The Journal of Nutrition Nutrient Physiology, Metabolism, and Nutrient-Nutrient Interactions

Dietary L-Homoserine Spares Threonine in Chicks1,2

Kasey I. Bryant, Ryan N. Dilger, Carl M. Parsons, and David H. Baker1,2*

Department of Animal Sciences and Division of Nutritional Sciences, University of Illinois, Urbana, IL 61802

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Four chick bioassays were conducted to evaluate the threonine (Thr) replacement value of L-homoserine (HS). Growth rate was increased (P , 0.05) by dietary addition of 800 mg L-HS/kg diet to a purified diet severely deficient in Thr or by the addition of 800 or 1000 mg of L-HS/kg diet to a corn-peanut meal diet distinctly deficient in Thr. The addition of an isomolar level of a-ketobutyrate, a catabolic product of both Thr and HS, did not elicit a response. Standard-curve methodology predicted a Thr replacement value of 38 6 9% for HS. Interactions (P , 0.01) were observed in assays 2 and 4 between dietary Thr adequacy and 800 or 1000 mg L-HS/kg supplementation. Thus, HS improved growth performance when added to a Thr-deficient diet (0.46 g Thr/100 g diet), but it decreased growth performance when added to the same diet containing surfeit Thr (0.80 g Thr/100 g diet). The results indicate that low levels of HS elicit a growth response in young chicks fed Thr-deficient diets. J. Nutr. 139: 1298–1302, 2009.

Introduction individual chicks and pen food intakes were measured at the beginning 3 Homoserine (HS) occurs free (unbound to protein) in a number and end of each chick assay. Body weight gain, food intake, and food of plants and it is particularly rich in pea seedlings (1). It is also efficiency (gain:food ratio) were calculated for each replicate pen of formed during catabolism of the carbon skeleton of methionine chicks. (Met) in higher organisms (1). It is well established (2–8) that Individual proteinaceous ingredients as well as basal diets were plants, bacteria, and yeast can synthesize not only threonine analyzed for crude protein (CP), Thr, Met, cyst(e)ine, and HS as (Thr) but also Met from HS. Watanabe and Shimura (2,3) previously described (11). The L-HS used herein was synthesized by showed that at least 2 separate enzyme fractions are needed in Evonik Degussa (Hanau-Wolfgang, Germany). Our amino acid (AA) analysis indicated the product was .99% pure HS and contained no yeast and Neurospora for synthesis of Thr from HS. The first detectable Thr. involves HS kinase, which catalyzes the phosphorylation of HS via ATP. The second key enzyme in Thr biosynthesis from HS is Assay 1. The objective of this assay was to determine whether L-HS Thr synthetase and this enzyme requires pyridoxal phosphate. could spare the need for Thr in young chicks. The purified basal diet, Evidence for these conversions in higher organisms, however, singly deficient in Thr, was supplemented with either 800 mg Thr/kg diet does not exist. We present evidence here that HS has Thr or isomolar (800 mg/kg) or twice isomolar (1600 mg/kg) concentrations replacement bioactivity in young chicks. of L-HS at the expense of cornstarch (Thr and HS have identical molecular weights). Five replicate pens of 4 chicks received each of the 4 experimental diets during a 12-d feeding period (d 8–20 posthatch). Materials and Methods Assay 2. This assay sought to determine whether L-HS or a-ketobutyrate All procedures were approved by the University of Illinois Animal Care (a-KB) could spare the need for Thr in young chicks fed a Thr-deficient, and Use Committee. Four assays were conducted using male chicks from corn-peanut meal diet. Analysis of both corn and peanut meal revealed the University of Illinois Poultry Farm following the same housing and that there was no detectable free or bound HS in either ingredient. rearing procedures described by Dilger and Baker (9). Graded doses of L-Thr (0, 400, or 800 mg/kg) were added to the basal Two separate basal diets (Table 1) were formulated to evaluate the diet to produce a standard curve. As a positive control, the basal diet was Thr replacement value of L-HS. The purified and the corn-peanut meal- also supplemented with 3400 mg L-Thr/kg diet to exceed the dietary Thr basal diets were formulated to be singly deficient in Thr but were requirement for this age chick. L-HS or a-KB was added to the basal diet otherwise nutritionally complete for chicks of this age (10). Experimen- or positive control diet at concentrations isomolar to 800 mg L-Thr/kg tal diets and tap water were freely available to chicks. Body weight of diet. Each of the dietary treatments (8 total) were assigned to 4 replicate pens of 4 chicks during a 9-d feeding period (d 8–17 posthatch).

