γ-Glutamyl transpeptidase of human amniotic fluid

y-Glutamy ANIL

G.

transpeptidase of human amniotic Auid

PALEKAR,

PH.D.

VADDANAHALLY PLATON JAMES

T.

J. N.

East Meadow

COLLIPP, MACRI,

and

Stony

MADDAIAH,

PH.D.

M.D. PH.D.

Brook,

New

York

y-Glutamyl transpeptidase (GGTP) activity in normal amniotic fluids and corresponding maternal sera obtained at various gestational periods was measured. The ontoget& pattern of enzyme activity in amniotic fluid is very similar to alpha f&protein (AFP). However, the levels of these two proteins behaved differently in corresponding maternal sera. Al?, in amniotic fluids obtaii from pregnancies with neural tube defects (NTD), only AFP concentration was abmally high whereas GGTP activity was normal. (AM. J. OBSTET. GYNECOL. 141:788, 1981.)

(GGTP) is a glycoprotein that catalyzes the metabolism of glutathione.’ In fetal liver, transpeptidase activity is much higher than in adult liver.‘. ’ 3 Alpha fetoprotein (AFP), which is also a glycoprotein, is a major protein component of fetal serum and amniotic fluid.4 Fetal liver can synthesize, besides AFP, a number of other plasma proteins, but only AFP has been abnormalty high in the amniotic fluid of a number of fetal anomalies including neural tube defects (NTD).” We have investigated GGTP activity in amniotic fluid and maternal serum obtained at different gestational ages. The pregnancies which eventually resulted in normal and NTD outcomes were classified as such and compared. Further studies were carried out on GGTP to better understand possible origins of the enzyme in amniotic fluid which might help explain the specific increase in amniotic fluid AFP levels in NTD. y-GLUTAMYLTRANSPEPTIDASE

From the Department of Pediatrics, Nassau County Medical Center and She Universi~ of New York; Stony Brook Health Sciences Center, and the Department of Obstetrics and Gynecology, State University of New York, Stony Brook Health Sciences Center. Supported by the National Long Island Division.

Foundation-March

of Dimes,

A preliminary report of this study was presented at a meet&g of the American Society of Biological Chemists, New Orleans, Louisiana, June I-6, 1980. Received for publication

March

17, 1981.

Revised June 30, 1981. Accepted July

13, 1981.

Reprint requests: Dr. Anil G. Palekar, Department of Pediatrics, Nassau County Medical Center, 2201 Hempstead Turnpike, East Meadow, New York 11554. 788

tllkimtetendm Concanavalin A-Sepharose was obtained from Pharmacia Fine Chemicals. Glutathione (GSH), oxidized glutathione (GSSG), y-glutamyl-p-nitroanilide, glycyglycine, cu-methyl-n-glucoside, neuraminidase attached to beaded Agarose, N-2-hydroxyethylpiperazine-N’-2ethanesulfonic acid (HEPES), vinyl pyridine, 5,5’dithiobis-(2-nitrobenzoate), p-nicotinamide adenine dinucleotide phosphate-reduced form, glutathione reductase, and Lubrol were purchased from Sigma Chemical Co. Samples of amniotic fluid were obtained by amniocentesis from patients with their prior consent for detection of NTD. At each gestational period, at least 25 different samples of amniotic fluid with normal outcome were analyzed. Estimation of AFP was carried out by radioimmunoassay as described.” GGTP activity in amniotic Huid and corresponding maternal serum was determined7 in the presence of 2.5 mM y-glutamyl-p-nitroanilide, 50 mM glycylglycine, and 0. 1M Tris-HCl, pH 8.0 at 37” C. One unit is expressed as a micromole of p-nitroaniline released per minute. Amniotic fluid containing 0.05M Tris-HCl, pH 8.0, 0.15M sodium chloride, 0.2 mM manganous chloride, 0.2 mM calcium chloride, and 0.2% Lubrol (TrisLubrol buffer) was obtained by mixing 0.1 ml of amniotic ffuid and 0.1 ml of twice-concentrated TrisLubrol buffer. It was then applied to a concanavalin A-Sepharose column (1 ml) and eluted with 2 ml of Tris-Lubrol buffer. GGTP that eluted in this fraction was called concanavalin A nonreactive. Enzyme that was absorbed on the column, called concanavalin A reactive, was eluted with 2 ml of Tris-Lubrol buffer containing 50 mg/ml of a-methyl-n-glucoside. In sepa0002-9378/81/230788+04$00.40/O

0

1981

The

C. V. Mosby

Co.

