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Department of Animal Sciences and Division of Nutritional Sciences, University of Illinois, Urbana, IL 61801
1To whom correspondence should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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1 mg/kg,
but not at PN doses of 1.2 or 1.4 mg/kg. Because dietary intakes of
both vitamin B-6 and methionine can affect plasma homocysteine levels,
dietary methionine (and protein) intake should be considered important
factors in setting safe and adequate requirement levels for vitamin
B-6.
KEY WORDS: • vitamin B-6 • methionine • protein • chicks • requirements
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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, Bender 1985
, Canham et al. 1969
, Driskell 1984
, Leklem 1991
,
Morgan et al. 1946
). The chick studies of Daghir and Shah (1973)
and Gries and Scott (1972)
,
together with the rat study of Okada et al. (1998)
, also
provided qualitative evidence that excess protein increases the dietary
requirement for vitamin B-6. Our recent chick work (Scherer and Baker 2000
) demonstrated that doubling the protein level from
200 to 400 g/kg, using methionine (Met)-fortified soy-protein
isolate, increased the vitamin B-6 requirement for maximal growth by
45%. We questioned whether this effect was due to protein (or excess
amino acids) per se, or whether there might be a single amino acid,
e.g., Met, that might be causing most of the effect.
Vitamin B-6 [as pyridoxal phosphate
(PLP)],2is intimately involved in sulfur amino acid (SAA) metabolism. In the
transsulfuration pathway, homocysteine (+ serine) conversion to
cystathionine, and cystathionine conversion to cysteine,
-ketobutyrate and ammonia require PLP. Of the homocysteine produced
from Met catabolism in mammals, an estimated 50% is remethylated to
Met, and roughly half of the homocysteine remethylation that occurs
uses 5-methyltetrahydrofolate as a methyl donor (Finkelstein 1990
). The biosynthesis of serine, with its subsequent
conversion to glycine, generates a methyl group, and this
PLP-requiring reaction is an important contributor to the folate
pool for use in remethylating homocysteine to Met (Martinez et al. 2000
). Thus, in the overall process of transsulfuration,
there are three key PLP-requiring reactions. In addition, several
S-adenosylmethionine–requiring reactions also require PLP as a
cofactor, e.g., the conversion of ornithine to putrescine, putrescine
to spermidine and spermidine to spermine. Moreover, one of the pathways
in cysteine catabolism involves transamination, which is a
PLP-dependent reaction.
Because vitamin B-6 status can affect the level of both homocysteine
(Leklem 1991
, Martinez et al. 2000
,
Rassin et al. 1977
, Ubbink et al. 1996
,
Wilcken and Wilcken 1998
) and cystathionine
(Andersson et al. 1990
, Leklem 1990
,
Linkswiler 1981
) in blood and urine, we attempted herein
to use the chick as a model for purposes of determining whether excess
dietary Met per se might increase the dietary need for vitamin B-6. In
a quantitative study involving both vitamin B-6 and Met, the chick is a
very useful animal model in that transsulfuration in avian species is
similar to that in mammals (Emmert et al. 1996
).
Moreover, chicks, unlike rats, do not practice coprophagy, a factor
that could confound interactive results of a vitamin B-6 dosing study.
It is well documented that an elevation in the circulating level of
homocysteine represents an independent risk factor for cardiovascular
disease in humans (Wilcken and Wilcken 1998
). Thus, if
excess Met ingestion caused by high protein diets were to exacerbate
vitamin B-6 deficiency, also a factor that causes homocysteinemia
(Martinez et al. 2000
, Miller et al. 1994
, Selhub et al. 1998, Smolin and Benevenga 1984
), high protein or high Met diets might
appropriately be added to the growing list of factors that contribute
to cardiovascular disease.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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All procedures were approved by the University of Illinois
Committee on Laboratory Animal Care. Two 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 vitamin
B-6. Chicks were fed a conventional 24% crude protein diet during the
first 7 d posthatching. On the morning of d 8 posthatching, after
16 h without either feed or 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. The two
experiments involved four pens of four chicks for each diet during a
12-d experimental feeding period of 8–20 d posthatching.
Basal diet.
The basal soy-protein isolate diet (Table 1
) was developed and characterized over several years for purposes of
studying utilization of several nutrients (Baker et al. 1999
, Emmert and Baker 1995 and 1997
,
Patel and Baker 1996
). The soy-protein isolate
product used was a functional alcohol-extracted soy product (Ardex
AF, ADM, Decatur, IL). Chemical analysis 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),
11.0 g/kg Met, 10.8 g/kg 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 24-h acid hydrolysis under a nitrogen
atmosphere. To quantify methionine and cyst(e)ine, performic acid
preoxidation (Moore 1963
) preceded acid hydrolysis. The
preoxidation procedure converts Met to Met sulfone and cyst(e)ine to
cysteic acid, both of which are stable under conditions of acid
hydrolysis. After quenching the excess performic acid with sodium
disulfite, the sample was hydrolyzed with concentrated HCl. Previous
work from our laboratory had established that 2 g/kg of supplemental
DL-Met3would meet the minimal level of SAA required for maximal growth of
chicks fed the basal diet containing a superadequate level (5 mg/kg) of
pyridoxine (PN) (Mavromichalis and Baker 2000
,
Scherer and Baker 2000
, Yen et al. 1976
).
