- Email: [email protected]
ROLE OF INHIBITORS OF FIBRINOLYSIS IN HEPATIC CIRRHOSIS
990 the tumour after radiotherapy, or without histological evidence of residual tumour. We therefore suggest that as little surgery as possible should be carried out after radical radiotherapy; and no major surgical procedure should be undertaken in under six months, unless the tumour is seen on repeat cystoscopy to be unaltered or growing rapidly, and then only after a positive biopsy.
To siliconised test-tubes was added 0-5 ml. of the test
Summary A series of 105 patients with carcinoma of the bladder treated by radical megavoltage radiotherapy is reported. The effectiveness of radiotherapy as the primary method of treatment is compared with that of radiotherapy following previous surgery. Repeated fulgurisation or partial cystectomy may give rise to fibrotic areas containing relatively anoxic tumour cells, thus accounting for the difference in the radio-curability of the tumours. This hypothesis is supported by a comparison of the results of the two methods of treatment. Surgery for radioresistant tumours is shown to be successful only in the earlier (T and T2) stages. I wish to thank Dr. J. A. C. Fleming and Dr. 1. ChurchillDavidson for their continual help and advice in the preparation of this paper; the urological surgeons for referring the patients; and the many secretaries and clerks of this and other helped in the follow-up and tracing of these patients.
hospitals
who
REFERENCES British Medical Journal (1961) ii, 692. Gray, L. H. (1961) Amer. J. Roentgenol. 85, 803. Pugh, R. C. B. (1957) Brit. J. Urol. 29, 222. Wallace, D. M. (1956) Ann. R. Coll. Surg. Engl. 18, 366.
ROLE OF INHIBITORS OF FIBRINOLYSIS IN HEPATIC CIRRHOSIS RALPH A. O’CONNELL M.D. Cornell
CARLO E. GROSSI Long Island, F.A.C.S.
M.D.
INTERN
ASSISTANT SURGEON
LOUIS M. ROUSSELOT M.D. Columbia, F.A.C.S. PROFESSOR OF CLINICAL
SURGERY,
NEW YORK UNIVERSITY SCHOOL OF MEDICINE
From the
Department of Surgery, St. Vincent’s Hospital of the City of New York University School of Medicine
serum
or
0’5
ml. physiological saline solution for a control; 0-2 ml. 1 % bovine
fibrinogen (Armour Laboratories,
Illinois); 0-1 ml. human
plasminogen
Fig. I-Plasminogen levels
in vitro.
(National American Red Cross, by courtesy of E. R. Squibb & Sons); 2 mg. per ml. distilled water; and 0’1 ml. streptokinase (Lederle Laboratories, New York) 500 units per ml. The above was clotted with 0’2 ml. bovine thrombin (10 N.I.H. units per ml.). The tubes were placed in a water bath at 37°C. The end-point was the completion of clot-lysis from the addition of streptokinase. A control blank substituting 0-5 ml. of physiological saline solution for serum was run simultaneously. The control blank of the lysis system was reproducible at 4 minutes ± 30 seconds, referred to as inhibitor-time "-a measurement of inhibition of fibrinolysis. This differs from " clot-lysis time ", which is a measurement of fibrinolysis itself. " Inhibitortime " and " clot-lysis time " are inversely proportional values -i.e., the lower figure represents either higher inhibitor activity "
enzyme activity. Variations of plasminogen concentration change the inhibitor-time of the control (fig. 1). Increasing the streptokinase concentration to 1000 units per ml. in the control blank gave the highest level of plasminogen activity (fig. 2). E-aminocaproic acid (Lederle Laboratories) was added to the
or
control system to test whether it could measure inhibition of clot lysis. The results are shown in fig. 3.
