Significant potassium ion accumulation at the external surface of Myxicola giant axons.

Potassium accumulation associated with outward membrane potassium current was investigated experimentally in Myxicola giant axon. During prolonged voltage-clamp pulses to positive transmembrane potentials, the K+ equilibrium potential may approach zero mV, suggesting massive K+ accumulation outside the axonal membrane to concentrations many-fold higher than those in the bathing medium. The potassium accumulation can be satisfactorily described by a three-compartment model, consisting of the nerve fiber, a restricted physiological periaxonal space and the bulk solution. The average thickness, theta, of the periaxonal space is calculated as 177 +/- 59 A, i.e., comparable to that in the squid, while the permeability coefficient of the external barrier, PKs, was calculated to be (1.4 +/- 0.4) X 10(-4) cm/s. These conclusions are well supported by morphological study.

Quantification of endotoxin inhibition in serum and plasma using a turbidimetric LAL assay.

A modified Limulus amebocyte lysate (LAL) test was developed which quantifies the inhibition associated with lipopolysaccharide (LPS) in serum and plasma. The assay utilized the LR50 value to determine relative inhibition. The LR50, measured in ng/ml, represented the concentration of endotoxin needed to elicit a turbidimetric response equal to 50% of the maximum above the control (no added endotoxin). The LR50 concept was used to compare plasma and serum from a normal donor population. Plasma LR50 values ranged from 115 to 5000 ng/ml, while serum samples ranged from 48 to 615 ng/ml. Endotoxin in saline measured in a similar manner yielded values averaging 0.07 ng/ml (LAL sensitivity). Various means to remove inhibition were also tested by this method. Heating, dilution, and chloroform extraction of serum were all found to be efficient in removing inhibition. Although no direct attempt was made to elucidate the biochemical nature of the inhibitor(s) present in serum and plasma, the results presented were consistent with other studies and indicate the involvement of a heat labile component(s). The LR50 may provide a tool for the elaboration of these components. It also was suggested that LR50 may be the best in vitro indicator of the overall ability of serum or plasma to neutralize endotoxin.

Sensitivity of Limulus amebocyte lysate (LAL) to LAL-reactive glucans.

The sensitivity of Limulus amebocyte lysate (LAL) to LAL-reactive glucans (LRGs) and lipid A was tested by using commercially available and experimentally formulated LAL reagents. The glucans included two kinds of beta-(1,3)-D-glucans, laminarin and curdlan, and cellulosic material, LAL-reactive material (LAL-RM), extracted from a hollow-fiber (Cuprophan) hemodialyzer. LAL-RM loses its LAL activity when it is digested with cellulase and thus appears to be a beta-(1,4)-D-glucan or a mixed glucan containing a substantial proportion of beta-(1,4) linkages. All LAL reagents tested were at least 1,000-fold more sensitive to endotoxin than to LRGs. The presence of the surfactant Zwittergent was shown to interfere with reactivity to LRGs; LAL reagents without added Zwittergent reacted more strongly to LRGs than did the same reagents containing Zwittergent. Chloroform extraction of LAL increased the reagents' sensitivity to both endotoxin and LRGs, but it was not responsible for LRG reactivity. The addition of Zwittergent significantly reduced the sensitivity of LAL reagents to lipid A. LAL without the surfactant was equally sensitive to endotoxin and lipid A. Both curdlan and LAL-RM amplified or enhanced the LAL response to endotoxin. Kinetic turbidimetric studies demonstrated that the enhancement was dependent on the glucan concentration.

Plastics, endotoxins, and the Limulus amebocyte lysate test.

A variety of polypropylene and polystyrene tubes have been tested for use with the Limulus amebocyte lysate (LAL) test. Polypropylene tubes tended to be more contaminated with endotoxin than polystyrene. One brand of polypropylene tube contained a water extractable inhibitor of the LAL test. Polystyrene tubes from some manufacturers caused enhancement of the LAL test. Other polystyrene tubes were not significantly different from glass for storage of endotoxin or dilution water. Results of these studies indicate that while some tubes are well suited for use with the LAL test, others are not.

Single-step, chromogenic Limulus amebocyte lysate assay for endotoxin.

