Determination of lead chelated with ethylenediaminetetra-acetic acid in blood after precipitation of protein with perchloric acid.
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With a quantitative blood endotoxin assay using a chromogenic substrate with a perchloric acid pretreatment (PCA-LCT), endotoxemia in various liver diseases was studied. With PCA-LCT, recovery of added endotoxin in human plasma was nearly 90%, as evidenced by an intra- and inter-assay coefficients of variation of 5.7% and 11%, respectively. Because the recovery of endotoxin was not affected in severely icteric plasmas, PCA-LCT proved to be applicable to patients with liver diseases where various degree of jaundice exist. In none of the plasmas from patients with chronic hepatitis, acute hepatitis without hepatic failure or liver cirrhosis without ascites did the endotoxin level exceed the normal range of less than 5 pg/ml. With the presence of ascites, however, endotoxemia became detectable, but at low levels and not in all cases. At the stage of hepatic failure complicated with renal failure or disseminated intravascular coagulation, endotoxemia was more frequent and endotoxin concentration greater. It is uncertain, at present, whether endotoxemia itself is deteriorating factor in hepatic failure or is merely concomitant phenomenon resulting from Kupffer cell failure.
Using with the newly modified perchloric acid pretreatment method and endotoxin specific assay (Endospecy), the plasma endotoxin in the patients with liver cirrhosis was investigated. The mean value of plasma endotoxin in control (n = 20) was below 9.8 pg/ml. The plasma endotoxin level in liver cirrhosis was 5.7 +/- 5.3 pg/ml (n = 70, mean +/- SD), and 20% of the patients (15 cases) showed above 9.8 pg/ml. Endotoxin level significantly correlated with the severity of liver function based on the Child-Turcotte classification (p less than 0.01). Plasma endotoxin positively correlated with total bilirubin (r = 0.417) and ICG clearance test (r = 0.298) and negatively correlated with prothrombin time (%) (r = 0.497) and HDL-cholesterol (r = 0.578) in the patients with liver cirrhosis. There was no correlation between esophageal varices and plasma endotoxin level. Plasma endotoxin was slightly detected in the patients with decompensated cirrhosis, and these data suggest that plasma endotoxin level in cirrhosis is not so elevated.
A novel protein was extracted with 5% perchloric acid from rat liver and kidney. It is absent from other rat organs. Its apparent molecular mass is 23 kDa as determined by HPLC gel filtration. A single band, corresponding to 10 kDa, was observed after SDS/PAGE, suggesting that the protein consists of two subunits with similar molecular masses. This protein can neither be phosphorylated by ATP, nor acetylated. The sequence of the cDNA encoding this protein was determined. Southern-blot analysis showed that the corresponding gene spanned at least 10 kb and contained at least five introns. Zoo-blot analysis at medium stringency strongly suggests that the gene has been conserved during evolution. The amino-acid sequence of this protein with a highly conserved region is similar to that of a heat-shock protein.
Commerically available Sephadex buffer-exchange columns for the separation of carcinoembryonic antigen (CEA) from perchloric acid were evaluated. Elution patterns of individual columns were found to be variable, but separation of acid from CEA could be accomplished. Repeatability of replicate elutions and agreement with dialysis results were acceptable. Nevertheless, the method is excessively time-consuming and inefficient for processing large numbers of patient samples.
The instability of pyruvate in neutralized perchloric acid extracts of erythrocytes is well-known from the literature. As shown in this study, the main reason for this phenomenon is a reaction of pyruvate with hydrogen peroxide, which is formed from oxyhemoglobin during acid precipitation. Thereby, pyruvate is oxidized to carbon dioxide and acetate. This decomposition is easily prevented by addition of catalase to the neutralized extracts, whereby authentic pyruvate can be determined precisely. Similarly, GSH and NAD(P)H oxidation in the extracts is prevented by addition of catalase.
High-resolution 1H NMR spectra were obtained from perchloric acid extracts of breast tumor specimens and adjacent non-involved tissue. Two-dimensional shift-correlated and homonuclear J-resolved spectroscopy were used to identify coupled resonances in the spectra. Chemical shifts, multiplicities and spin-spin coupling constants of several non-resolved resonances in the one-dimensional spectra could be determined by the two-dimensional methods. Several differences in the metabolite content of the two types of extracts were established. The spectra of extracts from non-involved tissue were dominated by signals from glucose and other carbohydrates, while most of the tumors had very low or no detectable levels of glucose. High concentrations of lactate, taurine and succinate, an increase of the phosphocholine level, and a very low phosphocreatine level were characteristic findings in the 1H spectra of tumor extracts. The variation in the level of myo-inositol follows the variation in glucose for the two types of tissue. Scyllo-inositol was for the first time observed in the NMR spectra from breast tissue. Uridine 5'-diphospho-N-acetylglucosamine and uridine 5'-diphospho-N-acetylgalactosamine have been identified and there is an increased level of these two hexoses in the tumor tissue. These results provide insight into breast tumor metabolism, by simultaneously detecting a large number of metabolites and demonstrate the potential for using 1H NMR spectroscopy for studying different metabolic pathways in breast tumors. At the same time they provide useful information for interpretation of in vivo 1H NMR spectra of breast tumors.