Assay 3. In this assay, the Thr-sparing effect of L-HS was evaluated 1 Supported by Evonik Degussa GmbH, Hanau-Wolfgang, Germany. 2 Author disclosures: K. I. Bryant, R. N. Dilger, C. M. Parsons, and D. H. Baker, no both directly and indirectly, i.e. via contribution from L-Met (i.e. Met conflicts of interest. conversion to HS). The Thr-deficient, corn-peanut meal-basal diet was 3 Abbreviations used: AA, amino acid; CP, crude protein; HS, homoserine; a-KB, supplemented with 0 or 800 mg L-Thr/kg diet or 800 mg L-HS/kg diet a-ketobutyrate. (i.e. isomolar to 800 mg L-Thr/kg diet). Additionally, 1000 or 2000 mg * To whom correspondence should be addressed. Email: [email protected]. L-Met/kg diet (isomolar and twice isomolar that of 800 mg L-HS/kg diet,

0022-3166/08 $8.00 ã 2009 American Society for Nutrition. 1298 Manuscript received January 8, 2009. Initial review completed February 27, 2009. Revision accepted May 5, 2009. First published online May 27, 2009; doi:10.3945/jn.109.104372. TABLE 1 Composition of Thr-deficient basal diets and multiplied by 100. Four of the diets in assays 2 and 4 formed a 2 3 2 factorial arrangement of treatments. Hence, these 4 treatment diets were Corn-peanut evaluated by single degree-of-freedom contrasts to assess the main effects Purified diet meal diet of HS supplementation and Thr concentration and also the interaction. Ingredient (assay 1)1 (assays 2, 3, and 4)2

