217 median for each week of gestation from 16 to 20 weeks. Fig. 2 in the report from Glasgow’ suggests that an average cut-off level of 2-8 times the median was used over this period so at 16 weeks it was somewhat higher (say 3-0) and at 20 weeks it was lower (say 2.7). This should not, however, make a great difference to the comparison of the results of the two studies shown in the table. The results from Glasgow’ are therefore consistent with those of the U.K. Collaborative Study.2 The expectations from any particular screening programme are dependent on the cutoff level, and this in turn will affect both the number of amniocenteses and the number of neural-tube defects detected-with a higher cut-off level fewer women would be referred for amniocentesis, but the detection-rate would also be lower. For example, less than half the number of women with unaffected pregnancies would require amniocentesis at a cut-off level of 3.0 times the median compared to 2.5 times the median at 16 to 18 weeks of gestation. At the same time, the detection-rate for all spina bifida would be reduced by 13%. In our opinion therefore it is unwise to argue that an amniocentesis-rate of less than 1% among women with unaffected pregnancies necessarily represents a better screening policy than one associated with a somewhat higher one (say, 1.5-2%). The factors which might influence the selection of cut-off levels in antenatal screening are discussed in the report of the U.K. Collaborative
screening from more than one centre to obtain better estimates of the detection-rate, and monitor the stability of the proportion of false positives. The U.K. Collaborative Study has shown that results from different centres cannot be compared directly but only by expressing them in multiples of each laboratory’s normal median; this must always be a second best, and can never tell us to what extent differences in the proportion of normal births with high serum-A.F.p. are due to assay imprecision rather than inherent differences in the screened population. A necessary condition for a national screening programme should be that the results from participating laboratories can be compared directly in terms of the same
reference preparation. Also, no such screening programme should be planned unless it is run in conjunction with a quality-control scheme to assess the precision and accuracy of the assays done in the various centres. Medical Computing and Statistics Unit, Medical School, University of Edinburgh, Edinburgh EH8 9AG
GILLIAN RAAB
Study. I.C.R.F. Cancer Epidemiology and Clinical Trials Department of the Regius Professor of Medicine, Radcliffe Infirmary, Oxford OX2 6HE
Department of Human Genetics, Western General Hospital, Edinburgh EH4 2XU
NICHOLAS J. WALD HOWARD S. CUCKLE DAVID J. H. BROCK
Paediatric Research Unit,
Guy’s Hospital, London SE1 9RT
ENDOGENOUS ACTIVATOR OF
Unit,
PAUL E. POLANI
SIR,-Professor Ferguson-Smith and his colleagues describe how their
screening of 11 500 pregnancies for raised serdetected 93% of those births with neural-tube defects, with a false-positive rate of only 1-4%. However, if their results are to be used to predict the consequences of a national screening programme, we need to know to what extent an equivalent intervention level might produce similar results in other centres, and in particular we must make an assessment of the possible biases and the standard errors of these estimates of percentages. Their detection-rate of 93% is based on the detection of 27 anencephalics out of 27, and 13 spina bifidas out of 16. These percentages should be examined separately rather than together, as there were more cases of anencephaly than spina bifida in this study although the incidence of the two defects is known to be similar; this difference in incidence-rates must itself cast some doubt on the adequacy of the follow-up in the Glasgow study. The true detection-rate for spina bifida should have been quoted as 12 out of 16, because 1 case was missed at screening and only retrospectively recognised as above the intervention limit. Thus the sensibilities of the screening test for the two defects should be given as 100% (95% confidence limits 87-100%) for anencephaly and 75% (95% confidence limit 47-92%) for spina bifida. The width of the confidence intervals is a result of the small numbers on which these percentages are based. In contrast the false-positive rate of 1.6% is based on large numbers and thus well determined for
um-A.F.p.