A new reagent for the chromogenic Limulus amebocyte lysate (LAL) assay is described. LAL was formulated for optimal performance in either an endpoint procedure or a kinetic procedure with the chromogenic substrate, buffer, and LAL components colyophilized as a single reagent. The kinetic chromogenic method required an incubating microplate reader coupled to a computer for collection and analysis of data. The kinetic method had a longer incubation time than the endpoint method and spanned a range of over 3 orders of magnitude compared with the 1-order-of-magnitude range of the endpoint assay. The kinetic method was less subject to operator error, since readings were continuous and automatic. The endpoint test was more operator intensive, requiring both addition of acetic acid to stop the reaction and transfer of the sample to the reading device. A single-step chromogenic reagent was also prepared without lyophilization by mixing reconstituted gel clot LAL with a buffer and a chromogenic substrate. The reagent prepared in this manner performed as well as the colyophilized agent.

Turbidimetric method for quantifying serum inhibition of Limulus amoebocyte lysate.

This study describes a method to quantify the inhibition of lipopolysaccharide (LPS) activity by serum with a turbidimetric Limulus amoebocyte lysate assay. Assays were performed in multiwell microplates, and turbidity was measured as the optical density at 380 nm with a microplate spectrophotometer. LPS potency was measured as the 50% maximal Limulus amoebocyte response (LR50) of LPS diluted with saline. By comparing LR50s in saline, LPSs from various species of bacteria were standardized against the U.S. Reference Standard Endotoxin, lot EC-5. The potency of Escherichia coli O113 and O18 and Serratia marcescens LPSs was found to be equal to that of the reference standard EC-5, whereas LPSs from two salmonella species were half as potent. The least potent LPSs tested, obtained from Klebsiella pneumoniae and E. coli rough mutant J5, were 5- and 10-fold less potent, respectively, than EC-5. As a measure of inhibition, the LR50 of LPS in serum was compared to the LR50 of LPS in saline. Serum inhibited the potency of LPS 103- to 6,400-fold compared with saline. A positive correlation was found between standardized potency in saline and serum inhibition of the various LPSs tested. Thus, LPSs from E. coli O113, O18, and EC-5 and S. marcescens, which exhibited the highest potency in saline, were inhibited the most by serum. Likewise, E. coli J5 and K. pneumoniae LPSs, which were the least potent tested, were the least inhibited. The degree of inhibition of all types of LPS tested increased with increasing serum concentration.

Polypeptide composition of squid neurofilaments.

Neurofilaments, 10 nm in diameter, from the axoplasm of the squid Loligo pealei have been isolated by a combination of sonication and Millipore filtration. The presence of neurofilaments during the isolation procedure was confirmed by negative staining and transmission electron microscopy. By use of this technique, which results in minimal or no chemical alteration of the native neurofilament proteins, it was shown that actin (43,000 daltons) and tubulin (56,000 daltons) are physically separable from intact neurofilaments. The neurofilament-rich retentate contained two major proteins of 200,000 and 63,000 daltons and larger polypeptides.

Quantitation of endotoxin in products using the LAL kinetic turbidimetric assay.

The data presented here show the kinetic turbidimetric LAL assay to be a highly quantitative and effective method for determining endotoxin concentrations in products. The assay allows for the accurate assessment of inhibiting or enhancing effects in products when related to a LRW standard curve. However, designating some products as inhibitors or enhancers can be both misleading and erroneous unless qualified as to the dilution and/or endotoxin concentration. Our results demonstrate that some products can yield both inhibiting and enhancing results when related to water. Due to the enhanced resolution of the kinetic turbidimetric assay, these complicating inhibition/enhancing effects can usually be avoided by diluting to the WED. Alternatively, products could be related to a PSC in which the endotoxin response is defined and quantified within the product itself. The practicality of a PSC, however, depends upon the pass/fail limit established, the "cleanliness" of the product used as a standard and the degree of product "lot to lot" variability. Current FDA Guidelines consider a PSC valid providing the value of the "unspiked" product extrapolated from the regression line of the PSC is less than 10% of lambda, the lowest endotoxin concentration used to construct the standard. All products in which LAL kinetics have not been previously analyzed will require a characterization similar to that used with the four products described. From these data, the optimal methodology for kinetically testing the product (dilution to a WED or generation of a PSC) can be determined. Although analysis of endotoxin in a product will always require a characterization of the kinetics of the LAL-endotoxin-product reaction, subsequent testing should be rapid and straightforward. More importantly, the kinetic turbidimetric assay allows the user to quantitatively assess product endotoxin levels with a degree of precision greater than that of any other methodology currently in use.

Neutralization of bacterial lipopolysaccharides by human plasma.