g/100 g Cornstarch 54.37 0.36 Results Corn, 8.4 g CP/100 g — 61.08 Peanut meal, 45.6 g CP/100 g — 27.50 Assay 1. Weight gain and gain:food increased (P , 0.05) when Casein, 84.8 g CP/100 g 2.50 — the Thr-deficient purified diet was supplemented with 800 mg Soy protein isolate, 82.4 g CP/100 g 4.00 — Thr/kg diet or an isomolar concentration (800 mg/kg) of L-HS AA mixture 19.563 2.264 (Table 2). The addition of 1600 mg HS/kg diet also increased Soybean oil 10.00 4.00 both gain and gain:food, but the response was no greater than Solka floc5 3.00 — that with 800 mg HS/kg. Purified mineral mix6 5.37 — Downloaded from https://academic.oup.com/jn/article/139/7/1298/4670474 by guest on 30 September 2021 Trace mineral mix6 — 0.15 Assay 2. Supplementation of the Thr-deficient corn-peanut Purified vitamin mix6 0.20 — meal diet with the first 3 doses of L-Thr resulted in linear (P , Vitamin mix6 — 0.20 0.01) responses in weight gain, food intake, and gain:food ratio Choline chloride 0.20 0.10 (Table 3). Adding 800 mg L-HS/kg to the basal diet improved P , 3 NaHCO3 1.00 0.40 ( 0.05) gain:food. Diets 1, 4, 5, and 6 represented a 2 2 Limestone — 1.40 factorial arrangement of treatments, and assessment of the Dicalcium phosphate — 2.10 interaction for these treatments revealed significance (P , 0.01) Sodium chloride — 0.40 for weight gain, food intake, and gain:food. Thus, when dietary Bacitracin premix7 — 0.05 Thr was deficient, 800 mg L-HS/kg produced a positive response, but when dietary Thr was superadequate, 800 mg L-HS/kg 1 Analyzed to contain (g/100 g diet): CP, 20.3; Thr, 0.22; Met, 0.32; cyst(e)ine, 0.20. depressed growth performance (Fig. 1). The addition of 686 mg 2 Analyzed to contain (g/100 g diet): CP, 19.0; Thr, 0.46; Met, 0.42; cyst(e)ine, 0.38. 3 Provided (g/100 g diet): L-Arg, 0.95; L-His, 0.33; L-Lys×HCl, 1.14; DL-Met, 0.20; a-KB/kg, isomolar to 800 mg HS/kg, did not affect growth L-cystine, 0.15; L-Phe, 0.50; L-Tyr, 0.45; L-Trp, 0.15; L-Leu, 1.00; L-Ile, 0.60; L-Val, 0.69; performance of chicks fed the Thr-deficient basal diet or the diet Gly, 1.00; L-Pro, 0.40; and L-Glu, 12.00. containing surfeit Thr. 4 Provided (g/100 g diet): L-Lys×HCl, 1.00; DL-Met, 0.20; L-cystine, 0.17; L-Val, 0.20; The gain:food ratio of chicks fed diets 1, 2, and 3 was used to L-Ile, 0.23; L-Arg, 0.06; L-Trp, 0.07; and Gly, 0.33. r 2 5 FS & D Corp., Urbana, OH. establish a linear regression equation (Y = 482.1 + 0.506X; = 6 See Dilger and Baker (33,34) for composition. 0.93, where Y = gain:food in g/kg and X = supplemental L-Thr 7 Provided 55 mg bacitracin methylene disalicylate/kg complete diet. intake in mg). This equation was then used as a standard curve to estimate the L-Thr replacement activity of L-HS for chicks receiving diet 5, which was 38.2 6 9.0%. respectively) was added to the Thr-deficient basal diet. Five replicate pens of 4 chicks were fed each experimental diet during an 8-d feeding Assay 3. period (d 8–16 posthatch). The addition of 800 mg L-Thr/kg to the Thr-deficient corn-peanut meal diet