Volume

141

Number 7

y-Glutamyl.

transpeptidase

in human

amniotic

fluid

789

Fig.

1. GGTP and AFP in amniotic Huid from normal pregnancies during gestation.

Fig. 2. GGTP and AFP in maternal serum from normal pregnancies during gest,ation.

rate

peak of 170 ngiml at 30 to 39 weeks. In contrast, GGTP activity remained at about the same level during the gestational period and was within the normal range reported for human serum.” Maternal AFP has been considered to be of fetal origin, passing either across the placenta or through membranes.” Since GGTP activity in maternal serum remains within tihe normal range throughout pregnancy, it seems unlikely that it has crossed the fetomaternal barrier. It was proposed that GGTP plays an important role in reabsorption of amino acids.” Levels of GGTP in amniotic Huid may reHect activity of this enzyme in fetal tissues where it is probably responsible for maintaining the supply of amino acids in the fetus during early gestation. Transpeptidase activities in amniotic Huid in pregnancies with normal outcomes as well as with NTD are listed in Table I. Although the variations of GGTP and AFP with gestational age were very similar, amniotic Huid with NTD disd not show enhanced levels of GGTP. It is possible that tissues other than fetal liver might contribute to amniotic Huid GGTP. Kottgen and co-workers”’ have shown thar depending on the developmental stage, GGTP exists in two forms in rat liver, namely, fetal and adult types, as a result of differences in glycosylation of the protein.

experiments aliquots of amniotic fluids were with neuraminidase, as described,x prior to concanavalin A-Sepharose chromatography. Glutathione oxidase activity was determined as described by Tate and colleagues.” One unit is expressed as a micromole of GSSG formed per minute. Reaction mixture (0.2 ml) containing 10 mM sodium phosphate, 20 mM HEPES, pH 7.4,O. 15M sodium chloride, 2 mM GSH, and 20 ~1 of amniotic fluid was incubated at 37” C for 30 minutes. Reaction was stopped with 20 ~1 of 50% sulfosalicylic acid and centrifuged for 10 minutes at 3,000 x g. Supernatant (50 ~1) was mixed with 50 ~1 of 0.5M sodium phosphate, pH 6.8, and 0.1 ml of O.lM triethanolamine buffer, pH 8.0, containing 0.1 pmole of vinyl pyridine. GSSG was determined with 5,5’dithiobis (2-nitrobenzoate) and glutathione reductase.“’ treated

Results end mmt Fig. 1 shows GGTP activity and AFP levels in normal human amniotic fluid collected at different times during gestation. Levels of both decrease with increasing gestational age, showing a very similar pattern between 15 weeks’ gestation and term. Levels of the two proteins in maternal serum obtained at different weeks of gestation are shown in Fig. 2. Concentration of AFP increased gradually from 45 rig/ml at 15 weeks to a

790

Palekar

Table

December Am. J. Obstet.

et al.

I. GGTP

activity in amniotic

fluid

(units/liter) Gestation

Tj$e of samples

I, I981 Gynecol.

