Genetic and Population Studies of Quantitative Levels of Adenosine Triphosphate in Human Erythrocytes

Size: px

Start display at page:

Download "Genetic and Population Studies of Quantitative Levels of Adenosine Triphosphate in Human Erythrocytes"

Barbra Sims
5 years ago
Views:

Bichemical Genetics 1:25-34 (1967) Genetic and Ppulatin Studies f Quantitative Levels f Adensine Triphsphate in Human Erythrcytes Gerge J. Brewer 1 Received 5 Dec. 66--Final 30 Jan.

1 Bichemical Genetics 1:25-34 (1967) Genetic and Ppulatin Studies f Quantitative Levels f Adensine Triphsphate in Human Erythrcytes Gerge J. Brewer 1 Received 5 Dec. 66--Final 30 Jan. 67 The mean cntent f ATP in red cells f American Negres is significantly less than the mean level in American Caucasians. This is cmpatible with the hypthesis that the quantitative level f ATP in red cells may be invlved in selective prcesses related t falciparum malaria. There is n evidence f a sex effect n levels f ATP in either ppulatin. Family studies cnducted in bth ppulatins indicate that the quantitative level f red cell ATP is at least partially inherited. Studies f a number f bichemical characteristics f red cells have been cnducted in an effrt t elucidate the mechanism f genetic and bichemical cntrl f quantitative levels f erythreytic ATP. These studies have been negative. Althugh ther studies have demnstrated that thalassemia trait influences the level f red cell ATP, the presence f sickle cell trait r G-6-PD deficiency, the ther tw systems pstulated t be invlved in malaria prtectin, did nt result in significant differences in mean red cell ATP cntent. INTRODUCTION Preliminary studies in ur labratry have indicated that the mean level f erythrcytic adensine triphsphate (ATP) in American Negres is significantly lwer than that f American Caucasians (Brewer, 1965b). This bservatin led t studies suggesting that relatively lw levels f red cell ATP in the Negr may be prtective against falciparum malaria (Brewer and PweU, 1965). It has als been fund that heterzygtes fr the beta thalassemia gene have significantly lwer levels f erythrcytic ATP than nrmal individuals (Ls et al., 1965; Brewer et al., in preparatin). It seems pssible that the pstulated prtective effect f thalassemia against malaria may result frm an effect f the thalassemia gene n levels f red cell ATP. The pssibility f an imprtant rle fr ATP in selective prcesses related t malaria makes The wrk reprted in this paper was supprted in part by the Research and Develpment Cmmand, Office f the Surgen General, Department f the Army, under cntract DA MD-2855 with the Department f Medicine, University f Michigan. With respect t this supprt, it is cntributin number 167 frm the Army Research Prgram n Malaria. The wrk was als supprted in part by USPHS grant AM and USPHS Career Develpment Award 1-K3-AM Departments f Medicine (Simpsn Memrial Institute) and Human Genetics, University f Michigan Medical Schl, Ann Arbr. 25

26 Brewer it imprtant t study the inheritance f quantitative levels f erythrcytic ATP and t investigate the pssible influence f ther erythrcyte enzymes n ATP cntent in hpes f elucidating mechanisms f

