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Article

Single Versus Multiple Deficiencies of Methionine, Zinc, Riboflavin, Vitamin B-6 and Choline Elicit Surprising Growth Responses in Young Chicks

Department of Animal Sciences and Division of Nutritional Sciences, University of Illinois, Urbana, IL 61801

¹To whom correspondence should be addressed.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
A soy-protein isolate diet that was deficient in methionine (Met), zinc (Zn), riboflavin, vitamin B-6 and choline for chick growth (Assay 1) was used to study individual or multiple deficiencies of several of these nutrients. In all cases, adding all three deficient nutrients together resulted in growth responses that were superior to those resulting from supplementation with any pairs of deficient nutrients. In Assay 2, single addition of Zn but not of methionine or riboflavin produced a growth response, but the combination of either Zn and Met or Zn and riboflavin resulted in growth responses that were greater than the response elicited by Zn alone. Assay 3 involved individual or multiple deficiencies of choline, riboflavin and vitamin B-6, and individual additions suggested that choline was first limiting. Choline + riboflavin supplementation, however, produced marked growth and gain:food responses that were far greater than those resulting from supplemental choline or riboflavin alone. Moreover, the growth response to a combination of choline + pyridoxine (PN) was also greater than that obtained from any of the three nutrients fed alone; even PN + riboflavin (in the absence of choline) produced responses greater than those observed with the unsupplemented negative-control diet. In Assay 4, chicks responded to individual additions of riboflavin, PN or Met, and in Assay 5, to either riboflavin or PN; all two-way combinations resulted in growth rates that were far greater than those occurring with any single addition. The data from these experiments show that unlike the situation with three deficient amino acids, the expected responses to first-, second- and third-limiting B-vitamins or deficient vitamins combined with deficient levels of Zn or Met do not follow the expected pattern of response to first-, further response to first- and second- and an even further response to first-, second- and third-limiting nutrients.

KEY WORDS: • choline • methionine • zinc • riboflavin • pyridoxine • chicks • multiple deficiency

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Diets with three deficient amino acids elicit growth responses to first- but not to second- or third-limiting amino acids; then, further responses to first- and second- but not to first- and third- or second- and third-limiting amino acids, and finally to all three in greater magnitude than to any pair of deficient amino acids (Biehl and Baker 1997 ). The soy-protein isolate (SPI)² diet developed in our laboratory (Emmert and Baker 1995 and 1997 , Emmert et al. 1996 , Mavromichalis and Baker 1999 , Patel and Baker 1996 ) to study various nutrient deficiencies was an ideal tool with which to evaluate chick growth responses to single vs. multiple deficiencies of various three-way combinations among methionine (Met), zinc (Zn), riboflavin, pyridoxine (PN) and choline.

Our objective was to determine whether the following multiple deficiencies would result in the same pattern of responses one would expect from diets containing three deficient amino acids: 1) riboflavin, Zn and Met; 2) riboflavin, PN and choline; or 3) riboflavin, PN and Met. The results were surprising and very different from what would be expected from growth responses to first-, second- and third-limiting amino acids.

	MATERIALS AND METHODS

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General procedures.

All procedures were approved by the University of Illinois Committee on Laboratory Animal Care. Five bioassays were conducted with male chicks from the cross of New Hampshire males and Columbian females (University of Illinois Poultry Farm, Urbana, IL). Chicks were housed in thermostatically controlled battery pens equipped with raised wire floors in an environmentally controlled laboratory room with 24-h continuous fluorescent lighting. All equipment, including batteries, feeders, water trays and feed mixing equipment was of stainless steel construction. Water and experimental diets were freely available, and diets were formulated to meet or exceed NRC (1994) requirements for all essential nutrients with the exception of those under investigation. Chicks were fed a conventional 24% crude protein diet during the first 7 d posthatching, except for Assay 2 in which a Zn-deficient pretest diet was fed. On the morning of d 8 posthatching, after 16 h without either diet and water, the chicks were wingbanded, weighed and then assigned to battery pens in a manner that ensured minimal variation in initial body weight among pens.

Assay 1 involved three pens of four chicks for each diet during an experimental feeding period of 8–22 d posthatching. Assays 2, 3, 4 and 5 employed four pens of four chicks per diet during experimental feeding periods of 8–20 d (Assay 2) or 8–21 d (Assays 3, 4 and 5) posthatching.

Basal diet.

