Usefulness of the endotoxin activity assay to evaluate the degree of lung injury

Yuichiro Sakamoto*

Department of Emergency and Critical Care Medicine Faculty of Medicine, Saga University, Saga, Japan

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What is ARDS?

The current concept of acute respiratory distress syndrome (ARDS) is a pathological response of the lung to an insult(s). The basis for this concept was first established in a landmark case report published by Ashbaugh and colleagues in 19671. This article recognized that ARDS is a group of related pathological abnormalities in the lung initiated by a wide variety of different insults, such as sepsis, trauma and aspiration of gastric contents. The initial description of ARDS was not specific enough to distinguish it from other lung diseases. Nevertheless, it served as a guiding principle for the diagnosis of this syndrome. In 1988, Murray and colleagues2 proposed a more precise definition of ARDS using a lung injury scoring system. However, this definition was not practical enough to be accepted globally. It was not until 1994 that the American-European Consensus Conference (AECC) on ARDS formulated the first clear definition of the syndrome to be adopted internationally3. The Committee recommended that ARDS be defined as “a syndrome of inflammation and increased permeability of the pulmonary capillaries caused by an insult(s).” In addition, the severity of hypoxemia necessary to make the diagnosis of ARDS was defined by the ratio of the partial pressure of oxygen in the patient’s arterial blood (PaO2) to the fraction of oxygen in the inspired air (FiO2). ARDS was defined as a PaO2/FiO2ratio of less than 200. When the impairment of oxygenation was less severe (i.e., PaO2/FiO2 201 - 300), the syndrome was classified as acute lung injury (ALI). This definition of ARDS remained unchanged until 2012, when it was further refined and given the name “Berlin definition.” In the new definition, ARDS was classified into the following three categories based on the degree of oxygenation (PaO2/FiO2 ratio): mild (PaO2/FiO2 201-300), moderate (101-200) and severe (≤100). According to the Berlin definition, ARDS is an acute diffuse, inflammatory lung injury, leading to increased pulmonary vascular permeability, increased lung weight, and loss of aerated lung tissue with the presence of bilateral opacities consistent with pulmonary edema. Another major change in the Berlin definition was that the term “acute lung injury (ALI)” was abandoned, thus eliminating the confusion derived from the previous AECC criteria4.

ARDS as pulmonary damage

Diffuse alveolar damage (DAD) is the pathologic hallmark of ARDS. However, among patients who met the Berlin definition of ARDS, as few as 12% of patients with mild ARDS had DAD, as did only 58% of patients with severe ARDS5. This indicates the limitations of the Berlin definition in predicting specific pathologic events in the lung. Irrespective of the definition of ARDS, it is critical for health care professionals to make an accurate diagnosis of the syndrome and initiate treatments that will improve clinical outcomes. The most important modalities in diagnosing ARDS are imaging modalities such as computed tomography (CT) of the chest. On CT scans, asymmetrical consolidation can be observed in the gravity-dependent, high-blood-flow regions of the lung in patients with ARDS. Importantly, the degree of diffuseness of heterogeneous opacities observed on CT scans is correlated with the severity of ARDS and the volume of the physiologic dead space6. Thus, the imaging result is also correlated with the outcome of patients with this syndrome. In addition, studies have shown the potential of imaging in identifying pathological features of DAD7. Therefore, imaging is useful for evaluating the effects of ARDS treatments and prognosticating the outcome of the syndrome. On the other hand, there are limitations with CT scan. Breathing function is not always correlated with the diagnostic imaging result. Therefore, the diagnostic image is not directly associated with the treatment strategy.

Objective diagnosis of pulmonary damage

Right ventricular dysfunction and pulmonary hypertension are reportedly correlated with poor outcome of ARDS8,9. These observations suggest that novel treatment strategies (for example, mechanical ventilation and pharmacological interventions) targeting these abnormalities could improve the outcome of ARDS. Thus, right ventricular dysfunction and pulmonary hypertension can be considered as objective, though supportive, diagnoses that directly lead to important clinical management decisions8,9.

Major approaches in studies on the pathogenesis of ARDS include analysis of blood and bronchoalveolar lavage fluid samples. Studies utilizing these approaches have reported that the pathogenesis of ARDS is associated with alterations in surfactant function, increases in proinflammatory cytokine levels, and damage to the endothelial surfaces of the lung10,11,12. Several researchers analyzed multiple biomarkers simultaneously to improve the accuracy of their diagnostic capabilities. In this article, we will discuss the usefulness of endotoxin activity assays in evaluating lung injury due to sepsis. Analysis of the levels of two biomarkers, i.e., endotoxin and neutrophil gelatinase-associated lipocalin, has been reported to increase the sensitivity and specificity for the diagnosis of sepsis-induced acute kidney injury13.

