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METABOLISM AND NUTRITION |
Department of Animal Sciences and Division of Nutritional Sciences, University of Illinois at Urbana-Champaign, Urbana, IL 61801
2 Corresponding author: dhbaker{at}uiuc.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-ketoadipic acid as an intermediary metabolite. An alternate pathway of Trp turnover leads to NAD synthesis. We hypothesized that excess Lys might improve the conversion of Trp to niacin by causing a buildup of -ketoadipic acid, thereby endproduct inhibiting the main Trp catabolic pathway and resulting in more niacin synthesis from Trp. Six bioassays were carried out in which 12 to 20 chicks were fed each experimental diet from d 8 to d 20 or 21 posthatching. The basal diet (4 mg/kg of bioavailable niacin) used for all assays was a semipurified corn gluten meal diet fortified with crystalline amino acids to 22.5% CP and 0.96% true digestible Lys. Assay 1 through 3 established the requirements for digestible Trp (0.16%) and bioavailable niacin (19.5 mg/kg) and showed that 0.96% digestible Lys was adequate for chick growth in the presence of adequate Trp and niacin. The fourth assay was done to determine the effect of 1% Lys (1.25% food-grade L-Lys·HCl) on niacin utilization. Excess Lys improved (P < 0.01) weight gain of niacin-deficient chicks. The fifth assay showed that 1% excess food-grade Lys improved weight gain in niacin-deficient (4 mg/kg) chicks but depressed weight gain in niacin-adequate (24 mg/kg) chicks (niacin x Lys interaction, P < 0.01). In assay 6, chicks fed 6 mg/kg of niacin gained faster (P < 0.01) than control chicks, but neither quinolinic acid (100 mg/kg) nor picolinic acid (4,200 mg/kg) elicited a response. These results suggest that excess Lys leads to an accumulation of -ketoadipic acid, which causes endproduct inhibition of the main Trp catabolic pathway to CO2, therefore increasing flux of 2-amino-3-carboxymuconate semialdehyde to NAD.
Key Words: niacin • tryptophan • lysine • picolinate • quinolinate
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Krehl et al. (1945, 1950) showed that excess Trp can be catabolized to niacin and that dietary Trp deficiency and overall poor protein quality contribute to niacin deficiency (i.e., pellagra) in populations subsisting on cereal-based diets. Further work in chicks revealed that under conditions of niacin deficiency and minimal Trp adequacy, excess Trp was converted to niacin with an efficiency (wt/wt) of approximately 2% (Baker et al., 1973; Oduho and Baker, 1993; Chen et al., 1996). Further work by Oduho et al. (1994) revealed that Fe deficiency reduced the conversion efficiency of Trp to niacin from 2.4 to 1.6% (wt/wt).
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-ketoadipic acid (Figure 1
). The branch point in Trp catabolism that leads to niacin production occurs at -amino-ß-carboxymuconate semialdehyde (ACS). Approximately 95% of ACS is ultimately catabolized to CO2 and H2O, producing -ketoadipic acid as an intermediate 3 steps down from ACS (Lan and Gholson, 1965; Pawlak et al., 2002). The rate of ACS decarboxylation is inversely related to the amount of NAD synthesized from Trp (Ikeda et al., 1965). Our hypothesis was that excess dietary Lys might lead to a build-up of -ketoadipic acid, which in turn would endproduct inhibit the catabolism of ACS to -ketoadipic acid, thus shunting more ACS (from Trp) toward NAD biosynthesis. |
MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Diet Development (Assays 1, 2, and 3)
Excess dietary Trp is converted to niacin with a wt/wt efficiency of approximately 2% and a M efficiency of 4% (Baker et al., 1973). Hence, tight control of dietary Trp is required in studies of niacin efficacy. A corn gluten meal (CGM) semipurified diet (Table 1) was formulated to meet or exceed the chick’s requirements for all nutrients except niacin and Trp. This diet contained an estimated 4 mg/kg of bioavailable niacin (Yen et al., 1977) and 0.06% true digestible Trp (Baker et al., 2002). This same basal diet (made adequate in niacin) was previously used to estimate the true digestible Lys requirement (0.96%) and true digestible Trp requirement (0.16%) of chicks. As a result, the true digestible Lys level was set at 0.96% in the basal diet shown in Table 1. With these levels of Lys and Trp, the basal diet either met or exceeded digestible amino acid levels prescribed in the Illinois ideal protein (Baker and Han, 1994; Baker et al., 2002). Also, when properly fortified with niacin, Trp, Lys, and other amino acids, the CGM diet shown in Table 1 produced weight gain and feed efficiency values that were equal to those obtained with a standard 23% CP corn-soybean meal diet (Peter et al., 2000).
