RAC§.-i. B§¥FERENCES 3N URENRRY WING AC1?) EXCRETEGN {N MAN Thesis for the Degree of M. S. MECHEGAN STATE UNIVERSITY NOW 8. SCOTT-EMUAKPOR 1968 1 H6519 Much? a Scam Umvdflly " ". : BINDING mI " {El , 1 HOME & SIINS' , ' I BUUK BINDERY INC. LIBRARY BINDERS SPIIIGPIIT. IICIIOII . .__.’- . .- ABSTRACT RACIAL DIFFERENCES IN URINARY AMINO ACID EXCRETION IN MAN By Ajovi B. Scott-Emuakpor A population of healthy white and Negro boys who live together and take all their meals together has been studied for possible differences in their patterns of uri- nary amino acid excretion. Consistent differences between the boys are assumed to be genetic, except for the unlikely possibility that previous experience influences urinary amino acid excretion patterns. An average of nine fasting urine samples (taken first thing in the morning) was taken from each of forty-two boys of each race. The urines were subjected to two-dimensional paper chromatography and the relative density was measured for a series of fourteen spots on each chromatogram. The consistency of pattern for each individual is sufficiently great, that many of the boys could be ruled out as donors of a randomly chosen chromatogram. Although there are no clear-cut racial differences between the white and the Negro boys in their pattern of urinary amino acid excretion, histidine and ornithine tend Ajovi B. Scott-Emuakpor to be denser spots for whites, and glutamine and beta-amino- isobutyric acid (BAIB)--in BAIB "non-excretors"--tend to be denser for Negroes. These differences can be used in making a proba- bility statement about the race of the donor of a urine sample. RACIAL DIFFERENCES IN URINARY AMINO ACID EXCRETION IN MAN by Ajovi B. Scott-Emuakpor A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Zoology 1968 To Papa (who left), Mother Bro. Lese, Brothers and Sisters and to Prof. Harman M. Slatis and Family ACKNOWLEDGMENTS The author would like to express special thanks to his professor, Dr. Harman M. Slatis, for suggesting this problem and also for his help and guidance during the course of experimental work, analysis of the data, and preparation of the manuscript. Special thanks are also due to Dean Armon F. Yanders and Dr. James V. Higgins of the Department of Zoology and to Dr. Richard L. Anderson of the Department of Biochemistry for their advice, while serving on my committee. Thanks are due to the Director of the Boys' Train- ing School, Lansing, Dr. Paul J. Spata, for making the boys available for this study; and to Mr. R. Allan Bancroft and Mr. Mike Marhanka for the preparation of the figures and the photographs. The author would also like to thank the Agency for International Development (AID) for providing the fellow- ship that made this study possible, and to the staff of the University of Nigeria Program office at Michigan State Uni- versity for their cooperation. ii TABLE OF CONTENTS Page ACKNOWLEDGWNTS O O O I O O O O O O O O O O O O 0 ii LIST OF TABLES O O O O O O O O O O O O O O 0 O O 0 iv LIST OF FIGURES O O O O O O O O O O O O O O O O 0 v LIST OF APPENDICES O I O O O O O O O O O O O O O 0 Vi INTRODUCTION 0 O O O O O O O O O O O O O O O O O O 1 REVIEW 0 O O O O O O O O O O O O O O O O O O O O O 3 MATERIALS AND METHODS . . . . . . . . . . . . . . 6 The S tudy subj e c ts O O O O O O O O O O O O O O 6 The Chromatographic Apparatus . . . . . . . . 6 The Solvent System . . . . . . . . . . . . . . 7 Method of Reading Spots . . . . . . . . . . . 7 RESULTS 0 I O O O O O O O O O O O O O O O O O O O 11 "High," "Normal" and "Low" Excretors . . . . . 11 Relative Positions of Glutamine and His tidine O O O O O O O O O O O O O O O 15 Distinguishing Power of G1 tamin . . . . . . 16 Analysis of Variance for Glutamine (Interpretation) 0 o o o o o o o o o o o o o 19 Discriminant Analysis (Equation) . . . . . . . 22 Discriminant Analysis (Interpretation) . . . . 22 DISCUSSION 0 O O O O O O O O O O O O O O O I O O O 25 REFERENCES 0 O O C O 0 I O O O O O O O C O O O O O 2 9 ADDITIONAL REFERENCES 0 O O O O O O O O I O O O O 32 APPENDICES O O O O O O I O O O O O O O O O O O O O 35 iii Table 1. LIST OF TABLES Comparison between elution technique and photographic light meter readings as a means of reading the relative density of spots on a chromatogram . . . . "High," "Normal" and "Low" excretors for the individual amino acids . . . . A table showing the number of urine samples that contain more glutamine than histidine for Overall racial means densities of spots Analysis of Variance both races . . for the relative for glutamine . iv Page 10 14 17 18 20 LIST OF FIGURES Figure Page 1. Picture of chromatographic system . . . . . 12 2.‘ Picture of a typical chromatogram . . . . . 12 3. Diagram showing relative position of antino aCidS O O O O O O O O O O O O O I I 13 4. Diagram showing relative position of mino aCidS O O O O O I O O O O O O 0 O O 2 1 5. Distribution of Negro and White discriminant functions . . . . . . . . . . 23 6. Distribution of Negro and White discriminant functions . . . . . . . . . . 24 LIST OF APPENDICES Appendix Page A.l Table of Means . . . . . . . . . . . . . . 36 A.2 Table of Means . . . . . . . . . . . . . . 38 A.3 The Chromatographic Rack . . . . . . . . . 