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Uncontrolled plasma proteolysis: A major threat to the septicemic patient
33
Resuscitation. 14 (1986) 33-42 Elsevier Scientific Publishers Ireland Ltd.
UNCONTROLLED PLASMA THE SEPTICEMIC PATIENT
A.O. AASEN,
N. SMITH-ERICHSEN
PROTEOLYSIS:
A MAJOR
THREAT
TO
and E. AMUNDSEN
Institute for Surgical Research, Rikshospitalet and Department Central Hospital, University of Oslo, Olso 1 (Norway)
of Anesthesia,
Akershus
SUMMARY
In order to further elucidate the pathophysiological significance of plasma proteolysis during septicemia, surgical patients with septicemia were studied by means of chromogenic peptide substrate assays. In fatal cases continuous low values for prekallikrein, plasminogen and antithrombin III were found until death. At autopsy a persistent septic focus was found in all but one of the fatal cases. Very low levels of prekallikrein during sepsis and reduced functional inhibition of plasma kallikrein in septic shock indicated a poor prognosis. In the survivors the parameters returned towards the normal range upon successful therapy. Furthermore the paper demonstrates the application of a new parameter, the proenzyme functional inhibition index (PFI-index) in patients with septicemia. The data reveal that by means of this parameter patients at high risk can be identified at an early stage of the disease. Key words: Chromogenic peptide substrate assays - Proenzyme functional inhibition-index - Prognosis - Septicemia
INTRODUCTION For more than two decades activation of the plasma proteolytic enzyme systems, i.e. the coagulation, fibrinolytic complement and kallikrein-kinin system, has been recognized as a frequent phenomenon in patients with septicemia (Rapaport et al., 1964; Mason et al., 1970; McCabe, 1973; Hirsch et al., 1974; O’Donnell and Clowes, 1976). During the last decade these pathophysiological events have been extensively studied by several groups (Aasen et al., 1980; Smith-Erichsen et al., 1982a; Smith-Erichsen et al.,
Abbreviations:
PFI-index,
0300-9572/66/$03.50 Printed and Published
proenzymes
functional
0 1966 Elsevier Scientific in Ireland
inhibition
index;
Plg, plasminogen.
Publishers Ireland Ltd.
34
1982b; Smith-Erichsen et al., 1983; KaIter et al., 1982; Fritz, 1980; Witt.e et al., 1982; McCann et al., 1982; Schipper et al., 1981; BiiIler et al., 1982; ten Cate, 1982). With the introduction of chromogenic peptide substrate assays, a new and important technique for investigation of proteolytic enzymes have been made available (Svendsen et al., 1972; Svendsen and Amundsen, 1973; Blomback et al., 1974; Amundsen et ai., 1977; Amundsen et al., 1978). In 1972, the first chromogenic peptide substrate assays were introduced for thrombin, trypsin and thrombin-like enzymes (Svendsen et al., 1972). Since then analyses have been developed for determination of active enzyme, proenzyme and functional inhibition of several proteases including components of the coagulation, fibrinolytic, complement and kallikrein-kinin system (Svendsen and Amundsen, 1973; Blomback et aI., 1974; Amundsen et al., 1977; Amundsen et al., 1978; Odegard et al., 1975; Aurel et al., 1977; Friberger et al., 1978; KIuft, 1978; Bergstriim and Blomback, 1974; Amundsen et al., 1979; Aasen et al., 1982; Friberger, 1982). The principle of chromogenic peptide substrate assays is the enzymatic cleavage of the peptide-pnitroaniline amid linkage of the chromogenic peptide substrate (Svendsen et al., 1972). This results in release of the chromophorep-nitroaniline. The reaction can be monitored at A4,,5nm in a spectrophotometer. Assays can be easily automated (Bergstrom and Blomb%ck, 1974; Aasen et al., 1982; Friberger, 1982). With automated, chromogenic peptide substrate assays, the possibility has emerged to rapidly obtain profiles of activity in various protease systems based on determination of level of proenzyme, enzyme activity and functional inhibitor capacity using the same chromogenic peptide substrate (Aasen et al., 1982). Using automated enzyme ana.Iysers only small plasma volumes are required (Aasen et al., 1982). Chromogenic peptide substrates are also available for determination of several components outside the plasma proteolytic enzyme systems including measurements of endotoxin content, activity of granuiocyte elastase, trypsin, chymotrypsin and glandular kallikrein activity (Aasen et al., 1982; Friberger, 1982). Using these techniques, we have studied surgical patients with septicemia (Aasen et al., 1980; Smith-Erichsen et al., 1982a; Smith-Erichsen et al., 198613; Smith-Erichsen et al., 1983). These patients still have a high mortality rate and are a major demand on the total costs of the intensive care unit (SmithErichsen et al., 1981). Our studies disclose that information obtained by chromogenic peptide substrates assays can give information of diagnostic, therapeutic and prognostic value in these critically ill patients. The present paper summarizes our findings. MATERIALS
AND METHODS
Thirty-nine surgical patients with septicemia were studied in two separate investigations (Smith-Erichsen et al., 1982a; Smith-Erichsen and Aasen, 1984).
