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PubMed1989

[Change of blood lentinan level in patients with advanced cancers].

A conventional limulus test can detect not only endotoxin but also beta(1----3) glucan. Therefore, with a quantitative limulus test, the limulus colorimetric test, we studied the pharmacokinetics of lentinan, an antitumor beta(1----3) glucan preparation, in the blood of 10 healthy volunteers and 20 patients with advanced cancers. The calibration curve of lentinan in the human plasma was linear in the range of 0 to 100 ng/ml. Two mg of lentinan was administered once a week and blood samplings for the measurement were done immediately before the next lentinan administration to document trough-levels serially at a week interval. Trough-levels tended to rise gradually and showed a rapid elevation between 4 and 8 weeks after beginning of administration. This phenomenon may correlate closely with the antitumor effect of lentinan and, therefore, deserves further investigations.

Antineoplastic Agents↗
PubMed1975

Endotoxin as a cause of aseptic meningitis after radionuclide cisternography.

The role of pyrogens in aseptic meningitis after radionuclide cisternography was studied by means of the Limulus test, a sensitive detector of endotoxin. During a 15-month period, 39 reactions associated with cisternography were reported. Ten samples of specific lots of the radioactive drugs implicated in 20 of these reactions were tested and all reacted strongly positive to the Limulus test. The less sensitive rabbit pyrogen test was negative for these preparations when tested on a dose-per-weight basis. Our findings apparently provide clinical evidence for the observation made in animals that endotoxin is at least 1,000 times more toxic intrathecally than intravenously. The data implicate endotoxin contamination as a cause of adverse reactions to radionuclide cisternography. We conclude that the USP pyrogen test is insufficiently sensitive for intrathecal injectables and should be supplemented by the Limulus test.

Arachnida↗
PubMed1980

Immunochemical and biological properties of the outer membrane-associated lipopolysaccharide and protein of Rochalimaea quintana.

The outer membrane complex of Rochalimaea quintana was isolated by use of ethylene-eiaminetetraacetate and was compared biochemically and biologically both with lipopolysaccharide (LPS) isolated by phenol-water extraction of whole organisms and with lipids isolated by chloroform-methanol extractions of the phenol-water insoluble residues. The outer membrane consisted of protein and LPS components, as distinguished by precipitin tests with sera from patients with trench fever or tests with hyperimmune animal sera. The outer membrane protein component, but not LPS, also reacted with sera from infections with Rickettsia tsutsugamushi. The LPS contained 2-keto-3-deoxy-octonate and heptose. The outer membrane and phenol-extracted LPS were reactive in the chick embryo lethality test, limulus assay, and complement activation. Outer-membrane activity was confined to the LPS component. The lipid extracts were reactive in chick embryo lethality and limulus assays, but did not activate complement.

Animals↗
PubMed1997

Endotoxic properties of free lipid A from Porphyromonas gingivalis.

The relationship between chemical structure and biological activity of the lipid A from Porphyromonas gingivalis, which we recently isolated and whose complete chemical structure was determined [Kumada et al. (1995). J Bacteriol 177, 2098-2106], was studied. The lipid A exhibited endotoxic activity in all the assay systems tested: Limulus gelation activity, lethal toxicity in galactosamine-sensitized mice, mitogenicity in mouse spleen cells and induction of nitric oxide (NO) and tumour necrosis factor alpha (TNF) release from both mouse peritoneal macrophages and the J774-1 mouse macrophage-like cell line. The activity was, however, about 100-fold less than that of Salmonella minnesota LPS used as a control. The moderate activity of the lipid A may be partially explained by its unique fatty acid composition and the lack of a phosphate group in position 4. In contrast, the lipid A as well as whole LPS of P. gingivalis unexpectedly exhibited an even stronger induction of TNF from the human monocytic THP-1 cell line than control LPS when measured by the minimum stimulatory dose. The difference in sensitivity of human and mouse cells to P. gingivalis lipid A suggests that the recognition mechanism, including that for the receptor for endotoxin, may be regulated in different ways in the two cells.

