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THE

PENTOSE

PHOSPHATE

PATHWAY

IN HUMAN

ROCYTES

Relationship

Between

the

Age

of

the

Subject

and

Enzyme

Activity

By Ruth T. Gross, M.D., and Ruth E. Hurwitz

Department of Pediatrics, Stanford University School of Medicine

(Accepted April 3, 1958; submitted January 9.) Supported by Public Health Service Grant RG 4754.

ADDRESS: (R.T.G.) 2351 Clay Street, San Francisco 15, California

PEDIATRICS, September 1958

453

E

ORMOUS advances in our knowledge

of carbohydrate metabolism have been

made since 1931 when Warburg and Chris-tian1 demonstrated the oxidation of

glucose-6-phosphate in yeast and erythrocytes. A

new pathway for the oxidation of carbo-hydrates, the pentose phosphate pathway, has been found to exist not only in

micro-organisms but in most animal tissues as well. The subject has been reviewed in

elegant fashion by many of the present in-vestigators in the field.26 Studies of human

erythrocytes have been reported by Glock and vIcLean7 and more recently by Marks8 as well as by the present authors.9

The pentose phosphate pathway and its relationship to the glycolytic scheme are

represented diagramatically in Figure 1.

These two pathways represent the only

known means by which glucose may be

metabolized in mature human erythrocytes.

The tricarboxylic acid cycle is active only

in reticulocytes and nucleated erythrocytes. The only means of formation of carbon

dioxide is via the first two

(

oxidative) steps of the pentose phosphate pathway (Fig. 1).

Assays of the enzymes involved in the oxidative process, glucose-6-phosphate de-hydrogenase (G6PD) and 6-phosphoglu-conic dehydrogenase (6PGD), are used in this study as a reflection of the activity of the pentose phosphate pathway in

erythro-cytes (Fig. 2).

The exact role of the pentose phosphate pathway is not known. The first two steps result in a source of reduced triphosphopy-ridine nucleotide (TPNH) and as such are

thought to be linked to glutathione

metab-olism in the erythrocyte (Fig. 3). If they

also represent the main route of ribose for-mation, one might expect increased activity via the pentose phosphate pathway in the liver of the actively growing subject. On the other hand, such a prediction need have no bearing on the metabolism of the eryth-rocyte. Investigations by others have

dealt with activities of entirely different enzyme systems which were found to be

low as a result of immaturity; for example, activities of carbonic anhydrase1#{176} and met-hemoglobin reductasehl in the erythrocyte and glucuronidaselz in the liver are low in the immature subject.

It was the purpose of this investigation to determine whether or not there was a difference in activity of certain enzymes of the pentose phosphate pathway in the

erythrocytes of full-term newborn and

pre-mature infants as compared to adults. In addition to measuring activity of G6PD and 6PGD, which are directly and exclusively involved in the pentose phosphate pathway, the following additional tests were done:

Stability of reduced glutathione

(

GSH) was measured because of its dependence on TPNH production (Fig. 3). Aldolase was chosen among the glycolytic enzymes (Fig. 2) because the product of the reaction, triose phosphate, may interact either with the glycolytic or with the pentose phosphate

pathway (Fig. 1). The possibility that

en-zyme activity might be related to content

of fetal hemoglobin was also explored.

SUBJECTS AND MEASUREMENTS

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con-TPN TPNH

GLUCOSE

‘IIV

GLUCOSE

-6-FRUCTOSE-6-P PHOSPHATE 6-PHOSPHOGLUCONATE

PATHWAY TPN

FRUCTOSE 1.6-P

tERYHROSE [TPNH

RIBULOSE-5-P

P YRUVATE

RIB OSE -5-P XYLULOSE -5-P

+

LACTATE

Frc. 1. Pathway of carbohydrate metabolism in the erythrocyte. TRIOSE P

sisted of 19 healthy newborn infants less than 5 days of age, 15 premature infants from 2

days to 1 year of age, 32 healthy infants

rang-ing in age from 53 weeks to 1 year and 15 adults without known hematologic disease.

