Effect of Dietary Mineral Composition on Nutritional Equivalency of Amino Acid Mixtures and Casein in Rats1 KIYOSHI EBIHARA, YUICHI IMAMURA ANDSH UH ACHÕKIRIYAMA Department of Agricultural Chemistry, Faculty of Agriculture, Ehime University, Tarami 3-5-7, Matsuyama, Japan ABSTRACT The effect of dietary mineral composition on the quality of amino acid mixtures and intact casein was examined in growing rats fed for 28 days under "conventional" conditions. When 25% demineralized casein (DC) was the sole dietary protein source, the widely-used mineral mixture by Harper (MMH) supported growth poorly (3.2 g/day), but a new formula mineral mixture (MM2) containing sufficient amounts of zinc gave a growth rate of over 8 g/day for 21 days. With a crude casein (CC) diet, rats grew at the same rate regardless of mineral mixtures. The growth-supporting power of amino acid mixtures, one of which simulated casein (AA-A) and another patterned after Rogers and Harper to give maximal growth of rats (AA-B), was compared with those of DC and CC with addition of MMH or MM2. When the protein source was DC, AA-A or AA-B, MM2 significantly improved the growth over that of rats fed MMH. The growth-stimulating effect of MM2 was greater when added to DC diet than when added to AA-A diet. When MM2 was added to AA-B diet, the growth rate greatly increased and reached that of rats fed DC diet containing MM2 (over 8 g/day), showing a nutritional equivalency between the amino acid mixture and intact protein. Drawbacks inherent in MMH did not appear with CC diet, because CC contained considerable amounts of zinc. When a highly purified amino acid-sucrose diet is used, dietary minerals become most limiting for growth even under conventional conditions. Thus, the previous conflicting results concerning nutritional equivalency of amino acid mixtures and protein can be explained by in adequate mineral sources. J. Nutr. 109: 2106-2116, 1979. INDEXING KEY WORDS purified diet •mineral mixture •zinc quality of proteins •quality of amino acid mixture •maximal growth of rat •amino acid requirement At the present stage of amino acid nutrition, there has been a lack of agreement on nutritional equivalency between intact protein and the corresponding amino acid mixtures even after the reports by Breuer et al. (1) and Rogers and Harper (2) appeared. Breuer et al. (3) compared the growth-stimulating effects in rats and found that amino acid mixtures were into intact

protein,

but

the

tation of asparagine to an amino acid diet improved growth rate to that obtained with a casein diet (1). They attributed the different effects on growth which were Receivedfor publicationMarch27. 1979. >A part of this study was presentedat the Reffi^/^^cÄ^^imÄÄ' Sfe'SSS ^tA'lf ^^y%S%$ F°0d """

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VALUE OF AMINO ACID DIET

TABLE 1 Composition of test diets Test diet used in Experiment 2 Constituent

cc

DC

Experiment 3

AA-A

CC

DC

AA-B

Crude casein1 Demineralized casein3 Amino acid mixture A4Amino 25.8— B5Corn acid mixture —5 oil6Mineral 54 mixture7MMH 41 MM2Vitamin or mixture8Choline 10.2 chloride'Vitamin 0.20.05 granule10Sucrose"21.62—5410.20.0522.62—5410.20.0518.62 E 0.05ti\ mÃ-tlrn IfiO18.62—5410.20.05=18.55410.20.05

1Purchased from Katayama Chemical Industries, Co., Osaka. * In each experiment, casein was added to the diet to give the same level of nitrogen as that of the amino acid diet. 3Crude casein was demineralized by washing 3 times with 0.5% Naz-EDTA solution as described in footnote 4 to the text. 4This was identical with the mixture used by Itoh et al. (4). For amino acid composition, see table 2. ' This was identical with the mixture used by Rogers and Harper (2) ; the composition is indicated in table 2. • Mixed with 0.2 ml of Chocóla A which contained retinyl palmitate and ergocalciferol dispersed in water to provide 6,000 and 600 IU, respectively, just prior to preparing the diet. Chocóla A was purchased from Eisai Co., Ltd., Tokyo. ' For the abbreviations and their mineral composition, see table 3. • This was identical with Harper's mixture (13). ' Four milliliters of a 50% solution in 50% aqueous ethanol were added. 10Trade name is "Juvela granule." Purchased from Eisai Co., Ltd., Tokyo and it contained 200 mg of all-rac-cr-tocopheryl acetate per gram. " Granulated sugar. Dietary supplement was added at the expense of sucrose.

previously observed between amino acid mixtures and intact protein to nutritionally inadequate composition of the former. Rogers and Harper (2) confirmed the growth-stimulating effect of asparagine in rats fed an amino acid diet. However, they failed to obtain maximum growth rate whenever the diets were fed dry, even if the amino acid mixture included aspara gine at a level comparable to that in the exeriments of Breuer et al. ( 1). Rogers and Harper (2) developed an amino acid-agargel diet which showed a significant growthstimulation as great as or greater than that of a casein diet supplemented with methionine if the amino acid diet contained addi tional asparagine (0.6% ) and a higher amount of arginine (over 1^)- The im provement in growth rate was accom panied with an increased food intake. They suggested that this effect was achieved by reducing the osmotic pressure caused by dietary constituents of low mo lecular weight such as amino acids and sucrose in a purified diet.