the Glasgow population. However, this rate is likely to vary both over time, and at different centres. The intervention level of 2.8 times the median used in this study was supposed to estimate the 97th percentile for normal singleton pregnancies and hence should have produced a false-positive rate of at least 3%. These points emphasise the need to combine the results of
PLASMA-INACTIVE-RENIN
SiR,—The apparent concentration of renin in human plasma can be increased by dialysis to pH 3.0-3.3 followed by dialysis to pH 7.4.1-3 This increase is due to activation of an inactive form of the enzyme. It has been argued that the activating process cannot be important physiologically since plasma is not exposed to a pH of 3.0in vivo. However, inactive renin is also activated by trypsin at pH 7.4,4,5 and an endogenous protease can activate the inactive renin of amniotic fluid.5 The experiments described below show that acid-activation of inactive renin in human plasma is probably mediated by an endogenous protease. In the course of this work I learned that Dr M. A. D. H. Schalekamp and his group had obtained similar results (see accompanying letter). Plasma from normal males was pooled. Samples were assayed for renin before and after the following procedures: (a) Dialysis to pH 3.0 and back to pH 7.4 (b) Dialysis to pH 3.0 in the presence of protease inhibitors, followed by dialysis to pH 7.4. ’Trasylol’ (aprotinin, Bayer) and soybean trypsin inhibitor (S.B.T.I.) in pH 7.4 phosphate-saline buffer were added directly to 10 x the volume of plasma and to the pH 3.0 glycine/HCl dialysis buffer at the concentrations stated in the table, and the mixture was incubated for 3 min at 37°C before acid dialysis. N-ethylmaleimide and o-phenanthroline were included in the pH 3.0 glycine/HCl buffer.3 (c) Plasma was also treated as in (a) and then assayed for renin in the presence of trasylol and S.B.T.I.
The kallikrein inhibitor trasylol and S.B.T.I. partially inhibited the acid-induced increase in renin concentration (table). Increasing concentrations of inhibitor increased its effect. N-ethyimaleimide had some inhibitory effect but o-phenanthroline had no effect. Neither trasylol nor S.B.T.I. affected the reaction between renin and renin substrate. These results suggest that acid-activation of inactive renin in plasma is mediated by a serine protease. The increase in plasma-renin 1. 2.
Skinner, S. L., Lumbers, E. R., Symonds, E. M. Clin. Sci. 1972, 42, 479. Leckie, B. J., McConnell, A.J. Endocr. 1975, 65, 7p. 3. Leckie, B. J., McConnell, A., Grant, J., Morton, J. J., Tree, M., Brown, J. J. Circulation Res. 1977, 40, suppl. 1, p. 46. 4. Cooper, R. M., Osmond, D. H., Scaiff, K. D., Ross, L. J. Fedn Proc. 1974,
33, 584. 5. Leckie, B. J., McConnell, A., Jordan, J. Adv. exp. Med. Biol. 1977, 95, 249. Morris, B. J., Lumbers, E. R., Biochim biophys. Acta. 1972, 289, 385. Millar, J. A., Leckie, B., J., Semple, P. F., Morton, J. J., Sonkodi, S., Robertson, J. I. S. Circulation Res. (in the press). 8. Osmond, D. H., Loh, A. Y. Lancet, 1978, i, 102.
6. 7.
,
218 there is no proof for activation in vivo. This study was undertaken to explore the possibility that plasma contains an enzyme which activates renin under physiological conditions. now
Pooled 6. 7. 8. 9.
plasma from fifteen normal
males
was
used. Renin
was
acti-
Osmond, D. H., Ross, L. J., Scaiff, K. D. Can. J. Physiol. Pharmac. 1973, 51, 705. Sealey, J. E., Moon, C., Laragh, J. H., Alderman, M. Am. J. Med. 1976, 61, 731. Atlas, S. A., Laragh, J. H., Sealey, J. E., Moon, C. Lancet, 1977, ii, 785. Cooper, R. M., Murray, G. E., Osmond, D. H. Circulation Res. 1977, 40, suppl. no. 1, p. 171.
(A) fl-units/ml of renin activated by acidification and by acidification in the presence of protease inhibitors. (B) Renin concentration in pre-acidified plasma assayed with and without protease inhibitors present in the incubation mixture. Mean +
s.E.
of n experiments.
Results in the presence of inhibitors out inhibitors by t test.
of varying pH at step B renin which is activated.
