632
RESEARCH NOTES
Nir, I., Z. Nitsan and A. Vax, 1973. The influence of force feeding and of protein supplementation to the diet on the metabolizable energy of diets, digestibility of nutrients, nitrogen retention and digestive enzymes output. Ann. Biol. Anim. Bioch. Biophys. 13: 465-479. Nir, I., and M. Perek, 1971. The effect of various protein levels in feed of goslings during the preparatory period on fatty liver production and blood plasma components. Ann. Biol. Anim. Bioch. Biophys. 11: 645-656. Stevenson, J. A. F., B. M. Box and A. J. Szlavko, 1964. A fat-mobilizing and anorectic substance in the urine of fasting rats. Proc. Soc. Exptl. Biol. Med. 115:424-429. Weil, R., and De W. Stetten, Jr., 1947. Urinary excretion of a fat-mobilizing agent. J. Biol. Chem. 168: 129-132.
A THEORETICAL METHOD FOR T H E DETERMINATION O F AMINO ACID A V A I L A B I L I T Y U T I L I Z I N G 14C L A B E L E D P R O T E I N S O U R C E S ' S. V. A M A T O , 2 R. M . FORBES AND H . M . SCOTT
Department of Animal Science, University of Illinois, Urbana, Illinois 61801 (Received for publication October 4, 1974)
ABSTRACT A theoretical method is described whereby the availabilities of amino acids from test proteins are determined relative to the availabilities of amino acids from egg albumen. The method requires that both the test protein and the egg albumen be ,4C labeled. The relative ' availabilities of the amino acids are determined from the change in specific activities of the dietary amino acids and those recovered in the feces. POULTRY SCIENCE 54: 632-634, 1975
INTRODUCTION
A
LTHOUGH the determination of availability of amino acids from protein supplements has been under study for many years, no completely satisfactory method has yet been developed. The most commonly used methods as well as the shortcomings
1. Based on a thesis submitted to the Graduate College of the University of Illinois is partial fulfillment of the requirement for the Ph.D. degree. 2. Present address: Gold Kist Inc., Atlanta, Georgia 30301.
of each have been reviewed by De Muelenaere et al. (1967). In the present communication a theoretical method for the determination of availability of amino acids is described which avoids many of the problems discussed by De Muelenaere et al. (1967). This method is based on the procedure described by Amato (1960) which involves the feeding of a ,4C labeled protein reference standard and l4C labeled test proteins to different groups of animals. The availability of the amino acids of the test proteins is determined relative to that of the reference protein and is based on the
Downloaded from http://ps.oxfordjournals.org/ at Library of Medical Center of Fudan University on May 3, 2015
urine for nutritional balance studies. Bull. Natl. Inst. Agr. Sci. Ser. G. 12, 37-44. Beaton, J. R., and A. J. Szlavko and J. A. F. Stevenson, 1966. Factors influencing the excretion of a fat-mobilizing substance in the urine of rats. Can. J. Physiol. Pharmacol. 44: 95-101. Chalmers, T. M., 1965. Lipid-mobilizing activity during fasting. In: Handbook of Physiology. Edited by A. E. Renold and G. F. Cahill, Jr. Waverly Press, Inc., Baltimore, Md. pp. 549-555. Mukaida, C. S., and I. J. Lichton, 1971. Some dietary influences on the excretion and biological activity of an anorexigenic substance in the urine of rats. J. Nutr. 101:767-774. Nir, I., M. K. Dimick and S. Lepkovsky, 1969. A fat-mobilizing substance in chicken urine. Can. J. Physiol. Pharmacol. 47: 435-443. Nir, I., and V. Levy, 1972. Influence of a fat-mobilizing substance from chicken urine on geese. Poultry Sci. 51: 1041-1042.
633
RESEARCH NOTES
R
=
W
=
Z
=
I
=
J
=
The specific activity of the lysine in the reference protein. The specific activity of the total lysine in the test diet. The specific activity of the total lysine in the reference diet. The specific activity of the total lysine recovered from the feces after feeding the test diet. The specific activity of the total lysine recovered from the feces after feeding the reference diet.
