食米蛋白質限制胺基酸研究方法之檢討―在等能量、等氮量對飼育條件下以老鼠肝臟細胞發育情形為指標的研究方法

Investigation on the Limiting Amino Acid of Rice protein-Using the Hepatic Cell Development as the Parameter under Isocaloric and Isonitrogenous Feeding Condition

楊祖馨(Tsu-Hsing Yang) ； 柯華雄(Hao-Shau Ko) ； 蔡玉吉(Yush-Chi Tsai) ； 許金土(Chien-Tu Sheu) ； 何寶珠(Bou-Ju Ho) ； 謝明哲(Ming-Jer Shieh) ； 潘自強(Elas Pan) ； R. Quentin Blackwell

《中華民國營養學會雜誌》 1卷1期 (1976/03) Pp. 1-11

https://doi.org/10.6691/JCNS.197603_1(1).0001

摘要

離乳之Sprague-Dawley幼鼠，經十二日調節飼養期後，視其生長情形及年齡，選擇體重相近之雄鼠（38~42克）按其血親關係平均分配為七組，每組六隻，各別飼以供試飼料，供試之基本飼料係以88.5%之BPI-76高蛋白質含量之白米（粗蛋白質含量為13%）配合必需之脂質、無機質、維生素及纖維素等所組成，並分別添加0%, 0.3%, 0.4%, 0.5%, 0.6% Lys, 0.4% Lys+0.2% Thr, 0.6% Lys+0.3% Thr，同時並以Ala, Asp, Glu, Glv等非必需胺基酸混合物調節至各組供試飼料之總氮量相等，飼養期間為80日，飼料之給予則採對飼育法，每隔七天測定體重一次，飼養期終了時，以乙醚麻醉，自心臟抽血後由頸靜脈放血，取其肝臟測定其重量，及其蛋白質，RNA, DNA之含量，血液則作血紅蛋白，血漿蛋白，及血球比容之分析，所得之結果如下：
(1)Lys之添加對被試老鼠體重增加有顯著促進效果（表三），其最適宜添加量，在前63天為 0.5%，嗣後及至80日則為0.4%。此添加量顯著高於以往的研究(4)(5)(6)，可能基因於本實驗比以往研究採用蛋白質含量甚高之食米飼料作更長期之飼養研究而引起。
(2)0.6%Lys添加組之體重增加率低於0.4%Lys添加組。但是0.6%Lys+0.3%Thr添加組在七個實驗組中顯示最高的體重增加率，此結果表示0.6%Lys之添加已超過適宜添加量而引起第二限制胺基酸Thr之不平衡，若同時再添加0.3%Thr時可消除其不平衡。此結果明顯地證明在本研究所採用之等能量，等氮量對飼育條件下，Thr乃是食米蛋白質之第二限制胺基酸。
(3)Lys或是Lys+Thr之添加對被試老鼠之血漿蛋白質量沒有重大的影響。血紅蛋白質量及血球比容等兩值隨Lys添加量之增加而增高，而以0.4~0.5%之添加量最適宜，0.6%之添加即過多，上記兩值反而顯示再下降，但除添加0.6%Lys外同時添加0.3%Thr時顯著地增加血紅蛋白質量及血球比容。而此兩值甚至比單添加0.5%Lys之適宜組為高，此結果明顯證明Thr為米蛋白質之第二限制胺基酸。（表五）
(4)肝臟總DNA含量除在0.6% Lys添加組顯著高於其他各組外，在其餘六試驗組略同（表六）。但肝臟之蛋白質／DNA及RNA/DNA值在0.6%Lys+0.3%Thr添加組很明顯比單獨添加0.6%Lys組為高（表六），單獨過量之添加Lys (0.6%Lys組）會引起肝臟細胞急驟分裂的異常現象。此異常現象可由同時添加0.3%Thr而消除。此結果更具體證明Thr為食米蛋白質第二限制胺基酸。又0.4%Lys添加組與0.4%Lys+0.2%Thr添加組之體重增加率及肝臟全DNA量略同。但關於蛋白質／DNA及RNA/DNA值顯然高於前者。此結果表示單由體重增加率而言第一限制胺基酸Lys添加以0.4%為適宜，但從細胞發育而言0.4%Lys之添加似為過量，需添加0.2% L-Thr來平衡，因此在蛋白質之營養研究上體重變化之觀察值僅為初步之研究，除此以外應再以細胞發育情形為營養狀態研究之指標。