40 vi INTRODUCTI ON Several studies on urinary amino acid excretion following-Harris's "high" and "low" excretor theory for beta-amino-isobutyric acid (Harris, 1953-54) have been carried out with little success. Sutton and Vanderberg (1953), Berry (1953) and Gartler 32331. (1955) found that- the excretion of glycine was under the control of a single autosomal recessive gene. At the same time, these same authors found the distinction made between "high" and "low" excretors of taurine by Dent and Harris (1951) relatively unsatisfactory. However, only very few studies have been made of racial differences in excretion patterns. Sutton and Clark (1955) found various differences between Chinese and Caucasians living in Michigan. In their study, the Chinese excreted significantly more beta-amino-isobutyric acid (BAIB), lysine, leucine, histidine, tyrosine and uric acid than the Caucasians. The Caucasians on the other hand excreted more of two unidentified substances that have-color reactions with acid bromocresol green (BCG acid) and basic bromocresol green (BCG basic) than the Chinese. McEvoy-Bowe and Lugg (1961) also found dif— ferences between Caucasians, Chinese, and Malays living in and near Singapore. They found that the Chinese sample excreted low cystine as compared to the others and the- l Senoi (a native Malay group) sample excreted low alanine and glutamine compared to the others. They also found notably higher glycine:tarine ratios in the Caucasian data than in the others. The distribution of BAIB was reason- ably different in the three groups as well. The Senoi sam- ple had the most "high" excretors and the Chinese the fewest excretors. Gartler gt_al. (1957) found more BAIB excretors among the Apache Indians than among Caucasians, with "Black Caribs" intermediate. None of these racial studies, how- ever, controlled sources of variability such as diet, age~ and sex, which probably produced some of the differences that have been observed. The present study was designed to search for pos- sible racial differences in amino acid excretion patterns between American Negroes and Caucasians. All the known sources of-variability were reasonably controlled. The boys chosen for this study were between 13 and 18 years old. They live together in an unsegregated training school for socially delinquent boys. Both racial groups are largely from socio-economically inadequate homes, though there are probably systematic differences between the races. Thus we cannot eliminate the (very unlikely) possibility that previous experience affects the pattern of excretion. All the boys were on the same diet, individuals rarely sup-~ plemented this diet in any way, and rules forbid the exchange of food items between boys, so that pattersn of food con-‘ sumption are roughly comparable for all individuals. REVIEW Mammalian urine is a complex mixture of substances. Consequently, an analysis of urine for its various constit- uents is also a complex process. Some of the substances present in urine, such as urea and creatinine, are excreted by all humans in quantities proportional to body mass and to the rate of metabolism. Others such as methylrhistidine do not form regular constituents of urine. They are ex- creted only after certain foods have been taken. It has been shown by Datta and Harris (1951) that methyl-histidine is a break-down product of anserine which is a prominent constituent of vertebrate muscle, and hence it is excreted in large amounts following a meal of meat. Yet other sub- stances are excreted in highly variable amounts and in this group belong most of the free amino acids. A variety of amino acids is excreted in the urine' of man. The relative amounts of each amino acid is charac- teristic of the individual, his state of health and his diet. The variations in the amounts of amino acid excreted may be due to two major factors. They may be due to the concentration of some amino acid in the blood, and they may be due to the height of renal threshold. Whatever the case, it has generally been presumed that these patterns are under genetic control, but only a few examples of this con- trol have been identified. The ability to excrete BAIB in large amounts is controlled by a common recessive gene (Harris, 1953), but there is an overlap between "high" and "low" excretors so that many peOple are in the intermediate region (Gartler g£_al., 1957). Persons who excrete rela- tively large amounts of certain other amino acids have 5 also been described, and these appear to be genetically determined, but such individuals are physically and/or J a physiologically abnormal in most cases and are also rare. tw! In this group are the excretors of phenylalanine (Medes, 1932), histidine (Ghadimi gt_al., 1961, 1962), tryptophan (Tada §E_31., 1963), cystine (Fox gt_21,, 1964, and Berry, 1959), methionine (Hooft gt_31., 1964), ornithine (Russel gE_31., 1962), proline (Schafer~g£_al., 1962, and Scriver gE_gl., 1961), and glutamic acid (Menkes gg_al., 1962). An autosomal recessive gene is said to control the excre- tion of all of the above amino acids except glutamic acid which is believed to be under the control of a sex-linked recessive gene. Disease conditions, such as cirrhosis and hepatitis, are also associated with high amino acid excretion (Kirsner §E_31., 1949, 1950). A considerable amount of disagreement exists as to whether the regular pattern of excretion of urinary amino acids is affected by diet. Eckhardt and Davidson (1948, 1949) feel that variations in the pattern are as a result of differences in urine preservation rather than by differences in the amount of food ingested. In support of this Fowler §E_al., (1956) argued that what might be excessive excretion by one criterion may be normal or even below normal by another. Berry (1953) suggested that uri— nary constituents are only slightly under dietary influence, and that most of the variance observed between individuals is genotypically conditioned. Age and sex have also been shown to affect the ex- cretion pattern of urinary amino acids. Jagenburg (1959) found that age has only