In the United States Pharmacopeia (USP), 0.1 N perchloric acid in acetic acid volumetric solution (hereafter HClO4 VS) used for non-aqueous titration has specified a water content between 0.02 and 0.05%. Preparing this titrant with such a narrow range of water content is very time consuming, precludes the use of commercially available titrants, and, consequently, prompted an investigation to try and expand the range up to 0.5%. In this study, the titrimetric results obtained using HClO4 VS containing more water were very close to those obtained using the USP specified titrants. A maximum assay difference of 0.7% in the titrations of three selected nitrogen-containing compounds, clonidine hydrochloride, dipyridamole, and adenosine were observed. The titrimetric results obtained using these titrants were also precise with RSDs of not more than 0.4%. Therefore, a wider range of water content in HClO4 VS between 0.02 and 0.5% is suggested for the USP potentiometric titration of nitrogen-containing compounds.
Perchloric acid adsorbed on silica gel efficiently catalyses acetylation of structurally diverse phenols, alcohols, thiols, and amines under solvent free conditions.
A simple automated method for the estimation of ammonia in perchloric acid supernate of blood or plasma using an ion-selective electrode (Orion Ammonia-selective electrode, Model 95-10) is described. The reliability of the proposed method has been checked against an ion-exchange resin procedure, which has been chosen as a standard procedure. Regression equation and correlation coefficient for the proposed method are y = 0.7 X + 10 and 0.945, respectively, as compared with the chosen standard method. Within-run and between-run precision are 2.1% and 3.5% respectively. The average percent recovery is 97.5% and a tentative range is 13-73 microgram/dl (9-52 micronmol/l) ammonia nitrogen.
Following a rapid review of techniques of mechanical and chemical emptying of the pulporadicular cavity, the use of perchloric acid and hypochlorite of sodium and oxygenated water is described by means of scan microscope histological slides.
Preexposure of blood samples to perchloric acid permitted an accurate, quantitative measurement of endotoxin levels as low as 1 pg/ml using a colorimetric limulus test. Conventional chloroform and dilution-heating methods were unsatisfactory because of high residual nonspecific amidolytic activity and poor recovery. The normal peripheral plasma endotoxin level was less than 10 pg/ml when Escherichia coli 0111:B4 endotoxin was used as a reference. One nanogram in this assay was equivalent to 2.9 endotoxin units of USP reference standard endotoxin (E. coli 0113). High values were noted in portal venous blood and in cases of acute hepatitis, liver cirrhosis, strangulation ileus, pyothorax, lung abscess, diffuse panbronchiolitis, and pneumonia. Normal human plasma and serum exhibited a high capacity to inactivate added endotoxin. E. coli 0111:B4 and Salmonella minnesota 9700 were more susceptible to inactivation than Pseudomonas aeruginosa endotoxin. This inactivating activity was temperature dependent, was maximal between 37 degrees and 45 degrees C, and disappeared completely after heating plasma or serum to 56 degrees C for 30 minutes prior to the addition of endotoxin. The E. coli 0111:B4 endotoxin-inactivating activity of normal platelet-rich plasma, platelet-poor plasma, and serum, all at 37 degrees C, was 8.1 +/- 3.1, 11.7 +/- 4.5, and 15.2 +/- 4.9 micrograms/min/ml (mean +/- SD; n = 4), respectively. Endotoxin-inactivating activity was markedly decreased in plasma from patients with endotoxemia, but returned to normal with recovery from the underlying illness.
The modernized limulus lysate assay on cerebrospinal fluid pretreated by the new perchloric acid (PCA) treatment was evaluated in one patient with Escherichia coli meningitis and four non-meningitis patients. The endotoxin (ET) concentration was much higher by the new PCA treatment than by the ordinary dilution method in the exacerbation phase of E. coli meningitis. The recovery rate of added ET also demonstrated the superiority of the new PCA treatment.