2 26 Brewer it imprtant t study the inheritance f quantitative levels f erythrcytic ATP and t investigate the pssible influence f ther erythrcyte enzymes n ATP cntent in hpes f elucidating mechanisms f genetic cntrl. In this paper we reprt: 1. The results f an expanded ppulatin study f the cntent f ATP in erythrcytes in a sample f American Negres and Caucasians. 2. The results f family studies f erythrcytic ATP cntent. 3. The results f studies crrelating varius bichemical features f erythrcytes with levels f ATP. METHODS Subjects fr the randm ppulatin survey came primarily frm tw surces. Mst male subjects were inmate vlunteers f the Suthern Michigan State Prisn at Jacksn, Michigan. The female subjects and the remainder f the male subjects were individuals wh had bld drawn at the University f Michigan University Hspital Outpatient Labratry. The latter grup were individuals wh were having preemplyment physical examinatins r wh were being seen in the Outpatient Clinic fr a variety f minr, nnhematlgic disrders. Family studies were perfrmed n the families f a subsample f the clinic grup and n the families f labratry persnnel. Individuals with G-6-PD deficiency r sickle cell trait were nt excluded except in the studies f crrelatin between bichemical parameters f erythrcytes and ATP levels. Adensine triphsphate f red cells was measured by a mdificatin f the methd f Krnberg (1950)as previusly described (Brewer and Pwell, 1966). Activities f glucse-6-phsphate dehydrgenase (G-6-PD) and 6-phsphglucnate dehydrgenase in hemlysates were measured by the methd f Glck and McLean (1953) as reprted by Zinkham and Lenhard (1959). Pyruvic kinase activity f hemlysates was assayed by the methd f Tanaka et al (1962) as mdified by Pwell and DeGwin (1965). Glutathine reductase activity f hemlysates was assayed by the methd f Lng and Carsn (1961). Hexkinase activity in hemlysates was measured as previusly reprted (Brewer et al., 1964). Inrganic phsphate in serum was measured by the methd f Fiske and Subbarw (1925). Electrphretic typing f red cells fr acid phsphatase, phsphglucmutase, and adenylate kinase was carried ut by the methds f Hpkinsn et al. (1964), Spencer et al. (1964), and Fildes and Harris (1966), respectively. Screening f Negr ppulatins fr G-6-PD deficiency was carried ut with the methemglbin reductin test (Brewer et al., 1960 and 1962). Hemglbin electrphretic types f sme Negr subjects were determined during starch gel electrphresis carried ut fr enzyme studies. RESULTS Ppulatin Studies In Fig. 1 the level f erythrcytic ATP in unrelated adult Negr males is cmpared t the level in an equal number f adult Caucasian males. All 402 f these subjects

3 Genetic and Ppulatin Studies in Human Erythreytes 27 were inmates f the Jacksn prisn, and hence were living under identical envirnmental cnditins. The mean f the values f the Negr subjects is significantly less than that f the Caucasians (P < 0.001). z Similar results were btained in a cmparisn f % Fig. 1. Cmparisn f frequency distributins f ~ r5-201 adult Negr males and 201 adult Caucasian males, all f whm were inmates f Suthern _~ E. Michigan Prisn at Jacksn, Michigan ,0 ATP CONTENT OF RBC (/4 mles/grn Hgb) NEGRO N Fig. 2. Cmparisn f frequency distributins i5 ~ ~ f 104 adult Negr females and the first 104 adult Caucasian females studied. ~ _ d ATP CONTENT OF RBC { A~ rnles/grn Hgb ) 51 adult Negr females with 104 adult Caucasian females (Fig. 2); the means f these distributins als differ significantly (P < 0.001). A summary f the ttal data cllected t date in the tw ppulatins and statistical evaluatin are shwn in Table I. There is n evidence f a sex effect n levels f ATP (Table I). Family Studies Studies f the inheritance f quantitative levels f ATP in red cells have been cnducted in bth Negr and Caucasian families. Analyses f cvariance (Tables II and 2 The differences f the means were tested fr statistical significance by Students "t" using the tables fr the t distributin presented by Fisher (1958).

4 28 Brewer III) demnstrate that after age adjustment, 3 between sibship variatin is significantly greater than within sibship variatin. Significant differences amng family means, adjusted fr age, indicate that either inheritance r envirnmental influences, r bth, influence levels f red cell ATP in bth ppulatins. One methd fr at least partially evaluating the rle f envirnmental influence is t determine the crrelatin cefficient between spuses (Table IV). The absence f psitive crrelatin between spuses suggests that envirnmental influences in the hme during adult life d nt play an imprtant rle in influencing levels f red cell ATP. Table I Sex Race Number f Mean S.D. c t a P subjects a ATP b M Negr < M Caucasian F Negr <0.001 F Caucasian M Negr <0.9 F Negr M Caucasian < 0.9 F Caucasian M & F Negr <0.001 M & F Caucasian All the subjects were adults. Expressed in micrmles f ATP per gram f hemglbin. c One standard deviatin. d t test f Fisher (1958). Studies f the Crrelatin Between Varius Bichemical Parameters f Erythrcytes and Variatin in Erythrcytie ATP Serum Inrganic Phsphrus Levels and Activities f Glutathine Reductase, G-6-PD and 6-PGD. It has been bserved that the increased level f inrganic phsphrus in the serum f uremic patients results in an elevatin f red cell ATP levels s Althugh nt presented in this paper, preliminary evidence f an age effect n erythrcyte ATP frm birth until age 21 (Gall, Sing, and Brewer, unpublished) suggested that family differences shuld be adjusted fr age. The analysis f cvariance (Steel and Trrie, 1960) f ATP adjusted fr age is given in Table II fr 17 Negr families and in Table III fr 9 Caucasian families. The mathematical mdel which is cnsidered may be written Y~s = lt+f~+bx~s+e~ where Y~I is the ATP level f thejth individual f the ith family,/z is the general mean, f~ is the fixed genetic effect f the ith family, b is the linear regressin f ATP n age, x~j is the deviatin (x~j-~) f the age f the jth individual f the ith family frm the average age, and etj is the randm errr effect assciated with the jth bservatin in the ith family. A reductin f 22% and 15% in the errr variance amng Caucasian and Negr individuals, respectively, was realized by emplying the cvariance analysis. The F rati was used t detect significant differences amng family means adjusted fr age.