resulted in increased (P , 0.05) gain, food Assay 4. To confirm the positive growth response to L-HS in chicks fed intake, and gain:food (Table 4). Likewise, chicks fed diets Thr-deficient diets as well as the negative growth response in chicks fed fortified with 800 mg L-HS/kg also responded with increased diets with surfeit Thr, 3 replicate pens of 4 chicks were fed 5 different (P , 0.05) weight gain, food intake, and gain:food, although the treatment diets in a 9-d growth bioassay. The Thr-deficient corn-peanut responses to L-Thr were greater (P , 0.05) than those to L-HS. meal diet (0.46 g Thr/kg diet) was fortified with either 1000 or 3400 mg An approximate calculation of Thr sparing by HS (gain response L-Thr/kg diet. As in assay 2, the Thr-deficient basal diet and the diet with to 800 mg HS/kg 4 gain response to 800 mg Thr/kg) resulted in surfeit Thr (3400 mg L-Thr/kg diet) were also supplemented with 1000 a HS replacement value of 40%. Supplementation of isomolar or mg L-HS/kg diet. At the end of the 9-d feeding period, heparinized blood twice isomolar levels of (excess) DL-Met did not affect chick was obtained via heart puncture from individual chicks. Plasma was prepared, after which an equal volume of plasma from each of the 4 performance. chicks in a pen-replicate was pooled and then deproteinized with an equal volume of sulfosalicylic acid. The 15 deproteinized plasma samples TABLE 2 Response of young chicks to HS when added to a (5 treatments 3 3 replicates) were then frozen until analyzed chromato- purified diet severely deficient in Thr (assay 1)1 graphically for AA (12). Diet Weight gain Food intake Gain:food Statistical analysis. All data were subjected to ANOVA using the General Linear Model procedure of SAS (13). Data were analyzed using g g g/kg pen means with procedures appropriate for a completely randomized 1. Thr-deficient basal diet2 6b 96b 62c design. Data are presented as mean values with pooled SEM estimates, 2. As 1 + 800 mg L-Thr/kg diet 23a 110a 210a and significance was set at a = 0.05. Differences among treatment means 3. As 1 + 800 mg L-HS/kg diet3 17a 103a,b 165b were evaluated using the least significance procedure (14). 4. As 1 + 1,600 mg L-HS/kg diet 16a 97b 165b In assay 2, the ability of L-HS to spare Thr was quantified using 4 standard-curve methodology (15,16). Briefly, gain:food (g/kg, dependent SEM 2.1 4.4 15 variable) was regressed on supplemental L-Thr intake (mg, independent 1 Data are means of 5 replicate pens of 4 male chicks during a 12-d feeding period variable) to produce a linear regression equation. Supplemental intake of from 8 to 20-d posthatch; mean initial weight was 89 g. L-Thr equivalents was then predicted for L-HS (i.e. diet 5) using the 2 Contained 0.22 g Thr/100 g diet. regression equation, after which this calculated value was divided by the 3 Isomolar to 800 mg Thr/kg diet. mean total supplemental intake of L-HS (i.e. 800 mg/kg 3 food intake) 4 Means in a column with superscripts without a common letter differ, P , 0.05.

