Endotoxemia in Human Septic Shock* Robert L. Danner; M.D.; Ronald] Elin, M.D., Ph.D.; Jeanette M. Hosseini, B.S.; Robert A. Wesley, Ph.D.; joseph M. Reilly, M.D.; and Joseph E. Parillo, M.D.

To evaluate the incidence, pattern and clinical importance of endotoxemia in septic shock, frequent, serial endotoxin determinations were made prospectively in patients with shock. Detectable endotoxin occurred in 43 of 100 patients with septic shock, but in only one of ten patients with shock due to nonseptic causes. During septic shock, endotoxemia frequently occurred in the absence of Gram-negative bacteremia. Using a logistic regression model, multiple organ failure occurred 10.3 times more frequently and depression of left ventricular ejection fraction (:545 percent) 4.8 times more frequently in endotoxemic patients. In

a lipopolysaccharide found in GramE ndotoxin, negative bacteria, has been considered by many to be the most important toxin involved in the development of septic shock. 1-5 However, direct measurement of circulating endotoxin in septic patients has not consistently correlated with important clinical events such as bacteremia, shock and death.?" Although the use of the limulus amebocyte lysate assay to measure endotoxin has been shown to be sensitive," relatively specificv" and to correlate with in vivo toxicity, II a number of problems have been identified.8.12-14 Many clinical studies on endotoxemia relied on a limulus amebocyte lysate assay that used gelation as an endpoint, h but clinically significant endotoxemia may occur below the sensitivity of this test.4.7 Further, endotoxin is rapidly cleared from the circulation in the normal animal or human host.ls. 16 Transient episodes of end~toxemia may go undetected when only single or infrequent endotoxin determinations are performed.

·From the Critical Care Medicine Department (Drs. Danner, Reilly and Parrillo), the Clinical Pathology Department (Drs. Elin and Hosseini), and the Information Systems Department (Dr. Wesley), Warren G. Magnuson Clinical Center, National Institutes of Health, Bethesda, Md. This study was approved by the Clinical Research Subcommittee of the National Institute of Allergy and Infectious Diseases and conforms to the ethical principles and guidelines for the protection of human subjects of research as set forth by the US Department of Health and Human Services, Written, informed consent was obtained from all subjects or their surrogates. Portions of this work were presented at the National Meeting of the American Federation lilr Clinical Research, Washington, DC, April 1988 (Clinical Research 1988; 36:370A and 1988; 36:453A) and at the Annual Meeting of the Society of Critical Care Medicine, Orlando, FL, May 1988 (Critical Care Medicine 1988; 16:397). Manuscript received June II; revision accepted July 30.

Reprint requests: Dr. Danner; Bldg 10, Rm 10048, National Institutes oJ Health, Bethesda 20892

patients with positive blood cultures, endotoxemia was associated with a high mortality. We conclude that endotoxemia occurs frequently in septic shock and is associated with severe manifestations of this syndrome, including cardiac depression and multiple organ failure. This study suggests that endotoxin is an important mediator of septic shock and supports efforts to develop anti-endotoxin therapies for treating patients with this disease.

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EU = endotoxin units

(Cheat 1991; 99:169-75)

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Several recent reports have avoided some of these limitations by using a chromogenic limulus amebocyte lysate assay that is very sensitive and quantifiable and serial sample collection. 17'20 These investigations revealed associations between endotoxemia and Gramnegative bacteremia,'? clinically defined septicemia!" and the development of ARDSI9 in various patient populations. Further, a recent report correlated endotoxemia with several important clinical endpoints (renal failure, ARDS, severe shock and death) but specifically only studied patients with systemic meningococcal disease. 20 . In this investigation, we prospectively evaluated the occurrence of endotoxemili in septic shock. Frequent, serial endotoxin determinations were made using a sensitive chromogenic limulus amebocyte lysate assay in 110 consecutive patients admitted to a medical intensive care unit with shock. One hundred patients were diagnosed as having septic shock, and ten patients were found to have nonseptic causes of shock. All patients were hemodynamically monitored and underwent sequential, comprehensive cardiovascular evaluations including radionuclide-determined left ventricular ejection fractions. The role of endotoxin in the laboratory and hemodynamic abnormalities, organ failure, cardiac depression and mortality of septic shock was examined. METHODS

lttients All patients admitted to the medical Intensive Care Unit of the National Institutes of Health between December 1984 and February 1987 were considered for this investigation. Prospective criteria for study entry included fever (temperature >38°C), hypotension (mean arterial pressure :560 mm Hg), a normal or high cardiac output (cardiac index ~3.0 Umin/m2 ) and no discernible cause for shock CHEST 1991 1 I JANUARY, 1991

169

other than septicemia. Of the 113 consecutive patients who met this clinical definition of septic shock, 100 consented to participate in the study. Ten additional patients with an identifiable nonseptic cause of shock (hemorrhage in five patients, adrenal insufficiency in two patients, and myocardial infarction, pulmonary embolus and neurogenic shock in one patient each) and without fever or positive blood cultures also gave informed consent and were evaluated.