The complete basal SPI diet (Table 1 ) was developed and characterized over several years for purposes of studying utilization of several nutrients (Emmert and Baker 1995 and 1997 , Patel and Baker 1996 ). The SPI product used was a functional alcohol-extracted soy product (Ardex AF, ADM, Decatur, IL). Chemical analyses of this product yielded the following results: 824 g/kg crude protein (macro-Kjeldahl), 49 g/kg lipid (chloroform-methanol extraction), 89 g/kg H₂O, 21.8 MJ/kg gross energy (bomb calorimetry), 21.8 g/kg Met + cystine, 31.7 g/kg threonine and 51.7 g/kg lysine (Emmert and Baker 1995 ). Amino acids were quantified by ion-exchange chromatography (Model 119 CL, Beckman Instruments, Palo Alto, CA) after performic acid preoxidation followed by 24-h acid hydrolysis under a nitrogen atmosphere. The Zn content of SPI (50.5 mg/kg) was determined by atomic absorption spectrophotometry (Model 306, Perkin-Elmer, Norwalk, CT) according to conventional wet-ashing procedures.

In the four bioassays involving multiple deficiencies of one, two or three essential nutrients (Assays 2, 3, 4 and 5), the remaining essential nutrients were at or in excess of NRC (1994) requirements. For levels of the B-vitamins not under investigation, the following percentages of NRC (1994) required levels were provided as supplements to the SPI basal diet: thiamin, 890%; nicotinic acid, 140%; D-pantothenic acid, 264%; vitamin B-12, 400%; D-biotin, 400%; and folacin, 730%.

Assay 2 was designed to test single vs. multiple deficiencies of riboflavin, Zn and Met. The data of Assay 1 had clearly shown that the SPI basal diet was severely deficient in both riboflavin and Met, and previous work in our laboratory (Edwards and Baker 1999 ) had demonstrated that the diet was also deficient in Zn. Indeed, it contained only 12 mg Zn/kg, and much of the Zn in SPI is bound to phytate and therefore unavailable. The chicks in this assay were pretested for 3 d on the SPI basal diet, which contained no supplemental Zn. This was done to deplete Zn stores and ensure that Zn supplementation would elicit a growth response during the subsequent 12-d assay period. The deficient levels of riboflavin (1.5 mg/kg), Zn (no added Zn) and Met (no added DL-Met) were selected to provide levels of these nutrients that would cause growth rates of between 25 and 50% of the growth rates produced by adequate levels of riboflavin (3.5 mg/kg, cf. Patel and Baker 1996 ), Zn (50 mg/kg) and Met (2000 mg/kg added DL-Met).

Deficiencies of choline (no added choline), riboflavin (1.5 mg/kg) and vitamin B-6 (0.5 mg/kg) in all possible combinations were studied in Assay 3. The results of previous research in our laboratory had suggested that the SPI diet with no supplemental choline, and containing these levels of riboflavin and PN, would result in a growth reduction of ~50% (Emmert and Baker 1997 , Lowry et al. 1987 , Patel and Baker 1996 , Yen et al. 1976 ).

In Assay 4, deficient vs. adequate levels of riboflavin (1.5 vs. 3.5 mg/kg), PN (0.5 vs. 2.5 mg/kg) and Met (0 vs. 2000 mg/kg added) were fed in all possible combinations. Our previous research had established that adequate levels of these nutrients were well above the minimal requirements of 2.5 mg/kg riboflavin (Chung and Baker 1990 , Patel and Baker 1996 ), 1 mg/kg PN (Yen et al. 1976 ) and 1100 mg/kg of supplemental DL-Met (unpublished data).

Because of the unusual nature of the responses in Assays 2, 3 and 4, a final assay (Assay 5) was conducted with deficient vs. adequate levels of riboflavin (1.5 vs. 3.5 mg/kg) and PN (0.5 vs. 2.5 mg/kg). After the 13-d feeding period, diets were removed for 6 h, after which the chicks were killed by CO₂ gas. In replicates 1 and 2, several tissues were quantitatively removed to determine whether riboflavin and/or vitamin B-6 deficiency influenced the weight of heart, liver, kidney, spleen, pancreas and bursa. Chicks in the remaining two replicates were used to determine whole-body protein. Ten representative chicks had been killed at the beginning of the 13-d assay so that protein accretion could be calculated. Procedures used in processing the chicks for assessment of whole-body protein (nitrogen x 6.25) have been outlined previously (Baker et al. 1996 ).

Statistical analyses.