In Group I the following determinations

were made with the erythrocytes: activity of G6PD and aldolase and concentration of fetal hemoglobin. The data could not be combined with data from Group II because activity of G6PD was measured in different units in the two groups and the figures could not be trans-posed.

Group II consisted of 22 healthy newborn infants less than 5 days of age and 14 adults without known hematologic disease. The labor-atory studies performed with subjects of Group II were determinations of activities of C6PD and 6PGD and concentration of GSH.

MATERIALS AND METHODS

Specimens of Blood

Five milliliters of heparinized venous blood were centrifuged for 10 minutes at 1,500 gray. The plasma and huffy coat were removed and the erythrocytes washed twice in an isotonic solution of potassium chloride buffered at pH 7.4 with 0.02 molar solution of potassium bi-carbonate. The erythrocytes were resuspended in approximately two volumes of this solution of potassium chloride. The preparation of eryth-rocytes was performed at 0#{176}C.Aliquots of the resuspended cells were lysed by freezing and thawing twice.

Chemical Determinations

The concentration of hemoglobin was meas-ured by pipetting a suitable quantity of sam-pie into 10 ml of an 0.4% solution of ammon-ium hydroxide and reading it in a spectropho-tometer at 540 m.t. The concentration of re-duced glutathione (GSH) was determined ac-cording to the method of Grunert and Phillips.13 Incubation with acetylphenylhydrazine for 2

hours was performed according to the method of Beutler et al.’ The concentration of fetal he-moglobin was measured according to the

method of Chernoff.15

Assays of Enzymes

The activities of glucose-6-phosphate dehy-drogenase (G6PD) and 6-phosphogluconic de-hydrogenase (6PGD) were assayed according to Marks’ modifications of the methods of Kornberg and Horecker6 and Horecker and Smyrniotis.’7 Activity of aldolase was assayed according to Beck’s modificationlS of the method of Sibley and Lehninger.’ The stand-ard used was a pure preparation of dihydroxy-acetone phosphate.*

Units of Measurement

For Group I, the activity of G6PD was ex-pressed as micromoles of TPNH per ml of erthyrocytes per minute. The extinction co-efficient for TPNH at 340 mp. was taken as

* This material was kindly supplied by Dr. C.

(3)

6.22

x

106 cm2/mole.bo The activity of aldo-lase was expressed in units according to the following definition: 1 unit = that amount of enzyme which transforms 1 mg of phosphorus

in 1 minute.

For Group II, determinations of the

con-centration of hemoglobin were done on all samples to be assayed so that all results were expressed in units per gram of hemoglobin. Activities of G6PD and 6PGD were reported as O.D. per gram of hemoglobin per minute.

RESULTS

Enzymes

ACTIVITY OF

G6PD:

In Group I, activity

of G6PD was measured 24 times with

eryth-rocytes of 19 full-term healthy newborns less than 5 days of age and 17 times with erythrocytes of a control group of 15 adults. In Group II, activity of G6PD was deter-mined by the same method but expressed in different units in studies of 23 full-term

healthy newborns less than 5 days of age and 14 healthy adults. In both groups, G6PD activity of erythrocytes was

signifi-cantly higher (p = <0.001) in newborn

infants than in adults (Table I).

Thirty-three determinations of activity of

G6PD were obtained with erythrocytes of

32 infants 53 weeks to 1 year of age. Not

until age 36 weeks did results with speci-mens from all of the infants fall within the range of results with specimens from adults.

(Fig. 4).

Activity of G6PD was found to be higher in premature infants than infants of com-parable age who were born at full term (Fig. 4). In the premature group, 23 deter-minations were done with specimens from 14 subjects from 2 days to 1 year of age.

AcTIvrn OF

6PGD:

Activity of 6PGD was measured in the erythrocytes of the 22 healthy newborns and 12 healthy adults in Group II. Activity of this enzyme was

G -6-P-D

G-6-P > 6-P---G+ TPNH

TPN

6-P-G-D

6-P-G > Ribose-5-P+ TPNH

TPN

Glut at hione

Glutathione reductase> G S H + TPN (oxidized) TPNH

Fic. 3. Formation of reduced glutathione (GSH).