35
Septicemia was diagnosed when at least four of the following criteria were fulfilled: temperature above 38°C; need of respiratory support; leukocytes above 15 X log/1 or below 5 X log/l; Trombocytes below 100 X lO’/l;Positive blood culture or an obvious septic focus. Septic shock was diagnosed when patients had hypotension, oliguria and need of vasopressor in addition to the criteria for septicemia.
Sampling
As soon as the diagnosis was ascertained, the first plasma sample was made from titrated blood and immediately stored at -70°C. Thereafter, plasma samples were collected with at least 24-h intervals until analyzed. Results were retrospectively compared to clinical course and outcome.
Assays Plasma prekallikrein(PKK), functional plasma kallikrein inhibition (KKI), plasminogen (Pig) and functional antiplasmin activity (AP) were measured with chromogenic peptide substrate assays as previously described (Aasen et al., 1980; Amundsen et al., 1978; Friberger et al., 1978; Aasen et al., 1982). Functional antithrombin III (AT III) was determined with a Quantichrome AT-III kit according to the manufacturers instructions (Abbott Diagnostics Ltd., North Chicago, IL). All values were expressed as the percentage of values obtained with a standard plasma pool from healthy donors. The PFI-index was calculated from the measured values for PKK, KKI, Plg, AP and AT III as defined by Aasen (1985).
Statistics To describe centrality and distribution of data, median, 25th and 75th percentiles were determined daily for all parameters during the first 4 days of septicemia, and for the recovery and terminal values. Statistical evaluation was performed with Wilcoxons rank sum test and P-values <0.05 were considered significant. RESULTS
AS shown in Figs. 1, 2, 3 and 4, values for plasminogen, antiplasmin, antithrombin III and plasma prekallikrein were significantly reduced in 18 surgical patients with persistent sepsis compared to values found in survivors. During the first week after diagnosis, value for these parameters were not significantly different in the two groups. During the second week after diagnosis, however, plasminogen and antithrombin III values were significantly higher in patients surviving septicemia than in fatal cases. Plasma prekallikrein and antiplasmin values were not significantly different in the two groups before the third week after diagnosis (Figs. 2 and 4). In patients with septic shock determination of functional plasma kallikrein inhibition revealed significantly lower values in fatal cases than in survivors (Fig. 5). At autopsy a persistent septic focus was found in all but one of the fatal cases.
36
% 120
-
llO= loo90 -
P
*
0.05
80 70 60 -
4
50 -
\
40 -
.
\
\
’
??
: .
30 203
Day 1
Third week
FII st week
Fig, 1. Plasminogen values during septicemia in survivors and fatal cases with a persistent septic focus. 0, survivors; 0, fatal cases.
Twenty-one patients with septicemia of whom 11 died, were studied by means of the PFI-index. As shown in Fig. 6, the PFI-index values were markedly negative in both groups from day 1. Values obtained in the fatal cases, however, were significantly lower than values found in the survivors from day 1 and during the whole observation period. No one of the individual parameters included in the index calculation could so clearly discriminate % 120110. loo90.
8070. 60.
1
I
I Day
1
First week
I
I
S$c,“,$”
Thor wee R
Fig. 2. Antiplasmin values during septicemia in survivors and fatal cases with a persistent septic focus. 0, survivors; 0, persistent sepsis.
37
1
Day
1
First week
I
I
I
I
S;c,“e;d
Thir wee ‘ii
Fig. 3. Antithrombin III values during septicemia in survivors and fatal cases with a persistent septic focus. 0, survivors, 0, persistent sepsis.
between patients who died and those surviving septicemia as the PFI-index (Smith-Erichsen and Aasen, 1984). The information which can be obtained by the PFI-index is further illustrated in Figs. 7 and 8 presenting data on individual cases. As shown in Fig. 7 the PFI-index was negative for the whole observation period of 14
I
I Day
I
1
First
week
I
scz”e$p:
I Tti r(!
wee*.