Fig. I-Effect were
compared
to
the results with-
Results (mean +s.D., n=5) are expressed achieved by standard technique. 2,5
on
the concentration of
as a
percentage of maxi-
mum
concentration induced by cold can be prevented by trasylol8,9 and may involve the action of a similar protease. The inactive renin in human plasma is not activated by incubation at 37°C either at pH 7.4 or pH 4.0.3 Plasma contains serine protease inhibitors which probably inhibit the action of the renin-activating enzyme(s), unless the inhibition is destroyed by acid or cold.9 Such enzymes might, however, be active in plasma from people with inherited o:i-trypsin-inhibitor deficiency, resulting in an abnormally high proportion of active renin. However, the concentrations of active and inactive renin in such a patient were: active renin 20 p.U/ml, inactive renin 146 p.U/ml; percent inactive renin 17%. These compare with 9-52 jU/m! for active renin and 38-180 p.U/ml for inactive renin in plasma samples from sixty normal subjects. Whether activation of inactive renin occurs in plasma is still unknown, therefore. The activation may take place in the kidneylO or blood-vessel walls, and the presence of a renin-activating system in circulating plasma may be due to "escape" of activating enzyme from these sites. M.R.C. Blood Pressure Unit, Western Infirmary, Glasgow G11 6NT
B. LECKIE
SIR,-Normally more than half the renin in plasma is inactive. It is activated under such unphysiological conditions as low pHl-5 and low temperature6-8 and also by trypsin.9 Until 9. 10.
Sealey, J. E., Laragh, J. H. Kidney Int. 1977, 6, 495. Inagami, T., Murakami, K., Misono, K., Workman,
R. J., Cohen, S., Suk1977, 95, 225. 1. Skinner, S. L., Cran, E. J., Gibson, R., Taylor, R., Walters, W. A. W., Catt, K. J.Am.J. Obstet. Gynec. 1975, 121, 626. 2. Derkx, F. H. M., Wenting, G. J., Man in ’t Veld, A. J., Van Gool, J. M. G., Verhoeven, R. P., Schalekamp, M. A. D. H. Lancet, 1976, ii, 496. 3. Leckie, B. J., McConnell, A., Grant, J., Morton, J. J., Tree, M., Brown, J. J. Circulation Res. 1977, 40, suppl. no.1, p. 46. 4. Boyd, G. W. Lancet, 1977, i, 215. 5. Derkx, F. H. M., Wenting, G. J., Man in ’t Veld, A. J., Verhoeven, R. P., Schalekamp, M. A. D. H. Clin. Sci. mol. Med. 1978, 54, 529. eta, Y. Adv. exp. Med. Biol.
I
I
I
Fig. 2-Effect (mean +S.D.) of trasylol, 800 kallikrein-inactivating units/ml on concentration of renin which is activated.
219 as before:2,5 (A) dialysis to pH 3.3 for 24 h at pH 7.5 for 24 h at 4°C, (C) incubation, with sheep renin-substrate at pH 7.5 for 3 h at 37°C, and (D) radioimmunoassay of angiotensin i. This standard procedure was modified as follows. Plasma pH at step B was rapidly adjusted to values ranging from 3.55 to 7.5 with sodium hydroxide solution (1 mmol/ml), followed by dialysis against buffers of the same pH (i.e., glycine/HCl buffers for the pH range 3.5-5.0 and sodium-phosphate buffers for the pH range 5.0-7-5). After 24 h dialysis at 4°C pH was readjusted to 7.5 by sodium hydroxide, and finally steps C and D were done. The kallikrein-inhibitor, aprotinin (’Trasylol’, Bayer) was added to plasma and dialysis buffer at step A or B, at concentrations stated in figs. 2 and 3. It was also added to the renin/renin-substrate mixture at C or to
vated and measured
4"C, (B) dialysis
to
the angiotensin-i/antibody mixture at D.
Activation of inactive renin occurred at neutral pH (fig. 1), but prior acidification was necessary. Addition of trasylol to plasma and the dialysis buffer during acidification inhibited this activation (fig. 2). Inhibition was reversed by further dialysis at pH 3.3 with no trasylol in the buffer. Thus trasylol must have exerted its inhibitory effect during one of the later steps when pH is higher. This was confirmed when trasylol was added after acidification and rapid neutralisation (step B, see fig. 2). The inhibitory effect of trasylol on the activation of inactive renin was proportional to the concentration of the inhibitor. Plotting the kinetic data by the method of Hill’O gave a straight line with slope 1 (fig. 3), indicating that one mole-
3--Concentrations ([LU/ml) of active renin generated in the absence (Co) and presence (C) of trasylol plotted according to
Fig.
