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The influence of plasma lipoproteins on the renin reaction in normal human plasma
BIOCHEMICAL
MEDICINE
21, 347-351 (1979)
The Influence of Plasma Lipoproteins on the Renin Reaction in Normal Human Plasma’ PETER Sepulveda
EGGENA, Hypertension California
JACK D. BARRETT,
AND MOHINDER
Division, Veterans Administration 91343 and Deparrment of Medicine, Los Angeles, California 90024
P. SAMBHI
Medical Center, University of California,
Received February 2. 1979
The presence of modifiers of the renin reaction in plasma has been demonstrated by several investigators. We as well as others have reported on modifiers in plasma which increase the rate of angiotensin I production in plasma (l-3). Lipid inhibitors of the renin reaction have also been reported (4-6). Several studies have also indicated that prostaglandins are potential inhibitors of the renin reaction (7,8). Recently, Kotchen et al. (9) demonstrated that plasma from normotensive human subjects contained an acetone-soluble neutral lipid which inhibited the renin reaction in vifro. In uremic patients with hypertension, the presence of this neutral lipid was less evident, leading these investigators to suggest that the absence of this neutral lipid inhibitor may in part contribute to the hypertension of these uremic patients. The present study was undertaken to establish whether the majority of the plasma lipids which are present in the form of lipoproteins in normal human plasma could alter the renin reaction.
METHODS Plasma was obtained with informed consent from healthy normotensive male subjects ranging in age from 25 to 60 years and pooled. Plasma lipoproteins were removed by flotation (10). The plasma was adjusted to a density of 1.195 g/ml by the addition of solid NaBr and centrifuged at 4°C for 45 hr at 360,OOOg. The lipoprotein fraction (25% of the total volume) containing the HDL, LDL, VLDL, and chylomicron moieties was removed by aspiration. The lipoprotein fraction and the infranate (delipoproteinated plasma) were dialyzed against 0.15 M NaCl at pH 7.4 to ’ This study was supported by Veterans Administration
Grant 7697.
347 000&2944/79/030347-05$02.00/O Copyri&t @ 1979 by Academic FTess, Inc. AU rights of reproduction in any form reserved.
348
EGGENA,
BARRETT.
AND SAMBHI
remove NaBr. As a control, an aliquot of the original plasma was stored with NaBr for 45 hr at 4°C and dialyzed as above. Plasma renin concentrations (PRC) were determined at pH 7.4 using the added renin method of Haas et al. (11). Added renin concentrations were chosen such that over the whole range of added renins linearity in angiotensin I generation was maintained; no more than 5% of total available substrate was consumed in any sample to assure initial rates. Exogenous renin substrate was not added to these samples since this method does not require zero-order kinetics with regard to renin substrate. The angiotensin I generation rate was determined at five increments of added renin (2.73 x lo-” to 1.365 x IO-” III/ml). Renin reactivity was defined as the slope of the linear, least-squares regression line of angiotensin I generation rate (ng/ml/hr) per 1 x lO-4 JU of added renin. Generated angiotensin I was determined by radioimmunoassay as previously described (10). The Michaelis-Menten constants (K,n and V,) were determined from Lineweaver-Burk plots by linear least-squares regression analysis of [angiotensin I production]-’ as a function of [renin substrate concentration]-‘. Triplicate determinations at five renin substrate concentrations were employed. Renin substrate was quantitated as previously described (12). In both the renin reactivity and kinetic measurements, less than 5% of total renin substrate was consumed to assure initial rate determination (13). RESULTS
The renin parameters of whole plasma and plasma devoid and enriched with lipoproteins are shown in Table 1. With respect to their controls (the addition of 0.15 M NaCl in place of the isolated lipoprotein fraction) no TABLE THE INFLUENCE
Whole plasma Delipoproteinated
OF NORMAL
plasma
PLASMA
1
LIPOPROTEINS
ON RENIN
REACTIVITY
(n =
12)
Plasma renin activity (ng AI/ml/hr)
Plasma renin concentration (X lo-4 IU)
Renin reactivity (ng AI/ml/hr/ IU x10-4)
(ng AI/ml)
10.4 +. 0.3 11.2 * 0.3
5.2 k 0.2 5.7 t 0.5
2.36 f 0.43 2.64 +- 0.40
2244 2 54 2045 -’ 56
Renin substrate
3 parts plasma: 1 part lipoprotein 3: 1 controla
(v/v)
8.8 + 0.2 8.9 ” 0.3
4.4 + 0.2 4.9 -t 0.3
2.31 -’ 0.23 2.15 + 0.28
1311 t 28 1374 k 26
1 part plasma: 1 part lipoprotein 1:1 control
(v/v)
5.6 _’ 0.2 6.4 k 0.2
3.8 + 0.5 3.0 ” 0.8
1.88 * 0.17 1.85 r 0.18
%1 ? 56 967 r 37
” Addition
of one part 0.15 M NaCl in place of the lipoprotein
fraction.