Fig. 4. PKK values during septicemia in survivors and fatal cases with a persistent septic focus. ??, survivors; 0, persistent sepsis.
38
* P-
00.5
c
-*-
R.S.S
FSS
Sepsis
Fig. 5. Functional plasma kallikrein inhibition during fatal septic shock (FSS), resuscitated septic shock (RSS), septicemia without shock (sepsis) and recovery values (Ret).
days in a severely injured patient who developed septicemia. At autopsy the patient was found to have an intraabdominal septic focus due to an unrecognized perforated duodenal ulcer. In Fig. 8 the PFI-index changes found in an 82-year-old women developing septicemia due to an acute cholecystitis is shown. After surgical treatment of the septic focus the PFI-index
I
I 1
I 2
I
I
3
4
Term. DAYS’
Fig. 6. The PFI-index during the early stage of septicemia, terminally (Term) and in the recovery (Ret) phase. Median , 25th and 75th percentiles are given. 0, survivors; 0, fatal cases.
39
AUTOPSY:
-w1
2
3
4 DaYs after
5
6
14
admission
Fig. 7. The PFI-index values in a 60-year-old man with
multiple trauma.
values became significantly less negative (Fig. 8). After stopping treatment with antibiotics on the fifth day after admission, however, she develops septicemia again. This was followed by a reduction of index values. The patient was successfully treated with gentamycine followed by a normalization of the PFI-indek (Fig. 8). DISCUSSION
The studies summarized in the present paper strongly emphasize the pathophysiological importance of disturbances of plasma proteolysis during septicemia in humans.
+30
*
+10
-
-10
_
-ANTI6lOTICS
-xl CHOLECYSTECTOMY -60
--
-70
-
DRENAGE GENTAMICIN
-SO-
-1lOL 0
2
4
6
6
10
12
14
Days afnr admission
Fig. 8. The PFI-index cholecystitis.
values in an &&year-old women developing septicemia due to
40
BY means of chromogenic peptide substrate assay technique this phebe closely monitored (Aasen et al., 1982; Friberger, 1982). Upon automation of the assays, data from chromogenic peptide substrate analyses now can be made available within a few hours after sampling (Aasen et al., 1982), and data on major functional parameters of the plasma proteolytic enzyme systems have been made available for the daily evaluation and treatment of patients with septicemia. The majority of studies performed in patients with septicemia using chromogenic peptide substrate assays including the present investigation, have been done retrospectively. Prospective investigations are under way by several groups in order to further evaluate the importance of these new tools in the treatment of severely ill patients. The importance of protease inhibitor control is strongly underlined by the fact that about 10% of the plasma proteins are protease inhibitors. The observation in the present study of insufficient proteases inhibition in septicemia patients likely disclose an aspect of therapy which today is not adequately addressed. A protease-antiprotease imbalance which also is reported in other conditions such as acute pancreatitis (Lasson, 1980), might be handled in different ways. Of particular importance is the fact that concentrates of both antithrombin III and C?-esterase inhibitor are available for use in humans (Schipper et al., 1978; van der Starre et al., 1980). Furthermore activated proteases might be controlled by removal of plasma compounds using plasmapheresis (Scharfman et al., 1979; Bjorratn et al., 1984). This procedure which presently is extensively studied might be a way of eliminating activators such as endotoxin and activated monocytes (Bjorvatn et al., 1984). Endotoxin which is released from the cell wall of gram-negative bacteria is thought to be an important component in the pathophysiology of septicemia (McCartney et al., 1983). This compound can now be quantitated using a chromogenic peptide substrate assay (Scully et al., 1980; Thomas et al., 1981). Thus the elimination of endotoxin can be monitored by a bed side analysis. Recently, constituents of gram-positive bacteria also have been demonstrated to be strong activators of plasma proteolysis (Kalter et al., 1983). These findings underline that in patients with septicemia the plasma is transformed to a ‘pathological perfusate’ which might be a precipitating factor for development of multiple organ failure frequently seen in septicemia. Pathological plasma proteolysis is a major phenomenon in patients with septicemia and therapy to control this process should be started in early stages of the disease. nomenon can
ACKNOWLEDGMENT
The financial support by The Laerdal Foundation acknowledged.
