This
is the twelfth
events in FASEB Annual Meeting.
in a series of articles recalling scientfic 50 years ago, including reminiscences of the 1942
Role of Amino Acids in Human Nutrition By Julius
E. Johnson
and
William
J.
Haines
IT WAS MAY 1942. PROF. WILLIAM C. ROSE asked to see us in his office. The proposal he outlined was exciting, challenging, and a bit overwhelming. He suggested that the two of us do the preparatory work, conduct the first experiments, perform the analyses, and serve as the first experimental subjects to determine the amino acid requirements of humans using mixtures of purified amino acids in place of proteins. This had been his ultimate objective since the discovery of threonine in 1935 (1). It did not take long for us (Bill Haines and Julius Johnson), both graduate students in the Department of Biochemistry, University of Illinois, Urbana-Champaign, to decide. We gave Dr. Rose an affirmative answer the next morning. Johnson was occupied during the summer months of 1942 selecting, purifying, and formulating the dietary ingredients. The vitamins were supplied daily in doses of cod liver oil and appropriate quantities of the other vitamins known then. Possible unidentified factors were obtained from an extract of liver powder. Ninety-five percent of the nitrogen of the diet was supplied in the form of a mixture of the 10 amino acids known to be indispensable for animals. Supplies of amino acids were recrystallized and reanalyzed to assure a minimum nitrogen content of unknown identity. Then came a major challenge: preparing a palatable diet that could be tolerated for several weeks. Cornstarch, purified cellulose flour (for bulk), sucrose, corn oil, unsalted butter (melted and centrifuged to remove protein), baking powder, distilled water, and sodium chloride and other minerals; this was the list of starting materials presented to Mrs. Rose, who was asked to prepare an edible wafer. After many hours in her kitchen during August of 1942, she developed the recipes suitable for baking in an oven or frying on a hot plate. Portions of the butter and sucrose could be eaten separately, spread on the wafers or pancakes. Dr. Rose even allowed the use of peppermint to flavor sugar cubes and maple extract or cinnamon to use with sugar on the pancakes, because these flavorings contributed a negligible amount of nitrogen. From the outset it was decided to dissolve the amino acid mixture in distilled water and to sip the mixture while eating the rest of the diet divided equally into three meals each day. The unmodified solution had a vile taste; barely tolerable. After many attempts it was finally decided to add sucrose and lemon juice strained and centrifuged to remove the pulp. A poor-tasting lemonade, but tolerable. Haines returned to the campus in September and joined the final critical stages of preparation for the experiments. The next weeks were spent calibrating and refining analytical procedures to be applied to urine, blood, and feces.
Vol. 6
March
1992
The first full meal, at noon October 1, 1942, supplied too much bulk, so the wafers were reduced by one-fourth that evening and the equivalent calories were supplanted with added sucrose and butter. By October 4 we had adjusted to the diet. Daily urine samples were collected and pooled for a 24-hour period starting at the same time each morning. At the beginning of each dietary regime feces were marked by ingesting charcoal tablets (stools were well formed and regulated with cellulose fiber). At the outset appetites were good but Johnson was losing weight. The caloric intake for both of us was increased by 300, adding butter and sucrose. In spite of this adjustment both of us excreted more nitrogen than we consumed. Dr. Rose asked us to increase our intake of the mixture of all 10 amino acids from 41.0 to 50.3 g per day. For the ensuing 8-day period Haines manifested a slight positive balance. Johnson was marginal; positive for 5 of the 8 days (2).