15

16

17

18

19

Normal outcome

614.6

611.1

528.3

517.6

19i.3 475.4

24;.2

NTD

17i.7 -

20;.7 517.2 -c

(wk)

Term

20

21

22

23

24-26

30-39

494.9

386.6

283.2

267.8

171.6

100.0

13.3

8.1

26.9

18+5.6 173.3

15+2.9 619.3 2

9i.l 126.0

5:.2 -

.;6

526.9

2Oi.5 303.1

4.1 -

32i.5 (4)

27i.Y (7)

lOi. (4)

activities

in amniotic

‘-’ -

3oG.3

W* *Numbers

Table

in parentheses

II. Concanavalin

(1)

are the numbers

A-nonreactive

of samples

analyzed.

and reactive

GGTP

Gestation Activity

5i.4 (5)

(1)

fluid

(wk)

15

16

17

18

19

20

21

22

Concanavalin nonreactive

A-

89.3

110.6

112.3

126.0

135.2

114.5

89.7

89.2

Concanavalin

A-

2Gl.1 525.3

2i.3 500.5

2+74 416:0

3i.l 391.6

*:3 359:7

2i.4 272.1

1:6 193:5

2:.4 178.6

reactive Concanavalin nonreactive

A($%)*

14;.0 14.5

14;.8 18.0

1 li.5 21.2

lOi. 24.3

lOi. 27.3

9i.6 29.6

92.1 31.6

9:.3 33.3

*Percentage

of total amniotic

fluid

GGTP

activity

not bound

Table III. Glutathione oxidase activity in amniotic fluid (units/liter) Gestation

(wk)

15

16

17

18

19

20

21

22

87.8

85.1 + 19.5 (7.2)

70.5 k 18.3 (7.5)

66.9

61.7

45.5

29.7

26.8

1:1 (7.7)

20 (8.0)

1:1 (8.5)

1;2 (9.5)

i5 (10.0)

2& (7.0*)

*Values in parentheses indicate ratio of GGTP one oxidase activity.

to

glutathi-

Separation of the two forms can be performed with the use of concanavalin A-Sepharose which does not bind the fetal form and binds only the adult-type GGTP. On this basis, the fetal type may be called concanavalin A nonreactive and the adult, concanavalin A reactive. The results of separation of the two forms of GGTP in amniotic fluid during gestation are shown in Table II. Concanavalin A-nonreactive GGTP remained fairly constant during the gestation. However, the concanavalin A-reactive form of the enzyme and total GGTP activity decreased gradually. This resulted in an increased percentage of concanavalin A-nonreactive GGTP from 14.5% at 15 weeks’ to 33.3% at 22 weeks’ gestation. Treatment with neuraminidase prior to concanavalin A-Sepharose chromatography did not alter

by concanavalin

A-sepharose.

the distribution. This pattern is similar to that found for AFP. Ruoslahti and colleagues” have found that amniotic Huid from early pregnancy contained mostly concanavalin A-reactive AFP (55% to 85%), and this diminished significantly in late pregnancy. They have also shown that AFP synthesized by yolk sac tissue and by liver are glycosylated differently. The yolk sac produced essentially the concanavalin A-nonreactive type, whereas AFP arising from liver was the reactive form. AFP from NTD has been shown to be mainly of the concanavalin A-reactive type.‘” These observations indicate that in the second trimester amniotic Huid AFP originates in the fetal liver and this is also true of amniotic Huid AFP in NTD. These tissue-similar origins of amniotic Huid AFP and GGTP may help explain why both proteins show a similar gestational pattern in normal pregnancy. The explanation for the fact that only AFP, not GGTP, is elevated in amniotic Huid from NTD is not clear. AFP in amniotic Huid after the second trimester is composed solely (about 96%) of the reactive

type,

arising

from

the

liver,

whereas

GGTP

at

this stage of gestation contains about 30% of the nonreactive type which could be arising from tissues other than the liver. Recently, GGTP has been reported to exhibit glutathione oxidase activity. I8 It is shown to be present in renal cortex, epididymal caput, jejunal villus tip cells,

