Plasma aminopeptidase activity (oxytocinase) in pregnancy and labor

OBSTETRICS

Plasma aminopeptidase activity (oxytocinase) in pregnancy and labor MARY ANN TITUS, A.B. D. RICHARD REYNOLDS, M.D. MARY BETH GLENDENING, PH.D. ERNEST W. PAGE, M.D. San Francisco, California

T H E ability of pregnancy plasma to inactivate oxytocin was first observed by Fekete1 in 1930 and has since been studied by a number of investigators. In 1946, Page 2 described an enzymatic unit for "pitocinase" (now an inappropriate term) based upon the time required for 50 per cent destruction of the substrate. The rate of oxytocin inactivation was determined by serial bioassays on the isolated rat uterus. Despite the inaccuracies of the bioassay, Page 3 noted in 1947 that pregnancy could be diagnosed as early as 4 weeks after conception; and that during the first half of normal pregnancy, the time of conception could be estimated with an accuracy of ±5 days. The values obtained at term were no doubt higher than the true inactivation rates

in vivo, because the plasma samples were diluted. Gonzalez-Panizza and associates4 demonstrated that a progressive dilution of pregnancy plasma produced less than the expected decrease of enzymatic activity. This is probably due to the presence of an inhibitor to oxytocinase in plasma, and dilution reduces the inhibitor activity more than it reduces the enzyme activity. Thus, the true increment of oxytocinase activity due to pregnancy is probably between 50 and 100 times, as described by Werie and Semm, 5 rather than a thousandfold as described earlier. 2• 3 Most observers have failed to find any changes in plasma oxytocinase prior to or during labor. In 1959, however, Hilton and Johnson, 6 utilizing the same type of rat uterus bioassay, reported that the oxytocinase activity of plasma decreased to zero or near zero in 6 of 9 women studied during the first stage of labor. A similar decline had been reported by Namiki 7 in 1952, and by Dicker and Tyler8 in 1954, although denied by Dicker and Whyley 9 in 1959. If true, this would be of considerable theoretical importance be-

From The Department of Obstetrics and Gynecology, University of California School of Medicine. This investigation was supported by Training Grant 2G-145 from the National Institutes of Health and by a grant from the Association for the Aid of Crippled Children.

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Plasma aminopeptidase activity 1125

cause it might explain the cause of labor in primates. Oxytocinase, incidentally, exists only in the plasma of pregnant women and monkeys, and not in other species. Cystine aminopeptidase

The structure of the oxytocin molecule was announced independently by Tuppyl 0 and by Du Vigneaud and co-workers11 in 1953. It is an octapeptide amide, and the relationship of cystine in the cyclic pentapeptide moiety is shown in Fig. 1. In 1957, Tuppy and Nesvadba12 demonstrated that when synthetic oxytocin is acted upon by human pregnancy plasma, the ring is split between cystine and tyrosine and the hormone thus became inactive. They proved this by adding performic acid to the inactivated product to split the disulfide linkage, thus liberating the cysteine fragment. These experiments led them to conclude that the primary oxytocin-inactivating enzyme of pregnancy plasma was a cystine aminopeptidase. In 1958 Miiller-Hartburg, Tuppy, and Nesvadba13 described a synthetic substrate for determining cystine aminopeptidase activity. This compound, cystine di-,8-naphthylamide, is illustrated in Fig. 2. When acted upon by pregnancy plasma, two molecules of ,8-naphthylamide are released which can be converted to a compound readily measured by colorimetry. The synthetic substrate is apparently split more readily than oxytocin by other nonspecific arninopeptidases found in nonpregnancy plasma, since nonpregnancy values are relatively high when compared to the biologic assay method NIH2 C t 2 . - CH

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which uses oxytocin as the substrate. The chemical method is unsatisfactory, therefore, for the diagnosis of early pregnancy. The colorimetric method, nevertheless, is vastly simpler and considerably more accurate, thus compensating for some loss of specificity. Method