for Acute Medicine is
41 REFERENCES Aasen, A.O. (1985) The proenzyme functional inhibition index. A new parameter for evaluation of the severely injured and septic patient. Acta Chir. Stand., Suppl. 522, 211-233. Aasen, A.O., Kierulf, P. and Stromme, J.H. (1982) Methodological considerations on chromogenic peptide substrate assays and application on automated analysers. Acta Chir. Stand., 509,17-23. Aasen, A.O., Smith-Erichsen, N., Gallimore, M.J. et al. (1980) Studies on the components of the plasma kallikrein-kmin system in plasma samples from normal individuals and patients with septic shock. Vol. IV, pp. l-10. Advances in Shock Research. Alan R. Liss Inc., New York, 1980. Amundsen, E., Gallimore, M.J., Aasen, A.O. et al. (1978) Activation of human plasma prekallikrein: Influence of activators, activation and temperatureandinhibitors.Thromb. Res., 13,625. Amundsen, E., Putter, J., Friberger, P. et al. (1979) Methods for the determination of glandular kallikrein by means of a chromogenic tripeptide substrate. In: Kinins II: Biochemistry, pathophysiology and clinical aspects. pp. 83-95. Editors: S. Fuji, H. Moriya and T. Suzuku. Plenum Publ. Corp., New York. Amundsen, E., Svendsen, L., Vennerod, A.M. et al. (1977) Determination of plasma kallikrein with a new chromogenic tripeptide derivative. In: Chemistry and biology of the kallikreinkinin system in health and disease. pp. 215-220. Proceedings No. 27, Washington DC, US Government Printing Office, Aurel, L., Friberger, P., Karlsson, G. et al. (1977). A new sensitive and highly specific chromogenic peptide substrate for factor Ka. Thromb. Res., 11, 595. Bergstrom, K. and Blombiick, M. (1974) Determination of plasma prothrombin with a reaction rate analyzer using a synthetic substrate. Thromb. Res., 4, 719. Bjorvatn, B., Bjertnaes, L. and Fadnes, H.O. et al. (1984) Meningococcal septicemia treated with combined plasmapheresis and leucopheresis or with blood exchange. Br. Med. J., 288,439-441. Blombiick, M., Blomtick, B. and Olsson, P. (1974) The assay of antithrombin using a synthetic chromogenic substrate for thrombin. Thromb. Res., 5, 621. Biiller, H.R., Bolwerk, C., ten Cam, J. et al. (1982) Postoperative hemostratic profile in relation to g&negative septicemia. Crit. Care Med., 10, 311. Friberger, P. (1982) Chromogenic peptide substrates. Their use for the assay of factors in the fibrinolytic and the plasma kallikrein-kinin system. Stand. J. Clin. Lab. Invest., 42,162. Friberger, P., Kniis, M., Gustavsson, S. et al. (1978) Methods for determination of plasmin, antiplasmin and plasminogen by means of substrate S-2251. Haemostasis, 7, 138. Fritz, H. (1980) Proteinase inhibitors in severe inflammatory processes (septic shock and experimental endotoxemia): Biochemical, pathophysiological and therapeutic aspects. In: Protein degradation in health and disease. pp. 351-379. CibaFoundationSymp. 75 (New Series). Amsterdam. Excerpta Medica. Hirsch, E.F., Nakajima, T., Oshima, G. et al. (1974) Kinin system responses in sepsis after trauma in man. J. Surg. Res., 17, 147. Kalter, E.S., Timmermanns, A. and Bouma, B.N. (1982) The kinin generating system during sepsis and bacterial shock:Agents Actions, 9,638-644. Kalter, E.S., van Dijk, W.C., Thnmermans, A. et al. (1983) Activation of purified human plasma prekallikrein triggered by cell wall fractions of Escherichia coli and Staphyiococens eureus. J. Infect. Dis., 1983. Kluft, C. (1978) Determination of prekallikrein in human plasma: Gptimal conditions for activating prekallikrein. J. Lab. Clin. Med., 91, 83.