All bioassays were completely randomized designs, and pen means in all cases were used in statistical analyses. Pooled SD were calculated for data within diet groups of Assay 1, because differential mortality occurred and also because an additional dietary treatment (Met supplementation) was evaluated in the SPI group but not in the group fed amino acid diets. The least significant difference (LSD) multiple comparison procedure (Carmer and Walker 1985 ) was used to establish differences among means within each diet group. The remaining bioassays involved either three-factor (Assays 2, 3 and 4) or two-factor (Assay 5) factorial arrangement of treatments. These assays were analyzed using orthogonal single df comparisons, but means separation using the LSD procedure was also done. Pooled SEM were computed for all data in Assays 2, 3, 4 and 5.

	RESULTS

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Assay 1 (nutrient deficiencies in SPI diet).

Among the supplemental nutrients deleted from either the nutritionally complete amino acid diet or the SPI diet, riboflavin and thiamin deletion resulted in the greatest reductions in growth and food utilization (Table 2 ). The growth depression caused by PN deletion was also severe. That the SPI diet contained essentially zero bioavailable riboflavin, thiamin and vitamin B-6 is evidenced by the comparison of growth depressions occurring in chicks within diet groups. Expressed as a percentage of the growth occurring in chicks fed the positive-control complete amino acid or SPI diet, the growth depressions occurring from deleting each of these B-vitamins were virtually the same for both diets. Mortality of chicks fed riboflavin-free diets during the 14-d feeding period was greater (P < 0.05) in fact for chicks fed the SPI diet than for those fed the amino acid diet.

Assay 2 (riboflavin, zinc, and Met deficiency).

Single addition of Zn, but not riboflavin or Met, elicited a small but significant (P < 0.05) increase in weight gain; this was due primarily to an increase in voluntary food intake (Table 3 ). Viewed in another way, a singular deficiency of Zn (diet 6) depressed both gain and gain:food ratio more (P < 0.05) than singular deficiencies of either riboflavin (diet 7) or Met (diet 5), and riboflavin deficiency depressed chick performance more (P < 0.05) than Met deficiency. This suggested that the basal diet was first limiting in Zn, second limiting in riboflavin, and third limiting in Met.

Assay 3 (choline, riboflavin and vitamin B-6 deficiency).

Single addition of choline (but not riboflavin or PN) resulted in a 50% growth response (P < 0.05) that was mediated by an increase in voluntary food intake (Table 4 ). Thus, choline supplementation resulted not only in increased choline intake but also in a 40% increase in the consumption of both riboflavin and PN. Adding riboflavin alone did not increase weight gain but did increase (P < 0.05) gain:food ratio. Viewed in the form of a deletion assay, single deletion of choline (diet 7) depressed gain and food efficiency more (P < 0.05) than single deletion of riboflavin (diet 6), which in turn depressed weight gain more (P < 0.05) than single deletion of PN (diet 5). These results suggested that choline, riboflavin, and PN were first-, second- and third-limiting nutrients, respectively.

Assay 4 (riboflavin, vitamin B-6 and Met deficiency).

The basal diet in this assay was designed to be approximately equally limiting in riboflavin, vitamin B-6 and Met (Table 5 ). Addition of any one of these nutrients to the basal diet increased (P < 0.05) weight gain, a significant portion of which could be attributed to increased food intake in the case of riboflavin or PN supplementation. Thus, the increased food intake resulting from either of these additions caused increased intakes of all three deficient nutrients. With Met supplementation, however, the 24% increase in weight gain occurred primarily as a result of improved food utilization. Evaluation of the data as nutrient deletions rather than additions provided some help in predicting the limiting order of nutrients in the basal diet. Thus, relative to the complete diet (diet 8), Met deficiency (diet 5) depressed gain and gain:food ratio more (P < 0.05) than singular deficiencies of either riboflavin (diet 7) or PN (diet 6). Performance reductions due to riboflavin deficiency or PN deficiency were similar. These results suggested that Met was first limiting and riboflavin and PN were equally second limiting.

Assay 5 (riboflavin and B-6 deficiency).

