrees more closely with their estimate of.17% for the 6- to 8-wk-old dog. The excellent gain (170 gd for 12- to 14-wk-old dogs) and feed efficiency (.497) reported here provided evidence that the crystalline amino acid diet met the desired palatability and growth-promoting needs of the rapidly growing puppy. In fact, other research in our laboratory with this diet indicates that when fed as is (i.e., dry) the amino acid purified diet allows better growth rates than commercial diets it has been tested against. Urea-N and total urinary N decreased with increasing dietary L-trp (table 3). These data are consistent with the fact that when one amino acid is limiting, other amino acids cannot be utilized as efficiently for protein synthesis and are, therefore, catabolized to urea and excreted in the urine. As dietary L-trp increased, trp became less limiting and, as expected, urea-n and total urinary N decreased and N-retention increased. In animals fed practicaltype diets, urea-n typically accounts for 75 to 80% of the urinary N (Oser, 1965). The urea-n values reported in table 3 account for only 45 to 68% of the urinary N. Low values of urea-n in the urine of dogs fed a purified amino acid diet have previously been reported (Milner, 1979; Burns and Milner, 1982) and are most likely due to the highly balanced amino acid patterns in these diets when compared with practical diets. What accounts for the balance of the urinary N in urine of puppies fed well balanced purified diets remains to be determined. As seen in table 3, neither creatinine nor ammonia can account for the discrepancy. Also, recent research in our laboratory provides evidence that free amino acid N accounts for only 1.75% of the total urinary N. In conclusion, it is apparent that.15% L-trp in the diet is more-than-adequate for dogs over 10 wk of age. Also, D-trp contains about 35% trp activity for puppy growth. Hence, DL-trp would be expected to possess 68% activity relative to L-trp. L iterature Cited Baker, D. H., N. K. Allen, J. Boomgaardt, G. Graber and H. W. Norton Quantitative aspects of D- and L-tryptophan utilization by the young pig. J. Anim. Sci. 33:42. Berg, C. P Physiology of the D-amino acids. Physiol. Rev. 33:145. Berg, C. P utilization of D-amino acids. In: A. A. Aibanese (Ed.) Protein and Amino Acid Nutrition. pp Academic Press, New York. Boebel, K. P. and D. H. Baker Comparative utilization of the isomers of phenylalanine and phenyllactic acid by chicks and rats. J. Nutr. 112:367. Burns, R. A. and J. A. Milner Threonine, tryptophan and histidine requirement of the immature dog. J. Nutr. 112:447. Celander, D. R. and C. P. Berg The availability of D-histidine, related imidazoles and D-tryptophan in the mouse. J. Biol. Chem. 202:339. Chaney, A. L. and E. P. Marback Modified reagents for determination of urea and ammonia. Clin. Chem. 8:130. Fawcett, J. K. and J. E. Scott A rapid and precise method for the determination of urea. J. Clin. Pathoi. 13:156. Hankes, L. V., R. R. Brown, J. Leklem, M. Schmaeler and J. Jesseph Metabolism of 14C labeled enantiomers of tryptophan, kynurenine and hydroxykynurenine in humans with scleroderma" J. Invest. Dermatol. 58:85. Langner, R. R. and C. P. Berg Metabolism of D-tryptophan in the normal human subject. J. Biol. Chem. 214:699. Loh, H. H. and C. P. Berg Production of D-kynurenine and other metabolites from D- tryptophan by the intact rabbit and rabbit tissue. J. Nutr. 101:465. Milner, J. A Assessment of the essentiality of methionine, threonine, tryptophan, histidine and isoleucine in immature dogs. J. Nutr. 109:1351. Morrison, W. D., T. S. Hamilton and H. M. Scott Utilization of D-tryptophan by the chick. J. Nutr. 60:47. Oesterling, M. J. and W. C. Rose Tryptophan requirement for growth and utilization of its optical isomers. J. Biol. Chem. 196:33. Ohara, I., S. I. Otsuka, Y. Yugari and S. Ariyoshi Comparison of the nutritive values of L-, DL- and D-tryptophan in the rat and chick. J. Nutr. 110:634. Oser, B. L Hawk's Physiological Chemistry. Mc- Graw-Hill Book Co., New York. Robbins, K. R., H. W. Norton and D. H. Baker Estimation of nutrient requirements from growth data. J. Nutr. 109:1710. Schayer, R. W Studies of the metabolism of tryptophan labeled with lsn in the indole ring. J. Biol. Chem. 187:777. Sugahara, M., T. Morimoto, T. Kobayashi and S. Ariyoshi The nutritional value of D-amino acid in the chick nutrition. Agr. Biol. Chem. 31:77. Steel, R.G.D. and J. H. Torrie Principles and procedures of Statistics. A Biometrical Approach (2nd Ed.). McGraw-Hill Book Co., New York. Triebwasser, K. C., P. B. Swan, L. M. Henderson and J. A. Budny Metabolism of D- and L-tryptophan in dogs. J. Nutr. 106:642. Wilkening, M. C. and B. S. Schweigert Utilization of D-tryptophan by the chick. J. Biol. Chem. 171:209.