42 Lasson, A. (1984) Acute pancreatitis in man: A clinical and biochemical study of pathophysiology and treatment. The University of Lund. Stand. J. Gastro enterol., 99. Mason, J.W., Kleeberg, U., Dolan, ,P. et al. (1970) Plasma kallikrein and Hagemann factor in gramnegativ bacteremia. Ann. Intern. Med., 73, 546. McCabe, W.R. (1973) Serum complement levels in bacteremia due to Gram negative organisms. N. Engl. J. Med., 288, 21-23. McCartney, A.C., Banks, J.G. and Clements, G.B. (1983) Endotoxemia in septic shock. Clinical and post mortem correlations. Intens. Care Med., 9, 117”121. McCann, R., Wasserman, F. and Haberland, G. (1982) The kallikrein-kinin system in the acutely ill: changes in plasma kinlnogen in acutely ill patients and the efficacy of pulmonary clearance of bradykinin. Role of chemical mediators in the pathophysiology of acute illness and injury. pp. 111-127. Raven Press, New York. Odegard, O.R., Lie, M. and Abildgaard, N. (1975) Heparin cofactor activity measured with an amidolytic method. Thromb. Res., 6, 287. O’Donnell, T.F. and Clowes, G.H.A,, (1976) Kinin activation in the blood of patients with sepsis. Surg. Gynecol. Obstet., 143, 539. R‘apaport, S.I., Tatter, D., Coeur-Barron, N. et al. (1964) Pseudomonas septicemia with intravascular clotting leading to the generalized Shwartzman reaction. N. Engl. J. Med., 271, 80-84. Scharfman, W.B., Tillofson, J.R. and Taft W. (1979) Plasmapheresis for meningococcemia with disseminated intravascular coagulation. N. Engl. J. Med., 300, 1277. Schipper, H.G., Kahle, L.H. and Jenkins, U.P. (1978) Antithrombin III transfusion in disseminated intravascular coagulation. Lance& 2, 854-858. Schipper, H.G., Roos, J., v.d. Meulen, F. et al. (1981) Antithrombin III deficiency in surgical intensive care patients. Thromb. Res., 21 73. Scully, M.F., Newman, Y.M., Clark, S.E. et al. (1980) Evaluation of a chromogenic method for endotoxin measurement. Thromb. Res., 20, 263-270. Smith-Erichsen, N. and Aasen, A.O. (1984) Evaluation of severity and prognosis in early stages of septicemia by means of chromogenic peptide substrate assays. Eur. Surg. Res., 2, 140-146. Smith-Erichsen, N., Aasen, A.O. and Amundsen, E. (1982a) Treatment of sepsis in the by means of chromogenic peptide substrate assays. Acta surgical patient evaluated Chir. Stand., 509, 33-38. Smith-Erichsen, N., Aasen, A.O. and Amundsen, E. (1983) The functional inhibition of plasma kallikrein. A critical factor in septic shock. In: Kinins III. Part B: 1049-1054. Editors: Fritz, Back, Dietze and Haberland. Plenum Press, New York. Smith-Erichsen, N., Aasen, A.O., Gallimore, M.J. et al. (1982b) Studies on components of the coagulation system in normal individuals and septic shock patients. Circ. Shock, 9,491-497. Smith-Erichsen, N., Loes, Oe and Osland, K. (1981) Treatment of septicemia in the intensive care unit. Acta Anaesthes. Stand., 72, 62. Svendsen, L. and Amundsen, E. (1973) Estimation of plasmin and brinase activities by means of a highly susceptible synthetic chromogenic peptide substrate. IVth International Congress on Thrombosis and Haemostasis, Vienna (Abstract). Svendsen, L., Blomblick, B., Blombiick, M. et al. (1972) Synthetic chromogenic substrates for determination of trypsin, thrombin and thrombin-like enzymes. Thromb. Res., 267. ten Cate J.W. (1982) Antithrombin-III: Clinical significance. Acta Chir. Stand., 509, 101-105. Thomas, L.L.M., Stouk, A.K., Kahle, L.H. et al. (1981) Quantitative endotoxin determination in blood with a chromogenic substrate. Clin. Chim. Acta, 116, 63-68. van der Starre, P., Sinclair, D. and Damen, J. (1980) Inhibition of the hypotensive effect of plasma protein solutions by Clesterase inhibitor. J. Thorac. Cardiovasc. Surg., 79, 738-741. Witte, J., Jochum, M., Scherer, R. et al. (1982) Disturbances of selected plasma proteins in hyperdynamic septic shock. Intens. Care Med., 8, 215-222.
Resuscitation. 14 (1986) 33-42 Elsevier Scientific Publishers Ireland Ltd.