To quantify the neutralization of bacterial lipopolysaccharide (LPS) by human plasma, dilutions of Escherichia coli O113 LPS were incubated with plasma, followed by the addition of Limulus amebocyte lysate (LAL). The reaction between the LPS and LAL was monitored spectrophotometrically, and the concentration of LPS resulting in 50% lysate response (LR50) was determined. Analysis of 145 outdated plasma samples yielded a range of LR50 between 6 and 1,500 ng/ml. Pools of plasma with high and low LR50 were prepared. The pool with high LR50 neutralized 166-fold more E. coli 0113 LPS, 190-fold more E. coli 0111B4 LPS, 42-fold more Klebsiella pneumoniae LPS, and 29-fold more Salmonella typhimurium LPS than did the pool with low LR50. Each pool had similar immunoglobulin G (IgG) and IgM antibody levels to homologous LPS, measured by an enzyme-linked immunosorbent assay. Analysis of 212 fresh-frozen plasma units revealed a range of LR50 between 48 and 6,000 ng/ml. Incubation of LPS in a pool of fresh-frozen plasma with high LR50 elicited significantly less fever in the rabbit pyrogen test than did LPS incubated in plasma with low LR50 (fever index, 2.68 +/- 0.61 degrees C X h and 3.52 +/- 0.66 degrees C X h, respectively; P = 0.003). We conclude that there is a 100-fold range in the endotoxin-neutralizing capacity of human plasma and that this variation is not due to LPS-specific IgG or IgM antibodies. Further investigations are needed to determine whether differing susceptibility of patients to the effects of LPS is due to differences in the endotoxin-neutralizing capacity of their plasma and whether plasma screened for high endotoxin-neutralizing capacity may be therapeutically useful in endotoxemia.

Role of interleukin-1 in augmenting serum neutralization of bacterial lipopolysaccharide.

We have previously described an assay to quantify the serum neutralization of bacterial lipopolysaccharide which is based on a spectrophotometric Limulus amoebocyte lysate test (T.J. Novitsky, P.F. Roslansky, G.R. Siber, and H.S. Warren, J. Clin. Microbiol. 21:211-216, 1985). Studies since have shown that serum samples drawn from patients with leukemia and fever, gram-negative or gram-positive bacterial infections, or shock caused by gram-negative bacteria neutralize approximately 10-fold more lipopolysaccharide than do samples from normal controls. These findings suggested that the increased neutralization might reflect an acute-phase response and raised the question of whether it might be under the control of interleukin-1. To answer this question, we studied the neutralization of lipopolysaccharide in serum samples drawn from rabbits before and after the administration of crude interleukin-1, prepared from activated macrophage supernatants, and recombinant human interleukin-1. Crude interleukin-1 induced a 5.7-fold increase in serum neutralization 24 h after intravenous injection, and cloned interleukin-1 induced a 3.0-fold increase (P less than or equal to 0.01 and 0.05, respectively). In individual rabbits given identical doses of crude interleukin-1 on a weight basis, the serum-neutralizing ability correlated significantly with three activities of interleukin-1: rise in temperature (r2 = 0.558; P less than or equal to 0.01), decrease in serum iron (r2 = 0.534; P less than or equal to 0.01), and increase in serum copper (r2 = 0.323; P less than or equal to 0.05). We conclude that the increase in neutralization of bacterial lipopolysaccharide by serum samples drawn from patients with inflammatory states is mediated, at least in part, by interleukin-1, presumably through the induction of acute-phase serum proteins.

Labor and infection. II. Bacterial endotoxin in amniotic fluid and its relationship to the onset of preterm labor.

We have previously reported the detection of endotoxin in the amniotic fluid of patients with gram-negative intraamniotic infection. Endotoxin or lipopolysaccharide is a potent biologic product capable of inducing prostaglandin release from several cell types and, therefore, may be involved in the onset of human parturition in the presence of intraamniotic infection. This article describes a technique for the quantification of endotoxin in amniotic fluid. The method uses a computer-assisted quantification of the turbidimetric reaction between the Limulus amebocyte lysate and endotoxin. Serial dilutions of Escherichia coli endotoxin in culture-negative amniotic fluid were prepared, and the samples were run in the assay. Amniotic fluid was found to enhance the reaction, and a dilution of 1:20 was required for this biologic fluid to behave similarly to pyrogen-free water. The sensitivity of this kinetic turbidimetric technique in the detection of endotoxin in amniotic fluid was 40 pg/ml. This method was applied to the quantification of endotoxin concentration in amniotic fluid in 26 patients with intraamniotic infection and premature rupture of membranes. Patients in active labor had higher concentrations of endotoxin (median = 47,514 pg/ml) than nonlaboring patients (median = 635 pg/ml) (p less than 0.025). Therefore, women with preterm labor had a higher median concentration of endotoxin in amniotic fluid than patients who were not in labor.