slight but characteristic effects on the amino acid pattern. He found that the excretion of ethanolamine and taurine were very high in the first 24 hours of life. Proline and hydroxy-proline were prominent in infants and not in adults, while histidine and methyl- histidine were excreted, in infancy, only in very small amounts and sometimes were absent. Glycine, in Jagenburg's study, seemed to be excreted in greater amounts during the 6th day of life than at any of the other age periods studied. Sex has been found to have somewhat less profound effects under normal physiological conditions. However, during pregnancy it has been found that histidine is considerably elevated (Honda, 1923; Voge, 1929; and Langley, 1941). Further studies by Lawrie (1947) and Christensen et_al., (1957) have shown that apart from elevated histidine, preg- nant women also exceed males in the excretion of free lysine, arginine, serine, threonine, tyrosine and tryptophan. MATERIALS AND METHODS The subjects were divided into four groups, each consisting of members of a different living unit. From each unit, ten or eleven boys of each race were chosen for study. Boys with a large amount of American Indian ancestry (determined visually) were excluded, as were light-pigmented Negroes, so that the "Caucasian" sample was almost entirely of European ancestry and the "Negro" sample was of predomi- nantly African ancestry. Individuals on drug therapy were also excluded. Urine samples were taken from all boys in a living unit so that excluded individuals did not realize that they were not part of the sample. Early morning urine samples (fasting samples) were taken from each group on ten mornings within a ten or eleven day period. Whatman 3mm chromatographic paper was used, and 100 lambda of the urine was slowly placed in a spot and dried with warm air using a hair drier. The chromatographic sys— tem used is shown in Figure 1. The rack is made of stain- less steel and consists of two flat square plates connected by four rods that are fastened to the plates with nuts (as described by Block, Durrum and Zweig, 1958). The Whatman paper is cut to be slightly larger than the size of the square plates and four holes are punches at its four corners. The paper sheets are then placed on the rack by stacking them on the rods through the holes. The sheets are separated from each other by l-inch spacers made of glass tubing. Ten sheets of Whatman paper were usually stacked on each rack. When placed in a chromatorgraphy jar, the steel racks were raised off the bottom of the jar by glass rods, and the papers extended below the racks into the solvent. The solvent was put in the air-tight chromatorgraphy jar (sealed with a greased glass plate) some two hours be- fore the insertion of the rack in order to allow thorough equilibration. The first direction of chromatography was in a solvent 100 ml. butyl alcohol, 100 ml. methyl alcohol, 50 ml. deionized water, and 1 ml. glacial acetic acid. The second direction was in a solvent of 150 ml. butyl alcohol, 150 ml. acetone, 75 m1. deionized water, 10 ml. diethylamine and 0.25 ml. 1M ammonium hydroxide. Ascending chromatography was run in the first direction for 20 hours, dried for at least four hours and then run in the second direction for another 20 hours. After drying, the papers were briefly immersed in a solution of 0.75 gm. of ninhydrin in 600 m1. of acetone and then dried in a fumehood for 5 minutes, and transferred into an oven held between 70° to 80° for 10 minutes. The density of each of 14 amino acid spots on the chromatograms was determined by measuring the amount of .f'YL'...‘ light transmitted through its center. The readings were done on a crude photographic light meter consisting of a photo-electric cell and a pointer that moves over a loga- rithymic scale. A series of tests has shown that this method gives consistent results and that these results are not affected by a wide range of variations in the amount of urine used. However it is not possible to convert these results into measurements of the concentration of each amino acid in the urine. This method of reading the spots ( is as efficient as, if not more efficient than, the elution technique which is very widely used. The elution technique involves cutting out a spot, eluting its color with 20% alcohol, and reading the light transmittance in a spectro- photometer. Table 1 gives the results obtained by these two methods from 100 lambda of the same urine sample chro— matographed three times. The two methods are highly cor- related as testified by the correlation coefficients of 0.97348, 0.95830 and 0.97560 for the first, second and third runs respectively. Although it is known that the intensity of color in the amino acid-ninhydrin reaction is directly proportional to the amount of amino acid present in the reaction, the results are only interpretable indirectly from the relative density of the spots produced by this method of analysis, rather than in values directly related to their true concentrations in the urine. 