並列摘要

Rice supplies 50% dietary energy and 40% protein in Taiwan Area, and of course it is the most important cereal of the world since it is the staple food of more than half the human race, The nutritive value of rice protein can be improved significantly by supplementation with the first limiting amino acid L-lysine and the second limiging amino acid L-threonine were reported by Pecora and Rosenberg. The results of Pecora’s and Rosenberg's were based on the different level of amino acid supplementation on the rats fed ad libitum with low-protein high-rice diets (diet protein level were 5-6%). In order to shorten the distance between the test dietary protein level and the protein requirement of the rats, high-protein high-rice diet (dietary protein level was 11.3%, composed of 88.5% BPI-76 rice, 5% vegetable oil, 4% salt mixture, 1% vitmain premix and 1.5% fiber) was used as the basal diet and supplemented with 0.3. 0.4, 0.5, 0.6% L-lysine HCI, 0.4% L-lysine HCI+0.2% L-threonine and 0.6% L-lysine HCI+0.3 L-threonine respectively and non-essential amino acid mixture compose of L-aspartic acid, L-glutamic acid, L-alanine and glycine were added so far as to adjusted the 7 test diets isonitrogenously. Male rats of the sprague-Dawley strain from U.S. Naval Medical Research Unit No.2, weighing from 38 to 43 grams were divided into 7 groups of 6 animals each. The test animal in each dietary gropp were pair fed with 7 test diets respectively.
Body weight change were measured. weekly and after 80 days isonitrogenous and isocaloric feeding the test rats were anesthetized with ether and exsanguinated. Plasma protein, hemoglonin and hematocrit were determined. The liver specimens were homogenized in ice-cold distilled water, Protein, RNA and DNA contents of the liver homogenates were determined by some modifications of the a1kaline hydrolysis procedure of Wannemacher et al.
The results revealed that:
(1) L-lysine HCI supplementation to basal high-rice diet remarkable improved body weight increase of the test rats (Table 3). The optimal supplementation of L-lysine HCI was 0.5% during the first 63 days of the test feeding while 0.4% became the optimal in the 64-80 day period. These figures are remarkab1e higher than that of the previous studies (4.6). It assumed that it might owed to the higher protein high-rice diet being used and the longer period being fed in present work than that of the previous studies.
(2) 0.6% L-lysine HCI supplemented group showed less body weight increase than that of the 0.4% group. Nevertheless, in 0.6% L-lysine HCI+0.3% L-threonine supplemented group showed the highest body weight increase among the seven test dietary groups. These results indicate that excess L-lysine supplementation gives rise second limiting amino acid imbalance and which can be further balanced by the 0.3%L-threonine supplementation. Therefore L-threonine is the second limiting amino acid of rice demonstrated by the present isocaloric and isonitrogenous feeding method.
(3) No considerable influence was observed by the supplemertation of L-lysine or L-lysine plus L-threonine on plasma protein level. Hemoglobin and hematocrit value were increased together with the increase amount of L-sysine HCI supplementation and showed maximum value by its 0.5% supplementation but its 0.6% supplementation become excess and both values significantly' dropped. This excess phenomenon could be corrected by the additional 0.3% L-threonine supplementation besides 0.6% L-lysine HCI and both valuss were enhanced to higher level in comparison with optimal 0.5% L-lysine HCI supplemented group. This can be considered as an another prove that L-threonine is the second limiting amino acid of rice protein.
(4) 0.6% L-lysine HCI supplemented group showed expectionally the highest liver total DNA value among the seven test dietary groups. But Liver Protein/DNA and RNA/DNA value was remarkable, dropped in this group. Nevertheless, significantly higher Liver Protein/DNA and RNA/DNA value were observed when 0.3% L-threonine was added instead of sole 0.6%L-lysine HCI supplementation. Such results indicate that excess L-lysine supplementation may give rise metabolic disorder which can be corrected by the second limiting amino acid supplementation. Thus the role of L-threonine as the second limiting amino acid rice of, protein is definitely established.