5 Genetic and Ppulatin Studies in Human Erythreytes 29 Table II. Analysis f Cvariance--Negr Families Surce f variatin Degrees f freedm Sum f prducts f Results ATP adjusted fr age y, y x, y x, x d.f. Sum f Mean squares square Ttal Amng families (unadjusted) Within families Within families a Amng families = Within families-within families ~ (adjusted) /~r.~ = Adjusted amng families M.S. F(16, 25) ~-- Adjusted within families M.S. =2.805 b a Errr assciated with the mdel, Yj = lt+b'xj+e'l which ignres family effects. b Significant at the prbability level. Table III. Analysis f Cvariance--Caucasian Families Surce f variatin Degrees f freedm Sum f prducts f Results ATP adjusted fr age y, y x, y x, x d.f. Sum f Mean squares square Ttal Amng families (unadjusted) Within families Within families a Amng families = Within families-within families" (adjusted) /~.x = Adjusted amng family M.S. Fts, 17) = Adjusted within family M.S. = b "Errr assciated with the mdel, Yj = lt+b'xj+e'j which ignres family effects. b Significant at the 0.05 prbability level. Table IV. Cefficients f Crrelatin Between Spuses in Levels f Erythrcytic ATP r N P Negr cuples >0.1 Caucasian cuples > 0.1

6 30 Brewer (Hurt and Chanutin, 1964). The rle f serum inrganic phsphrus in the regulatin f red cell ATP levels under physilgical circumstances was evaluated in 44 nrmal Negr males (Fig. 3). The crrelatin cefficient between serum inrganic phsphrus and red cell ATP (r = , N = 44) was nt significantly different frm zer. 40C 30(; >.> ~ 25C ~E g~ 7 el el a -~ 20C m ~,~ 250 2~ Q I ATP (~ redes/gin Hgb) 40 ] i ATP (IX mles/gm Hgb) ~I 5( <.c ~E.lj -.'E_ 4.0 Jx E ~ --rcz.n t 3~ ~ 3.0 :... ql 40l 0 2e ~g C,~ ~0 f I I I I "L.- b I I I t t ATP (IX mles/grn Hgb) ATP (u. rnnles/gm Hgb) 4.4 Fig. 3. Crrelatin diagrams shwing the lack f significant crrelatin between erythrcytic ATP and serum inrganic phsphrus, and between ATP and the activities f three red cell enzymes in male adult Negr subjects. Data crrelating the activity f each f three erythrcytic enzymes with ATP cntent f red ceils f Negr males are als shwn in Fig. 3. Crrelatin cefficients were nt significantly different frm zer.