Animals↗
PubMed1977

[Trial preparation and evaluation of a 81mKr-generator for medical use].

A prototype 81mKr-generator consisting of an ion exchange column and some attachments for handling was prepared on trial. Parent nuclide 81Rb obtained by the reaction of 82Kr(p, 2n)81Rb was absorbed on the resin, and radionuclidic purity, sterility and apyrogenicity of the generator eluate were examined. Analysis of gamma-ray spectrum obtained with a Ge(Li) detector and a multichannel pulse height analyzer revealed that the nuclidic purity of the 81mKr in the eluate was 99.997-99.999% with 0.001-0.003% of 79Kr at the start of the elution. Sterility and apyrogenicity of the eluate were proved by J.P. sterility test and limulus test respectively. All results obtained show that the 81mKr-generator is very suitable for medical application.

Evaluation Studies as Topic↗
PubMed2006

[Kinetic turbidimetric limulus assay of bacterial endotoxin in the replacement solution of on-line hemodiafiltration].

OBJECTIVE: To determine bacterial endotoxin in the replacement solution of on-line hemodiafiltration (on-line HDF) using kinetic turbidimetric limulus test. METHODS AND RESULTS: Validation test was performed with the replacement solution of on-line HDF in which quantified standard endotoxin was added. The recovery rates of endotoxin from the replacement solution and its dilutions at 1/5, 1/10, and 1/20 were 58.17%, 106.7%, 99.00% and 98.79%, respectively, suggesting that the optimal dilution was at 1/10. Standard endotoxin was added into the replacement solution of on-line HDF of 3 batches (040408, 040511,040527), and the recovery rates in their dilution at 1/10 were 76.32%, 99.00% and 96.24%, respectively. The standard endotoxin in the working curve was 1.00, 0.125, and 0.0156 Eu/ml (endotoxin unit/ml), and the dilution at 1/10 of the replacement solution is effective to eliminate the interference in limulus test. CONCLUSION: Kinetic turbidimetric limulus test provide a means to detect endotoxin in the replacement solution of on-line HDF.

Endotoxins↗
PubMed1980

Synthesis and pyrogenic effect of 3 alpha, 7 alpha-dihydroxy-5 beta-androstan-17-one and 3 alpha-hydroxy-5 beta-androstane-7, 17-dione.

The first chemical synthesis of 3 alpha, 7 alpha-dihydroxy-5 beta-androstan-17-one and 3 alpha-hydroxy-5 beta-androstane-7, 17-dione is reported. In this method, the 17 beta-side chain of commercial chenodesoxycholic acid was degraded in 6 steps after selective protection of the hydroxyl groups: 3 alpha-OH by a tert-butyldimethylsilyl group and 7 alpha-OH by an acetoxy group. The capacity of 3 alpha, 7 alpha-dihydroxy-5 beta-androstan-17-one and 3 alpha-hydroxy-5 beta-androstane-7, 17-dione to release a pyrogen by human leukocytes was investigated by two independent methods: supernatants from leukocytes incubated with a steroid are injected to rabbits whose fever is measured, or tested by the Limulus Test (a pyrogen detection technique). The 7-keto substituted etiocholanolone still possessed pyrogenic activity, while the 7 alpha-hydroxyl substituted one did not.

Animals↗
PubMed2005

Comparison of the rabbit pyrogen test and Limulus amoebocyte lysate (LAL) assay for endotoxin in hepatitis B vaccines and the effect of aluminum hydroxide.

