Angiotensins I and II, active and inactive renin, renin substrate, renin activity, and angiotensinase in human liquor amnii and plasma S. L.
SKINNER,
ELIZABETH
J.
ROBYN
R. W. K.
M.D. CRAN,
GIBSON,
J.
M.R.C.O.G.,
W.
WALTERS,
GATT,
Melbourne,
(HoNs.)
M.Sc.
TAYLOR, A.
B.Sc.
F.A.G.O. PH.D.,
M.D.,
F.R.C.O.G.,
F.A.G.O.
PH.D.
Australia
Most of the activated by gestation, 83 total amount
renin in liquor amnii and plasma exists in an inactive acidification to pH 3.3. In liquor collected by paracentesis per cent of renin was inactive (n =T 37). This proportion present. In first trimester plasma, the proportion inactive
form which can be at 34 to 37 weeks’ was unrelated to the (73 per cent) was
greater than in term plasma (65 per cent, P < 0.001) or in normal male and female control plasma (62 per cent) but all were less than in liquor (P < 0.001). Physiologically active renin was 16-fold more concentrated in liquor than in term plasma but renin substrate was only one fiftieth as high. Comparisons of endogenous renin activities and angiotensin levels in liquor and plasma liquor is lower than in plasma and (2) step in local angiotensin production.
indicate: that entry
(1) that clearance of substrate into
THE DE s CRIP ~10 N of extremely high concentrations of a renin-like enzyme in liquor amni+ ’ and its likely origin from chorion laeve2* 3 suggested that this enzyme subserves some local and possibly general role in the physiology of pregnancy.3, 4 More recently, however, it was reported that most of the renin in liquor probably occurs as an inactive pro-
renin from which activation can be accomplished by mild acid treatment.5 Angiotensin II, the physiologically active product of the reaction between renin and renin substrate has not been measured in liquor amnii where its presence would be pivotal in any claim that the conventional reaction has local physiologic importance. Accordingly, we have measured this end-product and its various precursors in the liquor of women during the last trimester of pregnancy. The contribution that each reactant makes to the angiotensin level in this fluid has been sought and comparisons have been made with maternal plasma and with plasma from nonpregnant control subjects.
From the Monash University Departments of Medicine, Prince Henry’s Hospital and of Obstetrics and Gynecology, Queen Victoria Hospital; Department of Physiology, University of Melbourne: Howard Florey Institute of .?&perimental’Physiology and* Medicine. . This
work
Australia
was supported by grants from the Health and Medical Research Council of and the Heart Foundation of Australia.
Received
for publication
Accepted
September
National
July
of angiotensin from liquor is the rate-limiting
Materials
2, 1974.
and
methods
Liquor amnii was collected by paracentesis from 37 women between the thirty-fourth and thirtyseventh weeks of pregnancy. Repeat paracenteses were performed on three women after intervals of
5, 1974.
Reprint requests: Dr. S. L. Skinner, Department of Physiology, University of Melbourne, Australia, 3052. 626
Volume Number
12 1 5
3 to 6 weeks. Twenty-nine women were judged to be clinically normal and nine displayed minor degrees of either hypertension (three), pre-eclamptic toxemia (four) , or placental insufficiency (two) . Venous samples were collected for renin assay from 20 clinically normal pregnant women. Eight were in the first trimester of pregnancy and twelve were at 36 to 40 weeks’ gestation. Plasma was also collected from normal male and female volunteers. Angiotensins I and II were measured in liquor by radioimmunoassay according to the method of Cain, Coghlan, and Catt.6 This method was originally described for angiotensin II but the production of a high titer of specific antibodies against angiotensin I permitted its unmodified application to the assay of the decapeptide. A 25 ml. sample of neat liquor was collected, immediately added to 75 ml. of cold ethanol, and stored at -15O C. for later extraction and radioimmunoassay. Renin concentration was measured in blood and liquor as the rate of angiotensin production during incubation with renin substrate from nephrectomized sheep at pH 7.5 under zero order kinetic conditions ( 1.1 pg of angiotensin content per milliliter), as previously described.4, 7 This method subjects human plasma or liquor to an acidifying step of pH 3.3 at 32O C. in order to destroy endogenous substrate and angiotensinase prior to incubation with the sheep substrate. Acidification has been shown by Lumbers,S however, to cause an increase in the assayed concentration of renin in liquor and plasma. The increase is apparently due to activation of an inactive prorenin and occurs only with treatment within the pH range of 3 to 4. The value obtained for renin after the acidification procedure is therefore referred to as “total renin.” Renin in liquor and plasma was also assayed without activating prorenin, in which circumstance adequate inhibition of angiotensinase was achieved by dialysis against buffers containing 0.005M ethylene diamine tetra-acetic acid (EDTA) in the pH range of 4.5 to 7.5 (Resuits) . The endogenous substrate remains unchanged through this sequence’ but does not interfere in the reaction between human renin and sheep substrate”, ’ because of extreme difference in affinity of each substrate for the enzyme.g The value obtained in the absence of acid activation is referred to as “active renin.” Total renin is composed of both active and inactive components. One unit of renin in this assay is equivalent to 1.0 x 10-S Goldblatt units of human renin (MRC standard renin, Mill Hill) .