(Abbreviations as in Fig. 2.)

ARTICLES 455

Glucose - 6- Phosphate Dehydrogenase

G-6-P-D

G-6-P > 6-P-G+ TPNH.

TPN

6-Phosphogluconic Dehydrogenase

6-P-G+ <6-P-G-Q> Ribose 5-P+TPNH TPN

,Aldolase

Aldolase

Fructose I. 6diP > Glyceraldehyde 3-P+ Dihydroxyacetone P

FIG. 2. Sites of action of the enzymes assayed. Abbreviations: G6P, glucose-6-phos-phate; G6PD, glucose-6-phosphate dehydrogenase; TPN, triphosphopyridine nu-cleotide; 6PG, 6-phosphogluconate; TPNH, reduced TPN; 6PGD,

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Age (wk) TABLE I

COMPARISON OF ENZYME LEVELS IN NEWBORN INFANTS AND ADULTS

Enzymes

(Mean value ±1 standard error)

Group I

Number of determinations

G6PD (pmole TPNH/ml erythrocytes/rnin) Aldolase (“units” of activity)

Adults

17 0.54 ± 0. O2

0.077 ± 0.004

Newborn Infants

Q4 0.79 ± 0.03 0. 126 ± 0. 005

Group II

Number of determinations

G6PD ( O.D./gm hemoglobin/mm) 6PGD ( O.D./gm hemoglobin/mm)

14 14.2 ± 0.5 16.6 ±0.9

23

20.4 ± 0.5 21.8 ±0.8

Abbreviations: G6PD, glucose-6-phosphate dehydrogenase; 6PGD, 6-phospliogluconic dehydrogenase.

also found to be significantly increased (p = <0.001) in the erythrocytes of the newborn (Table I).

Acrivrn OF ALDOLASE: Activity of al-dolase was measured 22 times with eryth-rocytes of 19 full-term healthy newborn in-fants less than 5 days of age and 11 times

with erythrocytes of nine healthy adults in

Group I. Significantly increased activity

(

p = <0.001) was again encountered in

erythrocytes of the newborn. The decline

in the activity of aldolase with age was followed in the same infants as was that of G6PD. Not until 48 weeks did all the

re-C

E 1*

o_

o

--o)

.4.,

> -.-%-.-I

UZ <ci

I-0

E

sults fall within the range encountered with studies of adults

(

Fig. 5).

Other Laboratory Determinations

The concentration of fetal hemoglobin was determined in the blood of 26 infants in this study. The percentage of fetal hemo-globin showed an inverse correlation with age, as was expected (correlation coeffi-cient = -0.802). There was a lower correla-tion between percentage of fetal hemo-globin and activity of G6PD

(

correlation

coefficient = +0.48). When the effect of age upon both of these variables was

ac-Fic. 4. Activity of glucose-6-phosphate dehydrogenase (G6PD) in erythrocytes of full-term and premature infants from 1 to 50 weeks of age with reference to the

(5)

Age (wk)

ARTICLES 457

C

0)

0

<0

“--C

0

Fic. 5. Activity of aldolase in erythrocytes of full-term and premature infants from 1 to 50 weeks of age with reference to the range in the first week of life and in adults.

counted for, the partial correlation coeffi-cient was + 0.39. The 95% confidence inter-val for the partial correlation coefficient ex-tends from - 0.03 to + 0.67, showing that

these data are insufficient to give a sharp estimate of the degree of relationship

be-tween concentration of fetal hemoglobin

and activity of G6PD.

The concentration of GSH and the ability of this compound to withstand incubation

with acetylphenylhydrazine was measured in erythrocytes of 16 newborns and 11 adults

in Group II. The amount of GSH remaining after incubation was expressed as a per-centage of the original value. The mean value for adults was 89.0% with a standard deviation of ± 10.0%. Ten newborns less

than 3 days of age had a mean value of 32.0 ± 10.0%, whereas six subjects 3 to 5

days of age had a mean value of 73.0 ± 7.1%.