Hill. 10 Data (mean +s.D.,
n=4) fall on a straight line with slope 1.
cule of the inhibitor is reacting with one molecule of the activator of inactive renin. Trasylol had no influence on the reaction of renin with its substrate nor on the radioimmunoassay
of angiotensin-i. The results demonstrate that normal human plasma contains a non-dialysable factor, proactivator, from which a proteolytic enzyme, activator, is generated by acidification. This enzyme activates inactive renin at physiological pH. In the course of their work Atlas et al.11 published a similar proposal in abstract. Cryoactivation of inactive renin may also act through an activator enzyme. 11, 12 Trasylol has been reported to interfere with cryoactivation of inactive renin. 12 Our results show that this also holds for acid activation. The kinetic data on the inhibitory effect of trasylol indicate that the activator of inactive renin is a single substance acting at one point of the activation process. Trasylol is a potent inhibitor of kallikrein and, as with the activator of inactive renin, one 10. 11. 12.
Loftfield, R. B., Eigner, E. A. Science, 1969, 164, 305. Atlas, S. A., Sealey, J. E., Laragh, J. H. Kidney Int. 1977, 12, 405. Osmond, D. H., Loh, A. Y. Lancet, 1978, i, 102.
molecule of kallikrein reacts with one molecule of the inhibitor to form an inactive complex which is dissociated at low pH.13 This raises the interesting possibility that kallikrein is the activator of inactive renin. Kallikrein is present in plasma in an inactive form, pre-kallikrein, which is activated by exposure to low pH, low temperature, or trypsin. The same procedures activate inactive plasma renin. This further supports the view that kallikrein is involved. Active kallikrein is present in those organs which also contain and secrete renin (i.e., kidney and salivary glandes).14 It is therefore tempting to speculate that kallikrein is the physiological activator-enzyme for inactive renin. Department of Internal Medicine I, University Hospital, Erasmus University, Rotterdam, Netherlands
F. H. M. DERKX H. L. TAN-TJIONG M. A. D. H. SCHALEKAMP
MULTIPLY RESISTANT PNEUMOCOCCUS
SIR,-Strains of Streptococcus pneumoniae resistant to penicillin’ or to chloramphenicol and tetracycline2 have been found in the U.K., while strains resistant to penicillin and chloramphenicol have been isolated in South Africa.3 We have isolated a pneumococcus resistant to all three antimicrobials from a holidaymaker from Spain, who was admitted to hospital in Plymouth with an acute skin condition. She had a productive. cough, and from mucopurulent sputum a pneumococcus (type 23, Danish nomenclature) was isolated. On routine testing this was found to be resistant to chloramphenicol and tetracycline, and the zone of inhibition round the penicillin disc (1-5units) was reduced from the expected 10 mm to 7 mm, measured radially from the edge of the disc. By the agar dilution method, using a minimal-growth endpoint, the minimum inhibitory concentration (M.LC.) of penicillin was 0.25 g/ml compared with an M.t.c. of 0-03 tg/ml for a sensitive strain. The M.i.c.s of tetracycline and chloramphenicol were both 20 g/ml. The inoculum used in these tests was about 106 organisms. Although this pneumococcus almost certainly was not indigenous to the U.K. its isolation makes it necessary for those treating serious pneumococcal disease to be aware that multiply resistant strains of the organism are close to, if not already on, our shores. A patient with a pneumococcal meningitis due to a strain with the M.i.c.s reported would be unlikely to respond adequately to treatment with penicillin or chloramphenicol. In respect of the treatment of such an infection we would add that its ampicillin M.i.c. was 0.15 p.g/ml compared with 0-07 for a penicillin-sensitive strain; however, experience elsewhere3has shown that much higher M.i.cs may be found. It has been said that testing pneumococci with high-concentration discs prevents the detection of significant penicillin resistance.4.5 Although we use low-concentration discs, the zone of inhibition round an 8 unit tablet (’Neo-sensitabs’, A/S Rosco, Denmark) with the strain reported was reduced from a radius measured from the edge of the disc of 14 mm for a sensitive strain, to 10 mm. We thank Dr M. T. Parker for
typing the
Public Health Laboratory Plymouth General Hospital, Plymouth PL4 8NN
strain.
P. D. MEERS R. B. MATTHEWS
***This letter is reprinted from
our issue of July 15, where it appeared without the authors’ names, a grave error by our printers for which we apologise to Dr Meers and Mr Mat-
thews.-ED.L. 13. Vogel, R., Werle, E. Handbk exp. Pharmac. 1970, p. 213. 14. Webster, M. E. ibid. p. 131.