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Many other previous data also indicate controversial results in the growth response of rats to purified amino acid diets (4-8). Recently, Itoh et al. (4) reported that the growth rate of rats fed a casein diet (5.2 g/day ) was always superior to those of rats fed ad libitum or pair-fed an amino acid diet at 3.2 and 4.8% levels of nitrogen whether the amino acid mixtures contained asparagine ( 3.7 g/day ) or not ( 3.4 g/day ). In spite "these of theseresults observations, cluded indicate they that con the amino acid equivalent mixture simulating nutritionally with intactcasein casein,"is because the growth rates were comparable (2.4 g/day) when meal-fed twice daily or force-fed the same amount of both diets. Salmon (5) stated that "apparently there was a growth-stimulating substance in testeddiets," that was not present in the the proteins amino acid a period of 20 years after Woolley's proposal of strepogenin (9, 10) and its refutation (11, 12). In all cases cited above, the significance of dietary mineral composition seems to

2108

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have been ignored and the investigators seem to have been accustomed to using only a mineral mixture familiar to them in diets containing either amino acid mixture or intact protein. Since the dietary constituents, particu larly carbohydrate and protein sources have increasingly been purified, some trace elements furnished by the prevailing min eral mixtures may be a limiting factor for growth of experimental animals. If this is the case, the discrepancy cited above can be explained. Therefore, it seemed desir able to formulate a new mineral mixture to give maximal growth in rats fed a puri fied diet. MATERIALS

AND METHODS

TABLE 2 Composition of amino acid mixtures Amino acid

AA-A1

AA-B2

dietb% of HC1b-Histidine-HCl-H2Ob-Isoleucineb-Leucineb-Lysine-HClb-Methionineb-Cystineb Arginine •

aniñeb-Aspartic Al acidb-Asparagine acidGlycineb-Prolineb-SerineDiammonium b-Glutamic 3.412.280.340.34—1.0918.52

Diet

Protein sources. The composition of diets is shown in table 1. Crude casein3 (CC), demineralized casein4 (DC), and two types of amino acid mixture r' ( AA-A and AA-B) were used as protein sources. The compositions of AA-A and AA-B are shown in table 2; AA-A was identical with the mixture used by Itoh et al. (4) which sim ulated the amino acid composition of casein, and AA-B was identical with the mixture used by Rogers and Harper (2) which lead to a maximal growth rate. In experiments 2 and 3 in which amino acid diets were used, all of the casein-contain ing diets were made isonitrogenous with the amino acid diet by adjusting the casein level. In experiments 1 and 4, 25% of DC was exclusively used as a protein source. Mineral mixtures. Three kinds of mineral mixture were used.6 First, mineral mixture H ( MMH ) shown in table 3 was identical with the mixture of Harper ( 13 ) which has been used most frequently by Japanese in vestigators in rat-nutrition studies.7 Min eral mixture H is characteristically low in zinc, manganese, calcium and phosphorus and high in copper, sodium and potassium as compared with the NRC requirements 8 (14). Second, mineral mixture 1 (MM1) was prepared by combining the newer trace elements (NEE) known or sus pected to be essential ( 16-18 ) with the salts of usual elements patterned on the basis of the NRC requirements ( 14). Here, NEE refer to the elements except Mo listed

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citrateSodium acetateTotal0.950.931.031.872.000.630.081.041.100.870.221.280.811.414.440.372.1

1All the crystalline b-amino acids were supplied by Ajinompto Co., Tokyo. Amino acid composition of AA-A is identical with the mixture used by Itoh et al. (4) which simulated the composition of casein. 2 Amino acid composition of AA-B is identical with the mixture used by Rogers and Harper (2) on which maximal growth rate was attained in rats. 3 Rogers and Harper (2) used b-histidine-HCl instead of b-histidine-HCl-H»O used here.