Results
The
sera
of 9
healthy volunteers and
cirrhosis of the liver normal
serum
were
tested. In all
inhibited the
test
lysis
15
patients with
0-5 ml. of system longer than cases
CIRRHOSIS of the liver has been shown to be associated with increased fibrinolytic activity (Grossi et al. 1961). Similarly, patients undergoing portacaval shunts have an increased tendency to operative and postoperative bleeding, owing to active fibrinolysis (Grossi et al. 1962). E-aminocaproic acid has given us a means of controlling this type of haemorrhagic defect, but the mechanism of the increased lysis remains obscure. In view of the known serum-protein aberrations in cirrhosis of the liver, we undertook to determine whether the observed fibrinolytic activity in patients with cirrhosis of the liver was related to von a decrease of fibrinolytic inhibitor in the serum. Kaulla (1964) has raised the same question. Patients and Methods from Blood healthy adult hospital personnel was used for controls against that of patients in hospital with a clinical diagnosis of cirrhosis of the liver and confirmatory laboratory reports. Serum was obtained by clotting blood for an hour at 37°C and centrifuging for 30 minutes at 4°C and 3000 r.p.m. The assay was carried out immediately. Previous methods for assaying serum inhibitors of fibrinolysis were used for detecting an increase in inhibitor (Nilsson et al. 1961, Correll and Sjoerdsma 1962). Our study was aimed at detecting a decrease in serum-inhibitor-level, by means of a new method of assay entailing the use of a completely exogenous clot-lysis system of streptokinase-activated human plasminogen acting on a standard bovine fibrinogen clot.
Fig. 2-Streptokinase
concentration.
Fig. 3-E-aminocaproic-acid concentration in vitro.
991 INHIBITOR-TIME AND CLOT-LYSIS TIME IN CIRRHOTIC PATIENTS AND CONTROLS
absence of fibrinolysis is related to the relative concentrations of activators and inhibitors present in the system. Sherry et al. (1959) showed that the antiplasmin content averaged the equivalent of 5-1 casein units per ml. as against an average plasminogen concentration of 3-8 casein units per ml.-indicating an imbalance favouring inhibition of fibrinolysis in the normal subject. Nevertheless, even with this imbalance, Fearnley et al. (1953) found low fibrinolytic activity in normal blood. Yet, even with a constant decrease in fibrinolytic inhibitors, only 2 of the cirrhotics showed active fibrinolytic activity when tested. The remaining patient manifested a striking decrease of inhibitor without any evident lysis. This suggests that in most cases the mechanism of the fibrinolytic system still maintains a balance between inhibitor and activator. The imbalance brought about by operation and release of activator, as shown by Macfarlane (1937), may account for the lysis in portacaval-shunt operations. The decrease in inhibitor levels in cirrhotics could also account for the decreased clearance of nicotinic-acidactivated lysis reported in cirrhotics by Fletcher (1964).
Summary A
*
5000 units per ml.
streptokinase.
hour. The tests were arbitrarily stopped at 1 hour for convenience. The inhibitor-time for the sera of all the cirrhotics was between 5 and 12 minutes (see table). Only 1 patient with cirrhosis of the liver had increased fibrinolytic activity. None of the controls evinced activity. 1 patient, with severe cirrhosis, had an inhibitor-time exceeding an hour. Increasing the streptokinase concentration in the test assay to 5000 units per ml. changed the inhibitor-time to 5 minutes. This patient also had chronic bronchitis, and, in view of the low inhibitor-time with higher streptokinase concentrations, we believe that the original high inhibitor-time reflected the presence of anti-streptokinase antibodies. Conversely, raising the streptokinase concentration in the normal sera had no effect on inhibitor-time. Oral ingestion of 1 g. of e-aminocaproic acid in 2 cirrhotics had a transient effect on serum-inhibitor levels as measured by inhibitor-times. This agrees with the recognised use of s-aminocaproic acid as a therapeutic inhibitor of abnormal fibrinolysis. an
Discussion