UNCONTROLLED PLASMA THE SEPTICEMIC PATIENT
A.O. AASEN,
N. SMITH-ERICHSEN
PROTEOLYSIS:
A MAJOR
THREAT
TO
and E. AMUNDSEN
Institute for Surgical Research, Rikshospitalet and Department Central Hospital, University of Oslo, Olso 1 (Norway)
of Anesthesia,
Akershus
SUMMARY
In order to further elucidate the pathophysiological significance of plasma proteolysis during septicemia, surgical patients with septicemia were studied by means of chromogenic peptide substrate assays. In fatal cases continuous low values for prekallikrein, plasminogen and antithrombin III were found until death. At autopsy a persistent septic focus was found in all but one of the fatal cases. Very low levels of prekallikrein during sepsis and reduced functional inhibition of plasma kallikrein in septic shock indicated a poor prognosis. In the survivors the parameters returned towards the normal range upon successful therapy. Furthermore the paper demonstrates the application of a new parameter, the proenzyme functional inhibition index (PFI-index) in patients with septicemia. The data reveal that by means of this parameter patients at high risk can be identified at an early stage of the disease. Key words: Chromogenic peptide substrate assays - Proenzyme functional inhibition-index - Prognosis - Septicemia
INTRODUCTION For more than two decades activation of the plasma proteolytic enzyme systems, i.e. the coagulation, fibrinolytic complement and kallikrein-kinin system, has been recognized as a frequent phenomenon in patients with septicemia (Rapaport et al., 1964; Mason et al., 1970; McCabe, 1973; Hirsch et al., 1974; O’Donnell and Clowes, 1976). During the last decade these pathophysiological events have been extensively studied by several groups (Aasen et al., 1980; Smith-Erichsen et al., 1982a; Smith-Erichsen et al.,
Abbreviations:
PFI-index,
0300-9572/66/$03.50 Printed and Published
proenzymes
functional
0 1966 Elsevier Scientific in Ireland
inhibition
index;
Plg, plasminogen.
Publishers Ireland Ltd.
34
1982b; Smith-Erichsen et al., 1983; KaIter et al., 1982; Fritz, 1980; Witt.e et al., 1982; McCann et al., 1982; Schipper et al., 1981; BiiIler et al., 1982; ten Cate, 1982). With the introduction of chromogenic peptide substrate assays, a new and important technique for investigation of proteolytic enzymes have been made available (Svendsen et al., 1972; Svendsen and Amundsen, 1973; Blomback et al., 1974; Amundsen et ai., 1977; Amundsen et al., 1978). In 1972, the first chromogenic peptide substrate assays were introduced for thrombin, trypsin and thrombin-like enzymes (Svendsen et al., 1972). Since then analyses have been developed for determination of active enzyme, proenzyme and functional inhibition of several proteases including components of the coagulation, fibrinolytic, complement and kallikrein-kinin system (Svendsen and Amundsen, 1973; Blomback et aI., 1974; Amundsen et al., 1977; Amundsen et al., 1978; Odegard et al., 1975; Aurel et al., 1977; Friberger et al., 1978; KIuft, 1978; Bergstriim and Blomback, 1974; Amundsen et al., 1979; Aasen et al., 1982; Friberger, 1982). The principle of chromogenic peptide substrate assays is the enzymatic cleavage of the peptide-pnitroaniline amid linkage of the chromogenic peptide substrate (Svendsen et al., 1972). This results in release of the chromophorep-nitroaniline. The reaction can be monitored at A4,,5nm in a spectrophotometer. Assays can be easily automated (Bergstrom and Blomb%ck, 1974; Aasen et al., 1982; Friberger, 1982). With automated, chromogenic peptide substrate assays, the possibility has emerged to rapidly obtain profiles of activity in various protease systems based on determination of level of proenzyme, enzyme activity and functional inhibitor capacity using the same chromogenic peptide substrate (Aasen et al., 1982). Using automated enzyme ana.Iysers only small plasma volumes are required (Aasen et al., 1982). Chromogenic peptide substrates are also available for determination of several components outside the plasma proteolytic enzyme systems including measurements of endotoxin content, activity of granuiocyte elastase, trypsin, chymotrypsin and glandular kallikrein activity (Aasen et al., 1982; Friberger, 1982). Using these techniques, we have studied surgical patients with septicemia (Aasen et al., 1980; Smith-Erichsen et al., 1982a; Smith-Erichsen et al., 198613; Smith-Erichsen et al., 1983). These patients still have a high mortality rate and are a major demand on the total costs of the intensive care unit (SmithErichsen et al., 1981). Our studies disclose that information obtained by chromogenic peptide substrates assays can give information of diagnostic, therapeutic and prognostic value in these critically ill patients. The present paper summarizes our findings. MATERIALS
AND METHODS
Thirty-nine surgical patients with septicemia were studied in two separate investigations (Smith-Erichsen et al., 1982a; Smith-Erichsen and Aasen, 1984).