Angiotensin
and
renin
in liquor
amnii
and
plasma
627
Neither of the values total and active renin does not relate directly to the physiologic activity of the system in vivo. In order to obtain such information endogenous renin activity was measured by allowing the already active component of renin to react with substrate naturally present in liquor. This incubation was performed after a single dialysis against the pH 7.5 buffer containing 0.005 M EDTA which in this fluid is sufficient to remove angiotensinase activity (Results). Plasma renin activity was measured as previously described’ by dialysis to pH 4.5, warming at 32O C., followed by dialysis to pH 7.5, and then incubation at 37O C., and measurement of formed angiotensin. As indicated above, this mild acid step to pH 4.5 neither activates prorenin nor denatures substrate but is necessary for complete removal of angiotensinase from whole plasma.7 Renin substrate was measured as angiotensin released (micrograms per milliliter) by 0.5 Goldblatt unit per milliliter of angiotensinase-free human renal renin during incubation to exhaustion. Angiotensinase was measured as per cent survival of added angiotensin I (200 ng. per milliliter; Schwarz Bio-research) and angiotensin II (200 ng. per milliliter; Hypertensin, Ciba) during a 60 minute incubation at pH 7.5 at 37O C. Because of the normal dependence of angiotensinase upon ionized calcium, liquor was dialyzed against pH 7.5 buffer containing 2.5 mM. calcium chloride before determination of angiotensinase. For renin, renin substrate, and angiotensinase measurements, the end-product was assayed by pressor action in the anesthetized ganglionblocked rat with the use of angiotensin II as the reference standard but with frequent checks of the sensitivity to angiotensin I.
Results Reactants in liquor and plasma. Table
I shows the mean values for total renin, active renin, the ratio of total to active, and also the renin activity and substrate in liquor, maternal plasma, and control plasma. Acid treatment (total renin) caused an increase in renin concentration above that already present (active renin) in both liquor and plasma. The ratio of total/active renin was, however, significantly higher for liquor (5.9) than firsttrimester plasma (3.7, P < 0.001) which was in turn higher than term plasma or control plasma (2.9 and 2.6, P < 0.001). In liquor 83 per cent of the total renin was inactive, whereas in firsttrimester, term, and control plasma the inactive component was 73, 65, and 62 per cent, respective-
Skinner et al.
628
March 1, 1975 Am. J. Obstet. Gynecol.
Table I. Renin-angiotensin system in liquor amnii and plasma (mean + 1 S.D.) Total
renin
(U./ml.)* Liquor
Active
renin
(U./ml.)
Total/active
Endogenous renin activity (n,g./ml./hr.)
Angiotensin Renin substrate (.tdnl.)
(p&ml.)
II (Pdml.)
amnii:
1,470 255 5.9 0.8 0.12 f860 +209 +-2.2 +0.5 +0.06 n = 37 n = 22 n= 22 n = 20 n= 22 Maternal plasma, 0 to 13 weeks’ gestation: 162 45 3.7 11.2 2.3 291.3 +26.5 to.6 24.2 Q.0 n=8 n=8 n=8 n=8 n=8 Maternal plasma, 36 to 40 weeks’ gestation: 43.3 15.4 2.9 10.1 6.0 +16.7 25.1 kO.7 +2.9 +2.3 n= 12 n= 12 n= 12 n= 17 n= 17 Control plasma: 22.8 8.9 2.6 1.7 1.1 211.1 24.2 +0.5 fO.5 20.3 n= 12 n= 12 n= 12 n = 45 n = 45 *One unit is equivalentto 1 x 10-SGoldblatt units (WHO standardhumanrenin).