Similar to the findings in Assay 4, addition of either riboflavin or PN increased (P < 0.05) weight gain and gain:food ratio, and roughly one half of the gain response could be attributed to increased food intake. Because riboflavin elicited a greater performance response over the negative-control diet (diet 1) than PN, and because deletion of riboflavin (diet 3) from the positive-control diet (diet 4) caused a greater growth depression than that caused by PN deletion (diet 2), it appeared that the basal diet was first limiting in riboflavin and second limiting in vitamin B-6. Thus, it was surprising that PN supplementation in the absence of riboflavin would increase weight gain by 56% and gain:food ratio by 21%. The riboflavin x PN interaction (P < 0.05) for weight gain is a reflection of the finding that PN supplementation in the presence of riboflavin increased weight gain more than PN supplementation in the absence of riboflavin. Protein accretion responses to riboflavin and/or PN supplementation paralleled the weight gain responses. The 53% increase in protein accretion resulting from supplemental PN alone, and in the absence of the first-limiting nutrient (riboflavin), was remarkable.

Among the tissues quantitatively removed and weighed at the end of the 13-d experimental feeding period, only heart, pancreas and bursa weight (mg/100 g fresh tissue) were affected (P < 0.05) by dietary treatment. Chicks fed diets deficient in riboflavin (diets 1 and 3) had smaller hearts and bursas than those fed riboflavin-adequate diets (diets 2 and 4). In addition, pancreas weight was greater in riboflavin-deficient chicks. Vitamin B-6 deficiency in the presence of riboflavin adequacy (diet 2) had no effect on any of the organs evaluated.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
We are unaware of any previous studies in which single, two-factor and three-factor deficiencies of various combinations of Met, Zn, choline, riboflavin and vitamin B-6 have been compared in a laboratory animal model. The five nutrients employed in our studies were selected for the following two reasons: 1) the SPI diet developed in our laboratory could easily be made deficient in any one, or any combination, of these five nutrients; and 2) all five nutrients have a multitude of well-established functions in the body. How these functions are prioritized during a nutrient deficit is not known, although it is known that phospholipid and/or acetyl choline synthesis are prioritized in avians over the methylation function (as betaine) of choline (Emmert et al. 1996 , Emmert and Baker 1997 , Lowry et al. 1987 ). Because choline via betaine supplies methyl groups for the synthesis of Met from homocysteine, and also to the folate pool (which also is used for Met synthesis from homocysteine), there is an obvious relationship between Met and choline. However, Met similarly supplies methyl groups for phosphatidyl choline biosynthesis, although the first of the three methylation reactions required in this process is very inefficient in the chick (Baker and Sugahara 1970 , Jukes et al. 1945 ).

There are many other functional interrelationships among the nutrients studied herein. Both of the reactions leading to Met synthesis from homocysteine are catalyzed by Zn-dependent enzymes, i.e., betaine-homocysteine methyltransferase (Millian and Garrow 1998 ) and methionine synthase (Goulding and Mathews 1997 ). Also, vitamin B-6 as pyridoxal phosphate (PLP) is required in two of the transsulfuration reactions leading to cysteine synthesis from methionine, and riboflavin is required in the conversion of PN to PLP (Kazarinoff and McCormick 1975 , Morisue et al. 1960 ). Specifically, Zn²⁺ is involved as a 1:1 chelate with ATP as cosubstrate in the kinase-catalyzed phosphorylation of each of the three B-6 vitamers [PN, pyridoxal (PL) and pyridoxamine] as shown by McCormick et al. (1961) , and riboflavin 5'-phosphate (FMN) is the coenzyme for the oxidase that converts the 5'-phosphates of PN and pyridoxamine to the functional coenzyme PL 5'-phosphate (Kazarinoff and McCormick 1975 ). Clearly, nutrients such as riboflavin, vitamin B-6 and Zn are needed in numerous metabolic reactions, both anabolic and catabolic, involving carbohydrate, lipid and protein metabolism.

In assays 2 and 3 (Tables 3 and 4) , riboflavin appeared to be second limiting in the basal diet after Zn (Assay 2) or choline (Assay 3). Supplementation with the first-limiting nutrient (alone) in these assays increased weight gain, and the growth response could be explained almost entirely by increased voluntary food intake. Because the basal diet contained 1.5 mg/kg riboflavin and 0.5 mg/kg PN, the increased diet consumption caused by adding the first-limiting nutrient also resulted in increased consumption of the second-limiting nutrient, riboflavin and the third-limiting nutrient (Met in Assay 2 and PN in Assay 3). Perhaps the most surprising findings in Assay 3 (Table 4) were the following: 1) that the combined supplement of choline (first limiting) and PN (third limiting) in the absence of added riboflavin (second limiting) would cause both gain and gain:food to increase over that resulting from choline supplementation alone; and 2) that the combination of supplemental riboflavin and PN, without added choline (first limiting), would increase weight gain by 48% over the basal diet. Although increased food intake accounted for some of these responses (Baker 1984 ), the vexing question remains of how (or why) does addition of second- and third-limiting nutrients in the absence of the first-limiting nutrient elicit such a marked increase in voluntary food intake.