7 Genetic and Ppulatin Studies in Human Erythrcytes 31 Activity fpyruvie Kinase in Red Cells. One f the steps in the glyclytic pathway f carbhydrate metablism in which ATP is regenerated is the reactin catalyzed by pyruvic kinase. The rle f variatin in activity f pyruvic kinase in regulating ATP levels f erythrcytes was evaluated in 35 Negr and 34 Caucasian individuals (Fig. 4). The crrelatin cefficients between hemlysate pyruvic kinase activity and ATP levels f red cells in Negres and Caucasians individually, and in the pled data, were nt significantly different frm zer. Fig. 4. Crrelatin diagram shwing the lack f significant crrelatin between erythrcytic ATP and hemlysate pyruvic kinase activity in 35 adult Negr and 34 adult Caucasian individuals. E Negr Value Caucasian Value. - a ~g, Z ~. ~e + 2.' ----~.~ ~2 ATP (Ix m01es/gm Hgb) i F. l J I E Negr Value ~" Caucasian Value ~ ~ ~ : ~ ~ ~" ~0 ~, ~ 21 3[0 +'. 51 "6 E ATP(I,k mles/gm Hgb) Fig. 5. Crrelatin diagram shwing the lack f significant crrelatin between erythrcytic ATP and hemlysate hexkinase activity in 14 adult Negr and 35 adult Caucasian subjects. Activity f Hexkinase in Red Cells. Hexkinase catalyzes the initial phsphrylatin f glucse, and may be rate-limiting in the human erythrcyte. The rle f variatin in hexkinase activity in regulating ATP levels was evaluated in 35 Caucasian and 14 Negr subjects (Fig. 5). The crrelatin cefficients fr the Negr, the Caucasian, and the pled data did nt differ significantly frm zer. Acid Phsphatase, Phsphgluemutase, and Adenylate Kinase Eleetrphretie Types in Red Cells. The mean cntent and standard deviatin f ATP in red cells f

8 32 Brewer Negr male subjects with varius electrphretic types f acid phsphatase, phsphglucmutase, and adenylate kinase are shwn in Table V. Significant effects f electrphretic type n erythrcytic ATP levels were nt detected (t test), althugh the sample size in the case f adenylate kinase was quite small. Table V A. Cmparisn f ATP Levels in Erythrcytes with Var. ius Acid Phsphatase Electrphretic Phentypes (Adult Negr Ppulatin Only). Acid phsphatase Mean level f phentype Number ATP + One S.D. A BA B CB B. Cmparisn f ATP Levels in Erythrcyte with Varius Phsphglucmutase Electrphretic Phentypes (Adult Negr Ppulatin Only). Phsphglucmutase Mean level f phentype Number ATP + One S.D C. Cmparisn f ATP Levels in Erythrcytes with Varius Adenylate Kinase Electrphretic Phentypes (Adult Caucasian Ppulatin Only). Adenylate kinase Mean level f phentype Number ATP ne S.D Sickle Cell Trait and G-6-PD Deficiency. The mean cntent f ATP in the red ceils f 19 male Negres with sickle cell trait (3.147 pmles/g Hb) did nt differ significantly frm the mean f nn-sickling Negr males (3.158 #mles). The mean cntent f ATP in erythrcytes f 35 G-6-PD deficient Negr males (3.284 #mles) did nt differ significantly (0.1 > p > 0.05) frm that f nndeficient Negr males (3.143 #mles), cnfirming ur earlier reprt n a smaller number f subjects (Brewer and Pwell, 1966). DISCUSSION It is clear that althugh there is cnsiderable verlap in the distributins, the mean level f erythrcytic ATP in Negres is significantly less than the mean level in

9 Genetic and Ppulatin Studies in Human Erythrcytes 33 Caucasians. Such bichemical differences between red cells f Negres and Caucasians may arise as a result f selective effects f falciparum malaria, since the gene pl f the American Negr is recently derived frm an African stck extensively expsed t malaria. The hypthesis that relatively lw levels f ATP in red cells is prtective against malaria has been supprted by preliminary studies with human vlunteers (Brewer and Pwell, 1965). Further, it appears that the rle f quantitative levels f ATP in the severity f the malarial attack may ffer an explanatin fr the hypthesized prtective effect f thalassemia against malaria. It was first bserved by Lts et al (1965) in the Netherlands and cnfirmed in Sardinia (Brewer et al, in preparatin) that individuals heterzygus fr beta thalassemia have lwer mean levels f ATP than nnthalassemics. Thus, beta thalassemia may exert its malaria prtective effect by acting as a majr genetic mdifier in cntrlling levels f erythrcytic ATP. Aside frm the effect f thalassemia n red cell ATP levels, and islated instances f ther majr genetic mdifiers (Lts et al., 1965; Brewer, 1965a), little is knwn abut the genetic cntrl f ATP levels. Analyses f cvariance in bth Negr and Caucasian sibships (Tables II and III), and the lack f a crrelatin between spuses (Table 4), suggest that levels f ATP are under at least partial genetic cntrl, Additinal evidence alng these lines is the relative cnstancy f ATP levels in erythrcytes f a given individual ver a perid f years (Brewer and Pwell, 1965). The mechanisms f this genetic cntrl are nt knwn with certainty; a priri ne might expect that this quantitative characteristic is determined by multifactrial inheritance, since the levels f ATP are prbably affected, at least slightly, by a number f enzymes which generate, synthesize, and utilize ATP. In the present study, significant crrelatins were nt detected between ATP levels and the cncentratin f serum inrganic phsphrus, the activity in hemlysates f several enzymes, r the electrphretic type f several additinal enzymes frm red cells. Either these factrs d nt influence ATP levels in red cells, r the effect f any ne is t slight t detect in the sample size emplyed. In view f the effect f thalassemia n levels f ATP, it is f interest that the tw ther majr systems hypthesized t have prtective effects against malaria, sickle hemglbin and G-6-PD deficiency (Beet, 1946; Mtulsky, 1960), d nt influence ATP levels. Their prtective influence is apparently mediated thrugh ther mechanisms. ACKNOWLEDGMENT The authr gratefully acknwledges the assistance f Dr. Charles Sing f the Department f Human Genetics, University f Michigan, in carrying ut the cvariance analysis. REFERENCES Allisn, A. C. (1954). Brit. Med. J. 1: 290. Beet, E. A. (1946). E. AJriean Med. J. 23: 75. Brewer, G. J., Tarlv, A. R., and Alving, A. S. (1960). Bull. Wrld Health Organ. 22: 622. Brewer, G. J., Tarlv, A. R., and Alving, A. S. (1962). J. Am. Med. Assc. 180: 386.