Fibrinolysis is a balanced system of activators and inhibitors. The activator system is well documented and includes such physiologically occurring substances as tissue activators, plasma activators, urokinase, trypsin, and plasmin (fibrinolysin) itself. Exogenous materials such as streptokinase, adrenaline, nicotinic acid, various pyrogens, and chloroform also activate fibrinolysis. The inhibitors of fibrinolysis are in two categories: (1) inhibitors of plasminogen activation; and (2) inhibitors of the activated enzyme, fibrinolysin (" antiplasmin "). Antiplasmin has been separated from the plasmalipids and lipoproteins in which it was found in the «2-globulin fraction of serum. Taylor et al. (1964) have characterised the plasma-inhibitor of fibrinolysin as a high-density protein with non-competitive activity. Martin (1949) and Satoskar et al. (1954) demonstrated a decrease in
assaying serum-inhibitors of fibrinolysis was applied to healthy controls, and to patients with cirrhosis of the liver. The patients with cirrhosis, in contrast to the controls, showed a striking decrease in serum-inhibitors. This finding may be helpful in understanding the mechanism of abnormal fibrinolysis and in the diagnosis of this condition. new
method for
This work was supported by a grant (AM-03897-04) from the National Institutes of Health, Bethesda, Maryland. We thank Miss Helen Zurawinsky for advice and assistance. REFERENCES
Correll, J. T., Sjoerdsma, A. (1962) Proc. Soc. exp. Biol., N.Y. 11, 274. Fearnley, G. R., Tweed, J. M. (1953) Clin. Sci. 12, 81. Fletcher, A. P., Biederman, O., Moore, D., Alkjaersig, N., Sherry, S. (1964) J. clin. Invest. 43, 681. Grossi, C. E., Moreno, A. H., Rousselot, L. M. (1961) Ann. Surg. 153, 383. Rousselot, L. M., Panke, W. F. (1962) Amer. J. Surg. 104, 512. Macfarlane, R. G. (1937) Lancet, i, 10. Martin, N. H. (1949) Brit. J. exp. Path. 30, 231. Nilsson, I. M., Krook, H., Sternby, N. H., Soderberg, E., Soderstrom, N. (1961) Acta med. scand. 169, 323. Satoskar, R. S., Lewis, R. A., Gaitonde, B. B. (1954) J. Lab. clin. Med. 44, 349. Sherry, S., Fletcher, A. P., Alkjaersig, N. (1959) Physiol. Rev. 39, 343. Taylor, F. B., Jr., Allen, L. W., Brickford, A. F., Jr. (1964) Arch. Biochem. Biophys. 104, 277. von Kaulla, K. N. (1964) Lancet, i, 1046. —
METYRAPONE AND PYROGEN IN THE ASSESSMENT OF PITUITARY-ADRENAL FUNCTION AFTER REMOVAL OF PITUITARY ADENOMA
J. S. JENKINS M.D. Cantab., M.R.C.P. SENIOR MEDICAL REGISTRAR
S. G. ELKINGTON M.B. Cantab., M.R.C.P. RESEARCH ASSISTANT
From the Medical Unit and
St.
Department of Neurosurgery, George’s Hospital, London, S.W.1
SURGICAL removal of a pituitary adenoma may be followed by hypopituitarism, the extent of which varies greatly in different patients according to the amount of destruction by the tumour itself, the damage to normal pituitary tissue at operation, and the radiotherapy usually carried out postoperatively. While some patients show overt signs of hypopituitarism, others claim to be well or complain only of vague symptoms short of the full clinical picture of anterior-pituitary deficiency. An accurate
To siliconised test-tubes was added 0-5 ml. of the test
Summary A series of 105 patients with carcinoma of the bladder treated by radical megavoltage radiotherapy is reported. The effectiveness of radiotherapy as the primary method of treatment is compared with that of radiotherapy following previous surgery. Repeated fulgurisation or partial cystectomy may give rise to fibrotic areas containing relatively anoxic tumour cells, thus accounting for the difference in the radio-curability of the tumours. This hypothesis is supported by a comparison of the results of the two methods of treatment. Surgery for radioresistant tumours is shown to be successful only in the earlier (T and T2) stages. I wish to thank Dr. J. A. C. Fleming and Dr. 1. ChurchillDavidson for their continual help and advice in the preparation of this paper; the urological surgeons for referring the patients; and the many secretaries and clerks of this and other helped in the follow-up and tracing of these patients.
hospitals
who
REFERENCES British Medical Journal (1961) ii, 692. Gray, L. H. (1961) Amer. J. Roentgenol. 85, 803. Pugh, R. C. B. (1957) Brit. J. Urol. 29, 222. Wallace, D. M. (1956) Ann. R. Coll. Surg. Engl. 18, 366.
ROLE OF INHIBITORS OF FIBRINOLYSIS IN HEPATIC CIRRHOSIS RALPH A. O’CONNELL M.D. Cornell
CARLO E. GROSSI Long Island, F.A.C.S.
M.D.
INTERN
ASSISTANT SURGEON
LOUIS M. ROUSSELOT M.D. Columbia, F.A.C.S. PROFESSOR OF CLINICAL
SURGERY,
NEW YORK UNIVERSITY SCHOOL OF MEDICINE
From the
Department of Surgery, St. Vincent’s Hospital of the City of New York University School of Medicine
serum
or
0’5
ml. physiological saline solution for a control; 0-2 ml. 1 % bovine
fibrinogen (Armour Laboratories,
Illinois); 0-1 ml. human
plasminogen
Fig. I-Plasminogen levels
in vitro.