DISCUSSION

It is clear from this study that activity of at least three enzymes (G6PD, 6PGD and aldolase) is significantly higher in eryth-rocytes of full-term newborn infants and, to an even greater extent in erythrocytes of premature infants than in those of adults.

The elevated activity of the enzymes does not merely represent a transient state associ-ated with birth, as it is not until well into the second 6 months of life that the activi-ties consistently fall within the lower range of values encountered in studies of adults. It cannot be said definitely that the high levels of activity of G6PD and 6PGD repre-sent increased activity in the pentose phos-phate pathway relative to glycolysis. Inas-much as activity of aldolase is equally ele-vated, it may be that the metabolism of glucose is accelerated via both pathways.

These findings are particularly interesting in the light of Marks’ report8 of high levels of activity of G6PD and 6PGD in very young cells and the decrement of enzyme

activity along with in-vivo aging of the cells. The high levels in young subjects may represent enrichment of the blood with younger cells and impoverishment with older cells. Alternatively, maturation of the metabolism of the erythrocytes may take

place gradually over a period of several

months after birth. In this study no effort

was made to determine the relative number of mature and immature erythrocytes.

(6)

% GSH

Protected

E11

Fic. 6. Effect of age on stability of reduced glutathione (GSH). The bars represent mean values for the percentage of GSH protected after 2 hours of incubation with

acetvlphenyl-hydrazine and mean values for activity of G6PD of ervthrocytes. 0-55 hours 55-95 hrs. ADULTS

458

G6PD were detected in two newborns, two older infants and one adult, all of whom were well and without evidence of hemato-logic disease. These findings were the basis for a study reported elsewhere9 in which it was shown that deficiency of G6PD is he-reditary and is associated with susceptibility to hemolytic anemia induced by certain chemical agents.

The results of the GSH stability test

war-rant further comment. The test has been used as a means of detecting subjects sus-ceptible to hemolytic anemia induced by such drugs as primaquine,14 naphthalene2’ and nitrofurantoin.22 Zinkham has reported similar findings in newborn infants.23 We have recently reported the observation that the ability to maintain normal concentra-tions of GSH after incubation with acetyl-phenylhydrazine is directly related to the activity of GGPD, except in newborn in-fants. In 40 children and adults the correla-tion coefficient between activity of G6PD

in erythrocytes and percentage of GSH pro-tected was +0.79 (p = <0.001). This may be compared with the results with 16 in-fants in this study less than 95 hours of age. The correlation coefficient for the

infants was -0.242. The difference be-tween the two groups is highly signfficant (p = <0.001). The instability of GSH which is characteristically present at birth continues for at least 55 hours but not long

after 95 hours

(

Fig. 6). Since all of the in-fants less than 95 hours of age had very

high levels of activity of G6PD, it is ap-parent that the ability to protect GSH in this age group was not related to activity of G6PD. Therefore, there must be some other as yet unidentified factor which is clearly related to age. The possibility of transient deficiency of glutathione reductase was considered but was not borne out by studies done in this laboratory in which the ample activity of the enzyme was de-tected.2

SUMMARY

Metabolism in erythrocytes was studied

in a group of premature and full-term, new-born, healthy infants by assay of the follow-ing enzymes : glucose-6-phosphate

dehy-drogenase, 6-phosphogluconic

dehydrogen-ase and aldolase. Concentrations of fetal hemoglobin and stability of GSH were also determined.

C

E

c

‘*- 0

OE

E

. CI)

(7)

ARTICLES 459

The studies indicate increased metabolic

activity in the erythrocytes of young

sub-jects. The pentose phosphate pathway ap-pears to be very active at birth with gradual decline, to values found for adult subjects, in the second 6 months of life.

GSH is unstable in newborns less than

3 days of age despite high levels of activity of G6PD in the same subjects.