HUMAN
PLASMA
LIPOPROTEINS
AND
RENIN
REACTION
349
statistically significant influence of either the presence or absence of lipoproteins was evident on the renin reactivity (slope of the linear leastsquares regression line of angiotensin generation rate as a function of added renin), plasma renin concentration, or plasma renin activity. Neither renin nor renin substrate was detected in the lipoprotein fraction. The parallel decrease in the PRA, PRC, and renin substrate concentration of plasma with either an addition of the lipoprotein fraction or NaCl is due to sample dilution. The decrease in renin reactivity is also due to the dilution of renin substrate, since renin substrate is the rate-limiting parameter in these samples; zero-order kinetics with respect to renin substrate would require substrate concentrations at least five times greater than K, (14). As is shown in Table 2, the plasma samples had K, values between 886 and 2470 ng angiotensin I equivalents per milliliter with renin substrate values in the same range (Table l), indicating therefore firstorder kinetics with respect to renin substrate. In Table 2 the Michaelis-Menten constants are shown as a function of added lipoproteins. As with the renin reactivity the lipoprotein fraction from normal human plasma failed to inhibit the renin reaction at normal physiological levels. Only at a ratio of 1:2, which represents a large excess of lipoprotein, was a marginally significant inhibition of the renin reaction evident: a 1.85fold decrease in K, and 2.1-fold decrease in V,. DISCUSSION
The lack of change of PRA, PRC, and renin reactivity following addition or the removal of native lipoproteins to plasma indicates that the TABLE
2
THE INFLUENCE OF POOLED NORMAL PLASMA LIPOPROTEINS ON THE MICHAELIS-MENTEN CONSTANTSOF NORMALPLASMA K&g AI/ml) 2464 2 105
Plasma
V,(ng
AI/mVmin) 6.3 + 0.4
r” 0.9993
3 parts plasma: 1 part lipoprotein 3: I control*
(“v)
2429 + 116 2470 r 76
6.9 + 0.4 6.7 f 0.1
0.9997 0.9999
1 part plasma: 1 part lipoprotein 1: 1 control
(“/v)
2075 k 360 2023 + 82
5.1 2 0.8 5.4 k 0.9
0.9985 0.9975
886 2 229e 1926 2 540
2.8 f 0.Y 4.9 ‘- 1.3
0.9884 0.9960
1 part plasma: 2 parts lipoprotein 1:2 control
(“lv)
a Correlation coefficient b Addition of one part c P < 0.10.
of Lineweaver-Burk 0.15 M NaCI in place
plot. of the lipoprotein
fraction.
350
EGGENA.