ly. Liquor from the limited number of women suffering from either hypertension, toxemia, or placental insufficiency displayed values within the normal range which are included in the statistical analyses. Liquor contained much higher levels of active renin than did plasma but in spite of this the endogenousrenin activity was lower in liquor than plasma. Substrate concentrations in control plasma and term pregnancy plasma were, respectively, 10 and 50 times higher than the level in liquor (Table 1). Angiotensins I and II were measured only in liquor. A wide range of concentrations was found but the mean levels were not significantly different (Table I). Correlations between reactants in individual liquor samples. Fig. 1 illustrates the results of a comparison in the 22 women shown in Table I between the active renin component and the total renin that could be assayed after acidification of liquor. The correlation between total and active renin in individual samples was significant (r = 0.88, P < 0.001). Endogenous renin activity and active renin (Table I) correlated at a low level of significance (r = 0.50, n = 20, P < 0.05) but, as might be expected for a first-order reaction,4, Q when correction was made for the substrate level in liquor by multiplying substrate concentration by active renin, the correlation with the directly measured endogenous
108 +76 n = 22
67 +43 n = 40
-
-
-
renin activity became more significant (r = 0.67, n = 20, P < 0.001). There was no consistent relationship between endogenous renin activity and either angiotensin I or II. Indeed, angiotensin did not correlate significantly with any reactant or combination of reactants. Nor did any other correlate of significance emerge between any of the reactants taken in every possiblecombination. Angiotensiuase. The 60 minute survival of both angiotensins I and II in clear liquor at 37’ C., pH 7.5, was 58 + 8 per cent (+S.D., n = 9). Addition of 0.005M EDTA increased survival to greater than 80 per cent at 24 hours in all samples. Comment
The level of angiotensin II in amniotic fiuid is not as high (3.5-fold normal plasma) as was expected from earlier reports of renin concentration in this fluid (loo-fold normal plasmalp“). The level of angiotensin II was virtually identical to that reported by Weir and associateslOfor last-trimester plasma and significantly higher than for normal human plasma (19 + 12 pg. per milliliter) .6 Analysis of the components of the renin-angiotensin system in liquor has to someextent elucidated the discrepancy. Thus, 80 per cent of liquor renin is normally inactive and renin substrate concentration is very low. Indeed, renin substrate is low enough to make it certain that the present value for endogenous renin activity must be an underestimate of the true
Volume Number
121 5
value. Up to 40 per cent of the substrate was consumed in obtaining this estimation and initial velocity was therefore not approximated. Conceding this error, and assuming that the true endogenous renin activity of liquor is higher by at most a factor of 2, than the present value of 0.8 ng. per milliliter per hour, then the comparison of measured angiotensin levels in liquor and plasma6 is of some interest. Liquor contains higher levels of angiotensins I and II than normal plasma, and yet renin activity is approximately the same. In addition, third-trimester plasmalU and liquor contain similar levels of angiotensin II and yet renin activity is lo-fold higher in the plasma. This raises the likelihood that the clearance of angiotensins I and II from liquor is less than in the circulation. Whether the production of angiotensin in liquor could constitute a significant source for maternal circulating angiotensin therefore seems unlikely, but the point could be established only by measurement of levels in maternal blood across the uterus together with amniotic fluid from the individual woman. The fact that the level of renin substrate in liquor is low when compared with term pregnancy plasma also has interesting implications. The possibility that the high local renin level digests substrate and keeps its concentration low seems discounted because a negative correlation did not emerge between active renin and renin substrate in liquor. It seems more likely that entry of the large protein substrate into liquor is slow and could be the ratelimiting step in the local generation of angiotensin. The significant correlation between endogenous renin activity of liquor and the multiple of substrate and active renin does not assist in clarifying which of the variables is the most important physiologic regulator of the system in liquor and simply emphasizes that, as in blood, both enzyme and substrate control the in vivo activity of the system. Angiotensinase activity in liquor was found to be low. Contrarily, Maebashi and Yoshinaga’l have demonstrated the complete absence of angiotensinases in liquor by incubation of synthetic angiotensin II-amide with amniotic fluid for 2 hours at 37’ C. They found by biologic assay no decrease in the pressor activity of the angiotensin following incubation. Both findings do, however, point to a low breakdown rate in liquor and therefore increase the likelihood that tissue clearance is the more important mode of removal. The antibody used for the radioimmunoassay of
Angiotensin
and
renin
in liquor
amnii
and
plasma
629
4000
0
3000 z -1
.
u) .?g
.
?
l
5 2000ifi LY
.
2 L IlOOO-
Ol 0
200 ACTIVE
400 RENIN
600
units/ml
Fig. 1. Renin
in liquor amnii collected by paracentesis from 22 women after 34 weeks of gestation. The mean difference between total renin (as measured after activation of prorenin following acidification to pH 3.3) and active renin (as measured without activation of prorenin) is 5.9 + 2.2 (1 S.D.). One unit is equivalent to 1 x 10-s Goldblatt units (WHO standard human renin).