Algebraically the following statements can be made: W = T U / ( B + T + G)
(1)
I =TU(100-t)/[A(100-a) + B(100 - b) + T(100 - t) + G(100 - g)]
(2)
Z = BR/(B + T + G)
(3)
J = BR(100-b)/[A(100-a) THEORY + B(100 - b) + T(100 - t)
The pertinent quantities involved in the determination are represented as follows:
+ G(100 - g)] A =
B
=
T
=
G
=
a b t g U
= = = = =
Fecal lysine originating from endogenous, bacterial and in the case of birds, urinary sources. Dietary lysine from the reference protein (egg albumen). Dietary lysine from the test ingredient. Dietary lysine from all sources except (B) and (T). The percentage absorption of A. The percentage absorption of B. The percentage absorption of T. The percentage absorption of G. The specific activity of the lysine in the test ingredient.
(4)
In the interest of brevity the denominator of Equations (1) and (3) will be represented by X and the denominator of Equations (2) and (4) will be represented by Y. Dividing Equation (2) by Equation (1) I
TU(100 - t ) / Y
W
TU/X
(100-t)X/Y
(5)
Dividing Equation (4) by Equation (3) J
BR(100-b)/Y
Z
BR/X
( 1 0 0 - b ) X / Y (6)
Downloaded from http://ps.oxfordjournals.org/ at Library of Medical Center of Fudan University on May 3, 2015
change in specific activity of the dietary • amino acids and the amino acids recovered from the feces or feces plus urine in the case of birds. All components of the protein sources would be uniformly labeled and since the method depends only on the specific activities of the individual amino acids, the availability of all the amino acids present in the protein could be determined from a single feeding trial. The proposed reference standard would be egg albumen prepared under controlled laboratory conditions thus assuring a standardized quality product. The diet containing the reference standard would be identical in every respect to that containing the test protein except that in one case the egg albumen would be l4 C labeled while in the other, the test protein would be labeled. Since the two diets would be identical in composition the change in specific activity of dietary and fecal amino acids can be related to the relative availability as described below. For ease of presentation the theory will be developed using lysine as the amino acid for which availability is being determined.
634
RESEARCH NOTES
Dividing Equation (5) by Equation (6) I/W J/Z
=
(100-t)X/Y (100-b)X/Y
=
(100-t) (100-b)
(7)
Equation (7) can then be rearranged to solve fort: ZI(100 - b) WJ
(8)
Now, if an arbitrary value is assigned to b (availability of reference lysine), availability of the test lysine, t, can be calculated. DISCUSSION Equation (8) shows that the availability of lysine from the test protein relative to that of the lysine from the reference protein can be determined from the specific activities of dietary lysine and lysine recovered from the feces. The advantages of the proposed method over methods currently in use are as follows: 1. Quantitative determination of either dietary or excreted lysine is not necessary. However lysine must be isolated from all other compounds and its specific activity determined. 2. The availability of all amino acids within the protein source can be determined with one feeding trial. The number of amino acids worked with is limited only by one's ability to separate the various amino acids. 3. Since the diets containing the reference
The primary disadvantage of the method is the need for radioactive protein sources. This limitation necessarily requires laboratory preparation of radioactive feed ingredients but this does not preclude the possibility that all of the usual feed ingredients can be prepared and subjected to analysis by this method. Due to the difficulty in preparing radio labeled feed ingredients this method could not be used in the routine determination of amino acid availability. However, it would appear that the method described would find utility as a check on other procedures as well as in studies designed to determine the effects of processing conditions on the availability of amino acids from protein sources. Since this method depends only on the specific activities of nutrients before and after feeding its use could be extended to the study of any nutrient which can be made radioactive and isolated in pure form. REFERENCES Amato, S. V., 1960. The absorption of free and protein-bound amino acids by the chick: Method and application to l4C lysine. Doctoral thesis. University of Illinois, Urbana Illinois. DeMuelenaere, J. J. H.,M-L. Chen and A. E. Harper, 1967. Assessment of factors influencing estimation of lysine availability in cereal products. J. Agr. Food Chem. 15: 310-317.
NEWS AND NOTES (Continued from page 614) Vice-President—William A. Adams, International Harvester Co., Agricultural/Industrial Equipment Engineering Center, Hinsdale, Illinois. Three new Technical Directors are: Electric Power
and Processing Division—George H. Foster, U.S.D.A. Grain Research Center, Manhattan, Kansas; Food Engineering Division—Dennis R. Heldman, Michigan State University, East Lansing, Michigan;
(Continued on page 637)
Downloaded from http://ps.oxfordjournals.org/ at Library of Medical Center of Fudan University on May 3, 2015
t = 100
and test protein are identical, the problems associated with amino acid imbalances, endogenous excretion and bacterial syntheses are eliminated because these effects would be the same across the two diets.