35
Septicemia was diagnosed when at least four of the following criteria were fulfilled: temperature above 38°C; need of respiratory support; leukocytes above 15 X log/1 or below 5 X log/l; Trombocytes below 100 X lO’/l;Positive blood culture or an obvious septic focus. Septic shock was diagnosed when patients had hypotension, oliguria and need of vasopressor in addition to the criteria for septicemia.
Sampling
As soon as the diagnosis was ascertained, the first plasma sample was made from titrated blood and immediately stored at -70°C. Thereafter, plasma samples were collected with at least 24-h intervals until analyzed. Results were retrospectively compared to clinical course and outcome.
Assays Plasma prekallikrein(PKK), functional plasma kallikrein inhibition (KKI), plasminogen (Pig) and functional antiplasmin activity (AP) were measured with chromogenic peptide substrate assays as previously described (Aasen et al., 1980; Amundsen et al., 1978; Friberger et al., 1978; Aasen et al., 1982). Functional antithrombin III (AT III) was determined with a Quantichrome AT-III kit according to the manufacturers instructions (Abbott Diagnostics Ltd., North Chicago, IL). All values were expressed as the percentage of values obtained with a standard plasma pool from healthy donors. The PFI-index was calculated from the measured values for PKK, KKI, Plg, AP and AT III as defined by Aasen (1985).
Statistics To describe centrality and distribution of data, median, 25th and 75th percentiles were determined daily for all parameters during the first 4 days of septicemia, and for the recovery and terminal values. Statistical evaluation was performed with Wilcoxons rank sum test and P-values <0.05 were considered significant. RESULTS
AS shown in Figs. 1, 2, 3 and 4, values for plasminogen, antiplasmin, antithrombin III and plasma prekallikrein were significantly reduced in 18 surgical patients with persistent sepsis compared to values found in survivors. During the first week after diagnosis, value for these parameters were not significantly different in the two groups. During the second week after diagnosis, however, plasminogen and antithrombin III values were significantly higher in patients surviving septicemia than in fatal cases. Plasma prekallikrein and antiplasmin values were not significantly different in the two groups before the third week after diagnosis (Figs. 2 and 4). In patients with septic shock determination of functional plasma kallikrein inhibition revealed significantly lower values in fatal cases than in survivors (Fig. 5). At autopsy a persistent septic focus was found in all but one of the fatal cases.
36
% 120
-
llO= loo90 -
P
*
0.05
80 70 60 -
4
50 -
\
40 -
.
\
\
’
??
: .
30 203
Day 1
Third week
FII st week
Fig, 1. Plasminogen values during septicemia in survivors and fatal cases with a persistent septic focus. 0, survivors; 0, fatal cases.
Twenty-one patients with septicemia of whom 11 died, were studied by means of the PFI-index. As shown in Fig. 6, the PFI-index values were markedly negative in both groups from day 1. Values obtained in the fatal cases, however, were significantly lower than values found in the survivors from day 1 and during the whole observation period. No one of the individual parameters included in the index calculation could so clearly discriminate % 120110. loo90.
8070. 60.
1
I
I Day
1
First week
I
I
S$c,“,$”
Thor wee R
Fig. 2. Antiplasmin values during septicemia in survivors and fatal cases with a persistent septic focus. 0, survivors; 0, persistent sepsis.
37
1
Day
1
First week
I
I
I
I
S;c,“e;d
Thir wee ‘ii
Fig. 3. Antithrombin III values during septicemia in survivors and fatal cases with a persistent septic focus. 0, survivors, 0, persistent sepsis.
between patients who died and those surviving septicemia as the PFI-index (Smith-Erichsen and Aasen, 1984). The information which can be obtained by the PFI-index is further illustrated in Figs. 7 and 8 presenting data on individual cases. As shown in Fig. 7 the PFI-index was negative for the whole observation period of 14
I
I Day
I
1
First
week
I
scz”e$p:
I Tti r(!
wee*.