Homoserine spares threonine 1299 TABLE 3 Response of young chicks to HS and a-KB when TABLE 4 Efficacy of HS or excess DL-Met to replace Thr in added to a Thr-deficient or Thr-surfeit corn-peanut young chicks fed a Thr-deficient corn-peanut meal meal diet (assay 2)1 diet (assay 3)1

Weight Food Diet Weight gain Food intake Gain:food Diet gain2 intake Gain:food2 g g g/kg g g g/kg 1. Thr-deficient basal diet2 100c 202c 495c 3 e,f b,c a a a 1. Thr-deficient basal diet 98 205 478e 2. As 1 + 800 mg L-Thr/kg diet 153 259 590 d b b b b 2. As 1 + 400 mg L-Thr/kg diet 122 228 535d 3. As 1 + 800 mg L-HS/kg diet 121 220 550 b a 3 c c c 3. As 1 + 800 mg L-Thr/kg diet 159 270 589c 4. As 1 + 1000 mg DL-Met/kg diet 96 193 498 a a 3 c c c 4. As 1 + 3,400 mg L-Thr/kg diet 180 270 666a 5. As 1 + 2000 mg DL-Met/kg diet 95 192 494 5. As 1 + 800 mg L-HS/kg diet4 111d,e 215b,c 516d SEM4 3.6 5.1 7.7 4 c bc 6. As 4 + 800 mg L-HS/kg diet 137 217 632b 1 4 f c Data are means of 5 replicate pens of 4 male chicks during an 8-d feeding period 7. As 1 + 686 mg a KB/kg diet 95 203 469e from 8 to 16 d posthatch; mean initial weight was 100 g. 4 a a 8. As 4 + 686 mg a KB/kg diet 176 265 664a 2 Contained (g/100 g diet): Thr, 0.46; Met, 0.42; cysteine, 0.38. 5 3 Downloaded from https://academic.oup.com/jn/article/139/7/1298/4670474 by guest on 30 September 2021 SEM 5.0 7.7 7.6 1000 and 2000 mg DL-Met/kg are isomolar and twice isomolar, respectively, to 800 mg L-HS/kg diet. 1 Data are means of 4 replicate pens of 4 male chicks during a 9-d feeding period from 4 Means in a column with superscripts without a common letter differ, P , 0.05. 8 to 17 d posthatch; mean initial weight was 88 g. 2 Diets 1, 4, 5, and 6 formed a 2 3 2 factorial arrangement of treatments. Statistical P , analysis indicated an interaction, 0.01, between Thr adequacy and HS 0.05) than the addition of 1000 mg L-HS/kg alone. No HS was supplementation. Response to L-Thr (diets 1, 2, and 3) was linear, P , 0.01. L 3 Contained 0.46 g Thr/100 g diet. found in plasma of chicks fed diets without added -HS. Plasma 4 Isomolar to 800 mg Thr/kg diet. Thr was similar in chicks receiving diets with no added Thr or 5 Means in a column with superscripts without a common letter differ, P , 0.05. those with 1000 mg Thr/kg, but the addition of supplemental L-Thr (3400 mg Thr/kg) beyond the chick’s requirement resulted in elevated (P , 0.05) plasma Thr. Also, dietary addition of Assay 4. Supplemental L-HS at 1000 mg/kg increased (P , 1000 mg L-HS/kg together with 3400 mg L-Thr/kg increased 0.05) both gain and gain:food but only when added to the Thr- (P , 0.05) plasma Thr more than addition of 3400 mg L-Thr/kg deficient diet (Table 5). A HS replacement value of 39% was alone. Changes in other plasma AA (e.g. glycine, serine, Met, calculated (as in assay 3) based upon gain:food responses to cyst(e)ine) due to dietary treatment were unremarkable. 1000 mg HS/kg compared with 1000 mg Thr/kg, respectively. The addition of 1000 mg HS/kg to the diet containing surfeit Thr decreased weight gain, food intake, and gain:food ratio. Thus, in agreement with the results of assay 2, the Thr concentration 3 Discussion HS interaction was significant (P , 0.01) for all growth criteria. HS was found in plasma of chicks fed the 2 diets containing The results herein clearly indicate that dietary L-HS has Thr supplemental HS, and the combination of 1000 mg L-HS/kg plus replacement bioactivity. Thr biosynthesis is a possible explana- 3400 mg L-Thr elevated plasma HS to a greater extent (P , tion, but whether this might be occurring in the liver or other body tissues or perhaps in the gut via gut microbes (17,18) is open to question. Plants and microbes synthesize Thr from HS (2–8). The results of assay 2 clearly point to the conclusion that the HS response results from HS per se and not from a-KB, the

TABLE 5 Growth performance and plasma free AA concentrations in chicks fed supplemental L-HS in corn-peanut meal diets either deficient or surfeit in Thr (assay 4)1

Weight Food Plasma AA Diet gain2 intake2 Gain:food2 HS Thr

g g g/kg umol/L 1. Thr-deficient basal diet3 71d 169d,e 418d 0c 44.5c b b b c c 2. As 1 + 1000 mg L-Thr/kg diet 136 243 559 0 70.5 a a a c b 3. As 1 + 3400 mg L-Thr/kg diet 175 270 649 0 531.4 4 c d c b c 4. As 1 + 1000 mg L-HS/kg diet 85 180 473 17.6 47.0 4 b c b a a 5. As 3 + 1000 mg L-HS/kg diet 131 221 592 31.9 873.1 SEM5 4.5 5.2 14 1.7 10.1

1 Data are means of 3 replicate pens of 4 male chicks during a 9-d feeding period from 8 to 17 d posthatch; mean initial weight was 75 g. 2 Diets 1, 3, 4, and 5 formed a 2 3 2 factorial arrangement of treatments. Statistical P , FIGURE 1 P , analysis indicated an interaction, 0.01, between Thr adequacy and HS Plot of the interaction ( 0.01) of dietary Thr supplementation. 3 concentration 800 mg/kg HS supplementation for weight gain of 3 Contained 0.46 g Thr/100 g diet. chicks in assay 2 (Table 3). Values are means of 4 pens of 4 chicks 4 Isomolar to 1,000 mg Thr/kg diet. (pooled SEM = 5.0 g). 5 Means in a column with superscripts without a common letter differ, P , 0.05.