Howes, V. J., Mitchell, R. G. Br. med.J. 1976, i, 996. Howard, A. J., Hince, C. J., reported by Hansman, D. Lancet, 1978, i, 1102. Appelbaum, P C., Scragg, J. N., Bowen, A. J., Bhamjee, A., Hallett, A. F., Cooper, R. C. ibid. 1977, ii, 995. 4. Hansman, D., Devitt, L., Riley, I. Br. med.J. 1973, iii, 405. 5. Dixon, J. M. S. Lancet, 1974, ii, 474.
1. 2. 3.
screening from more than one centre to obtain better estimates of the detection-rate, and monitor the stability of the proportion of false positives. The U.K. Collaborative Study has shown that results from different centres cannot be compared directly but only by expressing them in multiples of each laboratory’s normal median; this must always be a second best, and can never tell us to what extent differences in the proportion of normal births with high serum-A.F.p. are due to assay imprecision rather than inherent differences in the screened population. A necessary condition for a national screening programme should be that the results from participating laboratories can be compared directly in terms of the same
reference preparation. Also, no such screening programme should be planned unless it is run in conjunction with a quality-control scheme to assess the precision and accuracy of the assays done in the various centres. Medical Computing and Statistics Unit, Medical School, University of Edinburgh, Edinburgh EH8 9AG
GILLIAN RAAB
Study. I.C.R.F. Cancer Epidemiology and Clinical Trials Department of the Regius Professor of Medicine, Radcliffe Infirmary, Oxford OX2 6HE
Department of Human Genetics, Western General Hospital, Edinburgh EH4 2XU
NICHOLAS J. WALD HOWARD S. CUCKLE DAVID J. H. BROCK
Paediatric Research Unit,
Guy’s Hospital, London SE1 9RT
ENDOGENOUS ACTIVATOR OF
Unit,
PAUL E. POLANI
SIR,-Professor Ferguson-Smith and his colleagues describe how their
screening of 11 500 pregnancies for raised serdetected 93% of those births with neural-tube defects, with a false-positive rate of only 1-4%. However, if their results are to be used to predict the consequences of a national screening programme, we need to know to what extent an equivalent intervention level might produce similar results in other centres, and in particular we must make an assessment of the possible biases and the standard errors of these estimates of percentages. Their detection-rate of 93% is based on the detection of 27 anencephalics out of 27, and 13 spina bifidas out of 16. These percentages should be examined separately rather than together, as there were more cases of anencephaly than spina bifida in this study although the incidence of the two defects is known to be similar; this difference in incidence-rates must itself cast some doubt on the adequacy of the follow-up in the Glasgow study. The true detection-rate for spina bifida should have been quoted as 12 out of 16, because 1 case was missed at screening and only retrospectively recognised as above the intervention limit. Thus the sensibilities of the screening test for the two defects should be given as 100% (95% confidence limits 87-100%) for anencephaly and 75% (95% confidence limit 47-92%) for spina bifida. The width of the confidence intervals is a result of the small numbers on which these percentages are based. In contrast the false-positive rate of 1.6% is based on large numbers and thus well determined for
um-A.F.p.
the Glasgow population. However, this rate is likely to vary both over time, and at different centres. The intervention level of 2.8 times the median used in this study was supposed to estimate the 97th percentile for normal singleton pregnancies and hence should have produced a false-positive rate of at least 3%. These points emphasise the need to combine the results of
PLASMA-INACTIVE-RENIN
SiR,—The apparent concentration of renin in human plasma can be increased by dialysis to pH 3.0-3.3 followed by dialysis to pH 7.4.1-3 This increase is due to activation of an inactive form of the enzyme. It has been argued that the activating process cannot be important physiologically since plasma is not exposed to a pH of 3.0in vivo. However, inactive renin is also activated by trypsin at pH 7.4,4,5 and an endogenous protease can activate the inactive renin of amniotic fluid.5 The experiments described below show that acid-activation of inactive renin in human plasma is probably mediated by an endogenous protease. In the course of this work I learned that Dr M. A. D. H. Schalekamp and his group had obtained similar results (see accompanying letter). Plasma from normal males was pooled. Samples were assayed for renin before and after the following procedures: (a) Dialysis to pH 3.0 and back to pH 7.4 (b) Dialysis to pH 3.0 in the presence of protease inhibitors, followed by dialysis to pH 7.4. ’Trasylol’ (aprotinin, Bayer) and soybean trypsin inhibitor (S.B.T.I.) in pH 7.4 phosphate-saline buffer were added directly to 10 x the volume of plasma and to the pH 3.0 glycine/HCl dialysis buffer at the concentrations stated in the table, and the mixture was incubated for 3 min at 37°C before acid dialysis. N-ethylmaleimide and o-phenanthroline were included in the pH 3.0 glycine/HCl buffer.3 (c) Plasma was also treated as in (a) and then assayed for renin in the presence of trasylol and S.B.T.I.
