Red cell metabolism: A comparative study of some mammalian species

, pp. 515-520, 1984

0305-0491/84 $3.00+ 0.00 © 1984 Pergamon Press Ltd

IETABOLISM: A COMPA SOME MAMMALIAN S!

STUDY

T. SUZUKI,*N. S. AGAR~ and M. St ¢Iedicine, Faculty of Agriculture, Gifu Un

Gifu 501-11, Japan

(Received 10 April 1984) Abstract--l. The activities of six enzymes--glucose-6-pho! rmes--glucose-6-phosphate deh3

gluconate dehydrogenase (6-PGD), glutathione reductas~ tase (GR), gl catalase and superoxide dismutase (SOD)--and levels of reduced i glu eration rates were measured in the red blood cells of eight mammalia golden hamster, sheep, goat, cattle and human. 2. The results show that whereas the activities of G-6-PI2 G-6-PD, 6-PGD al the experimental animals, there is no such variation in the activity ot 3. There does not appear to be a relationship between GSH reger G-6-PD, 6-PGD and GR--the enzymes associated with GSH G metabc

INTRODUCTION he red blood cell, under physiological conditions, is mtinuously exposed to oxidants such as the super~ide radical q(O2-.). These radicals are also produced oxide under ader experimental and/or clinical use of some oxidizin tizing drugs such as phenylhydrazine and primauine. However, the red blood cell has a protective qulne. mechanism Lechanism against this oxidative damage by some ways of certain enzyme systems. Three main enzymes, glutathione peroxidase (GSH-Px), 3SH-Px), catalase and superoxide dismutase (SOD), are :e known to protect the red blood cell against such damage. Another enzyme, glutathione reductase talnln an adequate (GR) is of importance in maintain maintaining e (GSH). The other two level of reduced glutathione enzymes of pentose phosphate pathway (PPP), Lrogenase (G-6-PD) and glucose-6-phosphate dehydrog lrogenase (6-PGD) pro6-phosphogluconate dehydrog vide the NADPH requiredL for G R to convert oxidized glutathione (GSSG) to GSH. nerated ed in GSH can be rapidly regenerated iin normal human red blood cells treated with ith the oxidizing agent, methyl phenylazoformate (~ iazoester). This measurement is called "the GSH regq;eneration test" (Kosower et al., 1967). These authors suggested that the GSH ii) the rate of NADPH regeneration test reflects (i) D step, (ii) the rate of regeneration in the G-6-PD 6-P by conversion of glucose to G-6-P b, hexokinase (HK), uction of GSSG to GSH (iii) the efficiency of the reduction by the intact red blood cells. ~lls. Consequently, GSH regeneration has been investi tigated extensively in humans and animals (Smith, 1968; 968; Yawata and Tanaka, 1973; Agar and Smith, 1974~,; Agar et al., 1974). The red blood cells of G-6-PD deficient humans regenerate little GSH in this testt (Kosower et al., 1967). r~f Hum l--Inm~n *Present address: Department of Curtin School of Medical Researct tional University, Canberra, A.C.'I (Tel: 49-2550). tPresent address: Department of Physio

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-6-PD), 6-phosphooxidase (GSH-Px), 1) and GSH regenbit, guinea-pig, rat, ely different among nd the activities of

