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A modified micromethod for endotoxin assay is reported which involved incubation of plasma extract with Limulus amoebocyte lysate for 30 min followed by incubation with chromogenic substrate S-2423 for another 8 min. Absorbance is measured in microtitre plates. The sensitivity is less than 5 pg/ml (0.058 Endotoxin Units/ml).
A chromogenic assay for coagulation factor VII has been adapted for use on a Cobas Bio centrifugal analyzer. The interassay coefficients of variation ranged from 4.1% to 14%. There was no effect on the factor VII amidolytic activity after freezing and thawing of the samples. The test was linear up to 200% factor VII. The correlation with a chronometric method for prothrombin time was acceptable (r = 0.97, y = 1.05 x + 5.5). The reference ranges for males (54%-138%) and females (56%-200%) were significantly different. The factor VII amidolytic activities were significantly elevated in males with non-insulin-dependent diabetes mellitus and in male hypertensives. No statistically significant increase was observed for female patients rather a decrease in females with insulin-dependent diabetes mellitus. Only in the male hypertensive patients and in the male patients with non-insulin-dependent diabetes mellitus were noticeable proportions of increased values seen (39.4% and 12.0%). Nevertheless, the availability of a precise, rapid and easy to perform test for the determination of factor VII might be useful in the study of the role of this factor in the development of cardiovascular disease.
Endotoxin levels were measured in sterile and bacterially infected ascites in a rat model of phenobarbital and carbon tetrachloride induced cirrhosis was used. An improved chromogenic substrate assay was used to measure endotoxin. All rat ascites specimens were positive for endotoxin. In culture-negative ascites (n = 8), it ranged from 0.05 EU/ml to 0.14 EU/ml (0.08 +/- 0.04 EU/ml, mean +/- SD) (Escherichia coli 0111:B4 endotoxin was used as a reference). In culture-positive ascites (premortem n = 3, postmortem n = 1), it ranged from 0.78 EU/ml to 1.8 EU/ml (1.29 +/- 0.59 EU/ml, mean +/- SD). All rats with premortem culture-positive ascites died within two days. This model is useful to study ascites endotoxin levels. In this study, increasing levels of ascites endotoxin correlated with spontaneous bacterial peritonitis and death.
In hemostasis testing the development of chromogenic substrates provides an alternative to the traditional methods based on the detection of forming clots. The new technology has often replaced the clotting tests, especially in the area of single clotting factor and inhibitor assay, less frequently for global screening tests. We report studies of the validity and clinical application of two reagents for activated partial thromboplastin time (APTT) testing with chromogenic substrates in comparison with the conventional clotting method. Congenital deficiencies of the intrinsic coagulation pathway, other than hypo- and dysfibrinogenemia detected by chromogenic APTT, agreed with those detected by the clotting APTT. The results with the two methods for plasma under heparin treatment suggest a lesser responsiveness of the chromogenic methods to heparinization. The chromogenic methods demonstrated the presence of the lupus anticoagulant in the majority of tested samples of known lupus subjects, but with a lower responsiveness than the clotting method. In conclusion, we found chromogenic APTT suitable for hemostasis testing because it generally gives the same information as the conventional clotting method with the exception of heparin monitoring and lupus anticoagulant detection, where an improved sensitivity would be desirable.
A standardized chromogenic assay of prothrombin (factor II, FII) was compared with a conventional coagulation assay and global tests (Quick prothrombin time, Hepatoquick) in patients on oral anticoagulant therapy. Twenty patients with chronic liver disease were also investigated. Studies of precision showed a coefficient of variation ranging between 2.9 and 4.1% in both normal and anticoagulant samples. Accuracy and variability studies yielded satisfactory results. There was a highly significant positive correlation between chromogenic and coagulation assays, whereas correlation with global coagulation tests was much lower, particularly when normal plasmas were tested. In patients with liver disease, FII assay gave reduced values and appeared at least as sensitive as global coagulation tests in showing the synthetic defect occurring in liver disease. These findings suggest that the chromogenic assay can be employed in the clinical laboratory, even though prospective studies are warranted to establish its predictive value.