angiotensin II measures octa-, hepta-, hexa-, and pentapeptides with equal specificity. In the absence of angiotensinase activity, only the octapeptide would be present in liquor to be assayed. However, if some small amount of breakdown of the octapeptide occurs, hepta-, hexa-, and penta-peptides are measured as “octapeptide-like” substances. The total octapeptide measurement in this case would very closely approximate the true octapeptide va1ue.l” The antibody used for the radioimmunoassay of angiotensin I was specific for the decapeptide, showing less than 4 per cent cross-reactivity with angiotensin II. Because the mean levels of angiotensins I and II measured in amniotic fluid samples were not significantly different, the converting enzyme activity would appear to be low. The concentration of reactants in liquor seems adequate for both local and general effects but their role in liquor remains obscure. One possibility that seems worth considering is that entry of renin substrate is the important variable that alters on appropriate occasions. Thus, maternal plasma displays a progressive increase in renin substrate,
630
Skinner
March 1, 1975 Am. J. Obstct. Gynecol.
et al.
reaching six times the nonpregnant level at term.’ Lying within this environment is a highly concentrated local store of a physiologically active enzyme capable of degrading the substrate to biologically active end-products. The local store is separated from maternal substrate by only fetal membranes and decidua. A break in the integrity of the barrier would permit the mixing of reactants with local formation of an‘giotensin in appropriately high concentration, for instance in parturition. On the other hand, the specific proteolytic properties of renin may subserve some entirely different function in this fluid. A direct relationship was revealed between active and inactive renin over a wide range of concentrations in liquor. It has been proposed-and circumstantial evidence supports*-that both the active and inactive forms of renin in liquor originate from chorion. If this is correct, then the present findings indicate that secretion occurs in a relatively fixed proportion, unrelated to production rate or at least to the level in liquor. The fact that acidifica-
REFERENCES
1. Brown, J. J., Davies, D. L., Doak, P. B., Lever, A. F., and Robertson, J. I. S.: Lancet 2: 64, 1964. 2. Skinner, S. L., Lumbers, E. R., and Symonds, E. M.: AM. J. OBSTET. GYNECOL. 101: 529, 1968. 3. Symonds, E. M., Stanley, M. A., and Skinner, S. L.: Nature 217: 1152, 1968. 4. Skinner, S. L., Lumbers, E. R., and Symonds, E. M.: Clin. Sci. 42: 479, 1972. 5. Lumbers, E. R.: Enzymologia 40: 329, 1971. 6. Cain, M. D., Coghlan, J. P., and Catt, K. J.: Clin. Chim. Acta 39: 21, 1972. 7. Skinner, S. L.: Circ. Res. 20: 391, 1967. 8. Stockigt, J. R., Collins, R. D., and Biglieri, E. G.: Circ. Res. 28 (Suppl. II): 175, 1971.
Note
to Authors:
Change
The Editors and Publisher
in Reference
tion of plasma to pH 3.3, whether from normal males or pregnant or nonpregnant females, similarly leads to the measurement of greater quantities of renin suggests that renin secretion from kidney is also in both an active and inactive form. How the present findings relate to the acid-activation of renin described for extracts of rabbit’” or hog’* kidney or with pepsin in human liquorI is not clear at present, but the possibility that during normal secretion an inactive storage form is only partly activated by some process which is duplicated by acidification in vitro must be considered in future work. The present finding that a significantly greater degree of activation can be obtained in liquor than plasma might then suggest that the normal activation-secretion process is less complete from chorion than kidney. Furthermore, the greater activation in first-trimester plasma than at term might indicate a contribution of chorionic renin to maternal plasma in early pregnancy, as previously suggested,” but could equally well be composed exclusively of renal renin.
9. Skinner, S. L., Dunn, J R., Mazzetti, J. Campbell, D. J., and Fidge, N. H.: Aust. J. Exp. Biol. Med. Sci. 53: February, 1975. In press. 10. Weir, R. J., Brown, J. J., Fraser, R., Kraszewski, A., Lever, A. F., McIlwaine, G. M., Morton, J. J., Robertson, J. I. S., and Tree, M.: Lancet 1: 291, 1973. 11. Maebashi, M., and Yoshinaga, K.: Tohoku J. Exp. Med. 95: 393, 1968. 12. Cain, M. D., Catt, K. J., and Coghlan, J. P.: J. Clin. Endocrinol. 29: 1639, 1969. 13. Leckie, B.: Clin. Sci. 44: 301, 1973. 14. Rubin, I.: Stand. J. Clin. Lab. Invest. 29: 51, 1972. 15. Morris, B. J., and Lumbers, E. R.: Biochim. Biophys. Acta 289: 385, 1972.
Style
have agreed to add the article title to references in the AMERICAN GYNECOLOGY. References will now conform to the style of the Cumulated Index Medicus, viz., name of author, title of article, name of periodical, volume, page, and year. Authors are always encouraged to limit references to sixteen for the following JOURNAL sections: Obstetrics: Gynecology, and Fetus, Placenta, and Newborn.