The kallikrein inhibitor trasylol and S.B.T.I. partially inhibited the acid-induced increase in renin concentration (table). Increasing concentrations of inhibitor increased its effect. N-ethyimaleimide had some inhibitory effect but o-phenanthroline had no effect. Neither trasylol nor S.B.T.I. affected the reaction between renin and renin substrate. These results suggest that acid-activation of inactive renin in plasma is mediated by a serine protease. The increase in plasma-renin 1. 2.
Skinner, S. L., Lumbers, E. R., Symonds, E. M. Clin. Sci. 1972, 42, 479. Leckie, B. J., McConnell, A.J. Endocr. 1975, 65, 7p. 3. Leckie, B. J., McConnell, A., Grant, J., Morton, J. J., Tree, M., Brown, J. J. Circulation Res. 1977, 40, suppl. 1, p. 46. 4. Cooper, R. M., Osmond, D. H., Scaiff, K. D., Ross, L. J. Fedn Proc. 1974,
33, 584. 5. Leckie, B. J., McConnell, A., Jordan, J. Adv. exp. Med. Biol. 1977, 95, 249. Morris, B. J., Lumbers, E. R., Biochim biophys. Acta. 1972, 289, 385. Millar, J. A., Leckie, B., J., Semple, P. F., Morton, J. J., Sonkodi, S., Robertson, J. I. S. Circulation Res. (in the press). 8. Osmond, D. H., Loh, A. Y. Lancet, 1978, i, 102.
6. 7.
,
218 there is no proof for activation in vivo. This study was undertaken to explore the possibility that plasma contains an enzyme which activates renin under physiological conditions. now
Pooled 6. 7. 8. 9.
plasma from fifteen normal
males
was
used. Renin
was
acti-
Osmond, D. H., Ross, L. J., Scaiff, K. D. Can. J. Physiol. Pharmac. 1973, 51, 705. Sealey, J. E., Moon, C., Laragh, J. H., Alderman, M. Am. J. Med. 1976, 61, 731. Atlas, S. A., Laragh, J. H., Sealey, J. E., Moon, C. Lancet, 1977, ii, 785. Cooper, R. M., Murray, G. E., Osmond, D. H. Circulation Res. 1977, 40, suppl. no. 1, p. 171.
(A) fl-units/ml of renin activated by acidification and by acidification in the presence of protease inhibitors. (B) Renin concentration in pre-acidified plasma assayed with and without protease inhibitors present in the incubation mixture. Mean +
s.E.
of n experiments.
Results in the presence of inhibitors out inhibitors by t test.
of varying pH at step B renin which is activated.
Fig. I-Effect were
compared
to
the results with-
Results (mean +s.D., n=5) are expressed achieved by standard technique. 2,5
on
the concentration of
as a
percentage of maxi-
mum
concentration induced by cold can be prevented by trasylol8,9 and may involve the action of a similar protease. The inactive renin in human plasma is not activated by incubation at 37°C either at pH 7.4 or pH 4.0.3 Plasma contains serine protease inhibitors which probably inhibit the action of the renin-activating enzyme(s), unless the inhibition is destroyed by acid or cold.9 Such enzymes might, however, be active in plasma from people with inherited o:i-trypsin-inhibitor deficiency, resulting in an abnormally high proportion of active renin. However, the concentrations of active and inactive renin in such a patient were: active renin 20 p.U/ml, inactive renin 146 p.U/ml; percent inactive renin 17%. These compare with 9-52 jU/m! for active renin and 38-180 p.U/ml for inactive renin in plasma samples from sixty normal subjects. Whether activation of inactive renin occurs in plasma is still unknown, therefore. The activation may take place in the kidneylO or blood-vessel walls, and the presence of a renin-activating system in circulating plasma may be due to "escape" of activating enzyme from these sites. M.R.C. Blood Pressure Unit, Western Infirmary, Glasgow G11 6NT
B. LECKIE
SIR,-Normally more than half the renin in plasma is inactive. It is activated under such unphysiological conditions as low pHl-5 and low temperature6-8 and also by trypsin.9 Until 9. 10.