BARRETT,
AND
SAMBHI
lipoprotein fraction isolated from the plasma of normotensive subjects does not inhibit the renin reaction when present in normal physiological concentrations. The disparity of these results with those of Kotchen et al. (9) demonstrating the presence of an acetone-soluble lipid renin inhibitor at normal physiological concentrations in the plasma of normotensive subjects may be due to (a) the nonphysiological method of studying emulsified lipids rather than those bound to their native lipoprotein moiety which is soluble in plasma or (b) the addition of free fatty acids liberated from albumin by acetone. The marginally significant inhibition of the plasma renin reaction seen at extremely elevated lipoprotein concentrations, leading to a decrease in both K, and V,, is compatible with the hyperbolic mixed type of inhibition, if one assumes a single modifier (14). In certain pathological states of elevated plasma lipoproteins, therefore, plasma lipoproteins may partially suppress the renin reaction. At present however, it is not known whether the observed inhibition resides in a particular lipoprotein subspecies such as HDL, LDL, or VLDL or if the inhibition is due to the lipid or protein moiety of the lipoprotein fraction. Clarification will have to await studies with the individual lipoprotein classes and their respective apoproteins. In conclusion, this study has demonstrated that under physiological conditions, native plasma lipids of normal human subjects, when bound to their respective apoproteins, do not appear to inhibit the renin reaction at normal physiological concentrations. SUMMARY Several studies have indicated that lipids isolated from plasma and kidney may be potential inhibitors of the renin reaction and that the increased renin reactivity observed in hypertensive subjects may be due to the lack of a plasma lipid found in normotensive subjects. In the present study lipoproteins were removed from normal human plasma by using density gradient centrifugation (d = 1.195 g/ml, 360,OOOg x 45 hr). This experimental design more closely resembles physiological conditions. Following dialysis against 0.15 M NaCl, the kinetic parameters of plasma renin in delipidated plasma and fresh plasma which was enriched with lipoproteins were compared. The renin reactivity and Michaelis-Menten kinetics of normal human plasma were not altered by the removal of lipoproteins (K, = 2464 ng AI/ml, V,,, = 6.3 ng AI/ml/min). Only at abnormally high concentrations of added lipoproteins was inhibition of the renin reaction evident (K, = 886 ng AI/ml, V,,, = 2.8 ng AI/ml/min). These results indicate that lipids of normal human plasma bound to apolipoproteins at normal physiological concentrations do not have an inhibitory effect on the renin reaction.
f : ’ + : ; E
HUMAN
PLASMA
LIPOPROTEINS
AND RENIN REACTION
351
REFERENCES 1. Sambhi, M. P., and Wiedeman, C. E., Renin activation in venous plasma from the involved kidney in the patient with renal hypertension. .I. Clin. Invest. 51, 22 (1972). 2. Sambhi, M. P., Eggena, P., Barrett, J. D., Tuck, M., Wiedeman, C. E., and Thananopavarn, C., A circulating renin activator in essential hypertension. Circ. Res. 36, 37, Suppl. I, 28 (1975). 3. McDonald, W. J., Cohen, E. L., Lucas, C. P., and Conn, J. W., Renin-renin substrate kinetic constants in the plasma of normal and estrogen-treated humans. J. C/in. Endocrinol. Metabol. 45, 1297 (1977). 4. Sen, S., Smeby, R. R., and Bumpus, F. M., Isolation of a phospholipid renin inhibitor from kidney. Biochemistry 6, 1572 (1%7). 5. Smeby, R. R., Sen, S., and Bumpus, F. M., A naturally occurring renin inhibitor. Circ. Res. 21, Suppl. II, 129 (1%7). 6. Osmond, D. H., Renal weight and content of lipid and phospholipid renin preinhibitor in rats with renal hypertension. J. Lab. Clin. Med. So, 755 (1972). 7. Kotchen, T. A., and Miller, M. C.. Effects of prostaglandins on renin reactivity. Amer. J. Physiol. 226, 314 (1974). 8. Eggena, P., Barrett, J. D., Sambhi, M. P., and Wiedeman, C. E., Influence of prostaglandins A2 and Q on the kinetics of the renin reaction in the presence of normal and hypertensive plasma. Biochem. Med. 14, 290 (1976). 9. Kotchen, T. A., Talwalker, R. T., Miller, M. C., and Welch, W. J., Modification of renin reactivity by lipids extracted from normal, hypertensive and uremic plasma, J. Clin. Endocrinol. Metabol. 43, 9771 (1976). lo. DeLalla, 0. F., and Gofman, J. W., Ultracentrifugal analysis of serum lipoproteins. Methods Biochem. Anal. 1, 459 (1954). 11. Haas, E., Gould, A. B., and Goldblatt, H., Estimation of endogeneous renin in human blood. Lancer 1, 657 (1968). 12. Eggena, P., Barrett, J. D., Wiedeman, C. E., and Sambhi, M. P., The validity of comparing the measurements of angiotensin I generated in human plasma by radioimmunoassay and bioassay. J. Clin. Endocrinol. Metabol. 39, 865 (1974). 13. Webb, J. L., “Enzyme and Metabolic Inhibitors.” Academic Press, New York, 1963. 14. Segal, I. H., “Enzyme Kinetics.” Wiley, New York, 1975.