Fig. 4. PKK values during septicemia in survivors and fatal cases with a persistent septic focus. ??, survivors; 0, persistent sepsis.
38
* P-
00.5
c
-*-
R.S.S
FSS
Sepsis
Fig. 5. Functional plasma kallikrein inhibition during fatal septic shock (FSS), resuscitated septic shock (RSS), septicemia without shock (sepsis) and recovery values (Ret).
days in a severely injured patient who developed septicemia. At autopsy the patient was found to have an intraabdominal septic focus due to an unrecognized perforated duodenal ulcer. In Fig. 8 the PFI-index changes found in an 82-year-old women developing septicemia due to an acute cholecystitis is shown. After surgical treatment of the septic focus the PFI-index
I
I 1
I 2
I
I
3
4
Term. DAYS’
Fig. 6. The PFI-index during the early stage of septicemia, terminally (Term) and in the recovery (Ret) phase. Median , 25th and 75th percentiles are given. 0, survivors; 0, fatal cases.
39
AUTOPSY:
-w1
2
3
4 DaYs after
5
6
14
admission
Fig. 7. The PFI-index values in a 60-year-old man with
multiple trauma.
values became significantly less negative (Fig. 8). After stopping treatment with antibiotics on the fifth day after admission, however, she develops septicemia again. This was followed by a reduction of index values. The patient was successfully treated with gentamycine followed by a normalization of the PFI-indek (Fig. 8). DISCUSSION
The studies summarized in the present paper strongly emphasize the pathophysiological importance of disturbances of plasma proteolysis during septicemia in humans.
+30
*
+10
-
-10
_
-ANTI6lOTICS
-xl CHOLECYSTECTOMY -60
--
-70
-
DRENAGE GENTAMICIN
-SO-
-1lOL 0
2
4
6
6
10
12
14
Days afnr admission
Fig. 8. The PFI-index cholecystitis.
values in an &&year-old women developing septicemia due to
40
BY means of chromogenic peptide substrate assay technique this phebe closely monitored (Aasen et al., 1982; Friberger, 1982). Upon automation of the assays, data from chromogenic peptide substrate analyses now can be made available within a few hours after sampling (Aasen et al., 1982), and data on major functional parameters of the plasma proteolytic enzyme systems have been made available for the daily evaluation and treatment of patients with septicemia. The majority of studies performed in patients with septicemia using chromogenic peptide substrate assays including the present investigation, have been done retrospectively. Prospective investigations are under way by several groups in order to further evaluate the importance of these new tools in the treatment of severely ill patients. The importance of protease inhibitor control is strongly underlined by the fact that about 10% of the plasma proteins are protease inhibitors. The observation in the present study of insufficient proteases inhibition in septicemia patients likely disclose an aspect of therapy which today is not adequately addressed. A protease-antiprotease imbalance which also is reported in other conditions such as acute pancreatitis (Lasson, 1980), might be handled in different ways. Of particular importance is the fact that concentrates of both antithrombin III and C?-esterase inhibitor are available for use in humans (Schipper et al., 1978; van der Starre et al., 1980). Furthermore activated proteases might be controlled by removal of plasma compounds using plasmapheresis (Scharfman et al., 1979; Bjorratn et al., 1984). This procedure which presently is extensively studied might be a way of eliminating activators such as endotoxin and activated monocytes (Bjorvatn et al., 1984). Endotoxin which is released from the cell wall of gram-negative bacteria is thought to be an important component in the pathophysiology of septicemia (McCartney et al., 1983). This compound can now be quantitated using a chromogenic peptide substrate assay (Scully et al., 1980; Thomas et al., 1981). Thus the elimination of endotoxin can be monitored by a bed side analysis. Recently, constituents of gram-positive bacteria also have been demonstrated to be strong activators of plasma proteolysis (Kalter et al., 1983). These findings underline that in patients with septicemia the plasma is transformed to a ‘pathological perfusate’ which might be a precipitating factor for development of multiple organ failure frequently seen in septicemia. Pathological plasma proteolysis is a major phenomenon in patients with septicemia and therapy to control this process should be started in early stages of the disease. nomenon can
ACKNOWLEDGMENT
The financial support by The Laerdal Foundation acknowledged.
for Acute Medicine is
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