Julius
E. Johnson
In retrospect it was fortuitous that we happened to be the first subjects. Haines had a relatively low calorie requirement (48 cal/kg body weight); Johnson, somewhat higher. Later volunteers had an unexpectedly high requirement for 50 or more cal/kg body weight when on the amino acid diet. The investigations were tedious and expensive ($30 per man per day in 1942 for the amino acids only). We could have taken the path of adding other amino acids to the diet, one by one or in combination. Fortunately, because of the earlier increase in our calorie intake, nitrogen balance was achieved. (Dr. Rose soon thereafter initiated human studies (3) to investigate further the calorie relationships when casein was fed in comparison with either hydrolyzed casein or mixtures of pure amino acids. The results clearly demonstrated that diets containing mixtures of amino acids necessitate higher calorie intakes for nitrogen balance than like rations containing casein.) After the foreperiod described, valine was removed from the diet and the remaining nine amino acids were increased in the same proportion to compensate for the nitrogen contributed by the valine. One day later our appetites failed, we manifested a negative balance, became fatigued, irritable, and suffered headaches. When valine was restored, we again attained a positive nitrogen balance, our appetites and tolerable dispositions returned.
Correspondence may be addressed to Dr. Haines, 5 Bedford Dr., RD #2, Doylestown, PA 18901, USA, and/or to Dr. Johnson, 1111 Knollwood Ct., Midland, MI 48640, USA.
50 YEARS AGO
2361
w.fasebj.org by Iowa State University Serials Acquisitions Dept (129.186.138.35) on January 10, 2019. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNum
relief. Evening engagements frequently terminated suddenly when sample collections were necessary. Dr. Rose and his students worked diligently for about 12 years to complete this classical study of humans. More than 50 volunteers participated as experimental subjects. The background, results, and conclusions are described in a fascinating review published in 1957 (5). What a privilege it was to have worked under the guidance and inspiration of this remarkable man! “How did it happen?” This was the title of another paper (6), which Dr. Rose prepared in his 92nd year at the urging of his friends. In the last paragraph of that paper he answered the question in words that accurately revealed his spirit and motivation. “How did it happen? It happened when I saw a lowly rat on his way to oblivion because of histidine starvation, suddenly and dramatically change his course and begin to grow after consuming a tiny bit of the missing amino acid. Nothing in metabolism could be more spectacular or arouse more reverence for the marvelous chemistry that takes place in living things.” Grants from the Nutrition Foundation, Inc., the Rockefeller Institute, and the Graduate College Research Fund of the University of Illinois were of vital importance in support of these studies.
REFERENCES
William C. Rose at his 90th birthday celebration in 1977. In the background (left to right) is Dr. Rose after graduation from Davidson College; at his desk; Herbert E. Carter (ASBMB, AIN-R); and Dr. and Mrs. Rose.
After continuing another 6 days on the complete diet (7.04 g nitrogen per day), methionine was excluded from the food for 6 days. Again we lost more nitrogen than was ingested; however, the negative balances were not as large as with valine deprivation. The side effects also were less severe (4). During these experiments we were both dating the young ladies we married soon after our Ph.D. degrees were granted. The only advantage we enjoyed with our rigorous protocol was that courtship cost less-cokes for them, distilled water for us. On a stipend of $60 per month this was welcome
2362
1. McCoy, R. H., Meyer, C. E., and Rose, W. C. (1935) Feeding experiments with mixtures of highly purified amino acids. VIII. Isolation and identification of a new essential amino acid. j Biol. Chem. 112, 283-302 2. Rose, W. C., Haines, W. J., and Johnson, J. E. (1942) The role of the amino acids in human nutrition. j Biol. Chem. 146, 683-684 3. Rose, W. C., Coon, M. J., and Lambert, G. F. (1954) The amino acid requirements of man. IV. The role of caloric intake. J. Biol. Chem. 210, 331-42 4. Rose, W. C., Johnson, J. E., and Haines, W. J. (1950) The amino acid requirements of man. I. The role of valine and methionine. j Biol. C/tern. 182, 541-555 5. Rose, W. C. (1957) The amino acid requirements of adult man. Nuts Abstr. Revs. 27, 631-647. (The eight amino acids essential for establishment and preservation of nitrogen equilibrium in adult man are valine, methionine, threonine, isoleucine, leucine, phenylalanine, lysine, and tryptophan.) 6. Rose, W. C. (1979) How did it happen? Ann. N} Acad. Sci. 325, 229-234
50 YEARS AGO
Vol. 6
March
1992
w.fasebj.org by Iowa State University Serials Acquisitions Dept (129.186.138.35) on January 10, 2019. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNum
is the twelfth
events in FASEB Annual Meeting.
in a series of articles recalling scientfic 50 years ago, including reminiscences of the 1942
Role of Amino Acids in Human Nutrition By Julius
E. Johnson
and
William
J.