Sepsis and endotoxin

The most common cause of ARDS is sepsis, a life-threatening condition that arises when the body's response to infection injures its own tissues and organs. A recent study reported that the annual incidence of sepsis in Western countries is 377 cases per 100,000 population14. This is much higher than the annual rates of 223 cases for stroke and 208 cases for heart attacks. In the United States, about 200,000 people die from sepsis every year. Over the past decade, the number of patients with sepsis increased by 8 - 13% each year15. Possible reasons for this increase include population aging, multidrug-resistant organisms, malnutrition, poverty and shortage of vaccines. The increase in health care costs is another major factor.

Despite enormous efforts in developing novel compounds for the treatment of sepsis, no specific therapeutic agent is currently approved for sepsis in Japan. Activated protein C was once widely accepted as a therapy for sepsis. However, the use of this agent is not recommended in the present guidelines for the management of sepsis. In a review article, leading intensive-care physicians from many different Western countries recommended a select number of therapeutic agents and strategies for the treatment of sepsis16. These included statins, low-dose steroids and genetically-engineered thrombomodulin. Another promising treatment modality described in that article was an endotoxin adsorption column called PMX16. Endotoxin is one of the principal components of the outer membrane of Gram-negative bacteria. It is the most prominent alarm molecule sensed by the host's innate immune system following invasion by Gram-negative bacteria. PMX removes endotoxin from the circulating blood by adsorption, thus preventing the onset and progression of sepsis17.

Endotoxin removal

ToraymyxinTM, a PMX cartridge for hemoperfusion, was developed in Japan. It is comprised of polymyxin B covalently linked to the surface of chemically modified sea-island-type composite fibers. Polymyxin B is an antibiotic that binds endotoxin with high affinity. The use of fibrous materials made it possible to increase the adsorbing surface area. It also allowed for the construction of a column that exhibits a low blood pressure drop in the blood flow compartment. Animal studies showed favorable biocompatibility of Toraymyxin (18). They also demonstrated improved survival in animals with endotoxin treated with this device. These results suggested the potential of Toraymyxin for clinical application18.

In Japan, PMX has been clinically used in patients with sepsis since August 1994. And recently PMX has been used in patients with ARDS. PMX was used in approximately 1,000 sepsis cases during the first year, but two decades later, in 2013, the use of PMX has increased ten-fold. The cartridge is mainly used in intensive care units (ICUs), emergency medical centers and dialysis centers. PMX is most effective in treating septic shock from abdominal infections, such as diffuse peritonitis resulting from lower gastrointestinal tract perforation. However, it is also used for the treatment of severe sepsis and septic shock from respiratory tract infections (such as pneumonia), urinary tract infections and soft tissue infections. Toraymyxin was certified as being in compliance with the requirements of the European Medical Device Directives and obtained a CE mark in 1998. In 2003, clinical application of this cartridge began in Italy. Since then, its clinical use has expanded to several other European countries such as Spain, Russia and Switzerland and Asian countries such as India and Taiwan. In countries outside of Japan, similar endotoxin adsorption columns have been developed and marketed. Furthermore, different types of endotoxin removal devices have been invented. Thus, endotoxin is now universally accepted as a promising target for the treatment of sepsis19.

Measurement of endotoxin levels

A major problem in the techniques used for quantifying endotoxin levels has been their low sensitivity. To address this problem, a diagnostic kit called the Endotoxin Activity Assay (EAATM, Spectral Medical Inc., Toronto, Canada) was developed. In the EAATM, a monoclonal antibody against lipopolysaccharide (LPS) makes immune complexes with endotoxin in the whole blood. Upon opsonization of the complexes with complement, they are phagocytosed by neutrophils in the blood, leading to the production of reactive oxygen species (ROS). The production of ROS is enhanced by internalization of zymosan, which is included in the reagents.

The ROS then oxidize luminol in the reagents, resulting in light emission. Because of this chemiluminescence-based immunoassay platform, the EAATM shows a higher sensitivity than other endotoxin assay protocols20.