ACKNOWLEDGMENT

The authors wish to express appreciation to Mrs. Judith Beasley for her assistance in carrying out many of the chemical deter-minations.

REFERENCES

1. Warburg, 0., and Christian, W. : Uber Aktivierung der Robisonschen

Hexose-Mono-Phosphursaure in Roten Blutzel-len und die Gewinnung Aktivierender Ferment Losungen. Biochem. Ztschr.,

242:206, 1931.

2. Horecker, B. L., and Mehier, A. H. : Car-bohydrate metabolism. Ann. Rev.

Bio-chem., 24:207, 1955.

3. Wood, H. G. : Significance of alternate pathways in the metabolism of glucose. Physiol. Rev., 35:841, 1955.

4. Stetten, D., and Topper, Y.

J.

: The metab-olism of carbohydrates. Am.

J.

Med., 29:96, 1955.

5. Racker, E. : Micro- and macrocycles in

car-bohydrate metabolism. Harvey Lect.,

1955-56, p. 143.

6. Marks, P. A. : A newer pathway of

carbo-hydrate metabolism (the pentose

phos-phate pathway). Diabetes, 5:276, 1956.

7. Clock, G. E., and McLean, P. : Levels of

enzymes of the direct oxidative path-way of carbohydrate metabolism in

mammalian tissues and tumors.

Bio-chem.

J.,

56:171, 1954.

8. Marks, P. A. : Glucose-6-phosphate dehy-drogenase, 6-phosphogluconic

dehydro-genase and purine nucleoside

phosphor-ylase in erythrocytes. Science, to be published.

9. Gross, R. T., Marks, P. A., and Hurwitz, R. E.: An hereditary enzymatic defect in human erythrocytes.

J.

Clin. Invest.,

to be published.

10. Day, R., and DuPan, R. M.: Carbonic

an-hydrase in the blood and kidneys of premature infants (abstract). Am.

J.

Dis.

Child., 77:109, 1949.

ii. Lonn, L., and Motulskv, A.:

Electro-phoretic demonstration of a

non-hemo-globin protein (methemoglobin reduc-tase) in hemolysates. Clin. Res. Proc., 5:157, 1957.

12. Brown, A. K. : Studies on the neonatal

de-velopment of the glucuronide

conjugat-ing system (abstract). A.M.A.

J.

Dis.

Child., 94:510, 1957.

13. Grunert, R. R., and Phillips, P. H. : A modification of the nitroprusside method

of analysis for glutathione. Arch. Bio-chem., 30:217, 1951.

14. Beutler, E., Dern, R.

J.,

Flanagan, C. L., and Alving, A. S. : The hemolytic effect of primaquine. VII. Biochemical studies of drug-sensitive erythrocytes.

J.

Lab. & Clin. Med., 45:286, 1949.

15. Chernoff, A. I. : The human hemoglobins in health and disease. New England

J.

Med., 253:322, 1955.

16. Kornberg, A., and Horecker, B. L. : In Methods in Enzymology, Vol. I.

Cob-wick, S. P., and Kaplan, N. 0., editors.

New York, Acad. Press, 1955, p. 323. 17. Horecker, B. L., and Smyrniotis, P. Z.:

Phosphogluconic acid dehydrogenase from yeast.

J.

Biol. Chem., 193:371, 1951.

18. Beck, W. S. : Determination of triose phos-phates and proposed modifications in the aldolase method of Sibley and Lehn-inger.

J.

Biol. Chem., 212:847, 1955. 19. Sibley,

J.

A., and Lehninger, A. L. :

De-terminations of aldobase in animal

tis-sues.

J.

Biol. Chem., 177:859, 1949. 20. Horecker, B. L., and Kornberg, A. : The

extinction co-efficients of the reduced

bond of pyridine nucleotides.

J.

Biob.

Chem., 175:385, 1948.