1300 Bryant et al. immediate deamination product of HS. Because the carbon due largely to a 20% decrease in voluntary food intake (Tables skeleton of Met is metabolized to HS and then a-KB (1,19,20), 3 and 5). Previous research done in our laboratory 32 y ago we endeavored in assay 3 (Table 4) to show a growth response to involved graded dietary additions of L-HS or a-KB to a complete excess dietary Met, i.e. as a source of HS. We realized, however, crystalline AA chick diet. Supplemental L-HS (0, 4000, and 8000 that HS produced via metabolism is not the same as providing mg/kg) caused a precipitous and linear (P , 0.01) decrease in L-HS in the diet. Also, relative to the Thr deficiency in the basal daily weight gain, i.e. 12.1, 6.6, and 4.7 g/d, respectively; diet, this diet already contained a considerable excess of Met. equimolar additions of a-KB, however, did not affect growth Thus, the lack of a growth response to additional excess Met is performance. probably not surprising. We have recently supplemented the In conclusion, oral HS spares dietary Thr, but very modest Thr-deficient corn-peanut meal diet used herein with 5000 mg doses (800 mg/kg) of supplemental L-HS are noxious when L-Met/kg and were unable to detect free HS in blood plasma provided in diets adequate to superadequate in Thr. The sparing (unpublished data). Moreover, no HS was found in plasma of effect of HS may result from Thr biosynthesis from HS or from chicks receiving either Thr-deficient or Thr-surfeit diets not a HS-induced decrease in Thr catabolism. Why such a small containing supplemental L-HS in assay 4 herein (Table 5). Thus, dietary addition of L-HS reduces food intake and growth of Met catabolism may not yield HS per se. Cystathionine g-lyase chicks fed diets with adequate to surfeit Thr remains to be (EC 4.4.1.1), also known as HS deaminase-cystathionase, is the determined. Downloaded from https://academic.oup.com/jn/article/139/7/1298/4670474 by guest on 30 September 2021 enzyme that degrades cystathionine to cysteine, a-KB, and ammonia (1,19,20). The HS formed in this reaction is bound to the enzyme and, in fact, may never be released as free HS, only as Literature Cited a-KB. What remains to be done is to orally administer 1-14C– 1. Meister A. Biochemistry of the amino acids. 2nd vol. New York: Academic Press; 1965. p. 675–83. labeled HS (and Met) to see if the radiolabel can be identified in 2. Watanabe Y, Shimura K. Biosynthesis of threonine from homoserine. 5. body Thr or perhaps also in gut mucin, a protein rich in Thr that Nature of an intermediary product. J Biochem. 1956;43:283–94. decreases under conditions of Thr deficiency (21–23). Up to 3. Watanabe Y, Simura K. Mechanism of L-threonine synthesis from O- 30% of the AA content of mucin is comprised of Thr, and mucin phosphohomoserine. J Biochem. 1960;47:266–8. biosynthesis accounts for a significant portion of the total 4. Flavin M, Slaughter C. Purification and properties of threonine dietary Thr requirement (21–25). If either gut mucosal tissue or synthetase of Neurospora. J Biol Chem. 1960;235:1103–8. gut bacteria are capable of metabolizing HS to Thr to facilitate 5. Flavin M, Slaughter C. Threonine synthetase mechanism: studies with synthesis of gut mucins, this could (indirectly) allow more of the isotopic hydrogen. J Biol Chem. 1960;235:1112–8. Thr in portal circulation (or that made available from protein 6. Flavin M, Kono T. Threonine synthetase mechanism: studies with isotopic oxygen. J Biol Chem. 1960;235:1109–11. degradation) to be available for muscle protein synthesis and 7. Rinder J, Casazza AP, Hoefgen R, Hesse H. Regulation of aspartate- growth. derived amino acid homeostasis in potato plants (Solamun tuberosum) Klasing (26), in commenting on the lysine knockout work of by expression of E. coli homoserine kinase. Amino Acids. 