Sealey, J. E., Laragh, J. H. Kidney Int. 1977, 6, 495. Inagami, T., Murakami, K., Misono, K., Workman,
R. J., Cohen, S., Suk1977, 95, 225. 1. Skinner, S. L., Cran, E. J., Gibson, R., Taylor, R., Walters, W. A. W., Catt, K. J.Am.J. Obstet. Gynec. 1975, 121, 626. 2. Derkx, F. H. M., Wenting, G. J., Man in ’t Veld, A. J., Van Gool, J. M. G., Verhoeven, R. P., Schalekamp, M. A. D. H. Lancet, 1976, ii, 496. 3. Leckie, B. J., McConnell, A., Grant, J., Morton, J. J., Tree, M., Brown, J. J. Circulation Res. 1977, 40, suppl. no.1, p. 46. 4. Boyd, G. W. Lancet, 1977, i, 215. 5. Derkx, F. H. M., Wenting, G. J., Man in ’t Veld, A. J., Verhoeven, R. P., Schalekamp, M. A. D. H. Clin. Sci. mol. Med. 1978, 54, 529. eta, Y. Adv. exp. Med. Biol.
I
I
I
Fig. 2-Effect (mean +S.D.) of trasylol, 800 kallikrein-inactivating units/ml on concentration of renin which is activated.
219 as before:2,5 (A) dialysis to pH 3.3 for 24 h at pH 7.5 for 24 h at 4°C, (C) incubation, with sheep renin-substrate at pH 7.5 for 3 h at 37°C, and (D) radioimmunoassay of angiotensin i. This standard procedure was modified as follows. Plasma pH at step B was rapidly adjusted to values ranging from 3.55 to 7.5 with sodium hydroxide solution (1 mmol/ml), followed by dialysis against buffers of the same pH (i.e., glycine/HCl buffers for the pH range 3.5-5.0 and sodium-phosphate buffers for the pH range 5.0-7-5). After 24 h dialysis at 4°C pH was readjusted to 7.5 by sodium hydroxide, and finally steps C and D were done. The kallikrein-inhibitor, aprotinin (’Trasylol’, Bayer) was added to plasma and dialysis buffer at step A or B, at concentrations stated in figs. 2 and 3. It was also added to the renin/renin-substrate mixture at C or to
vated and measured
4"C, (B) dialysis
to
the angiotensin-i/antibody mixture at D.
Activation of inactive renin occurred at neutral pH (fig. 1), but prior acidification was necessary. Addition of trasylol to plasma and the dialysis buffer during acidification inhibited this activation (fig. 2). Inhibition was reversed by further dialysis at pH 3.3 with no trasylol in the buffer. Thus trasylol must have exerted its inhibitory effect during one of the later steps when pH is higher. This was confirmed when trasylol was added after acidification and rapid neutralisation (step B, see fig. 2). The inhibitory effect of trasylol on the activation of inactive renin was proportional to the concentration of the inhibitor. Plotting the kinetic data by the method of Hill’O gave a straight line with slope 1 (fig. 3), indicating that one mole-
3--Concentrations ([LU/ml) of active renin generated in the absence (Co) and presence (C) of trasylol plotted according to
Fig.
Hill. 10 Data (mean +s.D.,
n=4) fall on a straight line with slope 1.
cule of the inhibitor is reacting with one molecule of the activator of inactive renin. Trasylol had no influence on the reaction of renin with its substrate nor on the radioimmunoassay
of angiotensin-i. The results demonstrate that normal human plasma contains a non-dialysable factor, proactivator, from which a proteolytic enzyme, activator, is generated by acidification. This enzyme activates inactive renin at physiological pH. In the course of their work Atlas et al.11 published a similar proposal in abstract. Cryoactivation of inactive renin may also act through an activator enzyme. 11, 12 Trasylol has been reported to interfere with cryoactivation of inactive renin. 12 Our results show that this also holds for acid activation. The kinetic data on the inhibitory effect of trasylol indicate that the activator of inactive renin is a single substance acting at one point of the activation process. Trasylol is a potent inhibitor of kallikrein and, as with the activator of inactive renin, one 10. 11. 12.