34 Brewer Brewer, G. l., Pwell, R. D., Swansn, S. H. and Alving, A. S. (1964)., d. Lab. Clin. Med: 64: 601. Brewer, G. J. (1965a). Bichem. Biphys. Res. Cmmun. 18: 430. Brewer, G. J. (1965b). J. Lab. Clin. Med. 66: 858.

10 34 Brewer Brewer, G. l., Pwell, R. D., Swansn, S. H. and Alving, A. S. (1964)., d. Lab. Clin. Med: 64: 601. Brewer, G. J. (1965a). Bichem. Biphys. Res. Cmmun. 18: 430. Brewer, G. J. (1965b). J. Lab. Clin. Med. 66: 858. Brewer, G. J., and Pwell, R. D. (1965). Prc. Nat. Acad. Sci. 54: 741. Brewer, G. L, and Pwell, R. D. (1966). d. Lab. Clin. Med. 67: 726. Brewer, G., Siniscalc, M, Lenzarrini, L., and Latte, B. (In preparatin). Fildes, R., and Harris, H. (1966). Nature 209: Fisher, R. A. (1958). Statistical Methds fr Research Wrkers. Hafner, New Yrk. Fiske, C H., and Subbarw, Y. (1925). J. BiL Chem. 66: 375. Glck, G. E., and McLean, P. (1953). Bichem. d. 55: 400. Hpkinsn, D., Spencer, N., and Harris, H. (1964). Am. d. Human Genet. 16: 141. Hurt, G. A., and Chanutin, A. (1964). J. Lab. Clin. Med. 64: 675. Krnberg, A. (1950). J. BiL Chem. 182: 779. Lng, W. K., and Carsn, P. E. (1961). Bichem. Biphys. Res. Cmmun. 5: 394. Ls, J. A., Zurcher, C., and Prins, H. K. (1965). Presented at the IV Internatinal Sympsium n Erythrcytes, p Mtulsky, A. G. (1960). Human BiL 32: 28. Pwell, R. D., and DeGwin, R. L. (1965). Nature 205: 507. Spencer, N., Hpkinsn, D., and Harris, H. (1964). Nature 204: 742. Steel, R. G. D., Trrie, J. H. (1960). Principles and Prcedures f Statistics. McGraw-Hill Bk Cmpany, Inc., New Yrk. Tanaka, K. R., Valentine, W. N., and Miwa, S. (1962). Bldl9: 267. Zinkham, W. H., and Lenhard, g. E. (1959). J. Pediat. 55: 319:

Phenotypic and quantitative relationship of red cell

Phenotypic and quantitative relationship of red cell Jurnal f Medical Genetics, 1981, 18, 271-275 Phentypic and quantitative relatinship f red cell acid phsphatase with haemglbin, haptglbin, and G6PD phentypes N SAHA AND N PATGNARAJAH Frm the Faculty fmedicine,