JOURNAL
OF OBSTETRICS
AND
SKINNER,
ELIZABETH
J.
ROBYN
R. W. K.
M.D. CRAN,
GIBSON,
J.
M.R.C.O.G.,
W.
WALTERS,
GATT,
Melbourne,
(HoNs.)
M.Sc.
TAYLOR, A.
B.Sc.
F.A.G.O. PH.D.,
M.D.,
F.R.C.O.G.,
F.A.G.O.
PH.D.
Australia
Most of the activated by gestation, 83 total amount
renin in liquor amnii and plasma exists in an inactive acidification to pH 3.3. In liquor collected by paracentesis per cent of renin was inactive (n =T 37). This proportion present. In first trimester plasma, the proportion inactive
form which can be at 34 to 37 weeks’ was unrelated to the (73 per cent) was
greater than in term plasma (65 per cent, P < 0.001) or in normal male and female control plasma (62 per cent) but all were less than in liquor (P < 0.001). Physiologically active renin was 16-fold more concentrated in liquor than in term plasma but renin substrate was only one fiftieth as high. Comparisons of endogenous renin activities and angiotensin levels in liquor and plasma liquor is lower than in plasma and (2) step in local angiotensin production.
indicate: that entry
(1) that clearance of substrate into
THE DE s CRIP ~10 N of extremely high concentrations of a renin-like enzyme in liquor amni+ ’ and its likely origin from chorion laeve2* 3 suggested that this enzyme subserves some local and possibly general role in the physiology of pregnancy.3, 4 More recently, however, it was reported that most of the renin in liquor probably occurs as an inactive pro-
renin from which activation can be accomplished by mild acid treatment.5 Angiotensin II, the physiologically active product of the reaction between renin and renin substrate has not been measured in liquor amnii where its presence would be pivotal in any claim that the conventional reaction has local physiologic importance. Accordingly, we have measured this end-product and its various precursors in the liquor of women during the last trimester of pregnancy. The contribution that each reactant makes to the angiotensin level in this fluid has been sought and comparisons have been made with maternal plasma and with plasma from nonpregnant control subjects.
From the Monash University Departments of Medicine, Prince Henry’s Hospital and of Obstetrics and Gynecology, Queen Victoria Hospital; Department of Physiology, University of Melbourne: Howard Florey Institute of .?&perimental’Physiology and* Medicine. . This
work
Australia
was supported by grants from the Health and Medical Research Council of and the Heart Foundation of Australia.
Received
for publication
Accepted
September
National
July
of angiotensin from liquor is the rate-limiting
Materials
2, 1974.
and
methods
Liquor amnii was collected by paracentesis from 37 women between the thirty-fourth and thirtyseventh weeks of pregnancy. Repeat paracenteses were performed on three women after intervals of
5, 1974.
Reprint requests: Dr. S. L. Skinner, Department of Physiology, University of Melbourne, Australia, 3052. 626
Volume Number
12 1 5
3 to 6 weeks. Twenty-nine women were judged to be clinically normal and nine displayed minor degrees of either hypertension (three), pre-eclamptic toxemia (four) , or placental insufficiency (two) . Venous samples were collected for renin assay from 20 clinically normal pregnant women. Eight were in the first trimester of pregnancy and twelve were at 36 to 40 weeks’ gestation. Plasma was also collected from normal male and female volunteers. Angiotensins I and II were measured in liquor by radioimmunoassay according to the method of Cain, Coghlan, and Catt.6 This method was originally described for angiotensin II but the production of a high titer of specific antibodies against angiotensin I permitted its unmodified application to the assay of the decapeptide. A 25 ml. sample of neat liquor was collected, immediately added to 75 ml. of cold ethanol, and stored at -15O C. for later extraction and radioimmunoassay. Renin concentration was measured in blood and liquor as the rate of angiotensin production during incubation with renin substrate from nephrectomized sheep at pH 7.5 under zero order kinetic conditions ( 1.1 pg of angiotensin content per milliliter), as previously described.4, 7 This method subjects human plasma or liquor to an acidifying step of pH 3.3 at 32O C. in order to destroy endogenous substrate and angiotensinase prior to incubation with the sheep substrate. Acidification has been shown by Lumbers,S however, to cause an increase in the assayed concentration of renin in liquor and plasma. The increase is apparently due to activation of an inactive prorenin and occurs only with treatment within the pH range of 3 to 4. The value obtained for renin after the acidification procedure is therefore referred to as “total renin.” Renin in liquor and plasma was also assayed without activating prorenin, in which circumstance adequate inhibition of angiotensinase was achieved by dialysis against buffers containing 0.005M ethylene diamine tetra-acetic acid (EDTA) in the pH range of 4.5 to 7.5 (Resuits) . The endogenous substrate remains unchanged through this sequence’ but does not interfere in the reaction between human renin and sheep substrate”, ’ because of extreme difference in affinity of each substrate for the enzyme.g The value obtained in the absence of acid activation is referred to as “active renin.” Total renin is composed of both active and inactive components. One unit of renin in this assay is equivalent to 1.0 x 10-S Goldblatt units of human renin (MRC standard renin, Mill Hill) .