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Assay 1 was done to confirm the requirement estimate for true digestible Trp in the presence of excess niacin as done previously (Baker et al., 2002). The basal diet was supplemented with 20 mg of niacin/kg and 0.02% L-Trp to accommodate 6 graded doses of L-Trp, thus achieving final dietary true digestible Trp levels of 0.08, 0.10, 0.12, 0.14, 0.16, and 0.18%. The results indicated that a level of 0.16% true digestible Trp was adequate for maximum growth under conditions of niacin adequacy.
Assay 2 was done to determine the minimal requirement for niacin in a Trp-adequate diet (0.16% true digestible Trp). On d 8 posthatching, chicks were allotted to dietary treatments that consisted of 6 graded doses (0 to 20 mg/kg) of supplemental niacin. On d 21 posthatching, weight gain and feed intake were determined. The data revealed a requirement of 15.5 mg/kg of supplemental niacin (19.5 mg/kg of total bioavailable niacin) to support maximum growth.
The objective of assay 3 was to determine the adequacy of Lys in our experimental diet that was adequate in both Trp and niacin. The basal diet for this assay contained 0.96% digestible Lys, 24 mg/kg of bioavailable niacin, and 0.18% digestible Trp. Lysine adequacy was determined by comparing the basal diet to this same diet fortified with 0.10% supplemental L-Lys provided by FG L-Lys·HCl. Weight gain and feed intake were determined on d 20 posthatch.
Assay 4
This assay was done to determine the effect of 1% excess Lys on the biopotency of supplemental niacin and on Trp as a precursor for niacin. On d 8, chicks were assigned to dietary treatments consisting of a negative control (no added niacin or Trp) and 2 graded levels of niacin (4 and 8 mg/kg) and 2 graded levels of Trp (160 and 320 mg/kg), both in the absence and presence of 1% supplemental excess Lys. Based on previous research (Baker et al., 1973; Oduho and Baker, 1993), linear responses were expected to each supplement. Food-grade L-Lys·HCl was used in this bioassay to avoid any possible contaminating niacin activity that might have been present in the FG L-Lys·HCl used in the previous assays. On d 21, weight gain and feed intake were determined.
Assay 5
The objective of this assay was to determine the effect of excess dietary (food-grade) Lys on weight gain of chicks fed either niacin-deficient or niacin-adequate diets. Two levels of supplemental Lys (0 and 1%) from food-grade L-Lys·HCl were fed in the presence of 0 and 20 mg/kg of supplemental niacin. On d 20, weight gain and feed intake were determined.
Assay 6
This assay was carried out to determine the effect of quinolinic acid and picolinic acid—2 metabolites in the metabolism of Trp to niacin (Figure 1)—on weight gain of chicks fed a niacin-deficient diet. On d 8, chicks were randomly assigned to dietary treatments that included the basal niacin-deficient diet supplemented with niacin (6 mg/kg), quinolinic acid (100 mg/kg), or picolinic acid (4,200 mg/kg). Quinolinic acid was added to the diet at a level isomolar to 74 mg/kg of niacin. Picolinic acid was added to the basal diet at a level isomolar to 5,000 mg/kg of Lys. On d 21, weight gain and feed intake were determined.