MEDICINE
21, 347-351 (1979)
The Influence of Plasma Lipoproteins on the Renin Reaction in Normal Human Plasma’ PETER Sepulveda
EGGENA, Hypertension California
JACK D. BARRETT,
AND MOHINDER
Division, Veterans Administration 91343 and Deparrment of Medicine, Los Angeles, California 90024
P. SAMBHI
Medical Center, University of California,
Received February 2. 1979
The presence of modifiers of the renin reaction in plasma has been demonstrated by several investigators. We as well as others have reported on modifiers in plasma which increase the rate of angiotensin I production in plasma (l-3). Lipid inhibitors of the renin reaction have also been reported (4-6). Several studies have also indicated that prostaglandins are potential inhibitors of the renin reaction (7,8). Recently, Kotchen et al. (9) demonstrated that plasma from normotensive human subjects contained an acetone-soluble neutral lipid which inhibited the renin reaction in vifro. In uremic patients with hypertension, the presence of this neutral lipid was less evident, leading these investigators to suggest that the absence of this neutral lipid inhibitor may in part contribute to the hypertension of these uremic patients. The present study was undertaken to establish whether the majority of the plasma lipids which are present in the form of lipoproteins in normal human plasma could alter the renin reaction.
METHODS Plasma was obtained with informed consent from healthy normotensive male subjects ranging in age from 25 to 60 years and pooled. Plasma lipoproteins were removed by flotation (10). The plasma was adjusted to a density of 1.195 g/ml by the addition of solid NaBr and centrifuged at 4°C for 45 hr at 360,OOOg. The lipoprotein fraction (25% of the total volume) containing the HDL, LDL, VLDL, and chylomicron moieties was removed by aspiration. The lipoprotein fraction and the infranate (delipoproteinated plasma) were dialyzed against 0.15 M NaCl at pH 7.4 to ’ This study was supported by Veterans Administration
Grant 7697.
347 000&2944/79/030347-05$02.00/O Copyri&t @ 1979 by Academic FTess, Inc. AU rights of reproduction in any form reserved.
348
EGGENA,
BARRETT.
AND SAMBHI
remove NaBr. As a control, an aliquot of the original plasma was stored with NaBr for 45 hr at 4°C and dialyzed as above. Plasma renin concentrations (PRC) were determined at pH 7.4 using the added renin method of Haas et al. (11). Added renin concentrations were chosen such that over the whole range of added renins linearity in angiotensin I generation was maintained; no more than 5% of total available substrate was consumed in any sample to assure initial rates. Exogenous renin substrate was not added to these samples since this method does not require zero-order kinetics with regard to renin substrate. The angiotensin I generation rate was determined at five increments of added renin (2.73 x lo-” to 1.365 x IO-” III/ml). Renin reactivity was defined as the slope of the linear, least-squares regression line of angiotensin I generation rate (ng/ml/hr) per 1 x lO-4 JU of added renin. Generated angiotensin I was determined by radioimmunoassay as previously described (10). The Michaelis-Menten constants (K,n and V,) were determined from Lineweaver-Burk plots by linear least-squares regression analysis of [angiotensin I production]-’ as a function of [renin substrate concentration]-‘. Triplicate determinations at five renin substrate concentrations were employed. Renin substrate was quantitated as previously described (12). In both the renin reactivity and kinetic measurements, less than 5% of total renin substrate was consumed to assure initial rate determination (13). RESULTS
The renin parameters of whole plasma and plasma devoid and enriched with lipoproteins are shown in Table 1. With respect to their controls (the addition of 0.15 M NaCl in place of the isolated lipoprotein fraction) no TABLE THE INFLUENCE
Whole plasma Delipoproteinated
OF NORMAL
plasma
PLASMA
1
LIPOPROTEINS
ON RENIN
REACTIVITY
(n =
12)
Plasma renin activity (ng AI/ml/hr)
Plasma renin concentration (X lo-4 IU)
Renin reactivity (ng AI/ml/hr/ IU x10-4)
(ng AI/ml)
10.4 +. 0.3 11.2 * 0.3
5.2 k 0.2 5.7 t 0.5
2.36 f 0.43 2.64 +- 0.40
2244 2 54 2045 -’ 56
Renin substrate
3 parts plasma: 1 part lipoprotein 3: 1 controla
(v/v)
8.8 + 0.2 8.9 ” 0.3
4.4 + 0.2 4.9 -t 0.3
2.31 -’ 0.23 2.15 + 0.28
1311 t 28 1374 k 26
1 part plasma: 1 part lipoprotein 1:1 control
(v/v)
5.6 _’ 0.2 6.4 k 0.2
3.8 + 0.5 3.0 ” 0.8
1.88 * 0.17 1.85 r 0.18
%1 ? 56 967 r 37
” Addition
of one part 0.15 M NaCl in place of the lipoprotein
fraction.