Hemodynamic and lAboratory Evaluation All patients were monitored using systemic and pulmonary artery catheters. Patients were evaluated and treated using a standardized protocol as previously outlined...... Briefly, serial hemodynamic profiles were obtained on each patient every 4 hs from the time of admission to the intensive care unit until the shock resolved or the patient died. Hemodynamic profiles included heart rate, mean arterial pressure, central venous pressure, pulmonary capillary wedge pressure and thermodilution cardiac output. Systemic vascular resistance was calculated by the standard formula. "' In addition, serial left ventricular ejection fractions were determined using gated radionuclide scans."' Shock (mean arterial pressure S 60 mm Hg) was treated initially by fluid resuscitation to maintain a pulmonary wedge pressure of 12 to 15 mm Hg. If hypotension persisted, dopamine was started and titrated to a dose S20 p.g/kgf min as required. Ifhypotension still persisted, norepinephrine was started and the dopamine was decreased to renal perfusion levels (2 to 4 p.g/kgfmin). Routine laboratory tests were performed at least three times daily and included arterial blood gas determinations, serum electrolyte values, blood urea nitrogen concentration, creatinine level, lactate concentration, complete blood cell counts, prothrombin time, partial thromboplastin time and fibrinogen levels. Standard liver function tests were done daily. At study entry, three sets of blood cultures from different sites and a urine culture were obtained from all patients. Subsequent blood and urine cultures were collected as required for the evaluation of new or persistent fever. Sputum cultures were obtained on all intubated patients and as clinically indicated.

Endotoxin Determinations Serial blood samples were collected in sterile, pyrogen-free glass tubes containing preservative-free heparin (5 USP unitslml of blood) every 4 hs for the initial 24 hs, every 12 hs for the next 24 hs, and then daily for three days. The blood was centrifuged at 2,000 g for 10 min and the plasma stored at - 2(fC until assayed. A chromogenic limulus amebocyte lysate assay (Whittaker M. A. Bioproducts, Walkersville, MD) was used to measure endotoxin. In previous investigations, we have not observed a difference between plateletrich and platelet-poor plasma with this assay.t:> A total of 1,064 endotoxin determinations were performed on no patients by one technologist blinded to the clinical data. On the day of the assay, specimens were thawed, diluted 1:10 with pyrogen-free distilled water and then heated to l00"C for 10 min to remove nonspecific activators or inhibitors of limulus amebocyte lysate. The treated sample (50 p.l)and limulus amebocyte lysate (50 p.1) were then incubated together for 10 min at 37"C in pyrogen-free plastic microtiter tissue culture plates (Costar, Cambridge, MA). Chromogenic substrate (100 p.1) was added to the reaction mixture followed by 3 min of continued incubation. Endotoxin in a specimen activates a proenzyme in the lysate that then cleaves p-nitroaniline from the chromogenic substrate. The reaction was stopped by the addition of 50 percent glacial acetic acid (100 p.l) and absorhances were measured spectrophotometrically at 405 nm (1iterteck, Multiscan MC, Flow Laboratories, McLean, VA). The absorbance of an aliquot of each diluted plasma sample, processed without limulus amebocyte lysate or substrate, was subtracted from the absorbance of the measured aliquot to correct for the intrinsic color of individual specimens. The endotoxin

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concentration in endotoxin units per milliliter of plasma (1 EU = 100 pg of US standard endotoxin) was then read from a standard curve based on known amounts of purified reference endotoxin assayed in HIO. This method is sensitive to 10 pgfml of US standard endotoxin. All endotoxin-negative samples were retested using pyrogen-free glass tubes and a 3O-min incubation, which increases the sensitivity of the assay to 1 pgfml. Patient samples containing endotoxin concentrations higher than the upper limit of the standard curve were diluted further and retested. All positive patient specimens were confirmed by repeat analysis. A reference sample of pooled plasma spiked with a known quantity of endotoxin was run with each assay to assess the efficiency of endotoxin recovery. The plasma samples from the seven patients found to have Candida septicemia also were assayed using a limulus amebocyte lysate fraction that is nonreactive with yeast cell wall components (Endospecy, Seikagaku Kogyo Co Ltd, To\cyo, Japan)." Similar results were obtained fOr the two assays.