1 “Elm—.1 The densest amino acid on a chromatogram was given an arbitrary value of 30, the next densest was given a value of 28, the next a value of 26, and so on. A total of 14 amino acids were measured in this way, so that the least dense spot received a value of 4. If two or more spots had the same density, they received the same value which in this system is always an integer (for example, if two spots were of equal density and were denser than any other spot on the chromatogram, each would get a value of 29, and the third densest spot would get a value of 26). If an amino acid appeared to be missing, a reading would be made at the point at which it would be expected. Except for the "lower histidine" Spot, missing spots were rare. I 1:.mxv .Housnwuucoo “Once can no mdwmouhs £UH3 .mpmeoo o>fluflmomucfluomxififlu Hono>mm mo nomm xmameoo m on cu cmuommmsm ma “a .Hm>m30m .ucmfiwummxo.mnu.mo uumm menu cw omUSHocfi no: mm3 mawmonhu mm pmflmwusmow uomm m£a«« .socmfioconm mmdmuum was» new canon comb mun.c0mmmu oz .uammwnm on no: awe no woe poem Hm3oa on» use .ucmmmum m>m3am ma uomm women one .maflwflumflc How cmwMfludmoH on cmo muomm 039* 10 ommea.o ommmm.o memem.o ucmfioflmmmoo coflumamuuoo II II In II II II «*mdwmouma m.ma o.mm m.mH «.mm m.mH o.mm muam m.¢H ~.em m.¢a «.mm m.qH 0.5m mamamoumana o.mH m.hm o.mH H.mm o.ma m.km manamamaaamam m.¢a o.nm m.¢H o.mm m.ea «.mm mnflmumso o.vH ~.~m N.¢H m.¢m o.¢H m.~m mudguaa m.va o.mm ¢.¢H m.vm v.va o.mm mannuflcuo .. I- u- u- n- .. Isaac mcfloflumum ~.m m.me m.m «.mh m.m o.me «A.msv mcfloflumhm m.mH o.am m.ma o.mm m.mH o.om madaomnae o.NH o.mm o.~H o.ew m.HH o.mm manusma m.HH o.¢m H.HH «.mm o.HH H.¢m manumm m.m m.oe ~.m e.H> ~.m m.~e manowao m.oa 0.0m H.OH o.mm m.oa m.Hm maesmusao m.ma o.mm m.ma c.5m m.ma ~.mm chow onsmusao mcfipmmn cowmmfls mcHUmmu dowmmHE ocwvmmu cowmmflfi nwumfi Imamup Hmumfi Imcmuu “mama Imcmuu named a coausam prong m conusam unwed w coflusam manna cause cam wanna cam wcoomm cam umuflm .Emnmuoumsonno n so muomm mo muflmcmucw m>flumamu on» mnflommu mo mdmws 0 mm mmcacmwh.nmu08 unmwa ownmmumouonm can mdvacsomu dowusam nmmzfiwn GOmHHmmEOUII.H manna RESULTS Figure 3 shows the relative positions of the major spots at the time the colors are developed, and the chief amino acid that has been identified with each spot. Figure 2 is a picture of a typical chromatogram. The raw data for most of the boys consist of ten observations (average: 9.13). In general, there was a good deal of consistency for each individual from day to day and often quite a lot of differentiation between indi- viduals. The 14 amino acids selected for this study are glu- tamine, glycine, serine, taurine, threonine, histidine, ornithine, alanine, cysteine, phenylalanine, tryptophan, beta-amino-isobutyric acid (BAIB), and tyrosine. The spot identified for tyrosine is probably a complex spot that includes a lot of other ninhydrin-positive compounds in the' urine. Although the sample size is too small to allow any statement regarding "high" and "low" excretors to be valid, suggestive trends were observed. Table 2 gives the values of "high," normal" and "low" excretors for the various amino acids as computed from the data. Because the method of segregation of "high" and "low" excretors is a crude one by this technique, attention is drawn only to 11 12 9 o o o o s o o o o o a... 0. .‘...‘. I. O... Q. U...‘ o o o I . I O . . ‘ O . ' 0.....‘0. 0.0.0.0.... o O . .0.....\.. .l...‘. 0......‘....... ‘o 00.. Figure l.--Picture of chromatographic system. ...‘ r- . .‘ Figure 2.--Picture of a typical chromatogram. ‘3" FII’S?‘ D/rec ‘hon 13 79/“ BA IB , ’ \ / \ ' \. L—HLsf Second D/recf/on Figure 3.--Diagram showing relative position of amino acids. l4 .mmamEmm ocean m usonm How mmsHm> usmfla mo mommm>m may we Hmsofl>fitcw no mo some map can «memos Hmstfl>flocfl may mo smmfi cacao may .mmmo mambo ca .mumimxdmu some onae« m pmmma um .an xcmn some on» 30Hmn damn mosam> omos3 mmonu mum muoumuoxm =3oq= 30HOQ UGM ”>030 mflflg m qwfiu. mmmfl 0H0 WODHM> 0m0£3 GmOfi OHM mHOHOHUNm .muHc: .xcmn some on» =HMEHOZ: .much m ammoa pm .wn Mann some on» m>onm damn mdem>.mm0£3 mmozp mum muoumnoxm =£mflm=¥ . NN N . mm s . mm m N.m mHam H NN m m mm m .H He . o.OH mchmHmHmcmnm m as m N on e e mm m m.m cmnmoumaus H NN m N mm H N mm N m.mH mchummo N NN H N mm N m «m N «.mH mcHamHa NH Nm H m NN m w om m o.eH qunucho N Hm H H oe H H He . o.NN mcHeHumHm m we N H em v N Nm m m.HH mchomnna m mN N . He H m mm H N.HN maHusma H mm . . Ne . H He . m.mN mcHumm . em 1 . Ne I . Ne . «.mN mcHoaHo . em I I Ne . . Ne . m.eN manmusHo H Nm H H ow H . Ne . m.NH eHom OHEmusHo 30H HmEHoz (swam 30H Hmfinoz swam 30H Hmfiuoz boa: xcmm Hmpoe oumwz muHsz .«cmmz moH04 ocHem #mHoumHoxm mo muwnesz musHOmnm n E .moflom ocflem Hmscflbflocfl may now muoumuoxm =Soq= can =HmEHOZ= =.nmfim=nl.m manna 15 possibilities and not to facts. In general, the relative density of each amino acid is comparable from person to person. A "high" excretor is defined as a person who is 3 united or more above the mean rank, and a "low" excretor is defined as a person who is 3 units or more below the mean rank. The data presented in Table 3 show that although we have possibly one "high" and one "low" excretor for glu- tamic acid among the Negroes, no white boy showed either pattern. Another possible Negro-white difference that is apparent from the data is in the excretion of ornithine and phenylalanine. There seems to be a shift in the trend of "high" and "low" ornithine excretors from more "high" than "low" excretors among the whites to more "low" than "high" excretors among the Negroes. For phenylalanine, whereas there were no "high" excretors among the whites, there were 3 out of 42 (7.1 per cent) "high" excretors among the Negroes. Interestingly enough, there were no unusually low excretors for BAIB. For "high" excretors of BAIB, there were 3 out of 42 (7.1 per cent) whites and 4 out of 42 (9.6 per cent) Negores. Taking both groups together, we have 7 out of 84 (8.8 per cent) "high" ex- cretors of BAIB, which value is in reasonable agreement with Harris's 9.6 per cent (Harris, 1953). Two amino acids that tend to exchange positions in the two groups are glutamine and histidine. Among whites, histidine tend to be the second densest spot while ll‘l.ifl. ”HY?