A New Perchloric Acid Treatment of Human Plasma for Detection of Endotoxin by an Endotoxin-Specific Chromogenic Test

Inada, Katsuya; Yoshida, Masao; Takahashi, Toshio; Tamura, Shougo; Tanaka, Shigenori; Endo, Shunsuke; Yoshida, Tomoaki; Suda, Hidetoshi; Komuro, Takashi

doi:10.1007/978-1-4757-5140-6_20

Cited by 4 publications

(3 citation statements)

References 2 publications

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“…Finally, the LAL reagents widely used in the world react not only endotoxin but also (1,3)-β-D-glucan, component of cell wall of fungus [48], because they contain factor G reacting (1,3)-β-D-glucan in addition to factor C reacting endotoxin. Although endotoxin-specific reagents were produced early in Japan by removing G-factor from the lysate [48] proposed methods of sample pretreatment thereafter [52][53][54] were not adequate for the detection of minute amount of endotoxin in the blood of patients without bacteremia. Although some Japanese investigators have doubted the existence of portal endotoxemia, spillover endotoxemia or metabolic endotoxemia because of the negative results in their studies using the endotoxin-specific LAL tests, I disagree with them because we did detect endotoxemia in patients with chronic liver diseases by our improved chromogenic assay using endotoxin-specific substrate Endospecy (Seikagaku Kogyo Co., Tokyo, Japan) after the pretreatment of sample by perchloric acid and triethylamine [49,51].…”

Section: History Of Blood Endotoxin Assay and Their Problemsmentioning

confidence: 99%

Endotoxin and Other Microbial Translocation Markers in the Blood: A Clue to Understand Leaky Gut Syndrome

Fukui¹

2016

Cell Mol Med

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Gut affects various systems in the human body. The leaky gut hypothesis tells us that gut microbial products cause systemic low-grade inflammation, which enhances the progression of various human diseases. Microbial translocation attributable to intestinal barrier dysfunction and hyperpermeability have been proven in major human diseases. Among these microbial products, endotoxin/ lipopolysaccharide is most extensively studied in clinical situations. However, its detection in the blood and its impact in the clinical course still arouse much discussion. Overviewing the long-standing controversies in the assay system and the main results in various clinical situations, this review regards plasma endotoxin level as a feasible microbial translocation marker. Although the detection of bacterial DNA in the blood has been gradually accepted among other microbial products, uniformity of analytical methods and usefulness in the clinical site should be established. The results on peptidoglycan, flagellin, lipoteichoic acid, and (1→ 3)-β-D-glucan in the blood are still scarce. The analysis of Toll-like receptors has suggested that several microbial products act concomitantly as pathogen-associated molecular patterns (PAMPs) in the progression of various diseases. Collective efforts to read the whole story on leaky gut and its sequences from the side of circulating microbial products may lead future progress both in patient's care and preventive medicine.

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Section: History Of Blood Endotoxin Assay and Their Problemsmentioning

confidence: 99%

Endotoxin and Other Microbial Translocation Markers in the Blood: A Clue to Understand Leaky Gut Syndrome

Fukui¹

2016

Cell Mol Med

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“…To overcome this inhibitory activity, several methods of treating plasma before using it for the Limulus amoebocyte lysate test have been described. These methods include treatment of plasma by dilution-heating (5-8), Chloroform extraction (9), and acid oxidation with perchloric acid (10,11). However, these methods do not ftilly satisfy the basic requirement for elimination of interference.…”

Section: Immobilized Histidine Adsorbs Endbtoxins In Solutions Contaimentioning

confidence: 99%

“…Pretreatment of plasma samples by the new perchloric acid procedure Pretreatment of plasma samples by the new perchloric acid procedure was carried out by the method oflnada et al (11). To 100 μΐ of plasma sample, 100 μΐ of sodium hydroxide solution'(0.18 mol/1) were added and incubated at 37 °C for 5 min.…”

Section: Assaymentioning

confidence: 99%

Assay of Endotoxin in Human Plasma Using Immobilized Histidine, Limulus Amoebocyte Lysate and Chromogenic Substrate

Minobe

Nawata²,

Shigemori³

et al. 1994

Clinical Chemistry and Laboratory Medicine

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Summary:The Limulus amoeboeyte lysate test for endotoxin is inhibited or enhanced by many substances. It is particularly difficult to determine endotoxin in plasma. In order to overcome this problem, we have modified the specific endotoxin assay method by using a membrane fllter unit, a chromogenic Limulus amoeboeyte lysate reagent, and immobilized histidine (which is a specific adsorbent for endotoxins). This immobilized histidine method consists of the endotoxin adsorption Step on immobilized histidine, the Separation Step, in which Limulus amoeboeyte lysate-interfering substances are removed, and the Limulus amoeboeyte lysate test. Preheating of plasma samples (40-fold dilution with distilled water, at 100 °C for 7.5 min) was necessary, and it was necessary to dilute the sample more than 100-fold for the adsorption Step. Under these conditions, the fraction of endotoxin recovered frorii plasma by the immobilized histidine method was almost l. Moreover, by increasing the sample volume and extending the Limulus amoeboeyte lysate reaction time, the sensitivity could be increased. By using the immobilized histidine method, 50-200 units/1 of endotoxin in plasma samples can be accurately assayed. The method was used for the determination of plasma endotoxin in rabbits.

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Use of immobilized histidine in assay for endotoxin in patients with liver disease

et al. 1994

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The Limulus amebocyte lysate (LAL) test has the disadvantage of being influenced by various inhibitors and activators. We have developed a method for the LAL reaction that involves the specific adsorption and isolation of endotoxin using a membrane filter unit and immobilized histidine; in this present study we used the method to measure endotoxin in the plasma of patients with acute or chronic liver disease. The adsorbed endotoxins are separated from LAL-inhibitors or -activators by the membrane filter unit, and their activity is directly assayed with the LAL reagent in a filter cup without any inhibition or activation. The study population consisted of 23 subjects, 3 with fulminant hepatitis and 20 with cirrhosis (9 with esophageal varices and 11 without). All 3 (100%) of the samples of plasma from patients with fulminant hepatitis were positive for endotoxin, as were the samples of 7 (78%) of the 9 patients with cirrhosis and esophageal varices, and 2 (18%) of the 11 patients with cirrhosis but without such varices. The results suggested that this method appears to be useful for assaying the concentration of endotoxin in patients with fulminant hepatitis or cirrhosis of the liver.

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A New Perchloric Acid Treatment of Human Plasma for Detection of Endotoxin by an Endotoxin-Specific Chromogenic Test

Cited by 4 publications

References 2 publications

Endotoxin and Other Microbial Translocation Markers in the Blood: A Clue to Understand Leaky Gut Syndrome

Endotoxin and Other Microbial Translocation Markers in the Blood: A Clue to Understand Leaky Gut Syndrome

Assay of Endotoxin in Human Plasma Using Immobilized Histidine, Limulus Amoebocyte Lysate and Chromogenic Substrate

Use of immobilized histidine in assay for endotoxin in patients with liver disease

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