(National American Red Cross, by courtesy of E. R. Squibb & Sons); 2 mg. per ml. distilled water; and 0’1 ml. streptokinase (Lederle Laboratories, New York) 500 units per ml. The above was clotted with 0’2 ml. bovine thrombin (10 N.I.H. units per ml.). The tubes were placed in a water bath at 37°C. The end-point was the completion of clot-lysis from the addition of streptokinase. A control blank substituting 0-5 ml. of physiological saline solution for serum was run simultaneously. The control blank of the lysis system was reproducible at 4 minutes ± 30 seconds, referred to as inhibitor-time "-a measurement of inhibition of fibrinolysis. This differs from " clot-lysis time ", which is a measurement of fibrinolysis itself. " Inhibitortime " and " clot-lysis time " are inversely proportional values -i.e., the lower figure represents either higher inhibitor activity "
enzyme activity. Variations of plasminogen concentration change the inhibitor-time of the control (fig. 1). Increasing the streptokinase concentration to 1000 units per ml. in the control blank gave the highest level of plasminogen activity (fig. 2). E-aminocaproic acid (Lederle Laboratories) was added to the
or
control system to test whether it could measure inhibition of clot lysis. The results are shown in fig. 3.
Results
The
sera
of 9
healthy volunteers and
cirrhosis of the liver normal
serum
were
tested. In all
inhibited the
test
lysis
15
patients with
0-5 ml. of system longer than cases
CIRRHOSIS of the liver has been shown to be associated with increased fibrinolytic activity (Grossi et al. 1961). Similarly, patients undergoing portacaval shunts have an increased tendency to operative and postoperative bleeding, owing to active fibrinolysis (Grossi et al. 1962). E-aminocaproic acid has given us a means of controlling this type of haemorrhagic defect, but the mechanism of the increased lysis remains obscure. In view of the known serum-protein aberrations in cirrhosis of the liver, we undertook to determine whether the observed fibrinolytic activity in patients with cirrhosis of the liver was related to von a decrease of fibrinolytic inhibitor in the serum. Kaulla (1964) has raised the same question. Patients and Methods from Blood healthy adult hospital personnel was used for controls against that of patients in hospital with a clinical diagnosis of cirrhosis of the liver and confirmatory laboratory reports. Serum was obtained by clotting blood for an hour at 37°C and centrifuging for 30 minutes at 4°C and 3000 r.p.m. The assay was carried out immediately. Previous methods for assaying serum inhibitors of fibrinolysis were used for detecting an increase in inhibitor (Nilsson et al. 1961, Correll and Sjoerdsma 1962). Our study was aimed at detecting a decrease in serum-inhibitor-level, by means of a new method of assay entailing the use of a completely exogenous clot-lysis system of streptokinase-activated human plasminogen acting on a standard bovine fibrinogen clot.
Fig. 2-Streptokinase
concentration.
Fig. 3-E-aminocaproic-acid concentration in vitro.
991 INHIBITOR-TIME AND CLOT-LYSIS TIME IN CIRRHOTIC PATIENTS AND CONTROLS
absence of fibrinolysis is related to the relative concentrations of activators and inhibitors present in the system. Sherry et al. (1959) showed that the antiplasmin content averaged the equivalent of 5-1 casein units per ml. as against an average plasminogen concentration of 3-8 casein units per ml.-indicating an imbalance favouring inhibition of fibrinolysis in the normal subject. Nevertheless, even with this imbalance, Fearnley et al. (1953) found low fibrinolytic activity in normal blood. Yet, even with a constant decrease in fibrinolytic inhibitors, only 2 of the cirrhotics showed active fibrinolytic activity when tested. The remaining patient manifested a striking decrease of inhibitor without any evident lysis. This suggests that in most cases the mechanism of the fibrinolytic system still maintains a balance between inhibitor and activator. The imbalance brought about by operation and release of activator, as shown by Macfarlane (1937), may account for the lysis in portacaval-shunt operations. The decrease in inhibitor levels in cirrhotics could also account for the decreased clearance of nicotinic-acidactivated lysis reported in cirrhotics by Fletcher (1964).