HUMAN
PLASMA
LIPOPROTEINS
AND
RENIN
REACTION
349
statistically significant influence of either the presence or absence of lipoproteins was evident on the renin reactivity (slope of the linear leastsquares regression line of angiotensin generation rate as a function of added renin), plasma renin concentration, or plasma renin activity. Neither renin nor renin substrate was detected in the lipoprotein fraction. The parallel decrease in the PRA, PRC, and renin substrate concentration of plasma with either an addition of the lipoprotein fraction or NaCl is due to sample dilution. The decrease in renin reactivity is also due to the dilution of renin substrate, since renin substrate is the rate-limiting parameter in these samples; zero-order kinetics with respect to renin substrate would require substrate concentrations at least five times greater than K, (14). As is shown in Table 2, the plasma samples had K, values between 886 and 2470 ng angiotensin I equivalents per milliliter with renin substrate values in the same range (Table l), indicating therefore firstorder kinetics with respect to renin substrate. In Table 2 the Michaelis-Menten constants are shown as a function of added lipoproteins. As with the renin reactivity the lipoprotein fraction from normal human plasma failed to inhibit the renin reaction at normal physiological levels. Only at a ratio of 1:2, which represents a large excess of lipoprotein, was a marginally significant inhibition of the renin reaction evident: a 1.85fold decrease in K, and 2.1-fold decrease in V,. DISCUSSION
The lack of change of PRA, PRC, and renin reactivity following addition or the removal of native lipoproteins to plasma indicates that the TABLE
2
THE INFLUENCE OF POOLED NORMAL PLASMA LIPOPROTEINS ON THE MICHAELIS-MENTEN CONSTANTSOF NORMALPLASMA K&g AI/ml) 2464 2 105
Plasma
V,(ng
AI/mVmin) 6.3 + 0.4
r” 0.9993
3 parts plasma: 1 part lipoprotein 3: I control*
(“v)
2429 + 116 2470 r 76
6.9 + 0.4 6.7 f 0.1
0.9997 0.9999
1 part plasma: 1 part lipoprotein 1: 1 control
(“/v)
2075 k 360 2023 + 82
5.1 2 0.8 5.4 k 0.9
0.9985 0.9975
886 2 229e 1926 2 540
2.8 f 0.Y 4.9 ‘- 1.3
0.9884 0.9960
1 part plasma: 2 parts lipoprotein 1:2 control
(“lv)
a Correlation coefficient b Addition of one part c P < 0.10.
of Lineweaver-Burk 0.15 M NaCI in place
plot. of the lipoprotein
fraction.
350
EGGENA.