Kinetics of oxidation of reducing sugars D-galactose (Gal) and D-ribose (Rib) by N-bromoacetamide (NBA) in the presence of ruthenium(III) chloride as a homogeneous catalyst and in perchloric acid medium, using mercuric acetate as a scavenger for Br(minus sign) ions, as well as a co-catalyst, have been investigated. The kinetic results indicate that the first-order kinetics in NBA at lower concentrations tend towards zero order at its higher concentrations. The reactions follow identical kinetics, being first order in the [sugar] and [Ru(III)]. Inverse fractional order in [H(+)] and [acetamide] were observed. A positive effect of [Hg(OAc)(2)] and [Cl(minus sign)] was found, whereas a change in ionic strength (mu) has no effect on oxidation velocity. Formic acid and D-lyxonic acid (for Gal) and formic acid and L-erythronic acid (for Rib) were identified as main oxidation products of reactions. The various activation parameters have been computed and recorded. A suitable mechanism consistent with experimental findings has been proposed.
Heat inactivation has been proposed as an alternative to perchloric acid (PCA) precipitation for the extraction of carcinoembryonic antigen (CEA) from human plasma. We examined a commercial RIA kit using heat inactivation, and compared results with those obtained with PCA precipitation. Adequate sensitivity (1.5 micrograms CEA/l plasma), satisfactory analytical recovery of CEA added to plasma, and dilutional linearity of samples found to have elevated CEA concentrations, were demonstrated for the heat-inactivation assay. Between-assay precision was better with the heat inactivation than with the PCA assay. Although the absolute concentration of CEA estimated after heat inactivation was consistently lower than that estimated after PCA extraction of plasma specimens, there was excellent correlation between results obtained with the two methods in colon cancer patients free of disease, colon cancer patients with residual or recurrent disease, patients with benign gastrointestinal disease, and in patients with chronic renal failure. We conclude that the heat-inactivation assay is an excellent alternative to the PCA assay.
The Limulus test was carried out for endotoxin determination using a synthetic chromogenic substrate after perchloric acid(PCA) treatment to remove inhibiting substances in the blood. PCA treatment completely removed non-specific amidolytic activity and other factors which interfered with the Limulus test. The recovery of added endotoxin was about 100% irrespective of animal species. Endotoxin levels in PCA-treated blood plasma of healthy domestic animals were invariably below 1 pg ml-1. Many of the cows with gangrenous mastitis, from which Gram-negative rods were isolated, showed high endotoxin levels ranging from 22.8 to 138.0 pg ml-1. Endotoxin was also detected in the blood of cows affected with salmonellosis, colibacillosis, Haemophilus somnus infection and urethritis associated with Proteus.
The acid-catalyzed hydrolysis reactions of the antiinflammatory drugs indomethacin and acemethacin were investigated at 25.0 degrees C in a number of strongly concentrated perchloric acid media. The reaction rates were evaluated by UV measurements, and the intermediate species were detected by UV-vis, 1H NMR, 13C NMR, and mass spectroscopy measurements. A switchover from an A-2 to an A-1 mechanism as a function of the medium acidity is reported for the acid-catalyzed hydrolyses of the amide group of both indomethacin and acemethacin. In the A-2 hydrolysis, two water molecules are involved in the rate-determining step. An analysis of the kinetic data collected for acemethacin by the different techniques used reveals a complex mechanism, indomethacin being a metabolite intermediate species in the hydrolysis of acemethacin. The rate constants for the hydrolysis of the acemethacin ester group were considerably larger compared to those of the amide group.
Simple and inexpensive methods for the speciation of arsenite, arsenate, monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA) in environmental water samples were developed. In these methods a hydride generation-atomic absorption spectrometry (HG-AAS) technique was employed and perchloric acid (as a reaction medium), L-cysteine (as a pre-reducing agent for a certain contact time between its addition and analysis) and sodium tetrahydroborate(III) (NaBH4, as a reducing agent) were used. The use of L-cysteine greatly enhances the absorption signals of all four arsenic species at low acid concentration (0.001-0.04 M). The methods developed for the determination of total arsenic and total inorganic arsenic and speciation of the four arsenic species in environmental water samples are as follows. (i) DMA: 0.005 M acid and 0.04% NaBH4 in the absence of L-cysteine. DMA can also be speciated in the presence of L-cysteine as follows: 2 M acid, 2.5% L-cysteine after a contact time of approximately 5 min and 0.6% NaBH4. (ii) As(III): 5 M acid and 0.08% NaBH4 in the absence of L-cysteine. (iii) Total inorganic arsenic (As(III) + As(V)]: 8 M acid and 0.6% NaBH4 in the absence of L-cysteine. (iv) Total arsenic: 0.01 M acid, 5% L-cysteine after a contact time of 5 min and 2% NaBH4. (v) MMA: 8 M acid, 3% L-cysteine after a contact time of 50 min and 0.6% NaBH4. (vi) As(V): by difference. Detection limits and recoveries of added spikes for all analyses were found to be 0.5-1.7 ppb and 90-112% respectively.