Quantitationof Serum C.. -lilre Activity Serum samples were collected at the time of study entry and then daily for determination of C..-like activity. The C..-like activity was measured using a semiquantitative bioassay technique based on neutrophil aggregation as previously described.... Briefly, 50 p.1 of test serum was combined with 5 X 10"neutrophils in a siliconized cuvette. Aggregation of neutrophiIs at 37"C was quantitated using an aggregometer (model 300-5, Payton Associated, Buffalo, NY) that measured the increase in light transmittance associated with aggregation. Aggregation was expressed in units of deflection from baseline (1 unit=2.5 em) as recorded by a PVI0R-0 Recorder (Payton Associates). Normal control sera (n = 40) produced minimal neutrophil aggregation, 0.16± 0.02 units (mean ± SEM) with a range of 0.0 to 0.4 units. A deflection greater than or equal to 0.4 units was determined to be above the 95 percent confidence limits for this normal population.

Definitionof ClinicalEndpoints Clinical endpoints were defined prospectively and each determination was made by two clinicians without access to the endotoxin data. The development of renal insufficiency was defined as a serum creatinine of ~ 180 p.moVL (2 mgfdI) with an acute rise of ~50 percent over baseline, pre-shock creatinine concentrations. The ARDS was diagnosed by four criteria: hypoxemia requiring a fractional inspired oxygen concentration ~4O percent to maintain the partial pressure of arterial oxygen >60 mm Hg; roentgenographic findings of bilateral alveolar or interstitial infiltrates or both; normal intravascular volume with a pulmonary wedge pressure S 18 mm Hg, and an appropriate clinical setting (the absence of another specific pulmonary diagnosis). Patients were considered to have survived the episode of shock if they were discharged from the intensive care unit or were still alive without signs of shock two weeks after entering the study. Statistics

Means are expressed plus or minus the standard error of the mean in the text, tables and figures. In addition to the septic and nonseptic shock groups, septic patients were divided by the presence or absence of either detectable endotoxemia or documented bacteremia. For the purpose of analysis, certain variables that were determined more than once for each patient needed to be represented by one number which accurately reflected disease severity. Lactate concentrations, C..-like activity and bilirubin levels were summarized by using the single highest (peak)determination (1llble 1 to 4). The multiple fibrinogen concentrations, systemic vascular resistances and left ventricular ejection fractions obtained during an episode of septic shock were expressed with the single lowest Endomemia in HumanSepticShock (Denner et eI)

Table l-ClwracteriBtia of110 Ibtienta with Shock Evaluated for Endotoxemia Characteristics Age (yr) Lactate (mrnol/L) Left ventricular ejection fraction (%) Endotoxemia (%) Vasopressor therapy required (%) Renal insufficiency (%) ARDS(%) Mortality (%)

Nonseptic (n=10)

p Value*

Characteristics

Present

Absent

p Value*

46:t2 4.1 :tOA 4O:t 1

48:t2 7.6:t2.6 53:t5

NS 0.023 0.010

23 (62) 52:t3.0 6.1:t1.2 89:t 19

14 (38) 45:t0.6 2.8:t0.6 34:t 12

NS NS (0.061) 0.042

43 55

10 60

0.042 NS

430:t4O

7l0:t78

28 37 24

40 20

NS NS NS

No. of patients (%) Age (yr) Lactate (mmoVL) Bilirubin (....moVL Systemic vascular resistance (dyneS"S"CIll- O) Vasopressor therapy required (%) Renal insufficiency (%) ARDS(%) Both renal insufficiency andARDS(%) Mortality (%)

17 (74) 12 (52) 15 (65)

6 (43) 2 (14) 2 (14)

NS (0.059) 0.021 0.003

10 (44) 9 (39)

0 (0) 1 (7)

0.0038 0.034

50

(nadir) value for each patient (Table 1, 2, 4 and Fig 1). Comparisons among groups were performed using the chi-square statistic for frequency variables and unpaired t tests for continuous variables.