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Growing interest is observed in chromogenic substrate assays, because of their better precision, performance and possibilities for automation. Applied to a Cobas Bio centrifugal analyzer, we compared Nycotest-Chrom (N-test) and Thromboquant-PT (Tbq) with Thrombotest (TT). Precision tests were performed with samples from 4 plasma pools: normal (45 sec), low (100 sec), middle (150 sec) and high (200 sec) segments of the therapeutic range of TT. N-test had the best precision profile in both intraassay and interassay determinations compared with Tbq. Both chromogenic substrate assays were better than TT in this respect. By orthogonal regression a provisional therapeutic range was calculated from 312 determinations and later adjusted in a confirmation experiment with natural logarithm regression in 946 determinations. Compared with a TT range of 105-180 sec, the therapeutic ranges were 71-120 sec for N-test and 63-103 sec for Tbq. In a clinical therapeutic control phase, 110 patients were randomized in equal proportions to two groups A and B. Every 2 weeks for a period of 16 weeks, blood samples were tested for N-test, Tbq and TT. In group A, dose adjustment was based on N-test, and in group B on Tbq. The monitoring physician was blinded for Tbq and TT in group A and for N-test and TT in group B. No differences were found between the groups for mean TT, N-test, Tbq or mean dosage, nor differences were found in complication rate. A definite therapeutic range was complied using sensitivity/specificity curves together with their 95% confidence limits. Given TT 105-180 sec, the therapeutic range of N-test is from 80 (95% confidence limits: 77-82) to 110 (95% confidence limits: 108-115) sec, and of Tbq from 68 (95% confidence limits: 66-71) to 95 (95% confidence limits: 91-98) sec. It was concluded that the two chromogenic substrate assays performed equally safe in the monitoring of patients on oral anticoagulant therapy, despite differences in diagnostic correspondence with the reference TT.
The bioavailability of iron from foods is ultimately determined by interactions between iron and other components in the digestive milieu. Perhaps the most important factor is the concentration of Fe2+ during transit through the duodenum. During in vitro simulations of human digestion it is possible to probe the concentration of Fe2+, the rate of Fe2+ formation, and total iron concentration using ferrous chromogens. It is crucial, of course, that the chromogen not interfere with the redox reactions occurring during digestion. Accordingly, ferrozine was examined with regard to its ability to reduce complexes Fe3+, alter rates of Fe3+ production, detect Fe2+ present in the digestive mixture and differentiate the effects of chelating and reducing agents in the mobilization of iron from pinto beans. The chromogen was found to be free from apparent artefacts and to be a sensitive and reproducible probe of the state of iron in digestive mixtures.
Tabanus anticoagulant protein (TAP) was isolated from the whole body of the tabanus, Tabanus bivittatus, using three purification steps (ammonium sulfate fractionation, gel filtration on Bio-Gel P-60, and ion exchange chromatography on DEAE Sephadex gel). The purified TAP, with a molecular weight of 65 kDa, was assessed to be homogeneous by SDS-polyacrylamide gel electrophoresis, and an isoelectric point of 7.9 was determined by isoelectric focusing. The internal amino acid sequence of the purified protein was composed of Ser-Leu-Asn-Asn-Gln-Phe-Ala-Ser-Phe-Ile-Asp-Lys-Val-Arg. The protein was activated by Cu2+ and Zn2+, and the optimal conditions were found to be at pH 3-6 and 40-70 degrees C. Standard coagulation screen assays were used to determine thrombin time and activated partial thromboplastin time. Chromogenic substrate assays were performed for thrombin and factor Xa activity. TAP considerably prolonged human plasma clotting time, especially activated partial thromboplastin time in a dose-dependent manner; it showed potent and specific antithrombin activity in the chromogenic substrate assay. Specific anti-factor Xa activity in TAP was not detected. Overall, this result suggested that TAP has significant anticoagulant activity on blood coagulation system.