The chemical method of oxytocinase assay is based mainly upon the Bratton-Marshall reaction, as modified by Tuppy and Nesvadba. 12 It is described here with minor modifications and additions for those who might wish to carry out the procedure. Blood sample. Hemolysis of the drawn blood is avoided by emptying the syringe slowly and refraining from forcibly expelling any residual foam. Serum is usually used. Heparinized plasma and serum from the same sample of blood give identical values for aminopeptidase activity. The serum can be kept in the refrigerator for 48 hours or in the freezer for months with no change in the activity. Reagents. Substrate solution. L-cystine-di-,8-naphthylamide,* 135 mg., is dissolved in 50 mi. of 0.012 N HOI, with moderate warming. The clear solution is diluted to 100 mi. with distilled water and stored in the refrigerator. *May be obtained from Sanabo Company, Anton-Scharff· Gasse 7, Vienna.

1126 Titus et al.

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Buffer. (pH 7.9) 68.9 ml. of 0.1 N sodium Verona! (20.6 Gm. sodium diethyl-barbiturate per liter of boiled carbon dioxide- free dis tilled water ) is mixed with 31.1 mI. 0. 1 ~ HCI and 50 mi. distilled water. If the pH of the buffer is not 7.9, it is adjusted (glass electrode), then stored in the refrigerator. Other reagents. Trichloracetic acid, 10 per cent ( w/ v) ; hydrochloric acid, 0.36 normal acetone, reagent grade; sodium nitrite, 0.1 per cent aqueous; ammonium sulfamate, 0.5 per cent aqueous; ethylenediamine-dihydrochloride (Eastman Kodak ), 0.1 per cent, aqueous. Standard {3-naphth·y lamine. Eighty milligrams of {3-naphthylamine is dissolved in 500 mi. 0.012 N HCI with warming and stirring, and diluted to one liter with distilled water. The solution is stored in the dark. Method. Serum, 0.6 ml., is diluted with 0.9 ml. water and 3.0 ml. Verona! buffer; 0.75 mi. of the diluted serum is pipetted into each of 3 centrifuge tubes. The first of the

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Fig. 3. Shaded area includes 95 per cent of the I I 0 plasma cystine aminopeptidase determinations during normal pregnancy.

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December, 1960 Obst. & Gynec.

3 tubes serves as the zero time control. Into it is pipetted 0.25 mi. of substrate solution followed immediately by the addition of 1.0 mi. of 10 per cent trichloracetic acid. The remaining tubes (duplicates) arc placed in a water bath at 37° C . and 0.25 ml. substrate solution is added to each. After 4 hours at 37 ° C. , the reaction is stopped by the addition of 1 ml. of 10 per cent trichloracetic acid. After shaking, the tubes are centrifuged, and 1.0 ml. of the clear supernatant solution is pipetted into a 50 mi. Erlenmeyer flask. (If time does not permit the development of color on the same day as the enzyme reaction, these flasks may be stoppered and stored in the refrigerator overnight. ) A reagent blank and standards are set up as follows: 1.5 mi. of water is mixed with 3.0 ml. buffer, and 0. 75 mi. of the resulting solution is pipetted into each of 3 tubes. To the reagent blank is added 0.25 ml. of water and, to the standards, 0.25 ml. of the standard {3-naphthylamine solution. These tubes are then treated in the same manner as those containing serum. Color development. To each flask containing 1 ml. of filtrate is added 9 mi. of a mixture of 2 volumes of 0.36 N HCI and one Yolume of acetone. The flasks are carried to a dark room lighted only with a small red lam p. The following additions are made 3 minutes apart and the flasks are mixed by rotation after each addition: 1 mi. sodium nitrite, then 1 mi. ammonium sulfamate, and last, 1 mi. ethylenediamine dihydroch]oride. The flasks are stoppered and set in an incubator in the dark at 37 ° C. for 4 hours. The maximal color is reached after 3 to 4 hours and remains constant for several hours. After cooling to room temperature, the samples are read in a spectrophotometer at 565 m,u..