2008;34:213– Cleveland et al. (27), concluded that a reduction in the 22. catabolism of a given AA would likely result in a sparing (and 8. Kim YH, Park JS, Cho JY, Cho KM, Park YH, Lee J. Proteomic reduced requirement) for that AA. Hence, the Thr sparing effect response analysis of a threonine-overproducing mutant of Escherichia coli. of HS observed herein may have resulted from HS causing a Biochem J. 2004;381:823–9. reduction in Thr catabolism. Nishimura and Greenberg (28) 9. Dilger RN, Baker DH. DL-methionine is as efficacious as L-methionine, but modest L-cystine excesses are anorexigenic in methionine deficient found that HS decreased the activity of sheep liver Thr purified and practical-type diets fed to chicks. Poult Sci. 2007;86:2367– dehydratase (EC 4.2.1.19) and House et al. (29) showed that 74. 14 Thr 1- C-Thr oxidation in rat hepatocytes could be suppressed 10. NRC. Nutrient requirements of poultry. 9th ed. Washington, DC: by either cysteamine or a-cyanocinnamate. It may be possible National Academies Press; 1994. that these compounds, like HS, would elicit growth responses 11. Chung TK, Baker DH. Efficiency of dietary methionine utilization by when given to Thr-deficient chicks. Clearly, whether in hepato- young pigs. J Nutr. 1992;122:1862–9. cytes or the intact animal, a reduction in 1-14C-Thr oxidation 12. Fekkes D. State of-the-art high-performance liquid chromatographic analysis of amino acids in physiological samples. J Chromatogr B due to HS administration would offer support for the reduced Biomed Appl. 1996;682:3–22. catabolism mechanism of Thr sparing by HS. 13. SAS Institute. SAS/STAT user’s guide. Release 9.1. Cary (NC): SAS An explanation is not obvious for why 800 or 1000 mg/kg Institute, Inc; 2004. supplemental L-HS was growth depressing when added to a diet 14. Carmer SG, Walker WM. Pairwise multiple comparisons of treatment superadequate in Thr. Most of the HS flux involves its means in agronomic research. J Agron Educ. 1985;14:19–26. deamination to a-KB (1,19,20), but as shown in assay 2 (Table 15. Augspurger NR, Scherer CS, Garrow TA, Baker DH. Dietary S- 3), equimolar addition of 686 mg/kg a-KB to the diet containing methylmethionine, a component of foods, has choline-sparing activity surfeit Thr did not depress growth performance. Moreover, Thr in chickens. J Nutr. 2005;135:1712–7. conversion to a-KB via Thr dehydratase (EC 4.2.1.19) is only 16. Pahm AA, Scherer CS, Pettigrew JE, Baker DH, Parsons CM, Stein HH. Standardized amino acid digestibility in cecectomized roosters and 1 of 3 pathways of Thr degradation; the Thr dehydrogenase (EC lysine bioavailability in chicks fed distillers dried grains with solubles. 1.1.1.103) and Thr aldolase (EC 2.1.2.1) pathways are also Poult Sci. 2009;88:571–8. involved in Thr degradation (30–32). Hence, a buildup of a-KB 17. Torrallardona D, Harris CI, Fuller MF. Pigs’ gastrointestinal micro- from Thr (and HS) catabolism does not explain the HS-induced flora provide them with essential amino acids. J Nutr. 2003;133:1127– growth depression in chicks receiving diets containing surfeit 31. Thr. Does HS, representing the carbon skeleton of Met during 18. Raj T, Dileep U, Vaz M, Fuller MF, Kurpad AV. Intestinal microbial contribution to metabolic leucine input in adult men. J Nutr. Met catabolism, somehow disrupt transsulfuration? Is HS per se 2008;138:2217–21. toxic when added to diets adequate to superadequate in Thr and 19. Matsuo Y, Greenberg DM. A crystalline enzyme that cleaves homoser- all other AA? The decreased weight gain resulting from as little ine and cystathionine. IV. Mechanism of action, reversibility, and as 800 or 1000 mg/kg HS added to the diet with surfeit Thr was substrate specificity. J Biol Chem. 1959;234:516–9.