in the lower part of table 3. Since the levels of potassium and sodium were rather low in MM1, the final dietary level of 3 Purchased from Katayama Chemical Industries, Co.. Osaka. ' Demineralized casein was prepared by soaking crude casein In 0.5% EDTA (Na2) solution overnight. Subsequently the mixture was centrlfuged to remove the washings (this process was repeated three times). The remaining EUTA-metal complexes and excess KDTA were removed by washing with delonlzed(llstllled water and twice with 99% ethanol and then air-drving In a clean room. 5 All crystalline L-amlno acids were kindly sup plied from Ajlnomoto. Co.. Tokyo. 6 All the reagents used for preparing the mineral mixtures were JIS-speclal grade. ' For example, we Inspected the kinds of the min eral mixtures in the reports concerning rat-nutrltlon studies published In the Journal of Japanese Society of Food and Nutrition for 10 years from 1965 to 1!I74 and learned that about 40% of Japanese In vestigators had used the Harper's mineral mixture (13). 15% of them. McCollum's (No. 185), and the remainder were unclear about dietary mineral source. "The authors consulted 2nd edition of NRC (14). beennue they were not aware of its 3rd revision (15) In which NRC recommended that potassium level should be doubled. The authors are in debt to the Editors and Reviewers of J. Nutr. who Informed them of the revision.

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VALUE OF AMINO ACID DIET

TABLE 3 Comparison of mineral mixtures used1 Composition of mixture2 Salt

MM1

MM2

Dietary level of element provided Element

MMP

MM2«

MMH4

g or mg salt/kg mixture mg element/kg diet CaCO,CaHPO4-2H,OKH,P04MgSO«-7HsOFe(C,H5O,)-6H,OKIO,MnSO4-5H,OZnClzCuSO«-5H,ONaClNa,SeO,NaMoO4-2H2OCrCl,-6HjONaIB4

.015.93,943.46,080.60.54—————————

1 Mineral mixture l (MM1) was prepared by combining the newer trace elements (NEE) known or suspected to be essential (16-18) together with the salts of usual elements patterned on the basis of the NRC requirements (14). Mineral mixture 2 (MM2) was prepared by modifying the levels of several usual elements (I, Mn and Zn) in MMH and this mixture also contained NEE at about the same levels as in MM1. Mineral mixture H (MMH) was identical with Harper's mixture (13). 1The compounds listed in the upper and lower halves are expressed as g salt/kg of mineral mixture and mg salt/kg of mineral mixture, respectively. ' Indicates the dietary levels of elements attained when MM1 was added to the diet at 3.7% level. 4 In dicates the dietary levels of elements attained when MM2 or MMH was added to the diet at 4% level.

MM1 had to be 3.7% of the whole. Third, mineral mixture 2 (MM2) was prepared by modifying levels of several elements in MMH. This mixture also contained NEE at the same level as in MM1. Other dietary constituents. In experiment 3, we used a demineralized holocellulose fraction9 expecting an agar-like effect (2) since we have frequently observed stimu lated appetite and enhanced food efficiency in early stages of experimental feeding when a purified dry diet contained such dietary fiber (19). In all experiments, the vitamin mixture used was identical with that reported by Harper (13) which has also been used frequently in Japan. Sucrose was exclusively used as a dietary carbohydrate source.

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Animal care and analyses Weanling male rats of the Wistar strain10 were used in all experiments. They were housed in individual cages with screen-bottoms of stainless steel. After feeding a 25% casein-sucrose diet for 4 to 5 days, they were divided into groups of five rats and given test diets and deionized water ad libitum for 4 weeks in a room which was maintained at 23 ±1° and lighted from 0800 to 2000 hours; the en vironments were "conventional." Body weight and food intake were re corded daily in the morning. At the end of 8 Prepared by dellgnification with hypochlorite from crude dietary fiber obtained from the roots of edible burdock (Arctium lappa L.) and by demineralizing with EDTA solution in the same manner as in footnote 4. 10Purchased from Tokushima Jlkken-Dobutsu KenkyiiKho. Tokushima.

2110 250

EBIHARA,

IMAMURA AND KIRIYAMA

: ¿,'ì, MM2 : 2V. MM2 : 3.7 "/. MM1 •• 2°/oMM1

200

: 4% MMH : 2°/oMMH

gì '5

the diluted plasma sample ( X5 ) and for the wet-digested samples of selected or gans (liver and femurs) and test diets. The weight of several selected organs was also examined. All data were analyzed by analysis of variance and significant differ ences among means were separated by using Duncan's Multiple Range Test (21). All statements of significant differences refer to the 5% level of probability. RESULTS AND DISCUSSION

•o o 150 .o

Experiment 1. Figure 1 shows the growth rates of rats fed diets containing three dif ferent mineral mixtures at two different levels. Demineralized casein was used as a protein source in this experiment.