Table 2-ClwracteriBtU:s oj 100 Ibtienta with Septic Shock Evaluated for Endotoxemia Endotoxemia

No. of patients (%) Age (yr)t Underlying disease (%) Hematologic malignancy Solid tumor AIDS Liver disease Aplastic anemia Other Laboratory findings Positive blood culture(%) Lactate (mmollL) Co.-like activity (units of deflection) Bilirubin (....moVL) Fibrongen (gIL) Cardiovascular Evaluation Systemic vascular resistance (dynes-s-cm" 0) Left ventricular ejection fraction (%) Clinical Outcomes Vasopressor therapy required (%) Renal insufficiency ARDS Both renal insufficiency andARDS Mortality

Endotoxin

Septic (n= 1(0)

*Two-tailed chi square or t test as appropriate. NS denotes p 20.05.

Characteristics

Table 3-1btienta with Positive BloodCultures (n = 37)

p Value*

Present

Absent

43 (43) 49:t2.3

57 (57) 44:t2.3

NS

24 (56)

26 (46)

NS

13 (30) 1 (2.3) 2 (5) 2 (5) 1 (2.3)

16 8 0 0 7

(28) (14) (0) (0) (12)

NS NS (0.08) NS NS

23 (53)

14 (25)

0.003

5.5:t0.8 0.5:t0.06

3.0:t0.3 0.36:t0.03

0.002 0.017

68:t 12 2.8:t0.2

53:t 12 3.5:t0.2

NS 0.018

456:t3O

582:t33

0.013

34:t2

45:t2

0.0001

28 (65)

27 (47)

19 (44)

9 (16)

0.002

22 (51)

16 (37)

15 (26) 3 (5)

0.01 0.0006

12 (28)

12 (21)

0.001

*Two-tailed chi square or t test as appropriate. NS denotes p 20.05. All reported p values are two-tailed. Ejection fractions were first compared among groups by doing an analysis of variance. Since this result was statistically significant (p = 0.0001), group pairs were compared using t tests without inflating the experiment-wise error rate. Multivariate analysis was performed using a logistic regression model." The clinical outcomes of renal insufficiency, ARDS, or

Table 4-Multiooriate Analysis ojClinical Outcomes-

Variable

Outcome Death Renal insufficiency ARDS Both renal insufficiency and ARDS Vasopressor therapy required Left ventricular ejection fraction

{

Sex Lactate

{

Sex Endotoxin SVR

{

Age Sex Endotoxin

{

Sex Endotoxin SVR Sex WBC Lactate SVR Endotoxin

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Odds Ratio 3.5 2.5 2.6 3.6 2.9 2.8 1.9 2.1 2.8 10.3

504 3.2 2.8 3.8 3.9 4.8

95% Confidence Intervals

p Valuet

1.0, 12.6 0.8,7.5 0.8,8.1 1.1, 12.2 1.0,8.7 1.1,7.3 0.7,5.3 0.7,5.9 0.7,11.7 1.9,54.8 1.3,22.7 1.6,9.1 0.9,8.6 1.1, 12.8 lA, 11.1 1.6, 14.2

0.050 0.110 0.110 0.036 0.060 0.038 0.196 0.162 0.148 0.006 0.022 0.026 0.072 0.030 0.010 0.006

(:545%)t

NS (0.08)

NS

*Two-tailed chi square or t test as appropriate. NS denotes p2O.05. tContinuous variables are expressed as means:t SEM.

*Eight independent variables were used in each logistic regression model: age «50 years or 2:50 years); sex; the presence or absence of documented bacteremia; white blood cell count «LOX 10"1L or 21.0X 10"1L); serum lactate concentration «4 mmoVL or 2 4mmoVL); the presence or absence of detectable endotoxemia; systemic vascular resistance «500 dynes-s-cm:" or 2500 dynes-s-cm -0); and left ventricular ejection fraction (>45 percent or s45 percent). tlndependent variables with p values 20.2 are not shown in the table. tLeft ventricular ejection fraction not used as an independent variable in this regression. CHEST 1991 1 I JANUARY, 1991

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f---l

Nonseptic Shock

Septic Shock without Endotoxemia

H

Septic Shock with Endotoxemia

o

H

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10

20

30

40

50

60

Left Ventricular Ejection Fraction (%) FICURE

1. Nadir left ventricular ejection fractions in patients with

nonseptic shock (n =10), septic shock without endotoxemia (n =S7) and septic shock with endotoxemia (n = 43).

both, and severe shock requiring vasopressors (dopamine ~S ....gt Icgtmin or norepinephrine) were all significantly associated with mortality (p

Endotoxemia in human septic shock.

To evaluate the incidence, pattern and clinical importance of endotoxemia in septic shock, frequent, serial endotoxin determinations were made prospec...
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