EAATM for evaluating pulmonary damage

The degree of lung injury must be determined when formulating treatment strategies for patients with sepsis and prognosticating the outcome of this condition. Based on the observations described above, we hypothesize that endotoxin can serve as an important biomarker in evaluating lung injury. We expect that the roles of endotoxin in the pathogenesis of sepsis will be the focus of future studies, as LPS has been reported to stimulate the innate immune response even without binding to toll-like receptor21. Currently, lung injuries in patients with septic shock or ARDS can be assessed by cardiorespiratory monitoring.

One such monitoring system is called the pulse index contour cardiac output (PiCCO) system (Pulsion Medical Systems, Munich, Germany). It measures cardiac output (CO) using a thermodilution technique that employs a cold thermal indicator. It then calculates CO per beat using pulse contour analysis. The PiCCO system allows monitoring of the intravascular volume status and may be used to guide volume therapy in patients who have severe sepsis or are critically ill. The extravascular lung water (EVLW) and the pulmonary vascular permeability index (PVPI) have been shown to be strongly correlated with the severity of pulmonary edema22.

Using the PiCCO system, we monitored cardiorespiratory dynamics in 11 critically ill patients who required a respirator in our ICU. In the same patients, we also measured the levels of endotoxin (EAA units) and the inflammatory markers C-reactive protein (CRP) and procalcitonin (PCT). We analyzed the potential correlation between cardiorespiratory dynamics and these biomarkers. The results indicated that patients with high EVLW values tended to exhibit higher levels of EAA than those with normal EVLW values (0.46 ± 0.20 vs. 0.21 ± 0.19), although the difference was not statistically significant (p = 0.0664). Similarly, the PCT level tended to be higher in patients with high PVPI compared with those with normal PVPI (18.9 ± 21.8 vs. 2.4 ± 2.2, p = 0.0676). In contrast with these observations, PVPI was significantly higher in patients with high EAA levels than in those with normal EAA levels (3.55 ± 0.48 vs. 1.99 ± 0.68, p = 0.0029). In addition, the patient group with high PCT levels showed significantly lower cardiac indices than the group with normal PCT levels (3.40 ± 1.05 vs. 4.80 ± 0.39, p = 0.0325).

The EAATM was designed to measure the systemic level of endotoxin using whole blood. Although there is a possibility that activated neutrophils in the blood may reduce the sensitivity and specificity of the assay, clinical studies have so far demonstrated that the EAATM can be effectively used to evaluate the medical condition of patients with sepsis. The results described above indicate that the EAA level is closely correlated with the degree of lung injury assessed by the PiCCO monitor. This suggests that the EAATM could also be used as a valuable tool in monitoring lung injury. Further studies will be necessary to determine whether the EAATM can provide useful information in diagnosing the severity of ARDS and tailoring treatment strategies for this life-threatening lung condition. We have only a little data at present. However, the possibility of the usefulness of the EAATM is indicated.

DHP-PMX

In 1994, a polymyxin B-immobilized fiber column (DHP-PMX; Toray Industries Inc., Tokyo, Japan) was developed for direct hemoperfusion in Japan and has since been used for the control of endotoxemia in patients with septic shock. The use of a polymixin B-immobilized fiber column has been shown to reduce serum endotoxin levels23. However, adsorption materials using a DHP-PMX are not proved completely. Several clinical papers showed the effectiveness of this column have also been reported24,25; for example, promising clinical data showed an increase in the systolic blood pressure (SBP) and improved PaO2/FiO2 ratio26. Reduced levels of inflammatory cytokines and other mediators after DHP-PMX have also been reported27.

The PMX-DHP Clinical Study Group in Japan reported that the adsorption of plasma endotoxins with a PMX column may have beneficial effects on the symptoms and prognosis of patients with severe sepsis, including those with septic shock23-27. Because the PMX column had been developed to eliminate endotoxin from the peripheral circulation, reductions in many sepsis-related factors have also been reported with the use of the PMX column27, but adsorption of endotoxin is thought to be the main effect. The limulus test, which was established as a test to measure the endotoxin level in the blood, has several problems, including those posed by the presence of a response inhibitor factor and the longer time needed for the measurement of low concentrations. In the limulus test, limulus amoebocyte lysate (LAL) reacts with bacterial endotoxin to form gel clots. The gelation time for the entire LAL solution depends on the concentration of endotoxin in the sample. The LAL cascade reaction is extremely slow, and gelation does not proceed when the level of endotoxin is extremely low.

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