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Experiment 1.
Graded levels of excess supplemental DL-Met were added to
the basal diet made superadequate in vitamin B-6 (5 mg/kg supplemental
PN) to determine a level of excess Met that could be tolerated without
causing a growth depression. Previous results from our laboratory had
shown that 10 g/kg of excess DL-Met added to a conventional
corn-soybean meal diet would not depress either weight gain or food
efficiency (Han and Baker 1993
). Thus, 10, 20 or 30 g/kg
of added DL-Met were tested in the soy-protein isolate
semipurified diet to establish whether a 10 g/kg DL-Met
supplement would similarly be a tolerable excess in this diet.
Experiment 2.
A 2 x 7 factorial arrangement of treatments was used in this
bioassay, involving two levels of supplemental DL-Met added
to the Met-adequate basal diet (none, i.e., adequate, and 10 g/kg,
i.e., excess) and seven graded doses of PN ranging from 0.20 to 1.4
mg/kg. Our previous work with PN additions to the basal diet shown in
Table 1
indicated that these dosage levels of PN would cover both the
linear and plateau portions of the growth-response curve. The
objective of the bioassay was to determine whether excess Met might
depress growth at deficient but not at adequate levels of vitamin B-6,
and also to define dietary requirements for vitamin B-6 under
conditions of adequate and excess dietary Met.
Statistical analyses.
Both experiments were completely randomized designs. After ANOVA
of pen means data, orthogonal single df comparisons were made to
evaluate treatment differences (Steel and Torrie 1980
).
Linear and quadratic responses to excess Met were evaluated in
Experiment 1, and Met and PN (linear and quadratic) main effects and
their interaction were determined in Experiment 2. The weight gain data
in Experiment 2 were also fitted to a one-slope broken-line model
(Robbins et al. 1979
, Robbins 1986
) in
which gain was regressed on dietary PN level for chicks fed either an
adequate or an excess level of Met.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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) clearly established that 10 g/kg of supplemental DL-Met
represented a tolerable excess in that weight gain, voluntary food
intake and gain:food ratio were not reduced (P > 0.10)
from this level of excess Met. Excess Met doses of 20 or 30 g/kg,
however, depressed all measures of response. This bioassay established
that chicks fed the basal diet containing adequate Met and surfeit PN
could tolerate a 10 g/kg Met excess dose without exhibiting a growth
depression. The 10 g/kg level of excess Met was therefore used in
Experiment 2 to evaluate the effects of excess Met on responses to
graded increments of PN.
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in which the observed pen means data points for PN doses at each level
of Met were fitted to one-slope broken lines. The breakpoint
estimate of the PN requirement for maximal growth occurred at 0.73
± 0.04 mg/kg for chicks fed diets with adequate Met and at 1.05
± 0.04 mg/kg for chicks fed diets with 10 g/kg excess Met. The
difference was significant (P < 0.01) and represented
a 44% increase in the vitamin B-6 requirement of chicks fed excess
Met. The interaction of PN level x Met level was significant
(P < 0.01), and this is explained by the finding that
weight gain was depressed by excess Met at all PN dose levels 1.0
mg/kg, but the growth depression did not occur at higher PN dose levels
of 1.2 and 1.4 mg/kg. Food intake and gain:food ratio data are not
shown, but responses in these criteria generally followed the same
pattern as gain responses.
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It appears that the excess Met contained in excess protein may explain
a good portion of the excess protein–exacerbating effect on vitamin
B-6 utilization. On the basis of the recent work of Martinez et al. (2000)
, the need for PLP in catalyzing the serine
hydroxymethyltransferase and 
-cystathionase reactions may be more
important than the need for PLP in the cystathionine ß-synthase
reaction. Indeed, Sato et al. (1996)
showed that vitamin
B-6 deficiency in rats increases the proportion of hepatic
-cystathionase in apoenzyme form, and also increases the catabolism
of the enzyme. We did not measure plasma homocysteine in our chicks,
but deficiencies of vitamin B-6 are known to elevate plasma
homocysteine (Martinez et al. 2000
, Miller et al. 1994
, Selhub et al. 1993
, Smolin and Benevenga 1984
). Miller et al. (1994)
also found
that Met loading was additive with vitamin B-6 deficiency in causing
elevations of plasma homocysteine in rats.
That a level of Met that does not depress growth (when dietary vitamin
B-6 is superadequate) increases the vitamin B-6 requirement of chicks
by 44%, approximately the same as that caused by a doubling of the
protein (Scherer and Baker 2000
), implies that Met is
the primary component of excess protein that is causing this effect.