mga~ gar et al. (1! , reported that although new born hun~ ~her activities of G-6-PD and,GR the GSH regeneration rate rates are significantly lower than normal adult humans. T~ The enzymes of the Embden-/~ bden-Meyerhof pathway and the PPP of the red blood cells of various species have been investigated extensivel) ty (see reviews by Kant Kaneko, 1974; Agar and Board, 1983). 19 It is apparent from from these studies that there is a wit wide variation in the meta metabolic needs and capabilities ities of the red blood cell. Nonq None of these studies, however, has compared the inter: interspecies differences of all the six si enzymes associated with the PPP and regeneration ~eration of GSH required to protect the red blood cell from fror oxidation. The results presented here provide this information. METHODS MATERIALS AND METE Blood samples from rabbits ( Oryctolagus cuniculus ), alden hamsters (Mesoguinea-pigs (Cavia procellus), golden cricetus auratus ) and rats ( Rattus norveg'gicus) were obtained by cardiac puncture, from sheep (Ovis aries), goats ( Capra hircus) and cattle (Bos taurus) by jugul~ venepuncture and ~yjugular from humans (Homo sapiens) by brachial brachi~ venepuncture. All blood samples were collected into hep~ aarin tubes. Preparation of hemolysate and measurement mea~ of all enzymesactivitiesexcept SOD were carried carria out by the method of Beutler (1975). Hemoglobin (Hb) was w measured as the standard laboratory procyanmethemoglobin derivative by stand ymes is expressed as cedure. The activity of red cell enzyn with flavin IU/gHb (Beutler, 1975). GR activity measured m adenine dinucleotide (FAD) is referred to as "total GR", that without FAD as "active form" and the difference betweentotal GR and active form "it ~n as "inactive form" of the enzymes.SOD was measured by the met method of McCord and Fridovich (1969). GSH levels were measured by the method of Beutler mole/glib and mg/dl RBC. (1975) and are expressed as /~mole/gH GSH regeneration rates were measured measure as reported previously (Suzuki et al., 1983).

lc~hn

,TS -6-PD was highest in the ~.36 I.U./gHb) and lowest l.U./gHb) (Fig. 1).

T. SUZUKI el al. /gHb)

ts

in the red blood cells of ) and the lowest in those

total G R w~ guinea-pigs (19 of the rats (I.Z GSH-Px acti minants than which were clc Catalase: all of

20

onsiderably higher in ruanimals except for rats rots (Fig. 2). :t be subdivided into three

+ /glib) 15

Rat

Rabbit

Sheep

Goat

~1

Guinea pig

Cattle

+ I~

Camel

,

Rat

I-~ ~

I

Golden hamster

Buffalo

Human

20

Sheep

D

Goat Cattle

2.5

Buffalo

(IU/gHb)

6-PGD

5

7.5

Active form

10 Camel

N--b

}

Inactiveform

Rabbit

Human Guinea pig

'J,

Golden hamster

GSH-Px (IU/gHb) i.~

Rat

Sheep

0

100

150

200

Rabbit

}

Guinea pig

Goat

Cattle

'~

Golden hamster

Buffalo

"-~

Rat

Camel

" ~

Sheep

Human

50

I~

Fig. I. Enzyme activities of red ed cell glucose-6-phosphate dehydrogenase (G-6-PD) and 6-phosphogluconate dehyerent mammalian species drogenase (6-PGD) in different (mean_+ 95% confidence fidence limits).

Goat Cattle

I~

J

I

I

Buffalo

Camel

The maximum activity 0 f 6 - P G D was. . . .in . . . rats (8.01 l . U . / g H b ) and the minimum (C sheep (Fig. 1). G R activities including the activ active and inactive forms in the re

H. . . .

d cell glutathione reductase tase (GSH-Px) in different F 95°,i confidence limitsL

(ed cell metabolism in some mammalian ,, ivities lower than :ep; (ii) those with 04 I . U . / g H ~ a t t l e i) those with higher b--rabbits, humans • 3. Considering the arative values were the lowest (67~) in uniform among the ammals examined• Red blood cell GSH levels from different animal ecies are shown in Fig. 4. The range of their means as 70-120 mg/dl RBC (5-12/~mole/gHb). The rates ' GSH regeneration are shown in Fig. 4. It is evident at the mean rate using TBH is higher than that ing diamide in all animals. The previous data of buffalo (Bubalis bubalis) and mel (Camelus dromedarius) (Agar and Suzuki, ~82) are also shown in these figures to provide a mparison.