Loftfield, R. B., Eigner, E. A. Science, 1969, 164, 305. Atlas, S. A., Sealey, J. E., Laragh, J. H. Kidney Int. 1977, 12, 405. Osmond, D. H., Loh, A. Y. Lancet, 1978, i, 102.
molecule of kallikrein reacts with one molecule of the inhibitor to form an inactive complex which is dissociated at low pH.13 This raises the interesting possibility that kallikrein is the activator of inactive renin. Kallikrein is present in plasma in an inactive form, pre-kallikrein, which is activated by exposure to low pH, low temperature, or trypsin. The same procedures activate inactive plasma renin. This further supports the view that kallikrein is involved. Active kallikrein is present in those organs which also contain and secrete renin (i.e., kidney and salivary glandes).14 It is therefore tempting to speculate that kallikrein is the physiological activator-enzyme for inactive renin. Department of Internal Medicine I, University Hospital, Erasmus University, Rotterdam, Netherlands
F. H. M. DERKX H. L. TAN-TJIONG M. A. D. H. SCHALEKAMP
MULTIPLY RESISTANT PNEUMOCOCCUS
SIR,-Strains of Streptococcus pneumoniae resistant to penicillin’ or to chloramphenicol and tetracycline2 have been found in the U.K., while strains resistant to penicillin and chloramphenicol have been isolated in South Africa.3 We have isolated a pneumococcus resistant to all three antimicrobials from a holidaymaker from Spain, who was admitted to hospital in Plymouth with an acute skin condition. She had a productive. cough, and from mucopurulent sputum a pneumococcus (type 23, Danish nomenclature) was isolated. On routine testing this was found to be resistant to chloramphenicol and tetracycline, and the zone of inhibition round the penicillin disc (1-5units) was reduced from the expected 10 mm to 7 mm, measured radially from the edge of the disc. By the agar dilution method, using a minimal-growth endpoint, the minimum inhibitory concentration (M.LC.) of penicillin was 0.25 g/ml compared with an M.t.c. of 0-03 tg/ml for a sensitive strain. The M.i.c.s of tetracycline and chloramphenicol were both 20 g/ml. The inoculum used in these tests was about 106 organisms. Although this pneumococcus almost certainly was not indigenous to the U.K. its isolation makes it necessary for those treating serious pneumococcal disease to be aware that multiply resistant strains of the organism are close to, if not already on, our shores. A patient with a pneumococcal meningitis due to a strain with the M.i.c.s reported would be unlikely to respond adequately to treatment with penicillin or chloramphenicol. In respect of the treatment of such an infection we would add that its ampicillin M.i.c. was 0.15 p.g/ml compared with 0-07 for a penicillin-sensitive strain; however, experience elsewhere3has shown that much higher M.i.cs may be found. It has been said that testing pneumococci with high-concentration discs prevents the detection of significant penicillin resistance.4.5 Although we use low-concentration discs, the zone of inhibition round an 8 unit tablet (’Neo-sensitabs’, A/S Rosco, Denmark) with the strain reported was reduced from a radius measured from the edge of the disc of 14 mm for a sensitive strain, to 10 mm. We thank Dr M. T. Parker for
typing the
Public Health Laboratory Plymouth General Hospital, Plymouth PL4 8NN
strain.
P. D. MEERS R. B. MATTHEWS
***This letter is reprinted from
our issue of July 15, where it appeared without the authors’ names, a grave error by our printers for which we apologise to Dr Meers and Mr Mat-
thews.-ED.L. 13. Vogel, R., Werle, E. Handbk exp. Pharmac. 1970, p. 213. 14. Webster, M. E. ibid. p. 131.
Howes, V. J., Mitchell, R. G. Br. med.J. 1976, i, 996. Howard, A. J., Hince, C. J., reported by Hansman, D. Lancet, 1978, i, 1102. Appelbaum, P C., Scragg, J. N., Bowen, A. J., Bhamjee, A., Hallett, A. F., Cooper, R. C. ibid. 1977, ii, 995. 4. Hansman, D., Devitt, L., Riley, I. Br. med.J. 1973, iii, 405. 5. Dixon, J. M. S. Lancet, 1974, ii, 474.
1. 2. 3.