“ ~.y ." l" l6 glutamine is the third densest spot. Among Negroes gluta- mine tends to occupy the second position while histidine is third. Table 3 gives the number of white and Negro urine samples that contain more glutamine than histidine, those that have the same amount of glutamine and histidine and those that have more histidine than glutamine. It was H e found that of the 394 urine samples analyzed for whites, only 75 (19.035 per cent) showed more glutamine than his— j tidine while 300 (76.142 per cent) showed more histidine : g than glutamine. Among the Negroes, of the 373 samples ” I analyzed, 175 (46.917 per cent) showed more glutamine than histidine, while 173 (46.381 per cent) showed more histi- dine than glutamine. The unweighted means of the results for each boy were used as the information subjected to analysis in this study. The overall racial means for the relative density of spots are presented in Table 4. The results as presented in Table 4 do not show any clear distinction between the races. However, there are minor differences that are helpful in distinguishing boys of the two races. The major difference is that glu- tamine is relatively denser on the chromatograms of Negroes than on those of whites. The mean for Negroes, 26.871, is only 0.714 above the mean for whites, 26.157. The standard deviations, 0.655 and 0.541, respectively, are almost as large as this difference which indicates that a relatively 17 RUIN ”1...... H . MNH mN mNH mNm mmoummz oom ma mm «mm mwuflnz mcflsmusHm corp mHB mcwoflumfls swan monEmm ocflcwumwn mnoz . mcflfimuus who: mcHHD mo Hwnfisz mmomm ..mmomu nuon How mdflpfiumws away mcwfimusam once canvcoo umnu moamfimm means mo Hones: on» mcfi3osm mabmu «(l.m manna 18 Table 4.--Overall Racial Means for the Relative Density of Spots. Differences Amino Acids White Negro (white-Negro) Glutamic acid Glutamine Glycine Serine Taurine Threonine Histidine Histidine Ornithine Alanine Cysteine Tryptophan (Up.) (Lo.) Phenylalanine BAIB Tyrosine ¥ 18.3 26.2 29.8 23.2 21.1 15.2 18.3 26.9 29.8 23.5 21.3 11.7 26.7 13.4 +0.1 I“ IN I. u' “HQ—"F 19 large proportion of each race will fall into the mode set by the other race. If the midpoint of the two races, 26.514, is chosen as the decision point, 26.2 per cent of Negroes will be below this point and so will be misclassi- fied, while 23.8 per cent of the whites will be above this point and will be misclassified, as shown in Figure 4. An analysis of variance for glutamine is shown in Table 5. Individual variability within each race has F equal to 10.1860, which is significant at the 1 per cent level while a test of homogeneity between the races has F equal to 9.6207 which also is significant at the l per cent level. This means that although individuals of the same race dif— fer significantly from each other, the races also differ significantly. Therefore it appears that Negroes excrete relatively greater amounts of glutamine than do whites, although the two distributions overlap. Ornithine, which shows a greater mean.difference between the races, showed individual variability within each race to be significantly different at the 1 per cent level, but showed that both races constituted a homogeneous pOpulation. A discriminant analysis has been performed to de- termine the best formula for distinguishing members of the two races. A series of analyses found that the best dis- (crimination was obtained from the data for glutamine (GLN), serine (SER), threonine (THR), and cysteine (CYS). The discriminant function is: 20 Table 5.--Analysis of Variance for glutamine. if. u Sources of Error D.F. S.S. M.S.S. F Races (R) 1 111.6 111.6 R/P = 9.6207* People (P) 82 949.6 11.5805 P/S = 10.1860* Sample (S) 683 776.5 1.1369 Total 766 D.F. is Degree of freedom. 8.5. is Sums of squares. M.S.S. is Mean sums of squares. *Significant at l per cent level. 28— oo 00 000 275 000 NHDFKNAIT ooo 26.5/4 +5 o 264 oo 255 21 ++ ++ ++++++ + +++++ ++++ ++++ ++++hHH- +++ ++ Legend 0 ll 4.. ll Negro distribution White distribution RM. Figure 4.--Distrubution of Negro and White glutamine values. - ' I _ "A.. . ‘ ti- L"'-‘I‘VFJ 22 0.03123 GLN + 0.00479 SER + 0.00262 THR - 0.00360 CYS With this function, the mean value for Negroes is 0.92729 and that for whites is 0.90147, which is a difference of 0.02582. The standard deviations with each race are 0.01376, and 0.01687, respectively. Figures 5 and 6 represent a distribution of Negro and white discriminant values. Using a dividing point of 0.9127, 21.4 per cent of the Negroes will be below this point and hence will be misclassified as whites while the same percentage of whites will be above this point and will be misclassified as Negroes. ~m.-.-, , .96 —— .95- .94 — .93- SE— .995——+__ ”9,- .89~ .88 — Figure 5.--Distribution of Negro and White disciminant 000 O O 0 00000 0 08 O 0 000 0000080 00000 000 O 00 OO ++++ + + +4- ++++ 5~‘+ lkn+++ 1.. -+++++«+ ~+ + 4. ~+. H1. +~++ + 23 Legend 0. II + II Negro distribution White distribution v-A functions. RA .3. ""1 :4 n h . I "H... ..._.I .muouomm ucmcwfifluomap mpwnz can oummz mo soapsnfluumwall.m madman Nmmmm .693 81% 59mm 8.8. N95. 