517 se (x 104 IU/gHb) 10

G-6-PD: the present results are in confirmation ith previous reports (see review by Agar and Board, with 1983 )83) and indicate a wide variation in the G-6-PD activit •tivity in the red blood cells of different animal Sl~~ecies. Even in the same family (e.g. Bovidae) there is a wide variation in the level of this enzyme (Agar ad Suzuki, 1982). It may be of relevance that a and milar variability has been observed in the levels of similar lis enzyme in the red blood cells of different Austrathis m Macropididae. In this family, the level of G-6-PD lian nges from l l.30I.U./gHb in tammar wallaby ran (Macropus eugenii) to 38.07 I.U./gHb in eastern grey kangaroo (Macropus gigantieus) ieus) (Agar et al., 1976). This enzyme has been extensivel tensively investigated in humans where nearly 200 variants ariants have been characterized based on kinetics, electro ;lectrophoretic mobilities and substrate specificities (Yoshida and Beutler, 1983). The electrophoretic v~ ,ariants of G-6-PD in the als have been reported by red blood cells of farm animals McDermid et al. (1975). ~tigators have shown that 6-PGD: the previous invest1 the activity of this enzyme m in human red blood cells is approximately the same as ~s that of G-6-PD and is much lower in cattle, pigs, horses, dogs, guinea-pigs and rabbits (Agar and Board ard, 1983). The present results show that the four kinds of the laboratory animals have much higherr levels of the enzyme domestic animals and the activity than three kinds of C G-6-PD/6-PGD ratio is less ;s than 1 only in goats• Farnararo et al. (1980) stated tated that the ratio of G-6-PD/6-PGD is always hig] higher than 1 in the rat tissues and less than 1 in the le tissues of the chicken. GR: several investigators have examined G R activities in animals including human, aman, guinea-pig, sheep, rat, rabbit, cattle, goat, grey kang kangaroo, red kangaroo, horse, dog, pig and dromedar dry (Lankish et al., 1973; Agar et al., 1974)• However, exc~ Kaneko (1975) and Board and Petel these investigators measured G R a without FAD. The present results,

20

Rabbit

Guinea pi

Golden ham Rat

Sheep

Goat

Cattle Buffalo

+

Camel

DISCUSSION

15

Human

SOD (x 1O3unita/gHb) 103uni O

1

2

3

Rabbit

4

J

'

Guinea pig

Golden hamster

:

Rat

' I

:

Sheep

Goat Cattle

Buffalo

Came, "''--'--'1

Human

Fig. 3. Enzyme activities of red cell oxide dismutase (SOD) in different (mean + 95% confidence

catalase and super1mammalian species 1 limits).

in rats and sheep, are similar to the previous investigations of Lankish et al. (1973). Our results have saturation of G R with 11animals except rabbits, )wever, contrary to the (1976). Det~ ruination of t in riboflavin nutritional

T. SUZUKI el al. GSH ( y m o l e / g H b ) 2

10

8

6

4

GS~ 2

0

20

4(

30

120

+ + .F H--

Rat

+

Sheep

I

,

Goat

+

Cattle Buffalo

+

Came;

+

Human

T

GSH regeneration

rate

(pmole/min/gHb)

0.2

0.4

0.6

o.a

Rabbit Guinea

picj

I

]

I

Goldenhamster Rat GSH)

SheeD (high

Sheep

~l/lll~-q

(lowGSH)

Goat

~#1~--I

Cattle

~ ,

Buffalo

~

Camel H Um a n

I

I

~

TBHDiamIcel

1 ~/////////jf///I-~

t

I Fig. 4. Concentrationss of GSH (#mole/glib or mg/dl RBC) in different mammalian specie )ecles and GSH regeneration rate usin, ag diamide and t-butyl hydroperoxide (TBH) in different mamma mammalian species (mean +_ 95°/o confidence limits).

status in humans (Beutler, 1969 969) a n d in rats (Prentice and Bates, 1981). It is not known known whether the same relationship also holds true in othe especially ruminants, where micro~ F A D occurs in the gut. However, i the present results that the percent~

GSH-Px: the wide variation in tI the enzyme activity of G S H - P x in different m a mnmalian m a l i a r species observed in agreement with the Maral et al. (1977) and ;ince 1973, when it was eously but independently, nium (Se) as an integral