RESEARCH NOTES
Nir, I., Z. Nitsan and A. Vax, 1973. The influence of force feeding and of protein supplementation to the diet on the metabolizable energy of diets, digestibility of nutrients, nitrogen retention and digestive enzymes output. Ann. Biol. Anim. Bioch. Biophys. 13: 465-479. Nir, I., and M. Perek, 1971. The effect of various protein levels in feed of goslings during the preparatory period on fatty liver production and blood plasma components. Ann. Biol. Anim. Bioch. Biophys. 11: 645-656. Stevenson, J. A. F., B. M. Box and A. J. Szlavko, 1964. A fat-mobilizing and anorectic substance in the urine of fasting rats. Proc. Soc. Exptl. Biol. Med. 115:424-429. Weil, R., and De W. Stetten, Jr., 1947. Urinary excretion of a fat-mobilizing agent. J. Biol. Chem. 168: 129-132.
A THEORETICAL METHOD FOR T H E DETERMINATION O F AMINO ACID A V A I L A B I L I T Y U T I L I Z I N G 14C L A B E L E D P R O T E I N S O U R C E S ' S. V. A M A T O , 2 R. M . FORBES AND H . M . SCOTT
Department of Animal Science, University of Illinois, Urbana, Illinois 61801 (Received for publication October 4, 1974)
ABSTRACT A theoretical method is described whereby the availabilities of amino acids from test proteins are determined relative to the availabilities of amino acids from egg albumen. The method requires that both the test protein and the egg albumen be ,4C labeled. The relative ' availabilities of the amino acids are determined from the change in specific activities of the dietary amino acids and those recovered in the feces. POULTRY SCIENCE 54: 632-634, 1975
INTRODUCTION
A
LTHOUGH the determination of availability of amino acids from protein supplements has been under study for many years, no completely satisfactory method has yet been developed. The most commonly used methods as well as the shortcomings
1. Based on a thesis submitted to the Graduate College of the University of Illinois is partial fulfillment of the requirement for the Ph.D. degree. 2. Present address: Gold Kist Inc., Atlanta, Georgia 30301.
of each have been reviewed by De Muelenaere et al. (1967). In the present communication a theoretical method for the determination of availability of amino acids is described which avoids many of the problems discussed by De Muelenaere et al. (1967). This method is based on the procedure described by Amato (1960) which involves the feeding of a ,4C labeled protein reference standard and l4C labeled test proteins to different groups of animals. The availability of the amino acids of the test proteins is determined relative to that of the reference protein and is based on the
Downloaded from http://ps.oxfordjournals.org/ at Library of Medical Center of Fudan University on May 3, 2015
urine for nutritional balance studies. Bull. Natl. Inst. Agr. Sci. Ser. G. 12, 37-44. Beaton, J. R., and A. J. Szlavko and J. A. F. Stevenson, 1966. Factors influencing the excretion of a fat-mobilizing substance in the urine of rats. Can. J. Physiol. Pharmacol. 44: 95-101. Chalmers, T. M., 1965. Lipid-mobilizing activity during fasting. In: Handbook of Physiology. Edited by A. E. Renold and G. F. Cahill, Jr. Waverly Press, Inc., Baltimore, Md. pp. 549-555. Mukaida, C. S., and I. J. Lichton, 1971. Some dietary influences on the excretion and biological activity of an anorexigenic substance in the urine of rats. J. Nutr. 101:767-774. Nir, I., M. K. Dimick and S. Lepkovsky, 1969. A fat-mobilizing substance in chicken urine. Can. J. Physiol. Pharmacol. 47: 435-443. Nir, I., and V. Levy, 1972. Influence of a fat-mobilizing substance from chicken urine on geese. Poultry Sci. 51: 1041-1042.
633
RESEARCH NOTES
R
=
W
=
Z
=
I
=
J
=
The specific activity of the lysine in the reference protein. The specific activity of the total lysine in the test diet. The specific activity of the total lysine in the reference diet. The specific activity of the total lysine recovered from the feces after feeding the test diet. The specific activity of the total lysine recovered from the feces after feeding the reference diet.