540m 09% 8.8% mme NmHom mmHmm O /+ O +\. coausnfiuumfic onumz coHuanuumHu manz DISCUSSION The purpose of this study was to determine if ra- cial differences in urinary amino acid excretion can be detected between whites and Negroes living under identical conditions. The subjects were healthy adolescent boys, which gave the group a great deal of uniformity except for the racial difference. The chromatographic system that was used was cho- sen because it provides a large amount of data relative to the effort involved. It was possible for a single individual to analyze twenty urine samples per day as a routine procedure. High excretion of an amino acid may be as a result of one or more of several factors: 1. It may be due to overproduction of the amino acid. 2. It may be due to the inability of the system to utilize the particular amino acid. 3. It may be due to very low renal threshold. The first and second reason will concentrate the (amino acid in the blood, while the third reason will cause the urine to have too much of the amino acid without neces- sarily being too high in the blood. The genetic 25 26 interpretation that can be given to high excretion of an amino acid will depend upon the cause. If it is due to an enzyme defect, a single gene (which may be autosomal or sex-linked and may be recessive, semi-dominant or domi- nant) is often said to be responsible. On the other hand, if the defect is traced to renal malfunction, except if familial studies prove it to be genetic, it may be patho- logic. Interestingly enough, of the seven peOple who were classified as "high" excretors of threonine, six of them showed trends of excreting moderately higher amounts of glycine when expressed in terms of its light value rela- tive to the other amino acids and not in terms of its rank. The reason for this is easy to explain from the biochemical relationship between glycine and threonine. It is known that in the liver and kidney of mammals, the enzyme threo- nine aldolase (in the presence of a cofactor, pyridoxal phOSphate) catalyses the following reversible reaction. NH2 -CH-—CH-—COOH'——————> CH ——COOH + H N-—CH -—COOH V—-———— 3 2 2 OH CH3 Threonine Acetaldehyde Glycine 27 By this method of analysis (the ranking of light values), it was not possible to pick out "high" excretors of glycine. However, it would require a more quantitative analysis and thorough enzyme assays to confirm the pres- ence of such a relationship between glycine and threonine in the urine and to determine the position of the primary cause. There are no striking differences in excretion patterns between the two races. However, the minor differ— ence in the relative amounts of glutamine in the urine can be used to make a probability statement about the race of the donor of a given urine sample. Among the 30 per cent of the chromatograms that have glutamine as the densest or second densest spot, the probability that the chroma- togram comes from a Negro donor is 0.72, while among the six per cent of the chromatograms that do not have gluta— mine among the three densest spots, the probability is of the same magnitude that the donor is white. Similarly, among the 62 per cent of the chromatograms that have histi- dine as the densest or second densest spot, the probability that the chromatogram comes from a white donor is 0.64. The probability of guessing correctly about the’ race of an individual is increased by taking the average of several chromatograms, and this is slightly improved by a discriminant function analysis which combines infor- mation on the relative densities of glutamine, serine, 28 threonine and cysteine. The addition of information on the relative densities of the other amino acids did not increase the power of this method in separating the two races. The difference reported here may not be due to a major gene effect. It may be a result of the genetic back- ground of the individuals; in which case, it is a result of many genes. The racial differences that this study was designed to establish were not distinctly obvious. However, it was slightly obvious that the pattern of excretion of glutamine was different enough to separate the races, which separation is made slightly better by a discriminant analysis. The racial studies on urinary amino acid excretion by Sutton and Clark (1955), Gartler gt_§1. (1956-57), and McEvoy- Bowe and Lugg (1961) failed to show this racial difference. This difference may be made even more obvious if a much larger population is considered. REFERENCES Berry, H. K. Variations in urinary excretion patterns in a Texas pOpulation. Amer. J. phys. Anthrqp. 11, “M 559, 1953. ' F .91? "2' . Further studies on individual urinary and salivary amino acid patterns. Univ. Texas publ. No. 5109, 157, 1951. Berry, H. K., and Cain, L. Individual urinary excretion : . patterns of young children. Univ. Texas publ. v} No. 5109, 165, 1951. Berry, H. K. Cystinuria in mentally retarded siblings with a typical osteogenesis imperfecta. A.M.A. J. dis. Child. 97, 196, 1959. Block, R. J., Darrum, E. M., and Zweig, G. A manual of paper chromatography and paper electrophoresis. Academic Press, Inc., N.Y., 1958. Christensen, P. J., Date, J. W., Schonheyder, F., and Volqvartz, K. Amino acids in blood plasma and u urine during pregnancy. Scand. J. clin. Lab. Invest. 9, 54, 1957. Datta, S. P., and Harris, H. Dietary origin of urinary methyl-histidine. Nature 168, 296, 1951. Dent, C. E., and Harris, H. The genetics of cystinuria. Ann. Eugen. (Lond.) 16, 60, 1951. jEckhardt, R. D., and Davidson, C. S. Urinary excretion of amino acids following the rapid injection of a solution of amino acids in man. J. clin. Invest. 27, 727, 1948. . Urinary excretion of amino acids by a normal adult receiving diets of varied protein content. J. biol. Chem. 177, 687, 1949. Fowler, D. 1., Norton, P. M., Cheung, M. W., and Pratt, E. L. Observations on the urinary amino acid excretion in man: the influence of age and diet. Arch. Biochem. 68, 452, 1957. 