Angiotensin
and
renin
in liquor
amnii
and
plasma
627
Neither of the values total and active renin does not relate directly to the physiologic activity of the system in vivo. In order to obtain such information endogenous renin activity was measured by allowing the already active component of renin to react with substrate naturally present in liquor. This incubation was performed after a single dialysis against the pH 7.5 buffer containing 0.005 M EDTA which in this fluid is sufficient to remove angiotensinase activity (Results). Plasma renin activity was measured as previously described’ by dialysis to pH 4.5, warming at 32O C., followed by dialysis to pH 7.5, and then incubation at 37O C., and measurement of formed angiotensin. As indicated above, this mild acid step to pH 4.5 neither activates prorenin nor denatures substrate but is necessary for complete removal of angiotensinase from whole plasma.7 Renin substrate was measured as angiotensin released (micrograms per milliliter) by 0.5 Goldblatt unit per milliliter of angiotensinase-free human renal renin during incubation to exhaustion. Angiotensinase was measured as per cent survival of added angiotensin I (200 ng. per milliliter; Schwarz Bio-research) and angiotensin II (200 ng. per milliliter; Hypertensin, Ciba) during a 60 minute incubation at pH 7.5 at 37O C. Because of the normal dependence of angiotensinase upon ionized calcium, liquor was dialyzed against pH 7.5 buffer containing 2.5 mM. calcium chloride before determination of angiotensinase. For renin, renin substrate, and angiotensinase measurements, the end-product was assayed by pressor action in the anesthetized ganglionblocked rat with the use of angiotensin II as the reference standard but with frequent checks of the sensitivity to angiotensin I.
Results Reactants in liquor and plasma. Table
I shows the mean values for total renin, active renin, the ratio of total to active, and also the renin activity and substrate in liquor, maternal plasma, and control plasma. Acid treatment (total renin) caused an increase in renin concentration above that already present (active renin) in both liquor and plasma. The ratio of total/active renin was, however, significantly higher for liquor (5.9) than firsttrimester plasma (3.7, P < 0.001) which was in turn higher than term plasma or control plasma (2.9 and 2.6, P < 0.001). In liquor 83 per cent of the total renin was inactive, whereas in firsttrimester, term, and control plasma the inactive component was 73, 65, and 62 per cent, respective-
Skinner et al.
628
March 1, 1975 Am. J. Obstet. Gynecol.
Table I. Renin-angiotensin system in liquor amnii and plasma (mean + 1 S.D.) Total
renin
(U./ml.)* Liquor
Active
renin
(U./ml.)
Total/active
Endogenous renin activity (n,g./ml./hr.)
Angiotensin Renin substrate (.tdnl.)
(p&ml.)
II (Pdml.)
amnii:
1,470 255 5.9 0.8 0.12 f860 +209 +-2.2 +0.5 +0.06 n = 37 n = 22 n= 22 n = 20 n= 22 Maternal plasma, 0 to 13 weeks’ gestation: 162 45 3.7 11.2 2.3 291.3 +26.5 to.6 24.2 Q.0 n=8 n=8 n=8 n=8 n=8 Maternal plasma, 36 to 40 weeks’ gestation: 43.3 15.4 2.9 10.1 6.0 +16.7 25.1 kO.7 +2.9 +2.3 n= 12 n= 12 n= 12 n= 17 n= 17 Control plasma: 22.8 8.9 2.6 1.7 1.1 211.1 24.2 +0.5 fO.5 20.3 n= 12 n= 12 n= 12 n = 45 n = 45 *One unit is equivalentto 1 x 10-SGoldblatt units (WHO standardhumanrenin).
