Qualitative and quantitative detection of endotoxin in serum

Endotoxins are large (molecular weight, 200,000 to 1,000,000), heat-stable lipopolysaccharides (LPS) which are the major components of the cell wall of the gram-negative bacterium.There are more than 20 assays for the detection of endotoxin (McCabe, W. R., 1980), of which three have been used for the detection of endotoxin in clinical specimens: the rabbit pyrogen assay, the LAL

bioassay, and immunoassays. The method of choice would appear to be the LAL assay. The advantages of this assay are increased sensitivity, potential for quantitation, reactivity with the biologically active component lipid A, and relative convenience of operation.

The LAL Assay

In 1956, Bang (Bang, 1956) discovered that the endotoxin of a Vibrio species from seawater, pathogenic for the horseshoe crab (Limulus polyphemus), caused fatal intravascular coagulation and that endotoxin induced activation of this process in vitro. Levin, Bang, and coworkers subsequently showed that this coagulation was the result of an endotoxin-initiated reaction causing the enzymatic conversion of a clottable protein derived from the circulating blood cell (amebocyte) of the crab (Levin, et al.,1968; Young, et al., 1972). They recognized the potential for this biological reagent as a diagnostic tool and characterized its properties. A lysate from the amebocyte is extremely sensitive to the presence of endotoxin.

Coagulation system of L. polyphemus.

The coagulation system of L. polyphemus consists of several enzymes which are arranged in three pathways in a fashion which resembles the classic, alternate, and common mammalian coagulation cascade pathways, the components of which activate each other in a ‘‘cascade’’ sequence. The coagulation system of the Japanese horseshoe crab, T. tridentatus, which is considered homologous to the L. polyphemus American horseshoe crab, has been studied extensively (Fig.3) (Iwanaga, S., 1993; Iwanaga, et al., 1985). This

cascade sequence results in an amplification of the original stimulus which accounts for the sensitivity of the Limulus coagulation system to endotoxin at picogram-per-milliliter (10-12 g/ml) concentrations. An additional component of Limulus amebocytes is an anti-LPS factor which has anti-endotoxin properties (Warren, et al., 1992).
Gel clot LAL assay.
In the original version of the gel clot test, the endotoxin-activated clotting enzyme cleaves the coagulogen to form a clot. To perform this test, a small amount of LAL solution is added to an equal volume of a sample or a standard dilution in a small test tube. If, after an appropriate incubation time, a firm gel clot is formed, the test is scored positive. A firm gel clot is one that remains solid in the bottom of the reaction tube when the tube is inverted. Methods to

enhance the visualization of clot formation in microtiter volumes have been described (Gardi, et al, 1980; Hussaini, et al., 1987; Prior, et al., 1979). With all gel clotbased techniques, a semiquantitative result can be obtained through serial dilution of samples and standards.

Coagulogen-based LAL assay.
The limitations of the gel clot LAL test are the subjective endpoint and the relative lack of sensitivity. To overcome these limitations, various methods to quantitate the progress of the reaction leading to coagulogen conversion have been employed, for example, through monitoring the increase in turbidity (Dubczak, et al., 1979; Urbaschek, et al., 1985), the loss of coagulogen as the clot forms (Baek, 1983; Zhang, et al., 1988), the increase in precipitated protein (Nandan, et al., 1977; Nandan, et al, 1977), or the appearance of a peptide cleavage fragment of coagulogen (Zhang, et al., 1994).

Chromogenic LAL assay.
In the chromogenic LAL assay method (Iwanga, et al ., 1978), the coagulogen is completely or partially removed to be replaced by a chromogenic substrate (Scully, et al., 1980), a small synthetic peptide linked to a chromophore (para-nitroaniline) containing an amino acid sequence similar to that present at the site in the clotting protein cleaved by the clotting enzyme (X-Y-Gly-Arg-pNA). The chromogenic LAL assay usually has two stages: a LAL activation stage and, following the addition of the chromogenic substrate to the reaction mixture, a chromophore release stage. Release of the chromophore imparts a yellow color to the solution. The strength of the yellow color (as measured by optical density [OD] at 405 nm in a spectrophotometer) is a function of the amount of active clotting enzyme (and indirectly to the amount of endotoxin)

present in the solution. Both phases of the chromogenic reaction are critically time and temperature dependent, but within these limitations the chromogenic assay is sensitive to 10 pg/ml (Thomas, et al., 1981). A single-step chromogenic assay has been described (Duner, K. I., 1993; Lindsay, et al., 1989).