29 30 Fox, M., Thier, S., Rosenberg, L., Kiser, W., and Segal, S. Evidence against a single renal transport defect in cystinuria. New Eng. J. Med. 270, 556, 1964. Gartler, S. M., Firschein, I. L., Gidaspow, T. Some genet- ical and anthrOpological considerations of urinary beta-aminoisobutyric acid excretion. Acta Genet. (Basel) Suppl. No. 6, 435, 1956-57. Gartler, S. M., Dobzhansky, T., and Berry, H. K. Chroma- tographic studies on urinary excretion patterns on monozygotic and dizgotic twins. II. Heritability of the excretion rates of certain substances. Amer. J. hum. Genet. 7, 108, 1955. Ghadimi, H., Partington, M. W., and Hunter, A. A familial disturbance of histidine metabolism. New Eng. J. Med. 265, 221, 1961. . Inborn error of histidine metabolism. Pediatrics 29, 714, 1962. Harris, H. Familial studies on the urinary excretion of beta-amino isobutyric acid. Ann. Eugen. (Lond.), 18, 43, 1953-54. . Aminoaciduria in man. Third International Con- gress of Biochemistrx (abstract), 1955. ' Hooft, C., Timmermans, J., Snoeck, J., Antener, I., Oyaert, W., and Van den Hende, Ch. Methionine malabsorp- tion in a mentally defective child. Lancet ii, 20, 1964. Honda, M. Untersuchung des Harns gravider Frauen. J. Bio- chem. (Japan) 2, 351, 1923 (abstract). Jagenburg, R. The urinary excretion of free amino acids an and other amino compounds by the human. Scand. J. clin. Lab. Invest. suppl. 43, vol. 11, 1959. Kirsner, J. B., Sheffner, A. L., and Palmer, W. L. Studies on amino acid excretion in man. III: Amino acid levels in plasma and urine of normal men fed diets of varying protein contents. J. clin. Invest. 28, 716, 1949. Kirsner, J. B., Sheffner, A. L., Palmer, W. L., and Bergeim, 0. Amino acids in plasma and urine of patients with hepatitis before and after a single infusion of protein hydrolysate. J. Lab. clin. Med. 36, 735, 1950. 31 Langley, W. D. Urinary histidine. Determination of histi- dine in normal and in pregnancy urines. J. biol. Chem. 137, 254, 1941. Lawrie, N. R. The excretion of 1(-) tyrosine in urine. Biochem. J. 41, 41, 1947. McEvoy-Bowe, E., and Lugg: J. W. H. A direct quantitative paper chromatography of amino acids and application to the urinary excretion of some human ethnic groups. Biochem. J. 80, 616, 1961. Modes, G. A new error tyrosine metabolism: tyrosinosis, the intermediary metabolism of tyrosine and phenylala- nine. Biochem. J. 26, 917, 1932. ' Menkes, J. H., Alter, M.,Steigleder, G. K., Weakley, D. R., and Sung, J. A. A sex-linked recessive disorder with retardation of growth, peculiar hair, and V . ‘ focal cerebral and cerebellar degeneration. Pedi- _;f atrics 29, 764, 1962. "“2 Russel, A., Levin, B., Oberholzer, V. G., and Sinclair, L. Hyperammonaemia: a new substance of an inborn enzymatic defect of the biosynthesis of urea. Lancet ii: 699, 1962. Schafer, I. A., Scriver, C. R., and Efron, M. L. Familial hyperprolinemia, cerebral dysfunction, and renal anomalies occurring in a family with hereditary nephropathy and deafness. New Eng. J. Med. 267, 51, 1962. Scriver, C. R., Schafer, I. A., and Efron, M. L. New renal tubular amino acid transport system and a new hered- itary disorder of amino acid metabolism. Nature 192, 672, 1961. . Evidence for a renal tubular amino acid transport system common to glycine, L-proline and hydroxy L- proline. J. clin. Invest. 40, 1080, 1961. Sutton, H. E., and Clark, P. J. A biochemical study of Chi- nese and Caucasoids. Amer. J. phys. Anthrop. 13, N.S. 53, 1953. Sutton, H. E., and Vanderberg, S. G. Studies on the vari- ability of human urinary excretion pattern. Hum. Biol. 25, 318, 1953. Tada, K., Ito, H., Wada, Y., and Arakawa, T. Congenital tryptophanuria with dwarfism. Tohoku J. Exper. Med. 80, 118, 1963. 'Voge, C. A simple chemical test for pregnancy. Brit. med. ' J. 2, 829, 1929. ADDI TI ONAL REFERENCES Allan, J. D., Cusworth, D. C., Dent, C. E., and Wilson, V. K. A disease, probably hereditary, characterized by severe mental deficiency and a constant gross ab- normality of amino acid metabolism. Lancet i:182, 1958. Berry, H. K., Cain, L., and Rogers, L. L. A study of the urinary excretion patterns of six human individuals. Univ. Texas publ. No. 5109, 150, 1951. Clarkson, T. W., and Kench, J. E. Urinary excretion of amino acids by man absorbing heavy metals. Biochem. J. 62, 361, 156. Datta, S. P., and Harris, H. Urinary amino acid patterns of some mammals. Ann. Eugen. (lond.) 18, 107, 1953. Dent, C. E. Detection of amino acids in urine and other fluids. Lancet 2, 637, 1946. . A study of the behavior of some sixty amino acids and other ninhydrin-reacting substances on phenol- "collidine" filterpaper chromatograms, with notes as to the occurrence of some of them in biological fluids. Biochem. J. 43, 169, 1948. . Clinical applications of amino acid chromatography. Scand. J. clin. Lab. Invest. 10, suppl. 31, 122, 1957. .\ Dingham, H., and Wright C. A multivariate analysis*of amino acid excretion. J. Men. Def. Research 8(1), 77, 1964. Dustin, J. P., Moore, S., and Bigwood, E. J. Chromatographic studies on the excretion on the excretion of amino acids in early infancy. Metabolism 4, 75, 1955. Eckhardt, R. D., C00per, A. M., Faloon, W. W., and Davidson, C. S. The urinary excretion of amino acids in man. N.Y. Acad. Sci. Trans. 10, 284, 1948. 32 33 Evered, D. F. The excretion of amino acids by the human. A quantitative study of ion-exchange chromatography. Biochem. J. 62, 416, 1956. Gartler, S. M. A family study of urinary beta-aminoiso- butyric acid excretion. Amer. J. hum. Genet. 8, 120, 1956. Jagenburg, R. Amino acids and peptides in infant urine. Acta chem. scand. 9, 1047, 1955. Miller, S., Ruttinger, V., and Macy, I. G. Urinary excre- tion of ten amino acids by women during the repro- ductive cycle. J. biol. Chem. 209, 795, 1954. u‘..._. n 1!). 1““ I .’ Moore, S., and Stein, W. H. A modified ninhydrin reagent for the photometric determination of amino acids and related compounds. J. biol. Chem. 211, 907, 1954. ’ I.".hfif_ Mueller, G. C., Bowman, G., Herranen, A. Desalting amino acid solutions by ion exchange. Analyt. Chem. 27, 1357, 1955. Nasset, E. S., and Tully, R. H. Urinary excretion of essen- tial amino acids by human subjects fed diets con- taining proteins of different biological value. J. Nutr. 44, 477, 1951. Norton, P. M., Pratt, E. L., and Hasselmeyer, E. Urinary amino acid excretion by premature infants. Amer. J. dis. Child. 88, 659, 1954. Rubenstein, M., and Wolff, S. M. Amino acid excretion in familial mediterranean fever. Arch. Internal Med. 113(3), 409, 1964. Salisbury, P. F.,.Dunn, M. S., and Murphy, E. A. Apparent free amino acids in deproteinized plasma of normal and uremic persons. J. clin. Invest. 36, 1227, 1957. Sandler, M., and Pare, C. M. B. Starvation amino aciduria. Lancet 1, 494, 1954. Slatis, H. M. Normal variations in excreted amino acids in man. Proc. XI International Congress of Genetics, 1963 (aBStract). 34 Smith, I. Colour reactions on paper chromatograms by a dip- ping technique. Nature 171, 43, 1953. Soupart, P. Urinary excretion of free amino acids in normal adult men and women. Clin. chim. Acta 4, 265, 1959. Sutton, H. E. A further study of urinary excretion patterns in relation to diet. Univ. Texas publ. No. 5109, 173, 1951. APPENDICES m.mm m.mH o.mH m.n m.ha v.vH v.h >.v m.mm h.HH v.am ¢.¢N o.om n.5m m.mH couuflum o.H~ H.m m.mH m.> m.mH m.ma m.oa ¢.n H.wm v.oa m.H~ o.m~ o.om M.SN «.ma somuopad m.mH v.> >.HH m.m o.>H «.ma ¢.- m.m m.hm m.h o.om m.Hm ¢.mm w.mm n.5H mcwxpd o.w~ m.HH o.~H m.va m.va N.HH o.m m.m h.Hm m.ma h.m~ m.mm o.om H.0N H.nH umuz v.mH H.m m.m H.m m.mH m.mH ¢.oa H.OH m.mm quH N.HN m.m~ o.om m.m~ H.ma mmom m.Hm H.m v.m m.m o.mH o.¢H >.ma o.HH n.¢m o.mH m.mH m.mm o.om v.wm m.om (counoz >.mH m.o~ ~.> v.m m.MH m.mH m.HH m.m m.m~ m.oa o.Hm «.mm o.om m.>m mama Headmaoz N.Hm H.MH m.m m.m o.mH m.>H m.m m.h ~.mm m.NH v.om m.mm o.om o.>m Hyma comc£0h v.ma m.h m.m v.0H m.va m.nH m.oH m.NH m.m~ w.HH m.HN m.mm o.om H.>~ m.mH Gasman o.mm m.m m.m m.vH v.va H.mH m.oa n.0H o.m~ m.m m.o~ e.m~ o.om o.m~ m.mH whom o.m~ o.> H.m w.OH m.~H n.mH m.ma m.OH >.mm v.a m.mH m.mm o.om m.hm h.hH acmcom m.vH «.5 ~.m m.m m.NH ~.mH o.mH m.mH 0.0m .v-NH m.om v.mm o.m~ o.mm m.mH acmuumm m.m ¢.n w.HH b.HH m.mH m.mH m.mH m.hm >.m m.o~ «.mm m.m~ m.m~ m.mH osoHUoz o.mm m.m H.h o.m m.vH m.mH m.HH m.m m.mm o.~H o.HN o.m~ o.om 0.5m o.Hm mdw3uc¢ nu m.h m.m N.m m.~H m.mH .m.NH In m.h~ H.5H m.mm H.mm 0.0m >.mm H.mH uoxamz II H.MH N.HH o.HH m.mH m.mH o.m In w.mm o.HH H.mm ~.m~ o.om m.om m.mH mnmomm II H.0H m.m o.HH m.>H H.mH m.mH II v.5m m.OH m.HN m.mm «.mm m.mm m.o~ nuHEm II m.m m.OH «.0H m.wH v.mH m.mH In ~.n~ m.OH N.~N H.mm o.om w.mm m.ma Hmoz II o.m m.mH m.h m.~H «.mH m.mH II o.mm m.HH H.H~ m.mm H.m~ H.nm m.hH cmmHoz II H.0H H.¢H m.m m.vH m.ma m.HH In >.m~ OJHH m.mH m.mm m.m~ v.m~ m.H~ meson In o.oH m.~H m.HH m.oa H.vH m.ma nu o.m~ m.mH H.m~ m.¢m o.om o.wm o.mH .4.mwhmm II H.m o.oH o.m m.mH m.>H m.mH In >.mm m.m m.m~ o.mm 0.0m o.>m m.ha mfiaaflw II v.m m.m h.HH ~.wH v.~H h.HH an m.mm m.mH m.am «.mm m.m~ «.mm m.na Ems nmdflcdsu II o.n m.mH m.m H.mH m.mH H.~H In m.m~ m.mH o.- v.¢m 0.0m m.m~ o.om :SOHm II N.m o.mH H.mH m.vH- H.mH H.vH II N.n~ m.vH m.HN m.mm o.om m.mm m.hH oauumm .mwa mH..-I...!EJJJ Ilv‘u" N.NH m.m o.OH m.m m.mH N.mH 4.MH m.m N.mN N.HH N.HN m.MN 4.NN m.mN N.NH memo: mo can: N.NHm H.Nmm 4.34 N.Nmm m64m Home 4.3m m6: N.NNHHN.Nm4 o.mmm n.4mm 9me 4.NN: N.NE. sum o.NH N.N m.m m.m N.NH m.mH N.NH o.NH m.mN N.OH 4.HN 4.NN o.om N.NN m.mH nuance 4.NH m.m N.NH N.m m.NH 4.4H m.mH m.m N.NN N.oH 4.HN 4.NN o.om N.SN G.ON msHm 4.4H m.m m.m 0.4 m.NH G.mH 4.NH m.N m.mN 4.HH m.HN 4.4N o.om N.NN N.NH :msxumm o.NH 4.N m.OH m.m m.NH N.NH N.HH m.HH m.mN N.HH N.NH N.NN o.om 4.NN m.mH mucous N.NH 4.oH m.m N.m m.mH N.GH m.OH N.NH o.NN m.OH N.0N o.mN. o.om m.mN N.NH ooooz N.NH N.m N.oH m.m N.4H H.NH 4.5H N.OH m.mN N.m G.ON o.4N o.om N.NN m.mH mmunmssm N.GH N.HH m.N H.m 4.mH 4.mH 4.NH N.NH H.NN N.NH N.ON H.NN o.om N.SN H.NH nnmsom m.mH m.N m.m o.m 4.NH N.mH 4.NN 4.HH 4.NN o.HH o.o~ m.NN 0.0m H.oN m.mH mmcHseso 4.NH m.N m.OH H.N m.mH N.NH o.HH m.N N.mN 4.HH N.NN H.NN o.om N.NN 4.NH aocamo N.NH H.HH m.NH m.m 4.4H m.4H N.HH o.4 m.mN m.NH m.HN o.4N o.om N.NN N.NH uuHusm 4.NH m.m m.OH o.m m.mH N.NH 4.4H 4.4 o.NN m.oH H.HN 4.NN o.om o.NN m.NH amazoam 4.4H H.m N.m m.n m.mH N.NH N.eH N.4 o.mN H.NH H.HN o.mN o.om o.mN G.NH uHom m.mH N.N m.m 4.m 4.NH m.mH N.OH m.m o.mN m.HH H.NN 4.NN o.om o.NN G.NH ovum N.NH H.N H.0H 4.4 4.4H m.NH m.mH o.m N.NN m.mH 4.HN m.mN N.NN m.mN m.NH manna: m.mN m.OH m.m m.m 4.NH m.4H 4.HH o.m 4.4N m.HH o.ON m.mN o.om 4.NN 4.NH umHHHz N.ON N.m m.OH N.m N.NH m.4H N.NH H.m m.NN 4.NH N.NN m.NN o.om N.mN N.NH mason N.NH N.N 4.0H m.m N.4H N.mH m.ON m.m m.NN o.4H o.mH 4.NN o.om m.oN N.4H .o.czoum .mwa mHem .mmm .mmme .ammo . 0.0« 0.04 0.04 4.0 4.04 0.04 4.04 0.0 0.0« 0.0 «.04 «.H« 0400 0.0« 0.04 cemmafim 0.04 0.0 0.0 0.0 4.04 0.44 0.04 0.44 4.0« 0.04 0.«« 0.4« 0.00 0.0« 0.04 4405000 «.04 0.0 0.0 0.0 0.44 0.44 0.04 0.04 0.0« 0.0 0.4« 0.«« 0.00 0.0« 0.04 40004000 0.4« 4.44 0.04 0.0 0.04 0.04 0.44 0.0 0.0« 0.«4 0.0« 0.0« 0.00 «.0« 0.04 440440 0.4« 0.0 0.0 0.0 0.04 4.04 0.«H 0.04 «.0« «.04 0.0« «.0« 0.0« «.0« 0.04 Oncmumz 0.04 «.0 0.04 0.0 0.04 0.44 0.04 4.0 0.0« 4.04 0.0« H.«« 0.00 0.0« «.0« 40404450 .009 0H00 .000 .0000 49000 .404 .zmo.BmH0I0.BmH0ID.00000 .009 .000 .000 .zlaqw .009 404403 .045444coonu.«.4 44444004 40 THE RACK Appendix A. 3.--The Chromatographic Rack | ’.‘I HISAN STATE UHIVER’S YY’ L.~|FIP PIES III III II III | I I 174 5379