Haines
IT WAS MAY 1942. PROF. WILLIAM C. ROSE asked to see us in his office. The proposal he outlined was exciting, challenging, and a bit overwhelming. He suggested that the two of us do the preparatory work, conduct the first experiments, perform the analyses, and serve as the first experimental subjects to determine the amino acid requirements of humans using mixtures of purified amino acids in place of proteins. This had been his ultimate objective since the discovery of threonine in 1935 (1). It did not take long for us (Bill Haines and Julius Johnson), both graduate students in the Department of Biochemistry, University of Illinois, Urbana-Champaign, to decide. We gave Dr. Rose an affirmative answer the next morning. Johnson was occupied during the summer months of 1942 selecting, purifying, and formulating the dietary ingredients. The vitamins were supplied daily in doses of cod liver oil and appropriate quantities of the other vitamins known then. Possible unidentified factors were obtained from an extract of liver powder. Ninety-five percent of the nitrogen of the diet was supplied in the form of a mixture of the 10 amino acids known to be indispensable for animals. Supplies of amino acids were recrystallized and reanalyzed to assure a minimum nitrogen content of unknown identity. Then came a major challenge: preparing a palatable diet that could be tolerated for several weeks. Cornstarch, purified cellulose flour (for bulk), sucrose, corn oil, unsalted butter (melted and centrifuged to remove protein), baking powder, distilled water, and sodium chloride and other minerals; this was the list of starting materials presented to Mrs. Rose, who was asked to prepare an edible wafer. After many hours in her kitchen during August of 1942, she developed the recipes suitable for baking in an oven or frying on a hot plate. Portions of the butter and sucrose could be eaten separately, spread on the wafers or pancakes. Dr. Rose even allowed the use of peppermint to flavor sugar cubes and maple extract or cinnamon to use with sugar on the pancakes, because these flavorings contributed a negligible amount of nitrogen. From the outset it was decided to dissolve the amino acid mixture in distilled water and to sip the mixture while eating the rest of the diet divided equally into three meals each day. The unmodified solution had a vile taste; barely tolerable. After many attempts it was finally decided to add sucrose and lemon juice strained and centrifuged to remove the pulp. A poor-tasting lemonade, but tolerable. Haines returned to the campus in September and joined the final critical stages of preparation for the experiments. The next weeks were spent calibrating and refining analytical procedures to be applied to urine, blood, and feces.
Vol. 6
March
1992
The first full meal, at noon October 1, 1942, supplied too much bulk, so the wafers were reduced by one-fourth that evening and the equivalent calories were supplanted with added sucrose and butter. By October 4 we had adjusted to the diet. Daily urine samples were collected and pooled for a 24-hour period starting at the same time each morning. At the beginning of each dietary regime feces were marked by ingesting charcoal tablets (stools were well formed and regulated with cellulose fiber). At the outset appetites were good but Johnson was losing weight. The caloric intake for both of us was increased by 300, adding butter and sucrose. In spite of this adjustment both of us excreted more nitrogen than we consumed. Dr. Rose asked us to increase our intake of the mixture of all 10 amino acids from 41.0 to 50.3 g per day. For the ensuing 8-day period Haines manifested a slight positive balance. Johnson was marginal; positive for 5 of the 8 days (2).