BARRETT,
AND
SAMBHI
lipoprotein fraction isolated from the plasma of normotensive subjects does not inhibit the renin reaction when present in normal physiological concentrations. The disparity of these results with those of Kotchen et al. (9) demonstrating the presence of an acetone-soluble lipid renin inhibitor at normal physiological concentrations in the plasma of normotensive subjects may be due to (a) the nonphysiological method of studying emulsified lipids rather than those bound to their native lipoprotein moiety which is soluble in plasma or (b) the addition of free fatty acids liberated from albumin by acetone. The marginally significant inhibition of the plasma renin reaction seen at extremely elevated lipoprotein concentrations, leading to a decrease in both K, and V,, is compatible with the hyperbolic mixed type of inhibition, if one assumes a single modifier (14). In certain pathological states of elevated plasma lipoproteins, therefore, plasma lipoproteins may partially suppress the renin reaction. At present however, it is not known whether the observed inhibition resides in a particular lipoprotein subspecies such as HDL, LDL, or VLDL or if the inhibition is due to the lipid or protein moiety of the lipoprotein fraction. Clarification will have to await studies with the individual lipoprotein classes and their respective apoproteins. In conclusion, this study has demonstrated that under physiological conditions, native plasma lipids of normal human subjects, when bound to their respective apoproteins, do not appear to inhibit the renin reaction at normal physiological concentrations. SUMMARY Several studies have indicated that lipids isolated from plasma and kidney may be potential inhibitors of the renin reaction and that the increased renin reactivity observed in hypertensive subjects may be due to the lack of a plasma lipid found in normotensive subjects. In the present study lipoproteins were removed from normal human plasma by using density gradient centrifugation (d = 1.195 g/ml, 360,OOOg x 45 hr). This experimental design more closely resembles physiological conditions. Following dialysis against 0.15 M NaCl, the kinetic parameters of plasma renin in delipidated plasma and fresh plasma which was enriched with lipoproteins were compared. The renin reactivity and Michaelis-Menten kinetics of normal human plasma were not altered by the removal of lipoproteins (K, = 2464 ng AI/ml, V,,, = 6.3 ng AI/ml/min). Only at abnormally high concentrations of added lipoproteins was inhibition of the renin reaction evident (K, = 886 ng AI/ml, V,,, = 2.8 ng AI/ml/min). These results indicate that lipids of normal human plasma bound to apolipoproteins at normal physiological concentrations do not have an inhibitory effect on the renin reaction.
f : ’ + : ; E
HUMAN
PLASMA
LIPOPROTEINS
AND RENIN REACTION
351
REFERENCES 1. Sambhi, M. P., and Wiedeman, C. E., Renin activation in venous plasma from the involved kidney in the patient with renal hypertension. .I. Clin. Invest. 51, 22 (1972). 2. Sambhi, M. P., Eggena, P., Barrett, J. D., Tuck, M., Wiedeman, C. E., and Thananopavarn, C., A circulating renin activator in essential hypertension. Circ. Res. 36, 37, Suppl. I, 28 (1975). 3. McDonald, W. J., Cohen, E. L., Lucas, C. P., and Conn, J. W., Renin-renin substrate kinetic constants in the plasma of normal and estrogen-treated humans. J. C/in. Endocrinol. Metabol. 45, 1297 (1977). 4. Sen, S., Smeby, R. R., and Bumpus, F. M., Isolation of a phospholipid renin inhibitor from kidney. Biochemistry 6, 1572 (1%7). 5. Smeby, R. R., Sen, S., and Bumpus, F. M., A naturally occurring renin inhibitor. Circ. Res. 21, Suppl. II, 129 (1%7). 6. Osmond, D. H., Renal weight and content of lipid and phospholipid renin preinhibitor in rats with renal hypertension. J. Lab. Clin. Med. So, 755 (1972). 7. Kotchen, T. A., and Miller, M. C.. Effects of prostaglandins on renin reactivity. Amer. J. Physiol. 226, 314 (1974). 8. Eggena, P., Barrett, J. D., Sambhi, M. P., and Wiedeman, C. E., Influence of prostaglandins A2 and Q on the kinetics of the renin reaction in the presence of normal and hypertensive plasma. Biochem. Med. 14, 290 (1976). 9. Kotchen, T. A., Talwalker, R. T., Miller, M. C., and Welch, W. J., Modification of renin reactivity by lipids extracted from normal, hypertensive and uremic plasma, J. Clin. Endocrinol. Metabol. 43, 9771 (1976). lo. DeLalla, 0. F., and Gofman, J. W., Ultracentrifugal analysis of serum lipoproteins. Methods Biochem. Anal. 1, 459 (1954). 11. Haas, E., Gould, A. B., and Goldblatt, H., Estimation of endogeneous renin in human blood. Lancer 1, 657 (1968). 12. Eggena, P., Barrett, J. D., Wiedeman, C. E., and Sambhi, M. P., The validity of comparing the measurements of angiotensin I generated in human plasma by radioimmunoassay and bioassay. J. Clin. Endocrinol. Metabol. 39, 865 (1974). 13. Webb, J. L., “Enzyme and Metabolic Inhibitors.” Academic Press, New York, 1963. 14. Segal, I. H., “Enzyme Kinetics.” Wiley, New York, 1975.