Most amino acids require PLP in their catabolism, but Met catabolism
requires PLP in several steps. Also, Met is well established as being
among the most toxic of all amino acids when fed at excess levels in a
diet (Edmonds and Baker 1987
, Edmonds et al. 1987
).
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FOOTNOTES |
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3 Like rats, mice and pigs, avian species use the
D-isomer of Met almost as efficiently as the
L-isomer (Baker 1994).
Manuscript received June 22, 2000. Revision accepted August 31, 2000.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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1. Andersson A., Brattsttrom L., Israelsson B., Isaksson A., Hultberg B. The effect of excess daily methionine intake on plasma homocysteine after a methionine loading test in humans. Clin. Chim. Acta 1990;192:69-76[Medline]
2. Bai S. C., Sampson D. A., Morris J. G., Rogers Q. R. The level of dietary protein affects the vitamin B-6 requirement of cats. J. Nutr. 1991;121:1054-1061
3. Baker D. H. Utilization of precursors for L-amino acids. Amino Acids in Farm Animal Nutrition 1994:37-63 CAB International London, UK.
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Baker D. H., Edwards H. M., III, Strunk C. S., Emmert J. L., Peter C. M., Mavromichalis I., Parr T. M. Single versus multiple deficiencies of methionine, zinc, riboflavin, vitamin B-6, and choline elicit surprising growth responses in young chicks. J. Nutr. 1999;129:2239-2246
5. Bender D. A. The role of vitamin B-6 in amino acid metabolism. Amino Acid Metabolism 2nd ed. 1985:75-94 Wiley New York, NY.
6. Canham J. E., Baker E. M., Harding R. S., Sauberlich H. E., Plough I. C. Dietary protein—its relationship to vitamin B-6 requirements and function. Ann. N.Y. Acad. Sci. 1969;166:16-29[Medline]
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10. Edmonds M. S., Gonyou H. W., Baker D. H. Effect of excess levels of methionine, tryptophan, arginine, lysine or threonine on growth and dietary choice in the pig. J. Anim. Sci. 1987;65:179-185
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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
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14. Finkelstein J. D. Methionine metabolism in mammals. J. Nutr. Biochem. 1990;1:228-237[Medline]
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20.
Martinez M., Cuskelly G. J., Williamson J., Toth J. P., Gregory J. F. Vitamin B-6 deficiency in rats causes reduced serine hydroxymethyltransferase and cystathionine ß-synthase activity and in vivo impairment of protein turnover, homocysteine remethylation, and transsulfuration. J. Nutr. 2000;130:1115-1123
21. Mavromichalis I., Baker D. H. Pretest depletion of body reserves of riboflavin and vitamin B-6 in young chicks has little effect on the subsequent requirement for these B-vitamins. J. Anim. Sci. 2000;(in press)
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Moore S. On the determination of cystine as cysteic acid. J. Biol. Chem. 1963;238:235-237
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27. Patel K., Baker D. H. Supplemental iron, copper, zinc, ascorbate, caffeine and chlortetracycline do not affect riboflavin utilization in the chick. Nutr. Res. 1996;16:1943-1952
28. Rassin D. K., Longhi R. C., Sternowsky H. J., Sturman J. A., Gaull G. E. Homocysteine and cysteine loads in patients with homocysteinuria due to cystathionine synthase deficiency: effects of vitamin B-6. Clin. Chim. Acta 1977;79:197-210[Medline]
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31. Sato A., Nishioka M., Awata S., Nakayama K., Okada M., Horiuchi S., Okabe N., Sassa T., Oka T., Natori Y. Vitamin B-6 deficiency accelerates metabolic turnover of cystathionase in rat liver. Arch. Biochem. Biophys. 1996;330:409-413[Medline]
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33. Selhub J., Jacques P. F., Wilson P. W. F., Rush D., Rosenberg I. H. Vitamin status and intake as primary determinants of homocysteinemia in an elderly population. J. Am. Med. Assoc. 1993;70:2693-2698
34. Smolin L. A., Benevenga N. J. Factors affecting the accumulation of homocyst(e)ine in rats deficient in vitamin B-6. J. Nutr. 1984;114:103-111
35. Steel R.G.D., Torrie J. H. Principles and Procedures of Statistics: A Biometrical Approach 2nd ed. 1980 McGraw Hill New York, NY.
36. Ubbink J. B., van der Merwe A., Delport R., Allen R. H., Stabler S. P., Riezler R., Vermaak W. J. The effect of a subnormal vitamin B-6 status on homocysteine metabolism. J. Clin. Investig. 1996;98:177-184[Medline]
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38. Yen J. T., Jensen A. H., Baker D. H. Assessment of the concentration of biologically available vitamin B-6 in corn and soybean meal. J. Anim. Sci. 1976;42:866-870
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