Algebraically the following statements can be made: W = T U / ( B + T + G)
(1)
I =TU(100-t)/[A(100-a) + B(100 - b) + T(100 - t) + G(100 - g)]
(2)
Z = BR/(B + T + G)
(3)
J = BR(100-b)/[A(100-a) THEORY + B(100 - b) + T(100 - t)
The pertinent quantities involved in the determination are represented as follows:
+ G(100 - g)] A =
B
=
T
=
G
=
a b t g U
= = = = =
Fecal lysine originating from endogenous, bacterial and in the case of birds, urinary sources. Dietary lysine from the reference protein (egg albumen). Dietary lysine from the test ingredient. Dietary lysine from all sources except (B) and (T). The percentage absorption of A. The percentage absorption of B. The percentage absorption of T. The percentage absorption of G. The specific activity of the lysine in the test ingredient.
(4)
In the interest of brevity the denominator of Equations (1) and (3) will be represented by X and the denominator of Equations (2) and (4) will be represented by Y. Dividing Equation (2) by Equation (1) I
TU(100 - t ) / Y
W
TU/X
(100-t)X/Y
(5)
Dividing Equation (4) by Equation (3) J
BR(100-b)/Y
Z
BR/X
( 1 0 0 - b ) X / Y (6)
Downloaded from http://ps.oxfordjournals.org/ at Library of Medical Center of Fudan University on May 3, 2015
change in specific activity of the dietary • amino acids and the amino acids recovered from the feces or feces plus urine in the case of birds. All components of the protein sources would be uniformly labeled and since the method depends only on the specific activities of the individual amino acids, the availability of all the amino acids present in the protein could be determined from a single feeding trial. The proposed reference standard would be egg albumen prepared under controlled laboratory conditions thus assuring a standardized quality product. The diet containing the reference standard would be identical in every respect to that containing the test protein except that in one case the egg albumen would be l4 C labeled while in the other, the test protein would be labeled. Since the two diets would be identical in composition the change in specific activity of dietary and fecal amino acids can be related to the relative availability as described below. For ease of presentation the theory will be developed using lysine as the amino acid for which availability is being determined.
634
RESEARCH NOTES
Dividing Equation (5) by Equation (6) I/W J/Z
=
(100-t)X/Y (100-b)X/Y
=
(100-t) (100-b)
(7)
Equation (7) can then be rearranged to solve fort: ZI(100 - b) WJ
(8)
Now, if an arbitrary value is assigned to b (availability of reference lysine), availability of the test lysine, t, can be calculated. DISCUSSION Equation (8) shows that the availability of lysine from the test protein relative to that of the lysine from the reference protein can be determined from the specific activities of dietary lysine and lysine recovered from the feces. The advantages of the proposed method over methods currently in use are as follows: 1. Quantitative determination of either dietary or excreted lysine is not necessary. However lysine must be isolated from all other compounds and its specific activity determined. 2. The availability of all amino acids within the protein source can be determined with one feeding trial. The number of amino acids worked with is limited only by one's ability to separate the various amino acids. 3. Since the diets containing the reference
The primary disadvantage of the method is the need for radioactive protein sources. This limitation necessarily requires laboratory preparation of radioactive feed ingredients but this does not preclude the possibility that all of the usual feed ingredients can be prepared and subjected to analysis by this method. Due to the difficulty in preparing radio labeled feed ingredients this method could not be used in the routine determination of amino acid availability. However, it would appear that the method described would find utility as a check on other procedures as well as in studies designed to determine the effects of processing conditions on the availability of amino acids from protein sources. Since this method depends only on the specific activities of nutrients before and after feeding its use could be extended to the study of any nutrient which can be made radioactive and isolated in pure form. REFERENCES Amato, S. V., 1960. The absorption of free and protein-bound amino acids by the chick: Method and application to l4C lysine. Doctoral thesis. University of Illinois, Urbana Illinois. DeMuelenaere, J. J. H.,M-L. Chen and A. E. Harper, 1967. Assessment of factors influencing estimation of lysine availability in cereal products. J. Agr. Food Chem. 15: 310-317.
NEWS AND NOTES (Continued from page 614) Vice-President—William A. Adams, International Harvester Co., Agricultural/Industrial Equipment Engineering Center, Hinsdale, Illinois. Three new Technical Directors are: Electric Power
and Processing Division—George H. Foster, U.S.D.A. Grain Research Center, Manhattan, Kansas; Food Engineering Division—Dennis R. Heldman, Michigan State University, East Lansing, Michigan;
(Continued on page 637)
Downloaded from http://ps.oxfordjournals.org/ at Library of Medical Center of Fudan University on May 3, 2015
t = 100
and test protein are identical, the problems associated with amino acid imbalances, endogenous excretion and bacterial syntheses are eliminated because these effects would be the same across the two diets.