Homoserine spares threonine 1301 20. Carroll WR, Stacy GW, DuVigneaud V. Alpha-ketobutyric acid as a 27. Cleveland BM, Kiess AS, Blemings KP. a-Aminoadipate semialdehyde product in the enzymatic cleavage of cystathionine. J Biol Chem. synthase mRNA knockout reduces the lysine requirement of a mouse 1949;180:375–82. hepatic cell line. J Nutr. 2008;138:2143–7. 21. Faure M, Moe¨nnoz D, Montigon F, Mettraux C, Breuille´ D, Balle`vre O. 28. Nishimura JS, Greenberg DM. Purification and properties of L- Dietary threonine restriction specifically reduces intestinal mucin threonine dehydrase of sheep liver. J Biol Chem. 1961;236:2684–91. synthesis in rats. J Nutr. 2005;135:486–91. 29. House JD, Hall BN, Brosnan JT. Threonine metabolism in isolated rat 22. Nichols NL, Bertolo RF. Luminal threonine concentration acutely hepatocytes. Am J Physiol Endocrinol Metab. 2001;281:E1300–7. affects intestinal mucosal protein and mucin synthesis in piglets. J Nutr. 30. Bird MI, Nunn PB, Lord LAJ. Formation of glycine and aminoacetone 2008;138:1298–303. from L-threonine by rat liver mitochondria. Biochim Biophys Acta. 23. Horn NL, Adeola O. Implications of dietary threonine on crude mucin 1984;802:229–36. excretion in broiler chicks and ducklings. Poult Sci. 2007;86 Suppl 31. Balle`vre O, Cadenhead A, Calder AG, Rees WD, Lobley GE, Fuller MF, 1:781. Garlick PJ. Quantitative partition of threonine oxidation in pigs: effect 24. Schaart MW, Schierbeek H, van der Schoor SRD, Stoll B, Burrin DG, of dietary threonine. Am J Physiol. 1990;259:E483–91. Reeds PJ, Van Goudoever JB. Threonine utilization is high in the 32. Davis AJ, Austic RE. Dietary threonine imbalance alters threonine intestine of piglets. J Nutr. 2005;135:765–70. dehydrogenase activity in isolated hepatic mitochondria of chicks and 25. Bertolo RFP, Chen CZL, Law G, Pencharz PB, Ball RO. Threonine rats. J Nutr. 1994;124:1667–77. requirement of neonatal piglets receiving total parenteral nutrition is 33. Dilger RN, Baker DH. Cyst(e)ine imbalance and its effect on methio- considerably lower than that of piglets receiving an identical diet nine precursor utilization in chicks. J Anim Sci. 2008;86:1832–40. Downloaded from https://academic.oup.com/jn/article/139/7/1298/4670474 by guest on 30 September 2021 intragastrically. J Nutr. 1998;128:1752–9. 34. Dilger RN, Kobler C, Weckbecker C, Hoehler D, Baker DH. 2-keto-4- 26. Klasing KC. Minimizing amino acid catabolism decreases amino acid (methylthio) butyric acid (keto-analog of methionine) is a safe and effective requirements. J Nutr. 2009;139:11–2. precursor of L-methionine in the chick. J Nutr. 2007;137:1868–73.

1302 Bryant et al.