The most unusual findings were encountered in Assay 4 (Table 5) . On the basis of classical nutrient addition or deletion assay methodology and thinking, one would conclude that Met was first limiting in the basal negative-control diet and that riboflavin and vitamin B-6 were probably equally second limiting. If this is true, however, how does one explain that single addition of any one of the three deficient nutrients elicited a significant (P < 0.05) response in weight gain—mediated importantly by increased diet consumption, at least with riboflavin or PN supplementation, but mediated by improved diet utilization with Met supplementation? Equally perplexing is why voluntary food intake (hence weight gain) was so markedly increased when the two second-limiting nutrients (riboflavin and PN) were supplemented in the absence of the first-limiting nutrient (Met).

In the last bioassay, riboflavin and vitamin B-6 deficiency were evaluated (Table 6 ), and even though riboflavin appeared to be first limiting and PN second limiting in the basal diet, addition of either one elicited a large growth response that could be attributed to both increased food consumption and improved food utilization. That the growth responses to single addition of either riboflavin or PN were "true" tissue growth responses and were not attributable to water retention is indicated by the protein accretion responses to each nutrient. The finding that PN supplementation markedly increased weight gain and protein accretion when added to a diet that was first limiting in riboflavin indicates that the need for riboflavin in the form of FMN for the catalytic function of PN (pyridoxamine) 5'-phosphate oxidase (Kazarinoff and McCormick 1975 ) may represent a priority function for riboflavin (McCormick and Chen 1999 ). PN was the form of vitamin B-6 supplemented, but this is known to be converted in part to PN phosphate and then to PLP. The riboflavin growth response observed in our vitamin B-6–deficient chicks may thus have resulted from riboflavin functioning to convert the deficient level (0.5 mg/kg) of vitamin B-6 (all as PN) in our basal diet to its active coenzyme form, i.e., PLP. On the other hand, the reduced size of the heart and bursa as well as the pancreatic hypertrophy observed in our riboflavin-deficient chicks appeared to be due to riboflavin deficiency per se. Hence, only riboflavin supplementation normalized the relative size of these organs.

	FOOTNOTES

 
² Abbreviations used: FMN, riboflavin 5'-phosphate; LSD, least significant difference; Met, methionine; PL, pyridoxal; PLP, pyridoxal phosphate; PN, pyridoxine; SPI, soy-protein isolate; Zn, zinc.

³ The increased food intake in chicks fed diet 7 over those fed diet 1 also resulted in increased Met intake. However, unlike the situation in mammals, Met cannot replace (or spare) choline in avians (Molitoris and Baker 1976 ).

Manuscript received June 9, 1999. Initial review completed July 13, 1999. Revision accepted August 4, 1999.

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3. Baker D. H., Sugahara M. Nutritional investigation of the metabolism of glycine and its precursors by chicks fed a crystalline amino acid diet. Poult. Sci. 1970;49:756-760

4. Biehl R. R., Baker D. H. Microbial phytase improves amino acid utilization in young chicks fed diets based on soybean meal but not in diets based on peanut meal. Poult. Sci. 1997;76:355-360[Abstract/Free Full Text]

5. Carmer S. G., Walker W. M. Pairwise multiple comparisons of treatment means in agronomic research. J. Agron. Educ. 1985;14:19-26

6. Chung T. K., Baker D. H. Riboflavin requirement of chicks fed purified amino acid and conventional corn-soybean meal diets. Poult. Sci. 1990;69:1357-1363[Medline]

7. Edwards, H. M., III & Baker, D. H. (1999) Bioavailability of zinc in several sources of zinc oxide, zinc sulfate, and zinc metal. J. Anim. Sci. (in press).

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9. Emmert J. L., Baker D. H. A chick bioassay approach for determining the bioavailable choline concentration of normal and overheated soybean meal, canola meal and peanut meal. J. Nutr. 1997;127:745-752[Abstract/Free Full Text]

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16. Mavromichalis I., Baker D. H. Methodology for conducting B-vitamin growth requirement assays. FASEB J 1999;13:A230(abs)

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