Statistical Analysis
Data for all assays were subjected to ANOVA appropriate for completely randomized designs (Steel et al., 1997). Where appropriate, treatment means were separated using orthogonal comparisons. For assay 2, the requirement for supplemental niacin was estimated by fitting the data to a single-slope broken-line least-squares model (Robbins et al., 1979). Assays 1, 2, and 4 used graded dosing of Trp, niacin, or both, and single df linear and quadratic effects were evaluated for Trp dosing (assay 1) and niacin dosing (assay 2). In assay 4, in which a zero dose plus 2 graded levels of Trp and 2 graded levels of niacin were evaluated in the absence and presence of 1% excess Lys, linear and quadratic responses to Trp and niacin were quantified. Also, main effects of niacin, Trp, and Lys were assessed. The data of assay 4 were further subjected to common-intercept multiple linear regression (Finney, 1978; Oduho and Baker, 1993), and for this, weight gain (Y) was regressed on supplemental niacin intake (X1) and Trp intake (X2) in both the absence and presence of excess Lys. Assay 5 was analyzed as a 2 x 2 factorial, with main effects of niacin and Lys together with the interaction of niacin x Lys.
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RESULTS |
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) to supplemental Trp levels up to 0.08% (0.14% true digestible Trp). To provide a safety margin, however, subsequent assays involving niacin deficiency used diets with at least 0.16% true digestible Trp. Niacin addition to a Trp-adequate diet also resulted in a large linear increase (P < 0.01) in weight gain to up to 16 mg/kg of supplemental niacin (Table 3). Broken-line analysis (r2 = 0.97) revealed a minimal supplemental niacin requirement of 15.5 mg/kg, giving a total bioavailable niacin requirement estimate of 19.5 mg/kg. In assay 3, supplementation of 0.10% Lys to the basal diet with more-than-adequate Trp and niacin did not affect (P > 0.10) chick growth performance (Table 4).
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). The 1% level of supplemental food-grade Lys increased (P < 0.05) weight gain and gain:feed in chicks fed all of the (niacin-deficient) diets. The multiple linear regression equation of gain (Y in g) as a function of supplemental niacin (X1 in mg) or Trp (X2 in mg) intake was Y = 62.5 + 29.08 X1 + 0.55 X2 (r2 = 0.84) for the diets without excess Lys. The similar equation for the 5 diets containing excess Lys was Y = 72.41 + 34.13 X1 + 0.62 X2 (r2 = 0.89). The intercept value for the diets containing excess Lys was higher (P < 0.05) than that for the diets not containing excess Lys, suggesting that excess Lys was increasing utilization of the basal diet levels of either niacin (4 mg/kg) or digestible Trp (0.16%).
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). Thus, excess Lys (1%) improved growth performance in niacin-deficient chicks but decreased growth performance in niacin-adequate chicks.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-ketoadipic acid, which would cause endproduct inhibition of the main Trp catabolic pathway to CO2 (Figure 1), thereby increasing the flux of ACS to nicotinic acid mononucleotide and NAD. In assay 4 (Table 5), we did not observe an increase in the ratio of slopes (Trp:niacin) in chicks fed the diet series with 1% excess Lys. However, the basal level of digestible Trp (0.16%), although adequate for chicks fed niacin at its required level, would have been in considerable excess for chicks fed the niacin-deficient diets in this assay, in which growth rates ranged (by design) from about 30 to 60% of maximal. Thus, a portion of the basal level of Trp was likely catabolized, some of which would have gone to niacin. Excess Lys did increase the intercept value of the multiple linear regression equations, suggesting that the conversion of the basal diet level of Trp to niacin was indeed enhanced by excess dietary Lys.
Pellagra is the physiological outcome of niacin deficiency. This condition is prevalent in populations that depend on cereal-based diets that are low in bioavailable niacin and poor in protein quantity and quality (Goldsmith et al., 1952, 1961). Several studies have shown that excess dietary Trp can provide bioavailable niacin (e.g., approximately 50 mg of Trp is converted to 1 mg of bioavailable niacin; Krehl et al., 1945; Baker et al., 1973; Oduho and Baker, 1993), thereby showing that a dietary deficiency of Trp is a related factor in the etiology of pellagra in these populations. Although Trp is severely limiting in cereal-based diets, Lys is probably even more limiting, and this may also play a role in the physiological outcome of niacin deficiency. Our data certainly show that excess dietary Lys produces a growth response in chicks fed a niacin-deficient, Trp-adequate diet. It seems possible that with pellagragenic diets severely deficient in Lys, very little -ketoadipate (from Lys catabolism) would be available to endproduct inhibit the main catabolic pathway of Trp oxidation to CO2.
