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A procedure for the determination of a renin inhibitor in human plasma
Clinica Chimica Acta, 45 (1973) 67-71 0 Elsevier Scientific Publishing Company,
Amsterdam
- Printed in The Netherlands
67
CCA 5320
A PROCEDURE
FOR THE DETERMINATION
OF A RENIN INHIBITOR
IN HUMAN PLASMA
B. BAGGIO, S. FAVARO, A. BORSATTI
A. ANTONELLO,
S. TODESCO,
L. CAMPANACCI
AND
Istituto di Patologia Speciale Medica e Metodologia Clinica and Istituto di Chimica Biologica, Universith di Padova (Italy) (Received
July 19, 1972)
SUMMARY
An improved method for the determination of a phospholipid, which inhibits renin and is present in human plasma, is described. The phospholipid is extracted from plasma, added to a reconstituted renin-angiotensinogen system free from endogenous phospholipase(s) and then activated by the addition of an excess of exogenous phospholipase A. Angiotensin I produced in an assay medium containing renin-angiotensinogen and phospholipase A is compared with angiotensin I developed in an assay medium containing renin-angiotensinogen-phospholipid and phospholipase A, The inhibition of renin activity is expressed as percentage reduction in the production rate of angiotensin I, evaluated by radioimmunoassay of angiotensin I.
INTRODUCTION
A phospholipid isolated from dog plasma and kidney has been reported to inhibit the enzyme reninlgz. Even though this phospholipid had not been chemically identified, it was demonstrated that its action depended upon its previous transformation into a lysophospholipid. In fact, when this phospholipid was incubated with dog renin and its substrate (angiotensinogen), angiotensin production was inhibited and most of the phospholipid recovered at the end of the incubation appeared as a lysophospholipid2. Hence it seems that the phospholipase(s) responsible for this conversion are present as a contaminant either in renin or in angiotensinogen preparations. In the present paper, an improved procedure for the determination of the phospholipid inhibitor present in human plasma is described. This procedure is based on a radioimmunological assay of the angiotensin produced in the reconstituted “renin-angiotensinogen” system. Chemical identification of the phospholipid has still not been achieved, mostly because a very limited amount of it is available, but it has been confirmed that its inhibitory activity is dependent on a previous conversion into a lysophospholipid.
68
BAGGIO
et d.
MATERIALS
Angiotensinogen. Angiotensinogen was prepared from human plasma according to Haas et aL3. This preparation contained 42 mg protein/ml. In order to remove any residual lipid material, angiotensinogen was extracted three times with ether. Renin. Renin was prepared from human kidney according to the procedure A described by Haas et aL3. This preparation contained 7.20 mg protein/ml and 2.13 U.G.*/ml. Radioactive compounds. W-labelled [Ile6]angiotensin I, [Ilejlangiotensin I standard and anti-angiotensin specific antiserum were supplied by Sorin Research Center, Saluggia (Vercelli, Italy). Angiotensinase assay. The presence of angiotensinases in angiotensinogen, renin and phospholipase A has been controlled in two ways. I ml angiotensinogen, 0.1 ml renin and IO ,ul of phospholipase A were incubated for up to 8 h with 2.5 ,ug synthetic angiotensin II (Hypertensin, Ciba). The residual pressor activity evaluated in nephrectomized, pentolinium-treated rats ranged from g7-102%. The same amount of angiotensinogen, renin and phospholipase A were also incubated for up to 8 h with 4 and 8 ng of angiotensin I. Using the radioimmunoassay of Haber et aL5, the mean recovery of angiotensin I was 97%. Since at the concentrations we have employed there is no cross-reactivity between angiotensin I and II with specific antibody against angiotensin I, this control showed that our preparations had no convertingenzyme-like activity. EXPERIMENTAL
Blood sampling. 25 ml blood were collected from normal subjects in a test tube containing 0.3 ml 10% EDTA in water and immediately placed in ice. The plasma obtained after centrifugation at 5000 rev/min at 4’ for 15 min was stored at -20~. Renin inhibitor. 5 ml of plasma were extracted with chloroform-methanol (2 : I, v/v) and successively chromatographed through a silicic acid column as described by Ostovskj et aL4. The fraction containing the inhibitor in chloroformmethanol (4: I, v/v), assayed as described later, was concentrated under reduced pressure, resuspended in I ml chloroform-methanol (4: I, v/v) and diied under nitrogen. The dry weight of the product ranged from I to 2 mg. This product (containing the phospholipid inhibitor) was then resuspended in 0.5 ml of ethylene glycol and indirectly assayed in a medium containing 0.1 mM phosphate buffer, pH 6.9, 174 mg angiotensinogen, IO ,ug phospholipase A (n. 15057, Boehringer, Mannheim) and 0.360 mg renin in a total volume of 5 ml. Angiotensin I produced after 4 h of incubation at 37” was evaluated according to the radioimmunoassay described by Haber et a1.6, simplified as follows: 0.95 ml 0.10 M Tris-acetate buffer, pH 7.4, containing 2.4 mg lysozyme/ml, 50 ~1 [lz51]angiotensin I and 50 ,ul angiotensin antiserum were successively added to 5, IO, and 25 ,ul of the incubation mixture containing the produced angiotensin I. After 24 h incubation at 4”, 0.5 ml of a suspension of activated charcoal was added to each sample. After mixing, the tubes were centrifuged at 5000 rev/min for IO min and the radioactivity of the supernatant measured using a liquid scintillation counter. * Goldblatt
units.