. O.D . unknown- O .D. control C a Icu Iatwn. =-=----=---:---=--=---,-O.D. standard - O.D. blank x 5 ::::: milligrams of fi-naphthylamine liberated per 100 mi. serum per hour. The measure of aminopeptidase activity is expressed as the number of milligrams of .8-naphthylamine liberated per 100 mi. of

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Plasma aminopeptidase activity

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undiluted serum per hour from the substrate. The value of the factor 5 20.5 (average) when the Coleman Jr. spectrophotometer is used with 12 by 75 mm. round cuvettes. The standard solution described represents a value of 5.0 mg. ,8-naphthylamine per 100 ml. serum per hour, which is equivalent to the average value of cystine aminopeptidase activity found in the serum of pregnant women at term. Results When this reaction was applied to nonpregnancy and to pregnancy plasma samples, 95 per cent of 110 determinations fell within the shaded areas shown in Fig. 3. The activity of nonpregnancy plasma is due to a mixture of aminopeptidases, some or all of which show moderate increases in pregnancy. According to Tuppy/4 however, human pregnancy is characterized by the appearance of a new aminopeptidase which cannot be detected in the blood of nonpregnant individuals. The presumption is that this enzyme arises from the placenta or decidua, but this has not been proved.

Individual values obtained during the month prior to labor, during early or late labor, and during the early puerperium are shown in Fig. 4. No significant trends are in evidence. The wide scatter is due to differences between individuals and not to the presence or absence of labor. This is demonstrated in Fig. 5, in which serial values on the same individuals are shown before, during, and after labor. From these data, we may conclude that there are no declines in the oxytocinase activity of plasma at the end 10

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1128 Titus et al.

of pregnancy and that the onset of spontaneous labor in women cannot, therefore, be attributed to a diminution in the ability of the blood to destroy endogenous oxytocin. Summary

The primary oxytocinase activity of pregnancy plasma is apparently due to a cystine aminopeptidase. The latter activity may be measured by a chemical procedure which utilizes the synthetic substrate cystine-di-,8naphthylamide.

December, 1960

Am.

J. Obst. & Gynec.

The results of 110 determinations during pregnancy indicate a progressive rise of this aminopeptidase activity in plasma, paralleling the oxytocinase curves obtained by biologic assay methods. Contrary to some reports, there is no decline of activity during early or late labor, parturition, or the early puerperium. The onset of spontaneous labor in women can hardly be attributed to a diminished ability of the plasma to inactivate endogenous oxytocin.

REFERENCES

1. Fekete, K.: Endokrinologie 7: 364, 1930. 2. Page, E. W.: AM. J. OBsT. & GYNEC. 52: 1014, 1946. 3. Page, E. W.: Science 105: 2724, 1947. 4. Gonzalez-Panizza, V. H., Mendez-Bauer, C. ]., Carballo, M. A., Cabot, H. M., and Fielitz, C.: International Congress of Physiological Science (Abstracts and Communications), p. 109, 1959. 5. Werle, E., and Semm, K.: Arch Gynak. 187: 449, 1956. 6. Hilton, J. G., and Johnson, R. F.: AM. J. 0BST. & GYNEC. 78: 479, 1959. 7. Namiki, H.: J. Jap. Obst. & Gynec. Soc.

4: 10, 1952.

8. Dicker, S. E., and Tyler, C. M.: J. Obst. & Gynaec. Brit. Emp. 63: 690, 1956. 9. Dicker, S. E., and Whyley, G. A.: J. Obst. & Gynaec. Brit. Emp. 66: 605, 1959. 10. Tuppy, H.: Biochim. et biophys. acta II: 449, 1953. 11. Du Vigncaud, V., Ressler, C., and Trippett, S.: J. Bioi. Chern. 205: 949, 1953. 12. Tuppy, H., and Nesvadba, H.: Mh. Chem.

88: 977, 1957. 13. Miiller-Hartburg, W., Nesvadba, H., and Tuppy, H.: Arch. Gynak. 191: 442, 1959. 14. Tuppy, H.: Personal communication.