Our proposed mechanism for the Lys response in niacin-deficient chicks, though logical, has not been proven. Future research should be directed at measuring the activity of enzymes between ACS and -ketoadipic acid to establish whether they have indeed been inhibited by excess Lys ingestion. High activity of these enzymes has been shown to be correlated with the poor conversion of Trp to NAD in ducks (Chen et al., 1996) and the lack of Trp sparing of niacin in cats (DaSilva et al., 1952; Ikeda et al., 1965). Indeed, the decarboxylation of ACS catalyzed by picolinic carboxylase that leads to -aminomuconate semialdehyde biosynthesis (Figure 1) is a key enzyme in determining the yield of NAD from Trp turnover (Ikeda et al., 1965). Both cats and ducks have a very high activity of this enzyme relative to chickens, rats, and humans. Whether excess Lys by virtue of producing -ketoadipic acid as a metabolite might inhibit this enzyme remains to be determined. It would also be helpful to measure -ketoadipic acid per se in tissues of chicks fed diets with and without excess Lys.
We considered other possible explanations for the Lys response in niacin-deficient chicks. The FG Lys used in the basal diet might have had some niacin activity present as a contaminant, but the food-grade Lys used as a supplement in assays 4 and 5 was pure L-Lys·HCl. Thus, niacin activity contamination of our Lys product cannot explain the Lys response in niacin-deficient chicks. We also wondered about the HCl portion of L-Lys·HCl in terms of a possible beneficial effect on acid-base or cation:anion balance. However, we were able to obtain the same positive Lys response in niacin-deficient chicks given 1% excess Lys from L-Lys acetate (data not shown).
In assay 6, we evaluated a dietary concentration of 4,200 mg/kg of picolinic acid to observe whether it, like Lys, would produce a growth response in niacin-deficient chicks. Our reasoning was that this compound, being downstream from ACS, might endproduct inhibit ACS catabolism to CO2 and thereby increase the flux of ACS conversion to NAD. However, no response was obtained from this dose of picolinic acid. Perhaps the dose used (equimolar to 5,000 mg/kg of Lys) was not high enough to elicit a response.
The lack of an effect of dietary quinolinic acid on the growth of chicks fed a niacin-deficient, Trp-adequate diet is puzzling. Conversion efficiencies (wt/wt) of quinolinic acid to NAD in rats have been reported to be very low (Henderson, 1949; Krehl et al., 1950; Shibata and Murata, 1991). Dann et al. (1940) found that a quinolinic acid dose 25 times that of niacin was totally ineffective in preventing canine blacktongue. However, a dose 100 times greater than niacin gave complete protection. Krehl et al. (1950) observed that 3-hydroxyanthranilic acid was vastly superior to quinolinic acid as a niacin precursor for rats. In assay 6, we supplemented quinolinic acid at a molar ratio to niacin of 12.3:1, but no growth response occurred. The adequacy of dietary Trp in our assay, as opposed to the deficiency of Trp in the research referenced above, may lead to the production of nonlimiting amounts of quinolinic acid for NAD biosynthesis, the supplementation of which would not lead to increased production of niacin. Additionally, quinolinate transphosphoribosylase, the enzyme required to convert quinolinic acid to niacin, has been reported to be only in the liver of rats (Ikeda et al., 1965), and Ijichi et al. (1966) reported that labeled quinolinic acid penetrated into rat liver cells to a very small degree, indicating that, if these relationships hold true in poultry species, exogenous quinolinic acid would be poorly available for conversion to NAD. Although inefficient as a precursor, in vivo studies have shown growth responses from large quantities of oral quinolinate in niacin-deficient rats. Because quinolinic acid is present in foods (Shibata et al., 1985), the exact conversion efficiency of quinolinate to NAD needs more attention. Noteworthy, however, is that giving quinolinic acid as a dietary supplement probably does not reflect the same thing as quinolinic acid being synthesized from ACS in liver cells.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication August 25, 2006. Accepted for publication October 22, 2006.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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