Summary A
*
5000 units per ml.
streptokinase.
hour. The tests were arbitrarily stopped at 1 hour for convenience. The inhibitor-time for the sera of all the cirrhotics was between 5 and 12 minutes (see table). Only 1 patient with cirrhosis of the liver had increased fibrinolytic activity. None of the controls evinced activity. 1 patient, with severe cirrhosis, had an inhibitor-time exceeding an hour. Increasing the streptokinase concentration in the test assay to 5000 units per ml. changed the inhibitor-time to 5 minutes. This patient also had chronic bronchitis, and, in view of the low inhibitor-time with higher streptokinase concentrations, we believe that the original high inhibitor-time reflected the presence of anti-streptokinase antibodies. Conversely, raising the streptokinase concentration in the normal sera had no effect on inhibitor-time. Oral ingestion of 1 g. of e-aminocaproic acid in 2 cirrhotics had a transient effect on serum-inhibitor levels as measured by inhibitor-times. This agrees with the recognised use of s-aminocaproic acid as a therapeutic inhibitor of abnormal fibrinolysis. an
Discussion
Fibrinolysis is a balanced system of activators and inhibitors. The activator system is well documented and includes such physiologically occurring substances as tissue activators, plasma activators, urokinase, trypsin, and plasmin (fibrinolysin) itself. Exogenous materials such as streptokinase, adrenaline, nicotinic acid, various pyrogens, and chloroform also activate fibrinolysis. The inhibitors of fibrinolysis are in two categories: (1) inhibitors of plasminogen activation; and (2) inhibitors of the activated enzyme, fibrinolysin (" antiplasmin "). Antiplasmin has been separated from the plasmalipids and lipoproteins in which it was found in the «2-globulin fraction of serum. Taylor et al. (1964) have characterised the plasma-inhibitor of fibrinolysin as a high-density protein with non-competitive activity. Martin (1949) and Satoskar et al. (1954) demonstrated a decrease in
assaying serum-inhibitors of fibrinolysis was applied to healthy controls, and to patients with cirrhosis of the liver. The patients with cirrhosis, in contrast to the controls, showed a striking decrease in serum-inhibitors. This finding may be helpful in understanding the mechanism of abnormal fibrinolysis and in the diagnosis of this condition. new
method for
This work was supported by a grant (AM-03897-04) from the National Institutes of Health, Bethesda, Maryland. We thank Miss Helen Zurawinsky for advice and assistance. REFERENCES
Correll, J. T., Sjoerdsma, A. (1962) Proc. Soc. exp. Biol., N.Y. 11, 274. Fearnley, G. R., Tweed, J. M. (1953) Clin. Sci. 12, 81. Fletcher, A. P., Biederman, O., Moore, D., Alkjaersig, N., Sherry, S. (1964) J. clin. Invest. 43, 681. Grossi, C. E., Moreno, A. H., Rousselot, L. M. (1961) Ann. Surg. 153, 383. Rousselot, L. M., Panke, W. F. (1962) Amer. J. Surg. 104, 512. Macfarlane, R. G. (1937) Lancet, i, 10. Martin, N. H. (1949) Brit. J. exp. Path. 30, 231. Nilsson, I. M., Krook, H., Sternby, N. H., Soderberg, E., Soderstrom, N. (1961) Acta med. scand. 169, 323. Satoskar, R. S., Lewis, R. A., Gaitonde, B. B. (1954) J. Lab. clin. Med. 44, 349. Sherry, S., Fletcher, A. P., Alkjaersig, N. (1959) Physiol. Rev. 39, 343. Taylor, F. B., Jr., Allen, L. W., Brickford, A. F., Jr. (1964) Arch. Biochem. Biophys. 104, 277. von Kaulla, K. N. (1964) Lancet, i, 1046. —
METYRAPONE AND PYROGEN IN THE ASSESSMENT OF PITUITARY-ADRENAL FUNCTION AFTER REMOVAL OF PITUITARY ADENOMA
J. S. JENKINS M.D. Cantab., M.R.C.P. SENIOR MEDICAL REGISTRAR
S. G. ELKINGTON M.B. Cantab., M.R.C.P. RESEARCH ASSISTANT
From the Medical Unit and
St.
Department of Neurosurgery, George’s Hospital, London, S.W.1
SURGICAL removal of a pituitary adenoma may be followed by hypopituitarism, the extent of which varies greatly in different patients according to the amount of destruction by the tumour itself, the damage to normal pituitary tissue at operation, and the radiotherapy usually carried out postoperatively. While some patients show overt signs of hypopituitarism, others claim to be well or complain only of vague symptoms short of the full clinical picture of anterior-pituitary deficiency. An accurate