ly. Liquor from the limited number of women suffering from either hypertension, toxemia, or placental insufficiency displayed values within the normal range which are included in the statistical analyses. Liquor contained much higher levels of active renin than did plasma but in spite of this the endogenousrenin activity was lower in liquor than plasma. Substrate concentrations in control plasma and term pregnancy plasma were, respectively, 10 and 50 times higher than the level in liquor (Table 1). Angiotensins I and II were measured only in liquor. A wide range of concentrations was found but the mean levels were not significantly different (Table I). Correlations between reactants in individual liquor samples. Fig. 1 illustrates the results of a comparison in the 22 women shown in Table I between the active renin component and the total renin that could be assayed after acidification of liquor. The correlation between total and active renin in individual samples was significant (r = 0.88, P < 0.001). Endogenous renin activity and active renin (Table I) correlated at a low level of significance (r = 0.50, n = 20, P < 0.05) but, as might be expected for a first-order reaction,4, Q when correction was made for the substrate level in liquor by multiplying substrate concentration by active renin, the correlation with the directly measured endogenous
108 +76 n = 22
67 +43 n = 40
-
-
-
renin activity became more significant (r = 0.67, n = 20, P < 0.001). There was no consistent relationship between endogenous renin activity and either angiotensin I or II. Indeed, angiotensin did not correlate significantly with any reactant or combination of reactants. Nor did any other correlate of significance emerge between any of the reactants taken in every possiblecombination. Angiotensiuase. The 60 minute survival of both angiotensins I and II in clear liquor at 37’ C., pH 7.5, was 58 + 8 per cent (+S.D., n = 9). Addition of 0.005M EDTA increased survival to greater than 80 per cent at 24 hours in all samples. Comment
The level of angiotensin II in amniotic fiuid is not as high (3.5-fold normal plasma) as was expected from earlier reports of renin concentration in this fluid (loo-fold normal plasmalp“). The level of angiotensin II was virtually identical to that reported by Weir and associateslOfor last-trimester plasma and significantly higher than for normal human plasma (19 + 12 pg. per milliliter) .6 Analysis of the components of the renin-angiotensin system in liquor has to someextent elucidated the discrepancy. Thus, 80 per cent of liquor renin is normally inactive and renin substrate concentration is very low. Indeed, renin substrate is low enough to make it certain that the present value for endogenous renin activity must be an underestimate of the true
Volume Number
121 5
value. Up to 40 per cent of the substrate was consumed in obtaining this estimation and initial velocity was therefore not approximated. Conceding this error, and assuming that the true endogenous renin activity of liquor is higher by at most a factor of 2, than the present value of 0.8 ng. per milliliter per hour, then the comparison of measured angiotensin levels in liquor and plasma6 is of some interest. Liquor contains higher levels of angiotensins I and II than normal plasma, and yet renin activity is approximately the same. In addition, third-trimester plasmalU and liquor contain similar levels of angiotensin II and yet renin activity is lo-fold higher in the plasma. This raises the likelihood that the clearance of angiotensins I and II from liquor is less than in the circulation. Whether the production of angiotensin in liquor could constitute a significant source for maternal circulating angiotensin therefore seems unlikely, but the point could be established only by measurement of levels in maternal blood across the uterus together with amniotic fluid from the individual woman. The fact that the level of renin substrate in liquor is low when compared with term pregnancy plasma also has interesting implications. The possibility that the high local renin level digests substrate and keeps its concentration low seems discounted because a negative correlation did not emerge between active renin and renin substrate in liquor. It seems more likely that entry of the large protein substrate into liquor is slow and could be the ratelimiting step in the local generation of angiotensin. The significant correlation between endogenous renin activity of liquor and the multiple of substrate and active renin does not assist in clarifying which of the variables is the most important physiologic regulator of the system in liquor and simply emphasizes that, as in blood, both enzyme and substrate control the in vivo activity of the system. Angiotensinase activity in liquor was found to be low. Contrarily, Maebashi and Yoshinaga’l have demonstrated the complete absence of angiotensinases in liquor by incubation of synthetic angiotensin II-amide with amniotic fluid for 2 hours at 37’ C. They found by biologic assay no decrease in the pressor activity of the angiotensin following incubation. Both findings do, however, point to a low breakdown rate in liquor and therefore increase the likelihood that tissue clearance is the more important mode of removal. The antibody used for the radioimmunoassay of
Angiotensin
and
renin
in liquor
amnii
and
plasma
629
4000
0
3000 z -1
.
u) .?g
.
?
l
5 2000ifi LY
.
2 L IlOOO-
Ol 0
200 ACTIVE
400 RENIN
600
units/ml
Fig. 1. Renin
in liquor amnii collected by paracentesis from 22 women after 34 weeks of gestation. The mean difference between total renin (as measured after activation of prorenin following acidification to pH 3.3) and active renin (as measured without activation of prorenin) is 5.9 + 2.2 (1 S.D.). One unit is equivalent to 1 x 10-s Goldblatt units (WHO standard human renin).
angiotensin II measures octa-, hepta-, hexa-, and pentapeptides with equal specificity. In the absence of angiotensinase activity, only the octapeptide would be present in liquor to be assayed. However, if some small amount of breakdown of the octapeptide occurs, hepta-, hexa-, and penta-peptides are measured as “octapeptide-like” substances. The total octapeptide measurement in this case would very closely approximate the true octapeptide va1ue.l” The antibody used for the radioimmunoassay of angiotensin I was specific for the decapeptide, showing less than 4 per cent cross-reactivity with angiotensin II. Because the mean levels of angiotensins I and II measured in amniotic fluid samples were not significantly different, the converting enzyme activity would appear to be low. The concentration of reactants in liquor seems adequate for both local and general effects but their role in liquor remains obscure. One possibility that seems worth considering is that entry of renin substrate is the important variable that alters on appropriate occasions. Thus, maternal plasma displays a progressive increase in renin substrate,
630
Skinner
March 1, 1975 Am. J. Obstct. Gynecol.
et al.
reaching six times the nonpregnant level at term.’ Lying within this environment is a highly concentrated local store of a physiologically active enzyme capable of degrading the substrate to biologically active end-products. The local store is separated from maternal substrate by only fetal membranes and decidua. A break in the integrity of the barrier would permit the mixing of reactants with local formation of an‘giotensin in appropriately high concentration, for instance in parturition. On the other hand, the specific proteolytic properties of renin may subserve some entirely different function in this fluid. A direct relationship was revealed between active and inactive renin over a wide range of concentrations in liquor. It has been proposed-and circumstantial evidence supports*-that both the active and inactive forms of renin in liquor originate from chorion. If this is correct, then the present findings indicate that secretion occurs in a relatively fixed proportion, unrelated to production rate or at least to the level in liquor. The fact that acidifica-
REFERENCES
1. Brown, J. J., Davies, D. L., Doak, P. B., Lever, A. F., and Robertson, J. I. S.: Lancet 2: 64, 1964. 2. Skinner, S. L., Lumbers, E. R., and Symonds, E. M.: AM. J. OBSTET. GYNECOL. 101: 529, 1968. 3. Symonds, E. M., Stanley, M. A., and Skinner, S. L.: Nature 217: 1152, 1968. 4. Skinner, S. L., Lumbers, E. R., and Symonds, E. M.: Clin. Sci. 42: 479, 1972. 5. Lumbers, E. R.: Enzymologia 40: 329, 1971. 6. Cain, M. D., Coghlan, J. P., and Catt, K. J.: Clin. Chim. Acta 39: 21, 1972. 7. Skinner, S. L.: Circ. Res. 20: 391, 1967. 8. Stockigt, J. R., Collins, R. D., and Biglieri, E. G.: Circ. Res. 28 (Suppl. II): 175, 1971.
Note
to Authors:
Change
The Editors and Publisher
in Reference
tion of plasma to pH 3.3, whether from normal males or pregnant or nonpregnant females, similarly leads to the measurement of greater quantities of renin suggests that renin secretion from kidney is also in both an active and inactive form. How the present findings relate to the acid-activation of renin described for extracts of rabbit’” or hog’* kidney or with pepsin in human liquorI is not clear at present, but the possibility that during normal secretion an inactive storage form is only partly activated by some process which is duplicated by acidification in vitro must be considered in future work. The present finding that a significantly greater degree of activation can be obtained in liquor than plasma might then suggest that the normal activation-secretion process is less complete from chorion than kidney. Furthermore, the greater activation in first-trimester plasma than at term might indicate a contribution of chorionic renin to maternal plasma in early pregnancy, as previously suggested,” but could equally well be composed exclusively of renal renin.
9. Skinner, S. L., Dunn, J R., Mazzetti, J. Campbell, D. J., and Fidge, N. H.: Aust. J. Exp. Biol. Med. Sci. 53: February, 1975. In press. 10. Weir, R. J., Brown, J. J., Fraser, R., Kraszewski, A., Lever, A. F., McIlwaine, G. M., Morton, J. J., Robertson, J. I. S., and Tree, M.: Lancet 1: 291, 1973. 11. Maebashi, M., and Yoshinaga, K.: Tohoku J. Exp. Med. 95: 393, 1968. 12. Cain, M. D., Catt, K. J., and Coghlan, J. P.: J. Clin. Endocrinol. 29: 1639, 1969. 13. Leckie, B.: Clin. Sci. 44: 301, 1973. 14. Rubin, I.: Stand. J. Clin. Lab. Invest. 29: 51, 1972. 15. Morris, B. J., and Lumbers, E. R.: Biochim. Biophys. Acta 289: 385, 1972.
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have agreed to add the article title to references in the AMERICAN GYNECOLOGY. References will now conform to the style of the Cumulated Index Medicus, viz., name of author, title of article, name of periodical, volume, page, and year. Authors are always encouraged to limit references to sixteen for the following JOURNAL sections: Obstetrics: Gynecology, and Fetus, Placenta, and Newborn.
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