RENIN INHIBITOR IN HUMAN PLASMA
69
The amount of radioactivity present in the sample was obtained by means of a calibration curve set up by plotting the net counts against the corresponding concentration of standard angiotensin I. The use of different concentrations of the incubation mixture was necessary in order to avoid off-scale values, when using a concentration of angiotensin I standard ranging from 0.05 to 0.8 ng in the calibration curve. The concentration of angiotensin I produced was expressed as percent of a blank containing the same quantity of angiotensinogen, renin and phospholipase A as in the sample, but not the phospholipid renin preinhibitor. The addition of 5 and IO ng of angiotensin I to the renin-angiotensinogen-phospholipase mixture before incubation gave recoveries ranging from 94% to IOZ~/~. RESULTS AND DISCUSSION
The results obtained with the described procedure are summarized in Fig. I. It can be observed that the kinetics of the renin-angiotensinogen reaction remain linear up to 8 h. When only phospholipid is added to the renin-angiotensinogen system, no difference in angiotensin I production is observed. On the contrary, addi-
-0
2
4 TIME
6
8
(hours)
Fig. r. The amount of angiotensin I produced and determined as described in as a function of time. 0, the system contains renin and angiotensinogen. The obtained if phospholipid preinhibitor is present. 0, the system contains renin, and IO pg of phospholipase A. n, the system contains renin, angiotensinogen, and phospholipid preinhibitor.
the text is given same results are angiotensinogen phospholipase A
tion of phospholipase A to the phospholipid-renin-angiotensinogen system produces a significant fall in the rate of angiotensin production. The addition of different amounts of phospholipase A (up to 20 ,ug) showed that the degree of maximal inactivation was not modified by the phospholipase concentration. In other words, an amount of 5 pugcaused the same inactivation as 20 pg. This allowed the use of an excess of enzyme (IO ,ug) in the routine assay. Addition of phospholipase alone (up to 20 pg) to the renin-angiotensinogen mixture caused a fall in the rate of angiotensin I production not greater than 10%. However, errors due to the small inhibitory effect of phospholipase were avoided by using a mixture containing the same amount of phospholipase as a control. The results obtained in IO normal subjects are reported in Table I. In agreement with the data showing the presence of a phospholipid inhibiting
BAGGIO et ai.
7o TABLE
I
ACTIVITY
OF HUMAN
PLASMA
PHOSPHOLIPID
INHIBITOR
ON ANGIOTENSIN
1 PRODUCTION
The inhibition of renin activity is expressed as percentage reduction in the production rate of angiotensin I, after addition of the inhibitor extracted from 5 ml of plasma, as described in the text. o/0 of inactivation
Subjects I 2
50.9 61.6
3 4 2
41.9 44.8 38.6 45.3 55.1 49.7 52.4 14.6
ZG 9 IO
Average Standard deviation
+
48.49 6.81
the enzyme renin, we confirm the presence of such a phospholipid in normal human plasma. The inhibition of renin could be achieved only after conversion of the inactive phospholipid (preinhibitor) into the active lysophospholipid by endogenous or exogenous specific phospholipase(s). In fact, in our assay, addition of the preinhibitor to the renin-angiotensinogen system did not decrease the angiotensin I production rate (see Fig. I). On the contrary, when both phospholipid and phospholipase were employed together, angiotensin I production rate was strongly inhibited (see Fig. I). Our results were obtained without any contamination by phospholipase and therefore the method seems more specific and accurate than the one used by Smeby et a1.2, who utilized an assay system contaminated by endogenous phospholipase. Furthermore, our test, through the addition of an excess of exogenous phospholipase, A, ensures complete activation of the preinhibitor, which is a very important factor in the reliability of the assay. The addition of phospholipase A alone to the renin-angiotensinogen system causes a small fall in angiotensin I production rate, thus indicating that our preparations contain traces of phospholipid. This can be understood by considering that renin is extracted from human kidney and angiotensinogen from plasma which are both known to be rich in phospholipid inhibitore. However, errors due to this small amount of preinhibitor in the assay system can be avoided by employing as a control a mixture containing the same concentration of phospholipase A which is present in the sample. The radioimmunological method described here presents some advantages compared to the chemical and biological assays in use. In fact, the enzymatic reaction occurs in an extremely homogeneous system since renin, substrate and inhibitor are all human in origin. Moreover neither renin nor angiotensinogen preparations contain phospholipase(s) as contaminant(s). On the other hand, the concentration of preinhibitor which contaminates the assay system is very small and cannot affect the reliability of the procedure. In conclusion the method described in the present paper avoids the lack of specificity of the chemical assay and seems easier and more accurate than the biological tests.
RENIN INHIBITOR IN HUMAN PLASMA
71
Although the present procedure, as well as all currently used procedures, allows the determination of the preinhibitor concentration in plasma, the measurement of the true inhibitor requires a preliminary determination cjf the phospholipase(s) in the plasma. REFERENCES I 2 3 4 5
S. SEN, R. R. SMESY AND F. M. B~~~~~,Biochemist~y, 6 (1967) 1572. R. R. SMEBY, S. SEN AND F. M. BUMPUS, Ciw. Res., 21 (1967) 11-129. E. HAAS. H. GOLDBLATT, E. C. GIPSON AND L. LEWIS, Circ.Res., xg (1966) 739. D. OsTovsxY, S.SEPI,R. R. SMEBY AND F. M. BUMPUS,CbC. Res.,*I (1967) 497. E. HABER, T. KOERNER, L. B. PAGE, B. KLI~XAN AND A. PURNODE, J. C&z. Endocrino2., (1969) 1359. 6 Il. H. OSMOND, R. R. SMEBY AND F. M. BUMPUS, J. Lab. Clin. Med., 73 (1969) 795.
2~
Amsterdam
- Printed in The Netherlands
67
CCA 5320
A PROCEDURE
FOR THE DETERMINATION
OF A RENIN INHIBITOR
IN HUMAN PLASMA
B. BAGGIO, S. FAVARO, A. BORSATTI
A. ANTONELLO,
S. TODESCO,
L. CAMPANACCI
AND
Istituto di Patologia Speciale Medica e Metodologia Clinica and Istituto di Chimica Biologica, Universith di Padova (Italy) (Received
July 19, 1972)
SUMMARY
An improved method for the determination of a phospholipid, which inhibits renin and is present in human plasma, is described. The phospholipid is extracted from plasma, added to a reconstituted renin-angiotensinogen system free from endogenous phospholipase(s) and then activated by the addition of an excess of exogenous phospholipase A. Angiotensin I produced in an assay medium containing renin-angiotensinogen and phospholipase A is compared with angiotensin I developed in an assay medium containing renin-angiotensinogen-phospholipid and phospholipase A, The inhibition of renin activity is expressed as percentage reduction in the production rate of angiotensin I, evaluated by radioimmunoassay of angiotensin I.
INTRODUCTION
A phospholipid isolated from dog plasma and kidney has been reported to inhibit the enzyme reninlgz. Even though this phospholipid had not been chemically identified, it was demonstrated that its action depended upon its previous transformation into a lysophospholipid. In fact, when this phospholipid was incubated with dog renin and its substrate (angiotensinogen), angiotensin production was inhibited and most of the phospholipid recovered at the end of the incubation appeared as a lysophospholipid2. Hence it seems that the phospholipase(s) responsible for this conversion are present as a contaminant either in renin or in angiotensinogen preparations. In the present paper, an improved procedure for the determination of the phospholipid inhibitor present in human plasma is described. This procedure is based on a radioimmunological assay of the angiotensin produced in the reconstituted “renin-angiotensinogen” system. Chemical identification of the phospholipid has still not been achieved, mostly because a very limited amount of it is available, but it has been confirmed that its inhibitory activity is dependent on a previous conversion into a lysophospholipid.
68
BAGGIO
et d.
MATERIALS
Angiotensinogen. Angiotensinogen was prepared from human plasma according to Haas et aL3. This preparation contained 42 mg protein/ml. In order to remove any residual lipid material, angiotensinogen was extracted three times with ether. Renin. Renin was prepared from human kidney according to the procedure A described by Haas et aL3. This preparation contained 7.20 mg protein/ml and 2.13 U.G.*/ml. Radioactive compounds. W-labelled [Ile6]angiotensin I, [Ilejlangiotensin I standard and anti-angiotensin specific antiserum were supplied by Sorin Research Center, Saluggia (Vercelli, Italy). Angiotensinase assay. The presence of angiotensinases in angiotensinogen, renin and phospholipase A has been controlled in two ways. I ml angiotensinogen, 0.1 ml renin and IO ,ul of phospholipase A were incubated for up to 8 h with 2.5 ,ug synthetic angiotensin II (Hypertensin, Ciba). The residual pressor activity evaluated in nephrectomized, pentolinium-treated rats ranged from g7-102%. The same amount of angiotensinogen, renin and phospholipase A were also incubated for up to 8 h with 4 and 8 ng of angiotensin I. Using the radioimmunoassay of Haber et aL5, the mean recovery of angiotensin I was 97%. Since at the concentrations we have employed there is no cross-reactivity between angiotensin I and II with specific antibody against angiotensin I, this control showed that our preparations had no convertingenzyme-like activity. EXPERIMENTAL
Blood sampling. 25 ml blood were collected from normal subjects in a test tube containing 0.3 ml 10% EDTA in water and immediately placed in ice. The plasma obtained after centrifugation at 5000 rev/min at 4’ for 15 min was stored at -20~. Renin inhibitor. 5 ml of plasma were extracted with chloroform-methanol (2 : I, v/v) and successively chromatographed through a silicic acid column as described by Ostovskj et aL4. The fraction containing the inhibitor in chloroformmethanol (4: I, v/v), assayed as described later, was concentrated under reduced pressure, resuspended in I ml chloroform-methanol (4: I, v/v) and diied under nitrogen. The dry weight of the product ranged from I to 2 mg. This product (containing the phospholipid inhibitor) was then resuspended in 0.5 ml of ethylene glycol and indirectly assayed in a medium containing 0.1 mM phosphate buffer, pH 6.9, 174 mg angiotensinogen, IO ,ug phospholipase A (n. 15057, Boehringer, Mannheim) and 0.360 mg renin in a total volume of 5 ml. Angiotensin I produced after 4 h of incubation at 37” was evaluated according to the radioimmunoassay described by Haber et a1.6, simplified as follows: 0.95 ml 0.10 M Tris-acetate buffer, pH 7.4, containing 2.4 mg lysozyme/ml, 50 ~1 [lz51]angiotensin I and 50 ,ul angiotensin antiserum were successively added to 5, IO, and 25 ,ul of the incubation mixture containing the produced angiotensin I. After 24 h incubation at 4”, 0.5 ml of a suspension of activated charcoal was added to each sample. After mixing, the tubes were centrifuged at 5000 rev/min for IO min and the radioactivity of the supernatant measured using a liquid scintillation counter. * Goldblatt
units.
RENIN INHIBITOR IN HUMAN PLASMA
69
The amount of radioactivity present in the sample was obtained by means of a calibration curve set up by plotting the net counts against the corresponding concentration of standard angiotensin I. The use of different concentrations of the incubation mixture was necessary in order to avoid off-scale values, when using a concentration of angiotensin I standard ranging from 0.05 to 0.8 ng in the calibration curve. The concentration of angiotensin I produced was expressed as percent of a blank containing the same quantity of angiotensinogen, renin and phospholipase A as in the sample, but not the phospholipid renin preinhibitor. The addition of 5 and IO ng of angiotensin I to the renin-angiotensinogen-phospholipase mixture before incubation gave recoveries ranging from 94% to IOZ~/~. RESULTS AND DISCUSSION
The results obtained with the described procedure are summarized in Fig. I. It can be observed that the kinetics of the renin-angiotensinogen reaction remain linear up to 8 h. When only phospholipid is added to the renin-angiotensinogen system, no difference in angiotensin I production is observed. On the contrary, addi-
-0
2
4 TIME
6
8
(hours)
Fig. r. The amount of angiotensin I produced and determined as described in as a function of time. 0, the system contains renin and angiotensinogen. The obtained if phospholipid preinhibitor is present. 0, the system contains renin, and IO pg of phospholipase A. n, the system contains renin, angiotensinogen, and phospholipid preinhibitor.
the text is given same results are angiotensinogen phospholipase A
tion of phospholipase A to the phospholipid-renin-angiotensinogen system produces a significant fall in the rate of angiotensin production. The addition of different amounts of phospholipase A (up to 20 ,ug) showed that the degree of maximal inactivation was not modified by the phospholipase concentration. In other words, an amount of 5 pugcaused the same inactivation as 20 pg. This allowed the use of an excess of enzyme (IO ,ug) in the routine assay. Addition of phospholipase alone (up to 20 pg) to the renin-angiotensinogen mixture caused a fall in the rate of angiotensin I production not greater than 10%. However, errors due to the small inhibitory effect of phospholipase were avoided by using a mixture containing the same amount of phospholipase as a control. The results obtained in IO normal subjects are reported in Table I. In agreement with the data showing the presence of a phospholipid inhibiting
BAGGIO et ai.
7o TABLE
I
ACTIVITY
OF HUMAN
PLASMA
PHOSPHOLIPID
INHIBITOR
ON ANGIOTENSIN
1 PRODUCTION
The inhibition of renin activity is expressed as percentage reduction in the production rate of angiotensin I, after addition of the inhibitor extracted from 5 ml of plasma, as described in the text. o/0 of inactivation
Subjects I 2
50.9 61.6
3 4 2
41.9 44.8 38.6 45.3 55.1 49.7 52.4 14.6
ZG 9 IO
Average Standard deviation
+
48.49 6.81
the enzyme renin, we confirm the presence of such a phospholipid in normal human plasma. The inhibition of renin could be achieved only after conversion of the inactive phospholipid (preinhibitor) into the active lysophospholipid by endogenous or exogenous specific phospholipase(s). In fact, in our assay, addition of the preinhibitor to the renin-angiotensinogen system did not decrease the angiotensin I production rate (see Fig. I). On the contrary, when both phospholipid and phospholipase were employed together, angiotensin I production rate was strongly inhibited (see Fig. I). Our results were obtained without any contamination by phospholipase and therefore the method seems more specific and accurate than the one used by Smeby et a1.2, who utilized an assay system contaminated by endogenous phospholipase. Furthermore, our test, through the addition of an excess of exogenous phospholipase, A, ensures complete activation of the preinhibitor, which is a very important factor in the reliability of the assay. The addition of phospholipase A alone to the renin-angiotensinogen system causes a small fall in angiotensin I production rate, thus indicating that our preparations contain traces of phospholipid. This can be understood by considering that renin is extracted from human kidney and angiotensinogen from plasma which are both known to be rich in phospholipid inhibitore. However, errors due to this small amount of preinhibitor in the assay system can be avoided by employing as a control a mixture containing the same concentration of phospholipase A which is present in the sample. The radioimmunological method described here presents some advantages compared to the chemical and biological assays in use. In fact, the enzymatic reaction occurs in an extremely homogeneous system since renin, substrate and inhibitor are all human in origin. Moreover neither renin nor angiotensinogen preparations contain phospholipase(s) as contaminant(s). On the other hand, the concentration of preinhibitor which contaminates the assay system is very small and cannot affect the reliability of the procedure. In conclusion the method described in the present paper avoids the lack of specificity of the chemical assay and seems easier and more accurate than the biological tests.
RENIN INHIBITOR IN HUMAN PLASMA
71
Although the present procedure, as well as all currently used procedures, allows the determination of the preinhibitor concentration in plasma, the measurement of the true inhibitor requires a preliminary determination cjf the phospholipase(s) in the plasma. REFERENCES I 2 3 4 5
S. SEN, R. R. SMESY AND F. M. B~~~~~,Biochemist~y, 6 (1967) 1572. R. R. SMEBY, S. SEN AND F. M. BUMPUS, Ciw. Res., 21 (1967) 11-129. E. HAAS. H. GOLDBLATT, E. C. GIPSON AND L. LEWIS, Circ.Res., xg (1966) 739. D. OsTovsxY, S.SEPI,R. R. SMEBY AND F. M. BUMPUS,CbC. Res.,*I (1967) 497. E. HABER, T. KOERNER, L. B. PAGE, B. KLI~XAN AND A. PURNODE, J. C&z. Endocrino2., (1969) 1359. 6 Il. H. OSMOND, R. R. SMEBY AND F. M. BUMPUS, J. Lab. Clin. Med., 73 (1969) 795.
2~