The rabbit pyrogen test and Limulus amoebocyte lysate (LAL) assay have been used to detect endotoxins in vaccines, but interactions between the endotoxins and proteins or aluminum hydroxide can interfere with the results. Currently, the rabbit pyrogen test is used to detect endotoxin in hepatitis B (HB) vaccines even though the HB surface protein, the active ingredient, is over-expressed in and purified from eukaryotic cells which lack endotoxin. Therefore, we examined the possibility of replacing the animal tests with the more efficient LAL test. To this end, we determined whether the aluminum hydroxide in the HB vaccines affects the rabbit pyrogen test and the LAL assay. HB vaccines and HB protein solutions spiked with lipopolysaccharide (LPS) produced almost the same dose-dependent temperature rise in rabbits, indicating that the aluminum hydroxide in the HB vaccine does not interfere with the pyrogenic response in rabbit. In contrast, a spike recovery study showed that aluminum hydroxide interfered with the LAL clot and kinetic assays; however, the LAL clot assay was effective at detecting endotoxin without loss of LAL activity after serial dilution of the samples. Furthermore, there was good correlation in the LAL clot assay between the amount of LPS added and the amount recovered. However, both turbidimetric and chromogenic kinetic assays displayed no correlation between the LPS amount added and recovered. Our results suggest that the LAL clot assay is sensitive and reliable when samples are properly prepared, and can be used to replace the rabbit pyrogen test for the detection of endotoxin in HB vaccines.

Aluminum Hydroxide↗
PubMed1990

Endotoxin testing with limulus amoebocyte lysate in a radiopharmaceutical containing chelated metallic radionuclides and chelating agents.

The gel formation by endotoxin with limulus amoebocyte lysate (LAL) solution requires the presence of free calcium ions. The chelating agents and radiopharmaceuticals prepared from the chelating agents always reduce the available free calcium levels present in the kits used for the testing of apyrogenicity and thus inhibit gel formation of LAL with E. Coli endotoxin (ET). This inhibition of gel formation could be reversed by the addition of excess free calcium ions or the excessive dilution of radiopharmaceuticals and chelating agents. The tests of positive control (0.2 ml ET units and LAL), inhibition control (0.1 ml sample, 0.1 ml ET and LAL), and negative control (0.1 ml sterile saline and LAL) were carried out with the fresh preparation (0.1 ml) of samples (triplicate), tropolone, ACD anticoagulant, and 99mTc-labeled radiopharmaceuticals. In the Ca-supplemented tests, 0.1 ml of 60 mM sterile calcium chloride solution was added to the test solutions and incubated for 60 min at 37 degrees centigrade. The results of gel formation (+ve and -ve) and normalization of inhibition control tests with Ca-supplement indicate that commercial LAL kits need extra calcium ions for the correct testing of the apyrogenicity of chelate-containing radiopharmaceuticals and chelating agents.

Limulus Test↗
PubMed1977

Tests alternative to the rabbit bioassay for pyrogens.

The rabbit bioassay is currently the only legally acceptable method to test for pyrogenic contamination of parenteral preparations and medical devices designed to enter the parenterum. However, research efforts have recently made available alternative procedures to test for pyrogens which may have significant value in quality control. Part of the reluctance to accept tests other than the rabbit test may be due to the low visibility of emerging understanding of a significant theory of testing in which it is contended that the parameters of sensitivity and specificity are inversely related and that accuracy is an ideal. The rabbit pyrogen assay will detect 1 to 10 ng of enterobacteriaceal endotoxin (ET). The limulus test will detect 0.01 to 0.1 ng/ml of ET; some of the other tests approach the rabbit assay in sensitivity. Since it is current dogma that pyrogen is equivalent to ET, the basis for the use of ET to standardize pyrogen tests is rationalized. The source of ET in practice is bacterial contamination; therefore, numbers of bacteria that contaminate parenteral preparations can be directly related to potential pyrogenicity. Further, viable counts of bacteria in parenteral preparations, prior to sterilization, is a reliable test for pyrogens. Other tests such as nitroblue tetrazolium reduction and actinomycin-D enhancement of lethality of pyrogen for mice deserve consideration in quality control procedures. The limulus test, the most practical of currently available alternative tests for detection of ET and therefore, of pyrogen has application where the rabbit test cannot be used. Therefore, control personnel must learn of the availability, performance and interpretation of the limulus test. Practical considerations must govern the choice of an alternative test when the use of a test other than the rabbit bioassay is indicated.

Animals↗