Volume Number

14 I 7

choroid plexus, and retina but is absent in liver. Theretore, we investigated glutathione oxidase activity in amniotic Huid samples and resuhs are shown in Table III. Amniotic Huid showed significant oxidase activity, which decreased from X7.8 to 26.8 units from 15 weeks’ to 22 weeks’ gestation. The ratio of transpeptidase to glutathione oxidase in amniotic Huid remained between 7 and 10 at all gestational times tested. These results suggest that tissues other than liver, such as yolk

y-Glutamyl transpeptidase

in human amniotic fluid

791

sac, might be iadditional sources of ;urmiotic Huid GGTP. The relative contribution b! thesr ditf’erent sources would play a dynamic role in the maintenance of GGTP activit!. in amniotic fluid. .Uthottgh :iF‘p and GCTP follow a similar gestational pattern in normal pregnant! , their distribution differ3 in S I‘D, pocsibl\: because GGTP originates f’rom tissues besides liker. It is possible that this phenomenon may e.*trntl to othetproteins that are present in amniotic flu&.

REFERENCES

1. Meister, A., and Tate, S. S.: Glutathione and related y-glutamyl compounds: Biosynthesis and utilization, Ann. Rev. Biochem. 45:559, 1976. 2. Fiala, S., and Fiala, E. S.: Activation by chemical carcjnogem of y-glutamyl transpeptidase in rat and mouse liver, J. Natl. Cancer Inst. 51:151, 1973. 3. Kiittgen, E., and Lindinger, G.: Nachweis mol-kularer varianten der gamma-glutamyl-transferase mit differenter concanavalin-A affinitat, Hoppe Seylers Z. Physiol. Chem. 357:1439, 1976. 4. Norgaard-Pedersen, B.: Human alpha-fetoprotein, Stand. J. Immunol. 5:suppl. 4, 1976. 5. Brock, D. J. H.: Prenatal diagnosis-chemical methods, Br. Med. Bull. 32:16, 1976. 6. Ruoslahti. E., and SeppdP, M.: Studies of carcino-fetal proteins: Physical and chemical properties of human cu-fetoprotein, Int. J. Cancer 7:218, 1971. 7. Tate, S. S., and Meister, A.: Interaction of gamma glutamyl transpeptidase with amino acids, dipeptides, derivatives and analogs of glutathione, J. Biol. Chem. 249:7593, 1974. 8. Tate, S. S.. and Meister, A.: Subunit structure and isozymic forms of y-glutamyl transpeptidase, Proc. Natl. Acad. Sci. 73:2599, 1976. 9. Tate, S. S.. Grau, E. M., and Meister, A.: Conversion of glutathione to glutathione disulfide by cell membrane-

10.

11. 12. 14.

14. 15.

16.

bound oxidase activity, Proc. Nat]. Acad. Sci. 76:2715. 1979. Tietze, F.: Enzymatic method for quantitative determination of nanogram amounts of total and oxidized glutathione: Application to mammalian blood and other tissues, Anal. B&hem. 27:502, 1469. Szasz, G.: A kinetic photometric method for serum y-glutamyl transpeptidase, Clin. Chem. 15~124, 1969. Orlowski, M., and Meister, A.: The y-glutamyl cycle: A possible transport system for amino acids, Proc. Natl. Acad. Sci. 67:1248, 1970. Kottgen, E., Reutter, W., and Gerok, W.: Two different gamma-glutamyltransferases during development of liver and small intestine: A fetal (sialo-) and an adult (asialo-) glycoprotein, Biochem. Biophys. Res. Commun. 72:61, 1976. Ruoslahti, E., Engvall, E.. Pekkala, A., and SeppaIa, M.: Developmental changes in carbohydrate moiety of human alpha-fetoprotein, Int. J. Cancer 22:515, 197X. Smith, C. J., Kelleher, P. C., Belanger, L.. and Dallaire, L.: Reactivity of amniotic fluid alpha-fetoproteih with concanavalin A in diagnosis of neural tube defects, Br. Med. J. 1:920, 1979. Tate, S. S., and Orlando, J.: Conversion of glutathione to glutathione disulfide, a catalytic function ijf y-glutam$ transpeptidase, .J. Biol. Chem. 254:5573, 1979.