21. Zinkham, W. H., and Chibds, B. : Effect of

naphthalene derivatives on glutathione metabolism of erythrocytes from pa-tients with naphthalene hemolytic ane-mia (abstract). Proceedings of 49th an-nual meeting of the Am. Soc. for Clin. Invest., 1957, p. 74.

22. Kimbro, E. L. : The mechanism of

hemo-lytic anemia induced by nitrofurantoin

(Furadantin). Bull. Johns Hopkins

Hosp., 118:101, 1957.

23. Zinkham, W. H., and Childs, B. : Effect of vitamin K and naphthalene metabolites

on gbutathione metabolism of

erythro-cytes from normal newborns and

pa-tients with naphthabene hemolytic

ane-mia (abstract). A.M.A.

J.

Dis. Child., 94:420, 1957.

(8)

460

SUMMARIO IN INTERLINGUA

Le

Circuito

De

Phosphato

De

Pentosa

In Erythrocytos

Human

Le metabobismo de erythrocytos esseva studiate in un gruppo de infantes prematur e

nascite a termino per be essayage del sequente

enzymas : In be circuito de phosphato de

pentosa, dishydrogenase de glucosa-6-phos-phato (DG6P) e dishydrogenase 6-phospho-gluconic (D6PG), e in le circuito de Embden-Meyerhof, aldobase. Hemogbobina fetal e stabilitate de glutathiona esseva etiam investi-gate.

DG6P esseva mesurate 47 vices in 42

nor-mal neonatos nascite a termino a etates de

infra 5 dies e 31 vices in 29 normal adultos de

controbo. Le activitate del enzyma esseva sig-nificativemente plus alte in be infantes que in le adultos.

DG6P esseva etiam mesurate 33 vices in 32 normal infantes a etates de inter 53 septimanas

e 1 anno. Il esseva sobmente post be etate de 36 septimanas que omne be infantes attingeva vabores intra be limites del norma pro adultos.

Le mesuration esseva effectuate 23 vices in 14 prematuros a etates de inter 2 dies e 1 anno.

Esseva constatate que be activitate del enzyma

esseva plus alte in prematuros que in infantes nascite a termino e studiate a etates compara-bile.

D6PG esseva mesurate in le erythrocytos de

22 normal neonatos e de 12 normal adultos. Illo

esseva augmentate significativemente in le ery-throcytos del neonatos.

Aldolase esseva mesurate 22 vices in 19 normal neonatos nascite a termino a etates de

infra 5 dies e 1 1 vices in 9 normal adultos. De novo, un activitate augmentate esseva trovate in le neonatos. Il esseva solmente a! etate de 50 septimanas que be valores cadeva intra be limites del norma pro adubtos.

Hemogbobina fetal esseva determinate in 26

infantes. Su procentage se monstrava

inverse-mente correlationate con le etate, sed be datos non establiva un relation definite inter be

activitate del enzyma e be hemoglobina fetal. Dece-sex infantes a etates de infra 92 horas

monstrava instabilitate de glutathiona in

de-specto de alte nivelbos de DG6P.

Iste studios indica le presentia de augmentate nivebbos de activitate metabolic in be

erythro-cytos de juvene subjectos. Le circuito de

phosphato de pentosa es apparentemente multo

active al tempore del nascentia e descende

graduabmente a vabores adulte durante be se-cunde 6 menses del vita.

ANTIBODY RESPONSE TO POLIOMYELITIS VACCINE ADMINISTERED BY JET INJECTION, M. J. Lipson et a!. (Am. J. Pub. Health, 48:599, May, 1958.)

(9)

1958;22;453

Pediatrics

Ruth T. Gross and Ruth E. Hurwitz

Relationship Between the Age of the Subject and Enzyme Activity

THE PENTOSE PHOSPHATE PATHWAY IN HUMAN ERYTHROCYTES:

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1958;22;453

Pediatrics

Ruth T. Gross and Ruth E. Hurwitz

Relationship Between the Age of the Subject and Enzyme Activity

THE PENTOSE PHOSPHATE PATHWAY IN HUMAN ERYTHROCYTES:

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References

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