Red cell metabolism in some mammalian L, 1973; Flohe et al., has been used as a ts and of identifying ans. High variability bserved in our study f differences in seleowever, no attempt n the blood samples 65) and Maral et al. 977) have shown that catalase activities are ex'emely variable among different mammals and birds, he present results, however, show that there is not inch variation in catalase in different animals. It is at clear why birds' catalase activities are so much ~wer than mammals. Paniker and Iyer (1965) rearted that red blood cells of birds and dogs have the .west catalase activity, SOD: the enzyme activities were considerably uni)rm among various animals (Fig. 3). Tolmasoff et al. L980) have shown that, although no general cor,qation is found in the tissue SOD levels and life span f primate species, the long-lived species may have a igher degree of protection against by-products of xygen metabolism. Such a relationship does not ppear to exist in the present study, GSH and GSH regeneration rate: it is evident from Fiig. 4 that GSH regeneration rate was the highest in rabbits (0.712#mole/min/gHb) and the lowest in goats (0.102/~mole/min/gHb) using diamide. The rate Lte of GSH regeneration using TBH is higher than that lat using diamide in four animal species used. The present resent result confirms the findings of Agar and Smith mith (1974) that GSH regeneration rate of rabbits ).694/~mole/min/gHb) was much higher than those (0.694 of other animals. Both high and low GSH sheep (Board and Agar, 1983) had similar rates, when diamide was used to oxidiz~ ze GSH. However, when TBH was used as oxidant, the he GSH regeneration rate was almost twice as much ina the red blood cells from high GSH sheep compared1 to that from low GSH sheep. It has been reported by Yawata awata and T a n a k a (1973) and Agar et al. (1974) that although G R activity is higher in h u m a n cord redrl blood cells, the GSH regeneration rate is markedl tly lower than that from adult humans. Higher enzyme aae levels of both G-6-PD and G R in newborn ruminants rots are, however, associeration rates compared to ated with higher GSH regeneration the adult animals (Agar et al., 1974). The relationttion and red cell enzyme ship between GSH regeneration Jdied by Agar and Smith activities have also been studied (1973). Of the 11 enzymes assa ~sayed, six were positively or negatively correlated with GSH regeneration rates in sheep. In the list of these six enzymes, G-6-PD and G R were also included (the le other four were HK, aldolase, pyruvate kinase and glyceraldehyde 3-phosphate dehydrogenase). e). These authors concluded "We cannot explain the association between GSH regeneration and enz~yme activities". Our resuits suggest that there is no relationship between G S H regeneration and the enzyme ac and GR. In summary, the present study h~ activities of six enzymes (G-6-PI3

seven different results have cc in the literatur great variatior PD) while nol variation shou the metabolic r stress which th even in physi( trations and tl play an impor met~ metabolism in ular. The level cells of most m at the th present t and~G R may n and GSE GSH-Px and S prot~ ~rotection ag~ cells of mamrc

519 animals and human. The 'ious fragmentary reports ,Iso shown that there is a zyme activities (e.g. G-6others (e.g. SOD). This 'effect a wide variation in acity to face the oxidative ells undergo continuously itions. Also the concenle N A D P / N A D P H must the regulation of red cell nzyme activities in partic~nzymes in the red blood ecies are largely u n k n o w n ;ible that G-6-PD, 6-PGD liting enzymes in PPP but aauch more important for ,e damage in red blood

Ackn Acknowledgemer

: was supported by a grant from Gifu Univ md by a Student Exchange Scholarship fron 3f Education, Japanese GovSchol ernment (T.S.). visiting fellow supported by ernm the ,~ ience and the Japan Society the Australian A for the tl Promotion of Science and was on leave from the University of New England.

REFERENCES

ar N. S., and Board P. G. (1983) R Red cell metabolism. Agar M (Edited by In Red Blood Cells o f Domestic Mammals Agar Ag~ N. S. and Board P. G.), pp. 227-252. Elsevier, Amsterdam. Agar N. S., Gruca M. A. and Harley J. D. (1974) Studies on glucose-6-phosphate dehydrogem genase, glutathione redu( ductase and regeneration of reduced:1 glutathione in the red blood cells of various mammali aammalian species. Aust. J. exp. Biol. Med. 52, 607-614. Agar N. S., Gruca M. A., Mulley A., A Stephens T. and Harley J. D. (1976) Red cell enzymesmes--IV. A comparative various species of Marstudy of red blood cells from vario supials in Australia. Comp. Biochem. Bioc~ Physiol. 53B, 455-460. regeneration Agar N. S. and Smith J. E. (1973) Glutathione Gluta related to enzyme activities in eryt ythrocytes of sheep. Enzyme 14, 82-86. Agar N. S. and Smith J. E. (1974) Enzyme En: and glycolytic intermediates in the rabbit erythr( 'ythrocytes. Enzyme 17, 205-209. Agar N. S. and Suzuki T. (1982) Red cell metabolism in buffaloes and camels. J. exp. Zool. 223, 25-28. pounds on glutathione Beutler E. (1969) Effect offlavin compo studies. J. clin. reductase activity: in vivo and in vitro vi Invest. 48, 1957-1966. Beutler E. (1975) Red Cell Metabolism sin: A Manual o f Biochemical Methods. 2nd edn, Grune and Stratton, New York. Board P. G. and Agar N. S. (1983) Glutathione Glut metabolism in erythrocytes. In Red Blood Cells o f~Domestic Mammals (Edited by Agar N. S. and Board P P. G.), pp. 253-270. Elsevier, Amsterdam. Board P. G. and Peter D. W. (1976) Res Iponse of plasma and mammalian red cell glutathione reductase of several ,, :ns to flavin adenine dinu7. uni P. (1980) The G-6-PD/6narker in comparative bio"hysiol. 66B, 427-429. Schock H. H. (1973) Glu-

"[. SUZUKt et al. yme. FI£BS Lett. 32, ) Mammalian erythroz constants and satur•es. 36, 1511 1513. ythrocyte metabolism. 7 153. d London I. M. (19671 ithione in normal and e deficient human red arian M. and Iyer G. Y. N. (1977) Oxidant effect of acetylphenylhydrazine: a comparative study with erythrocytes of several animal species. Can. J. Biochem. 55, 597-69. mkish P. G., Schroeter R., Lege L. and Vogt W. (19731 Reduced glutathione and glutathione reductase-comparative study of erythrocytes from various species. Comp. Biochem. Physiol. 46B, 639-641. cCord J. M. and Fridovich I. (1969) Superoxide dismutase--an enzymatic function for erythrocuprein (hemocuprein). J. biol. Chem. 244, 6049-6055. cDermid E. M., Agar N. S. and Chai C. K. (1975) Electrophoretic variation of red cell enzyme systems in farm animals. Anim. Blood Grps Biochem. Genet. 6, 127-174. ahaffey E. and Smith J. E. (1975) Species difference in erythrocyte glutathione reduction rate after oxidation with t-butyl hydroperoxide. Int. J. Biochem. 6, 853 854. Mar aral J., Puget K. and Michelson A. M. (1977) Comparatlve tive study of superoxide dismutase, catalase and glu-

tathione pero n crythrocytcs of different animals. Bioc Res. Commun. 77, 15251535. Paniker N. V. an • (1965) Erythrocyte catalase and detoxifiat n peroxide. Can. J. Biochem. 43, 1029-103g Prentice A. M. ?. J. (1981) A biochemical evaluation of e reductase (EC 1.6.4.21 test for riboflavin and specificity of response in acute deficien~ • 45, 37-52. Rotruck J. T., r tther H. E., Swanson A. B., Ha Haferman D. ~tra W. G. (1973) Selenium: bic biochemical r ponent of glutathione peroxi oxidase. Scien 10. Smitt J. E. (19 Smith arocyte glucosc-6-phosphate dehydrogenast det primaquine insensitivity in she sheep. J. Lab. , 826-833. Suzul M•, O'D Suzuki ki T. and Agar N. S. (19831 Re generation :athione in human and ruminal red bloc nant !-deoxyglucose as substrate. Co Comp. Biochei B, 195 197. Tolm ,lmasoff J. M. Cutler R. G. (1980) Superoxi oxide dismuta with life-span and specific metabolic rate ecies. Proc. Natn. Acad. Sci. me U.S.A. 77, 27 U.; Yaw~ Y. and q Yawata 1973) Studies on glutathione red reductase and of reduced glutathione in normal human adult and cord red cells. Clinica Chim. no1 Acta 46, 267 275. G-6-PD variants: another Yoshida A. and Beutler E. (19831 G-6-F Yosh up~-date. Ann. Hum. Genet. 47, 25 338.