Julius
E. Johnson
In retrospect it was fortuitous that we happened to be the first subjects. Haines had a relatively low calorie requirement (48 cal/kg body weight); Johnson, somewhat higher. Later volunteers had an unexpectedly high requirement for 50 or more cal/kg body weight when on the amino acid diet. The investigations were tedious and expensive ($30 per man per day in 1942 for the amino acids only). We could have taken the path of adding other amino acids to the diet, one by one or in combination. Fortunately, because of the earlier increase in our calorie intake, nitrogen balance was achieved. (Dr. Rose soon thereafter initiated human studies (3) to investigate further the calorie relationships when casein was fed in comparison with either hydrolyzed casein or mixtures of pure amino acids. The results clearly demonstrated that diets containing mixtures of amino acids necessitate higher calorie intakes for nitrogen balance than like rations containing casein.) After the foreperiod described, valine was removed from the diet and the remaining nine amino acids were increased in the same proportion to compensate for the nitrogen contributed by the valine. One day later our appetites failed, we manifested a negative balance, became fatigued, irritable, and suffered headaches. When valine was restored, we again attained a positive nitrogen balance, our appetites and tolerable dispositions returned.
Correspondence may be addressed to Dr. Haines, 5 Bedford Dr., RD #2, Doylestown, PA 18901, USA, and/or to Dr. Johnson, 1111 Knollwood Ct., Midland, MI 48640, USA.
50 YEARS AGO
2361
w.fasebj.org by Iowa State University Serials Acquisitions Dept (129.186.138.35) on January 10, 2019. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNum
relief. Evening engagements frequently terminated suddenly when sample collections were necessary. Dr. Rose and his students worked diligently for about 12 years to complete this classical study of humans. More than 50 volunteers participated as experimental subjects. The background, results, and conclusions are described in a fascinating review published in 1957 (5). What a privilege it was to have worked under the guidance and inspiration of this remarkable man! “How did it happen?” This was the title of another paper (6), which Dr. Rose prepared in his 92nd year at the urging of his friends. In the last paragraph of that paper he answered the question in words that accurately revealed his spirit and motivation. “How did it happen? It happened when I saw a lowly rat on his way to oblivion because of histidine starvation, suddenly and dramatically change his course and begin to grow after consuming a tiny bit of the missing amino acid. Nothing in metabolism could be more spectacular or arouse more reverence for the marvelous chemistry that takes place in living things.” Grants from the Nutrition Foundation, Inc., the Rockefeller Institute, and the Graduate College Research Fund of the University of Illinois were of vital importance in support of these studies.
REFERENCES
William C. Rose at his 90th birthday celebration in 1977. In the background (left to right) is Dr. Rose after graduation from Davidson College; at his desk; Herbert E. Carter (ASBMB, AIN-R); and Dr. and Mrs. Rose.
After continuing another 6 days on the complete diet (7.04 g nitrogen per day), methionine was excluded from the food for 6 days. Again we lost more nitrogen than was ingested; however, the negative balances were not as large as with valine deprivation. The side effects also were less severe (4). During these experiments we were both dating the young ladies we married soon after our Ph.D. degrees were granted. The only advantage we enjoyed with our rigorous protocol was that courtship cost less-cokes for them, distilled water for us. On a stipend of $60 per month this was welcome
2362
1. McCoy, R. H., Meyer, C. E., and Rose, W. C. (1935) Feeding experiments with mixtures of highly purified amino acids. VIII. Isolation and identification of a new essential amino acid. j Biol. Chem. 112, 283-302 2. Rose, W. C., Haines, W. J., and Johnson, J. E. (1942) The role of the amino acids in human nutrition. j Biol. Chem. 146, 683-684 3. Rose, W. C., Coon, M. J., and Lambert, G. F. (1954) The amino acid requirements of man. IV. The role of caloric intake. J. Biol. Chem. 210, 331-42 4. Rose, W. C., Johnson, J. E., and Haines, W. J. (1950) The amino acid requirements of man. I. The role of valine and methionine. j Biol. C/tern. 182, 541-555 5. Rose, W. C. (1957) The amino acid requirements of adult man. Nuts Abstr. Revs. 27, 631-647. (The eight amino acids essential for establishment and preservation of nitrogen equilibrium in adult man are valine, methionine, threonine, isoleucine, leucine, phenylalanine, lysine, and tryptophan.) 6. Rose, W. C. (1979) How did it happen? Ann. N} Acad. Sci. 325, 229-234
50 YEARS AGO
Vol. 6
March
1992
w.fasebj.org by Iowa State University Serials Acquisitions Dept (129.186.138.35) on January 10, 2019. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNum
