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Active and inactive renin in normal human plasma. Comparison between acid activation and cryoactivation
227
Clinica Chimica Acta, 95 (1979) 227-234 0 Elsevier/North -Holland Biomedical Press
CCA 1043
ACTIVE AND INACTIVE RENIN IN NORMAL COMPARISON BETWEEN ACID ACTIVATIdN
PAUL J. LIJNEN,
ANTOON
K. AMERY
and ROBERT
HUMAN PLASMA. AND CRYOACTIVATION
H. FAGARD
Laboratory of Hypertension, Department of Physiopathology, University of Leuven, B-3000 Leuven (Belgium) (Received
January
16th,
Faculty of Medicine,
1979)
Summary
Inactive renin in human plasma is converted to active renin in vitro by acid activation or by cryoactivation. Renin activity was measured at pH 5.5 and renin concentration at pH 7.4. The plasma renin activity before and after cryotreatment is termed active (APRA) and total (TPRA) plasma renin activity; the plasma renin concentration before and after acid treatment active (APRC) and total (TPRC) plasma renin concentration. In this study we demonstrated that in normal subjects the proportion of active to total renin after cryo-treatment averaged 61%, which was significantly (p < 0.001) higher than the mean percentage active renin of 34 found with the acid activation procedure. Plasma angiotensin II correlated significantly with APRA, TPRA, TPRC and plasma angiotensin I (PA I), but not with inactive renin, which suggests that inactive renin does not produce angiotensin II in vivo. Cold treatment after acid activation and acid treatment after cryoactivation did not provoke a significant change in the measured renin concentration. Our data support the view that acidification of the plasma activates more than does cryo-treatment, and that inactive renin does not contribute to plasma angiotensin II.
Introduction
Inactive forms of renin in human plasma can be converted in vitro into active renin by acidification of plasma [l-6], by trypsin treatment [7] or by cold treatment [ 8,9]. Optimal acid activation of plasma renin is obtained at pH 3.3-3.5 [ 10-121. Reprint requests should be addressed to: Secretary of the Hypertension and Cardiovascular Unit. University Hospital St. Raphael, Kapucijnenvoer 35. B-3000 Leuven, Belgium.
Rehabilitation
228
The greatest cryoactivation of inactive renin is obtained between pH 7 and 9. No activation has been found in the frozen state [8]. Exogenous trypsin activates inactive renin in plasma at normal pH and at 4, 23 or 37°C proving that neither low pH nor cold are essential [ 71. To study further the proportion of active to total renin, we have compared the acid activation and cryoactivation of plasma renin in normal subjects on an unrestricted diet. The different forms of active, inactive and total renin were related to simultaneously determined plasma levels of angiotensin I and II, aldosterone and angiotensin converting enzyme (kininase II) activity. Methods
and materials
1. Plasma renin activity Plasma renin activity (PRA) was measured by radioimmunoassay of the angiotensin I generated during a l-h incubation of the plasma samples with the endogenous renin substrate at pH 5.5 and at 37°C according to the method of Vallotton [ 131. The results of these determinations were expressed in ng angiotensin I generated per ml plasma and per h (ng * ml-’ * h-l). The in vitro cryoactivation of renin was investigated by measuring the plasma renin activity in plasma samples immediately after thawing and after storage of the plasma at 0°C and at pH 7.4 for 7 days. The value of PRA before and after the cold treatment of the plasma is referred as active (APRA) and total plasma renin activity (TPRA). The difference (TPRA - APRA) is taken as the concentration of inactive plasma renin activity (IPRA) which is called pro-renin activity by Sealey et al. [8]. The inter-assay variation of our PRA method is 6.5%. 2. Plasma renin concentration The total plasma renin concentration (TPRC) was measured as the rate of angiotensin I generated during a l-h incubation with renin substrate from a nephrectomized sheep at pH 7.4 under zero order kinetic conditions, according to the method of Skinner [ 141, adapted for radioimmunoassay [ 151. This assay consists first of a denaturation of endogenous substrate by dialysis against a 0.05 mol/l aminoacetic acid buffer pH 3.3 containing 5 mmol/l EDTA and 90 mmol/l NaCl and of an inactivation of the angiotensinases by heating at 32°C for 1 h. The active plasma renin concentration (APRC) was determined after dialysing the plasma against a 0.05 mol/l aminoacetic acid buffer pH 4.5 and after heating the dialysate at 32°C for 1 h to produce adequate inhibition of the angiotensinases. Incubation was performed with excess sheep renin substrate at pH 7.4 for 1 h. The difference (TPRC-APRC) will be presented as the ‘inactive’ plasma renin concentration (IPRC). The inter-assay variation of the PRC determinations was 10.8% [ 151. 3. Assay of plasma aldosterone, angiotensin and converting enzyme A radioimmunoassay method was used for the measurement of the plasma aldosterone concentration (PAC) and of the plasma angiotensin I and II concentration (PAI, PAII) as previously described by Lijnen et al. [16-181. Plasma
229
angiotensin converting enzyme (kininase II) activity (ACE) was measured spectrophotometrically as described by Lijnen and Amery [19]. The inter-assay variation is 5.7% for PAC [18], 7.6% for PAI [16], 6.4% for PA11 [ 171 and 7.3% for ACE [19]. 4. Blood sampling Blood was withdrawn from an antecubital vein in 20 normal subjects on an unrestricted diet in the sitting position between 2 and 3 p.m. Anticoagulants were EDTA with o-phenantroline for the determination of PAI, PA11 and PRA, EDTA for PRC and PAC and heparin for estimation of ACE. 5. Statistical analysis The statistical methods used were regression analysis and Student’s twotailed t-test for paired data. The dispersion of the data is given by standard error of the mean (S.E.M.). Results 1. Activation studies The normal values for active, inactive and total plasma renin activity and concentration, plasma angiotensin I and II, aldosterone and angiotensin converting enzyme are given in Table I for the subjects aged 22-44 years (mean: 31) on an unrestricted diet. The total plasma renin concentration was significantly @ < 0.001) higher than the active plasma renin concentration in all subjects (Fig. 1A). The proportion of active to total renin produced by the acid activation procedure ranged from 17 to 52% and averaged 34 2 2.6% (Fig. 2). The total plasma renin activity was also higher (JJ < 0.001) than the active plasma renin activity in these normal subjects (Fig. 1B). The percentage of active to the total renin found after cryoactivation ranged from 22 to 99% and averaged 61 + 2.6%, which is significantly 0, < 0.001) higher than the % active renin found with the acid activation procedure. 2. Correlation studies Active plasma renin
concentration
correlated
OF THE NORMAL
VOLUNTEERS
significantly
with total
and
TABLE I BIOCHEMICAL APRA (ng . ml-’ * h-‘) TPRA (ng . II+’ . h-1) IPRA (ng . mlel . h-l) PAI (pg. ml-9 PAC (ng%)
PARAMETERS
0.96 i 0.13 1.63 ? 0.19 0.67 t 0.14 49.8 f 5.2
APRC (ng . ml-’ . h-‘) TPRC (ng . ml-‘. h-‘) IPRC (ng . I@‘. h-l) PA11
ON AN UNRESTRICTED 11.2 i 2.3 50.5 f 4.7 33.3 * 2.9 27.9 r 3.6
(pg. ml-9 6.0 + 0.8
n = 20. Mean t S.E. is given.
ACE (U/ml)
38.3 f 2.1
DIET
ACtD
ACTIVATION
PROCEDURE
n = 20 0*x
4% * SEM p<0.001
compared
APRC
to APRC
TPRC
CRYOACTIVATION
PROCEDURE
5-
l-
-
__--
_*--
-’
c-
_*-
_*
-0
/=--
_ -.-
n = 20
c
$ P*SEM
.-. *.-’
* 10 p
I
0.1
I
I
APRA
Fig. 1. (A) Active and total plasma activity in normal subjects.
renin
to APRA
TPRA rain
concentration
in normal
subjects.
(B) Active
and total
plasma
231
80 CRYOACTIVATION
ACID
z $
ACTIVATION
20
Fig. 2. Proportion of active to total renin in normal subjects.
inactive plasma renin concentration and with total plasma renin activity in these normal subjects (Table IIA). Active plasma renin activity is only significantly related with total plasma renin activity and not with inactive plasma renin activity. However active plasma renin activity correlated significantly with plasma angiotensin I and II (Table IIB). All the other correlations in Table II were not significant (p > 0.10). Plasma angiotensin II correlated significantly with APRA (r = 0.543 ; p < O.OZ), TPRA (r = 0.597; p < O.Ol), TPRC (r = 0.439; p = 0.05) and PA1 (r = 0.445;p < 0.05). Masma angiotensin I was also significantly related to APRA (r = 0.783; p < O.OOl), TPRA (r = 0.642;~ < 0.01) and PA II (r = 0.445;~ < 0.05). 3. Renin concentration after sequential acid and cold treatment The active plasma renin concentration determined in 5 normal TABLE
II
CORRELATION
STUDIES IN THE NORMAL r
Equation
SUBJECTS (n = 20) P
A. Acid activation APRC =-2.62 + 0.40 = 4.30 + 0.40 = 8.87 + 5.01 = 10.71 + 7.06 = 11.73 + 0.21 = 1.10 + 0.19 = 19.77 + 0.38 = 13.61 + 0.09
TPRC IPRC TPRA APRA PA11 PAI PAC ACE
0.839 0.550 0.455 0.392 0.340 0.428 -9.142 0.088
O.lO >O.lO >0.05 >O.lO >O.lO
B. Cryoactivation APRA = 0.34 + 0.36 = 0.97 -0.02 = 0.54 + 0.01 = 0.57 + 0.02 = 0.71 + 0.01 = 0.44 + 0.02 = 0.01 + 0.02 = 1.20-0.04 = 0.24 + 0.02
TPRA IPRA TPRC APRC IPRC PA11 PAI PAC ACE
0.585 -0.031 0.307 0.392 0.182 0.543 0.783 0.275 0.310
O.lO >O.lO >O.lO >O.lO <0.02 O.lO >O.lO
subjects
on
232 TABLE
III
RENIN CONCENTRATION AFTER SEQUENTIAL ACID ACTIVATION AND CRYOACTIVATION PLASMA FROM 5 HEALTHY SUBJECTS ON AN UNRESTRICTED DIET ---After acid treatment ~ APRC (ng . ml-‘. h-l) % Active to total renin
APRC (ng . ml-*~ h-‘) % Active to total renin a 0.01
>p
12.66 -
12.66 -
t 3.45
i 3.45
35.9 36
+ 6.30 a
-___
After sequential acid and/or cold treatment 33.80 38
t 5.56 a
After cold treatment
After sequential cold and acid treatment
22.26 58
21.34 61
i 5.08
IN
---
2 4.20 _I_-
> 0.001.
an unrestricted diet averaged 12.66 t 3.45 ng * ml-’ * h-i and increased significantly after acid or cold treatment (Table III). The proportion of active to total renin after acid or cold treatment of the plasma was 36 and 58%. Cold treatment after acid activation and acid treatment after cryoactivation did not provoke a significant change in the assayed renin concentration. The % active renin averaged 38 and 61%. The 2%fold increase in the renin concentration after acid treatment was significantly higher than the 1.7-fold increase found after cold treatment of the plasma. Discussion Many assays of plasma renin involve .an -acidification step which is likely to activate inactive renin. In these assays two-thirds of the total renin in normal human plasma consists of an inactive, acidactivable form [ 31. In normal human plasma we found on average 34% of active to total renin. Accordingly to Leckie et al. [ll] the percentage of inactive renin varied from 36 to 71% of the total renin concentration in normal subjects on an unrestricted diet. Boyd [6] also reported that active renin represents about 50% of the total renin concentration and that inactive renin is present in all subjects. Dray et al. [20,21], however, described an inactive form of renin found only in the plasma of patients with Wilm’s tumor, in diabetic patients and in hypertensive patients with proteinuria. They found no inactive renin in normal subjects or in the plasma of hypertensive patients without proteinuria, whereas Lijnen et al. [22] found one-third of the total renin to be present as active renin and two-thirds as inactive renin both at rest and during exercise in hypertensive patients on placebo treatment. The discrepancy in the study of Dray [21] may be due to diuretics given prior to blood sampling to increase the renin concentration. The data of Boyd [6] clearly indicate that diuretics greatly reduce the proportion of inactive renin in the acute stage. Morris ]23] has provided evidence that acid activation in human amniotic fluid is primarily due to hydrolysis of inactive renin by endogenous pepsin sub-
233
sequent to autocatalytic activation of pepsinogen as the H’ concentration increase with maximal destruction of the peptide inhibitor of pepsin being achieved when the pH reaches 3.3 [ 241. An inactive form of renin, called prorenin activity by Sealey et al. [8] or IPRA in this study, can also be determined in plasma from the total renin activity after cryo-treatment minus the prior endogenous plasma renin activity (TPRA-IPRA). The % active renin (34%) found after acidification of the plasma was significantly lower (p < 0.001) than the % active renin after cryoactivation (61%). Shulkes et al. [lo] also found that acidification of plasma activates more than does cold storage. No further increase in renin concentration was observed after sequential cold and/or acid treatment (Table II), As a possible explanation for the activation of inactive renin in the cold, Osmond et al. [25] postulated that endogenous protease was involved in the process of cryoactivation, having been present in the plasma from the outset, and subsequently induced (de-inhibited) by cold exposure. The presence of inactive renin has implications for renin assay methodology. According to Leckie et al. [ 111 there exists a significant correlation (r = 0.86; p < 0.001) between active plasma renin concentration and angiotensin II, but no correlation between inactive plasma renin concentration and angiotensin II, which is confirmed in this study. This suggests that inactive renin does not produce angiotensin II in vivo. However, the correlation between plasma angiotensin II and total renin concentration or activity can be poor because of the presence of the large percentage of inactive renin which is measured by the method but which does not contribute to plasma angiotensin II. Under various conditions total renin concentration is not a good index of the level of circulating active renin. Proportions of active and total renin are not only variable in different clinical conditions [26,27] but also in normal subjects studied under different sodium regimens. Inactive renin is a larger fraction of total renin in plasma of salt-loaded healthy subjects (82%) than salt-depleted subjects (62%). Thus changes in sodium intake stimulated larger changes in active plasma renin and smaller changes in inactive renin [ 281. The presence of inactive renin in human plasma thus has some implications for the interpretation of results of renin assay. Acknowledgements We gratefully acknowledge the technical assistance of Miss L. Lommelen, Miss S. Taelemans, Mr. L. Cockx and Mr. J. Huysecom, and the secretarial help of Mrs. M. Cober-Stinissen. This work was supported by a grant from the National Fund for Medical Research, Belgium. References 1
Lumbers.
2
Morris,
3
Skinner, Gynecol.
E.R. B.J. S.L..
(1971)
and
Enzymologia
Lumbers,
Cran,
121,626-630
E.J..
E.R. Gibson,
40.321-326
(19’72) R.,
Biochim. Taylor,
R.,
Biophys.
Acta
Walters,
W.A.W.
289, and
385591 Catt,
K.J.
(1975)
Am.
J. Obstet.
234 4 Leckie, B.J. and McConnell, A. (1975) Circ. Res. 36, 513-519 5 Derckx, F.H.M., Wenting, G.J., Man in ‘t Veld, Ad., Van Goal. J.M.G.. 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28
Verhoeven, R.P. and Schale.
kamp, M.A.D.H. (1976) Laneet ii, 496499 Boyd, G.W. (1977) Lancet i, 215-218 Cooper, R.M., Murray, G.E. and Osmond. D.H. (1977) Circ. Res. 40, SuPPI. 1.171-179 Sea.ley,J.E., Moon, C., Laragh, J.H. and Alderman, M. (1976) Am. J. Med. 61,731-737 Seaiey, J.E., Moon,C.. Laragh, 3.H. and Atlas, S.A. (1977) Circ. Res. 40, SuPPi. 1.4145 Shulkes, A.A., Gibson. R.R. and Skinner, S.L. (1978) Clin. Sci. Mol. Med. 55.41-50 Leckie, B.J., McConnelI, A., Grant. J., Morton, J.. Tree. M. and Brown, J.J. (1977)
Circ. Res. 40,
Suppl. 1.4661 Liinen, P. and Amery, A. (1977) Arch. In?.. Pbysiol. Biochim. 85, 183-184 Vallotton, M.B. (1971) in Immunological Methods in Endocrinology (Levine. R. and Pfeifer, E.F.. eds.), pp. 94-100, Academic Press, New York Skinner, S.L. (1967) Circ. Res. 20, 391402 Lijnen, P., Amery, A. and Fagard, R. (1976) FEBS Lett. 61, 32-33 Lijnen, P., Amery, A. and Fagard, R. (1978) J. Lab. Clin. Med. 92,353-362 Lijnen, P.. Amery, A., Fagard, R. and Katz, F. (1978) Clin. Chim. Acta 88,403412 Lijnen, P., Amery, A., Fagard, R. and Corvol. P. (1978) Clin. Chim. Acta 84,305-314 Lijnen, P. and Amery, A. (1978) Arch. Int. Physiol. Biochim. 86,436~-437 Dray. R.P. and Luetscher, ,J.A. (1975) J. Clin. Endocrinol. Metab. 40.1085-1093 Dray, R.P., Luetscher, J.A. and Gonzales, C.M. (1975) J. Clin. Endocrinol. Metab. 40, 1078-1084 Linen, P., Amery, A., Fagard, R., Reybrouck, T., Moerman, E. and De Scbaepdryver. A. (1979) Br. J, Clin. Pharmaeoi. 7.175-181 Morris, B.J. (1978) J. Clin. Endocrinoi. Metab. 46, 153-157 Herriott, R.M. (1941) J. Gen. Physiol. 24,325 Osmond, U.H. and Loh, A.Y. (1978) Lancet i, 102 Derckx, F.H.M., Wenting, G.J., Man in ‘t Veld, Verhoeven, R.P. and Schalekamp, M.A.D.H. (1978) Clin. Sci. Mol. Med. 54, 529-538 bijnen, P., Amery, A., Fagard, R. and Reybrouek, T. (1978) Angiology 29,354-366 Hsueh, W.A., Luetseher, J.A., Carlson, E.J. and Grislis, G. (1978) Lancet i, 1281-1284
Clinica Chimica Acta, 95 (1979) 227-234 0 Elsevier/North -Holland Biomedical Press
CCA 1043
ACTIVE AND INACTIVE RENIN IN NORMAL COMPARISON BETWEEN ACID ACTIVATIdN
PAUL J. LIJNEN,
ANTOON
K. AMERY
and ROBERT
HUMAN PLASMA. AND CRYOACTIVATION
H. FAGARD
Laboratory of Hypertension, Department of Physiopathology, University of Leuven, B-3000 Leuven (Belgium) (Received
January
16th,
Faculty of Medicine,
1979)
Summary
Inactive renin in human plasma is converted to active renin in vitro by acid activation or by cryoactivation. Renin activity was measured at pH 5.5 and renin concentration at pH 7.4. The plasma renin activity before and after cryotreatment is termed active (APRA) and total (TPRA) plasma renin activity; the plasma renin concentration before and after acid treatment active (APRC) and total (TPRC) plasma renin concentration. In this study we demonstrated that in normal subjects the proportion of active to total renin after cryo-treatment averaged 61%, which was significantly (p < 0.001) higher than the mean percentage active renin of 34 found with the acid activation procedure. Plasma angiotensin II correlated significantly with APRA, TPRA, TPRC and plasma angiotensin I (PA I), but not with inactive renin, which suggests that inactive renin does not produce angiotensin II in vivo. Cold treatment after acid activation and acid treatment after cryoactivation did not provoke a significant change in the measured renin concentration. Our data support the view that acidification of the plasma activates more than does cryo-treatment, and that inactive renin does not contribute to plasma angiotensin II.
Introduction
Inactive forms of renin in human plasma can be converted in vitro into active renin by acidification of plasma [l-6], by trypsin treatment [7] or by cold treatment [ 8,9]. Optimal acid activation of plasma renin is obtained at pH 3.3-3.5 [ 10-121. Reprint requests should be addressed to: Secretary of the Hypertension and Cardiovascular Unit. University Hospital St. Raphael, Kapucijnenvoer 35. B-3000 Leuven, Belgium.
Rehabilitation
228
The greatest cryoactivation of inactive renin is obtained between pH 7 and 9. No activation has been found in the frozen state [8]. Exogenous trypsin activates inactive renin in plasma at normal pH and at 4, 23 or 37°C proving that neither low pH nor cold are essential [ 71. To study further the proportion of active to total renin, we have compared the acid activation and cryoactivation of plasma renin in normal subjects on an unrestricted diet. The different forms of active, inactive and total renin were related to simultaneously determined plasma levels of angiotensin I and II, aldosterone and angiotensin converting enzyme (kininase II) activity. Methods
and materials
1. Plasma renin activity Plasma renin activity (PRA) was measured by radioimmunoassay of the angiotensin I generated during a l-h incubation of the plasma samples with the endogenous renin substrate at pH 5.5 and at 37°C according to the method of Vallotton [ 131. The results of these determinations were expressed in ng angiotensin I generated per ml plasma and per h (ng * ml-’ * h-l). The in vitro cryoactivation of renin was investigated by measuring the plasma renin activity in plasma samples immediately after thawing and after storage of the plasma at 0°C and at pH 7.4 for 7 days. The value of PRA before and after the cold treatment of the plasma is referred as active (APRA) and total plasma renin activity (TPRA). The difference (TPRA - APRA) is taken as the concentration of inactive plasma renin activity (IPRA) which is called pro-renin activity by Sealey et al. [8]. The inter-assay variation of our PRA method is 6.5%. 2. Plasma renin concentration The total plasma renin concentration (TPRC) was measured as the rate of angiotensin I generated during a l-h incubation with renin substrate from a nephrectomized sheep at pH 7.4 under zero order kinetic conditions, according to the method of Skinner [ 141, adapted for radioimmunoassay [ 151. This assay consists first of a denaturation of endogenous substrate by dialysis against a 0.05 mol/l aminoacetic acid buffer pH 3.3 containing 5 mmol/l EDTA and 90 mmol/l NaCl and of an inactivation of the angiotensinases by heating at 32°C for 1 h. The active plasma renin concentration (APRC) was determined after dialysing the plasma against a 0.05 mol/l aminoacetic acid buffer pH 4.5 and after heating the dialysate at 32°C for 1 h to produce adequate inhibition of the angiotensinases. Incubation was performed with excess sheep renin substrate at pH 7.4 for 1 h. The difference (TPRC-APRC) will be presented as the ‘inactive’ plasma renin concentration (IPRC). The inter-assay variation of the PRC determinations was 10.8% [ 151. 3. Assay of plasma aldosterone, angiotensin and converting enzyme A radioimmunoassay method was used for the measurement of the plasma aldosterone concentration (PAC) and of the plasma angiotensin I and II concentration (PAI, PAII) as previously described by Lijnen et al. [16-181. Plasma
229
angiotensin converting enzyme (kininase II) activity (ACE) was measured spectrophotometrically as described by Lijnen and Amery [19]. The inter-assay variation is 5.7% for PAC [18], 7.6% for PAI [16], 6.4% for PA11 [ 171 and 7.3% for ACE [19]. 4. Blood sampling Blood was withdrawn from an antecubital vein in 20 normal subjects on an unrestricted diet in the sitting position between 2 and 3 p.m. Anticoagulants were EDTA with o-phenantroline for the determination of PAI, PA11 and PRA, EDTA for PRC and PAC and heparin for estimation of ACE. 5. Statistical analysis The statistical methods used were regression analysis and Student’s twotailed t-test for paired data. The dispersion of the data is given by standard error of the mean (S.E.M.). Results 1. Activation studies The normal values for active, inactive and total plasma renin activity and concentration, plasma angiotensin I and II, aldosterone and angiotensin converting enzyme are given in Table I for the subjects aged 22-44 years (mean: 31) on an unrestricted diet. The total plasma renin concentration was significantly @ < 0.001) higher than the active plasma renin concentration in all subjects (Fig. 1A). The proportion of active to total renin produced by the acid activation procedure ranged from 17 to 52% and averaged 34 2 2.6% (Fig. 2). The total plasma renin activity was also higher (JJ < 0.001) than the active plasma renin activity in these normal subjects (Fig. 1B). The percentage of active to the total renin found after cryoactivation ranged from 22 to 99% and averaged 61 + 2.6%, which is significantly 0, < 0.001) higher than the % active renin found with the acid activation procedure. 2. Correlation studies Active plasma renin
concentration
correlated
OF THE NORMAL
VOLUNTEERS
significantly
with total
and
TABLE I BIOCHEMICAL APRA (ng . ml-’ * h-‘) TPRA (ng . II+’ . h-1) IPRA (ng . mlel . h-l) PAI (pg. ml-9 PAC (ng%)
PARAMETERS
0.96 i 0.13 1.63 ? 0.19 0.67 t 0.14 49.8 f 5.2
APRC (ng . ml-’ . h-‘) TPRC (ng . ml-‘. h-‘) IPRC (ng . I@‘. h-l) PA11
ON AN UNRESTRICTED 11.2 i 2.3 50.5 f 4.7 33.3 * 2.9 27.9 r 3.6
(pg. ml-9 6.0 + 0.8
n = 20. Mean t S.E. is given.
ACE (U/ml)
38.3 f 2.1
DIET
ACtD
ACTIVATION
PROCEDURE
n = 20 0*x
4% * SEM p<0.001
compared
APRC
to APRC
TPRC
CRYOACTIVATION
PROCEDURE
5-
l-
-
__--
_*--
-’
c-
_*-
_*
-0
/=--
_ -.-
n = 20
c
$ P*SEM
.-. *.-’
* 10 p
I
0.1
I
I
APRA
Fig. 1. (A) Active and total plasma activity in normal subjects.
renin
to APRA
TPRA rain
concentration
in normal
subjects.
(B) Active
and total
plasma
231
80 CRYOACTIVATION
ACID
z $
ACTIVATION
20
Fig. 2. Proportion of active to total renin in normal subjects.
inactive plasma renin concentration and with total plasma renin activity in these normal subjects (Table IIA). Active plasma renin activity is only significantly related with total plasma renin activity and not with inactive plasma renin activity. However active plasma renin activity correlated significantly with plasma angiotensin I and II (Table IIB). All the other correlations in Table II were not significant (p > 0.10). Plasma angiotensin II correlated significantly with APRA (r = 0.543 ; p < O.OZ), TPRA (r = 0.597; p < O.Ol), TPRC (r = 0.439; p = 0.05) and PA1 (r = 0.445;p < 0.05). Masma angiotensin I was also significantly related to APRA (r = 0.783; p < O.OOl), TPRA (r = 0.642;~ < 0.01) and PA II (r = 0.445;~ < 0.05). 3. Renin concentration after sequential acid and cold treatment The active plasma renin concentration determined in 5 normal TABLE
II
CORRELATION
STUDIES IN THE NORMAL r
Equation
SUBJECTS (n = 20) P
A. Acid activation APRC =-2.62 + 0.40 = 4.30 + 0.40 = 8.87 + 5.01 = 10.71 + 7.06 = 11.73 + 0.21 = 1.10 + 0.19 = 19.77 + 0.38 = 13.61 + 0.09
TPRC IPRC TPRA APRA PA11 PAI PAC ACE
0.839 0.550 0.455 0.392 0.340 0.428 -9.142 0.088
B. Cryoactivation APRA = 0.34 + 0.36 = 0.97 -0.02 = 0.54 + 0.01 = 0.57 + 0.02 = 0.71 + 0.01 = 0.44 + 0.02 = 0.01 + 0.02 = 1.20-0.04 = 0.24 + 0.02
TPRA IPRA TPRC APRC IPRC PA11 PAI PAC ACE
0.585 -0.031 0.307 0.392 0.182 0.543 0.783 0.275 0.310
subjects
on
232 TABLE
III
RENIN CONCENTRATION AFTER SEQUENTIAL ACID ACTIVATION AND CRYOACTIVATION PLASMA FROM 5 HEALTHY SUBJECTS ON AN UNRESTRICTED DIET ---After acid treatment ~ APRC (ng . ml-‘. h-l) % Active to total renin
APRC (ng . ml-*~ h-‘) % Active to total renin a 0.01
>p
12.66 -
12.66 -
t 3.45
i 3.45
35.9 36
+ 6.30 a
-___
After sequential acid and/or cold treatment 33.80 38
t 5.56 a
After cold treatment
After sequential cold and acid treatment
22.26 58
21.34 61
i 5.08
IN
---
2 4.20 _I_-
> 0.001.
an unrestricted diet averaged 12.66 t 3.45 ng * ml-’ * h-i and increased significantly after acid or cold treatment (Table III). The proportion of active to total renin after acid or cold treatment of the plasma was 36 and 58%. Cold treatment after acid activation and acid treatment after cryoactivation did not provoke a significant change in the assayed renin concentration. The % active renin averaged 38 and 61%. The 2%fold increase in the renin concentration after acid treatment was significantly higher than the 1.7-fold increase found after cold treatment of the plasma. Discussion Many assays of plasma renin involve .an -acidification step which is likely to activate inactive renin. In these assays two-thirds of the total renin in normal human plasma consists of an inactive, acidactivable form [ 31. In normal human plasma we found on average 34% of active to total renin. Accordingly to Leckie et al. [ll] the percentage of inactive renin varied from 36 to 71% of the total renin concentration in normal subjects on an unrestricted diet. Boyd [6] also reported that active renin represents about 50% of the total renin concentration and that inactive renin is present in all subjects. Dray et al. [20,21], however, described an inactive form of renin found only in the plasma of patients with Wilm’s tumor, in diabetic patients and in hypertensive patients with proteinuria. They found no inactive renin in normal subjects or in the plasma of hypertensive patients without proteinuria, whereas Lijnen et al. [22] found one-third of the total renin to be present as active renin and two-thirds as inactive renin both at rest and during exercise in hypertensive patients on placebo treatment. The discrepancy in the study of Dray [21] may be due to diuretics given prior to blood sampling to increase the renin concentration. The data of Boyd [6] clearly indicate that diuretics greatly reduce the proportion of inactive renin in the acute stage. Morris ]23] has provided evidence that acid activation in human amniotic fluid is primarily due to hydrolysis of inactive renin by endogenous pepsin sub-
233
sequent to autocatalytic activation of pepsinogen as the H’ concentration increase with maximal destruction of the peptide inhibitor of pepsin being achieved when the pH reaches 3.3 [ 241. An inactive form of renin, called prorenin activity by Sealey et al. [8] or IPRA in this study, can also be determined in plasma from the total renin activity after cryo-treatment minus the prior endogenous plasma renin activity (TPRA-IPRA). The % active renin (34%) found after acidification of the plasma was significantly lower (p < 0.001) than the % active renin after cryoactivation (61%). Shulkes et al. [lo] also found that acidification of plasma activates more than does cold storage. No further increase in renin concentration was observed after sequential cold and/or acid treatment (Table II), As a possible explanation for the activation of inactive renin in the cold, Osmond et al. [25] postulated that endogenous protease was involved in the process of cryoactivation, having been present in the plasma from the outset, and subsequently induced (de-inhibited) by cold exposure. The presence of inactive renin has implications for renin assay methodology. According to Leckie et al. [ 111 there exists a significant correlation (r = 0.86; p < 0.001) between active plasma renin concentration and angiotensin II, but no correlation between inactive plasma renin concentration and angiotensin II, which is confirmed in this study. This suggests that inactive renin does not produce angiotensin II in vivo. However, the correlation between plasma angiotensin II and total renin concentration or activity can be poor because of the presence of the large percentage of inactive renin which is measured by the method but which does not contribute to plasma angiotensin II. Under various conditions total renin concentration is not a good index of the level of circulating active renin. Proportions of active and total renin are not only variable in different clinical conditions [26,27] but also in normal subjects studied under different sodium regimens. Inactive renin is a larger fraction of total renin in plasma of salt-loaded healthy subjects (82%) than salt-depleted subjects (62%). Thus changes in sodium intake stimulated larger changes in active plasma renin and smaller changes in inactive renin [ 281. The presence of inactive renin in human plasma thus has some implications for the interpretation of results of renin assay. Acknowledgements We gratefully acknowledge the technical assistance of Miss L. Lommelen, Miss S. Taelemans, Mr. L. Cockx and Mr. J. Huysecom, and the secretarial help of Mrs. M. Cober-Stinissen. This work was supported by a grant from the National Fund for Medical Research, Belgium. References 1
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