Specificity of the LAL Assay

The two pathways leading to the coagulation of LAL, one activated by endotoxin triggered by factor C and the other activated by b-glucans triggered by a glucan-reactive factor G, can be specifically blocked by polymyxin and laminarin, respectively (Zhang, et al., 1994). Hence, reactivity with the LAL assay that is inhibited by polymyxin B can be used as specific evidence for endotoxin. LAL derived from the Japanese horseshoe crab and from which this factor G has been removed has been promoted as an endotoxin-specific reagent (Obayashi, et al., 1985, Obayashi, et al., 1986).

LAL Endotoxin Assay for Blood Samples

When the LAL assay is used to detect endotoxin in blood, two obstacles are encountered: (i) the complex and poorly understood inhibitory factors and (ii) the levels of endotoxemia generally being at the limit of test detection. Schematic overview 1. illustrates the complex interaction among components of blood, endotoxin, and LAL. Endotoxin interacts with several components of plasma, including bile salts, proteins, and lipoproteins, leading to disaggregation, some inactivation, and the formation of complexes. These multiple effects of plasma on the activity of endotoxin are not always apparent as inactivation. (Beller, et al., 1963).


Inhibition by plasma and serum.
The loss of reactivity to LAL on addition of endotoxin to plasma or serum is partly reversible, in that reactivity can be restored by dilution with distilled water or saline (Levin, et al., 1970), and partly irreversible (Johnson, et al., 1977). The ability of plasma or serum to inhibit endotoxin activity is time dependent and temperature sensitive, being maximal at 37 to 45°C and abolished after plasma or serum is heated at 60°C for 5 min, and varies in proportion to the endotoxin potency. These characteristics imply an enzymatic inactivation of endotoxin by native plasma (Johnson, et al., 1977; Novitsky, et al., 1985, Novitsky, et al., 1985, Obayashi, T., 1984, Olofsson, et al., 1986, Webster, et al., 1980), although this has yet to be definitively demonstrated.

Endotoxemia without Sepsis
Liver disease. Endotoxemia has been suspected of having pathogenic properties in patients with liver disease even in the absence of overt gram-negative sepsis (Nolan, J. P., 1975). The origin of endotoxin in this setting is also believed to be from the gastrointestinal tract because several studies have found a portal-to-systemic gradient of endotoxin level, with higher level in portal venous blood than in peripheral blood ( Bigatello, et al, 1987; Jacob, et al, 1977; Lumsden, et al, 1988; Prytz, et al, 1976).

Hemodialysis. pyrogenic reactions are an important problemwith hemodialysis, and there is concern that this is due to contamination of the dialysis water with bacteria or endotoxin (Pegues, et al, 1992; Raij, et al, 1973) or contamination resulting from the use of reprocessed dialyzers (Flaherty, et al, 1993; Gordon, et al, 1988). There is uncertainty as to whether endotoxin is able to cross the different types of dialyzer membranes and also whether the LAL-

reactive material (LAL-RM) found in the plasma of patients undergoing hemodialysis is something other than endotoxin. It is suggested that the LAL-RM is a cellulose-based material, possibly (1-3)-β-D-glucans, which has properties distinct from endotoxin (Roslansky, et al, 1991) and reacts with the factor G-drive pathway of LAL (Zhang, et al,1994). An endotoxin specific assay which does not react with (1-3)- β-D-glucans has been developed and applied (Taniguchi, et al, 1990). In any event, LAL testing of plasma of hemodialysis patients has limited ability to detect pyrogenic reactions, having positive and negative predictive values of less than 70% (Gordon, et al, 1992).

Intestinal endotoxemia. An origin from the gastrointestinal tract has often been presumed for endotoxemia in patients with gastrointestinal diseases (Cooperstock, et al, 1985; Wellmann, et al, 1984) and also in patients receiving radiotherapy to the abdomen in association with symptoms of nausea (Maxwell, et al, 1986).

Other conditions. Transient endotoxemia occurs in patients undergoing minimally invasive procedures of the urinary (Garibaldi, et al, 1973; Robinson, et al, 1975; Tanaka, et al, 1988), biliary (Lumsden, et al, 1989), or gastrointestinal (Kelley, et al, 1985) tract. In general, the severity of symptoms and the degree or frequency of detection of endotoxemia in these patients are higher when gram-negative bacteria are found at the sites of these procedures. In premature neonates, there is an association between endotoxin in cord blood and growth of gram-negative bacteria from placental samples (Scheifele, et al, 1984).