2-(2-Quinolinylazo)-5-dimethylaminobenzoic acid (QADMAB) is proposed as a new sensitive and selective chromogenic reagent for spectrophotometric determination of cobalt. The QADMAB reacts with cobalt in the presence of cetyl trimethylammonium bromide (CTMAB) medium to form a violet complex of molar ratio 1:2 (cobalt to QADMAB) in the pH range 3.2-5.2. The molar absorptivity of the complex is 1.28x10(5) L mol(-1) cm(-1) at 625 nm. Beer's law is obeyed in range 0.01-0.32 micro g mL(-1). The relative standard deviation for eleven replicate samples of 0.2 micro g mL(-1) is 0.76%. This method was applied to the determination of cobalt in biological samples, Vitamin B(12), alloys and water with good results.
An automatic reagentless photometric procedure for the determination of ethanol in red wine is described. The procedure was based on a falling drop system that was implemented by employing a flow injection analysis manifold. The detection system comprised an infrared LED and a phototransistor. The experimental arrangement was designed to ensure that the wine drop grew between these devices, thus causing a decrease in the intensity of the radiation beam coming from the LED. Since ethanol content affected the size of the wine drop this feature was exploited to develop an analytical procedure for the photometric determination of ethanol in red wine without using a chromogenic reagent. In an attempt to prove the usefulness of the proposed procedure, a set of red wines were analysed. No significant difference between our results and those obtained with a reference method was observed at the 95% confidence level. Other advantages of our method were a linear response ranging from 0.17 up to 5.14 mol L(-1) (1.0 up to 30.0%) ethanol (R = 0.999); a limit of detection of 0.05 mol L(-1) (0.3%) ethanol; a relative standard deviation of 2.5% (n = 10) using typical wine sample containing 2.14 mol L(-1) (12.5%) ethanol; and a sampling rate of 50 determinations per hour.
The first potassium optode based on a protic chromoionophore immobilized in a hydrogel matrix is presented. The highly selective protic chromoionophore consists of a cryptohemispherand moiety and a trinitroanilino chromophore part. The acidifying power of potassium ions over sodium ions is 0.6 pH units. This correlates with the findings in solution. In contrast to several crown and aza-crown based chromophores the highly pre-organized moiety allows ion detection even in aqueous environment. The detection limit for potassium ions at pH 7.7 is 5 microM.
A flow system designed by computer-controlled discrete commutation devices is proposed for the multidetermination of metallic ions with a single chromogenic reagent. Intermittent addition of masking agents and changes of the reaction pH were exploited to implement selective determination of four species. Multidetection with an optical-fiber CCD-array spectrophotometer enabled simultaneous measurements at different wavelengths for multidetermination. The proposed system was applied to the determination of iron, copper, nickel and zinc in alloys and pharmaceutical preparations, yielding results in agreement with those obtained by flame atomic absorption spectrophotometry at the 95% confidence level. The sampling rate was estimated to be 80 determinations per hour. Coefficients of variation lower than 2% (n = 20) were achieved for all species.
An alpha-galactosidase from the erythromycin-producing bacterium Saccharopolyspora erythraea was purified to near homogeneity. The enzyme has an apparent molecular mass of 45 kDa as determined by SDS-PAGE. The pH optimum, K(m) for p-nitrophenyl-alpha-D: -glucopyranoside (pNPalphaG), K(m) for melibiose and the V(max) are similar to those of other studied alpha-galactosidase enzymes. The N-terminal amino-acid sequence of this protein was determined. PCR amplification was used to generate a 640-bp product using oligonucleotide primers based on the N-terminal amino-acid sequence and a downstream region that is conserved in other related alpha-galactosidase enzymes. This fragment was used as a probe to clone the alpha-galactosidase gene, designated melA, from a S. erythraea lambda phage chromosomal library. S. erythraea appears to possess an unique alpha-galactosidase enzyme, encoded by melA, that can utilize galactopyranosides as carbon sources. Furthermore, the ability to use the product of melA as a reporter enzyme in S. erythraea has been demonstrated. The alpha-galactosidase uses the substrates 5-bromo-4-chloro-3-indoyl-alpha-D: -galactosidase (X-alpha-gal) on agar media and pNPalphaG in liquid media.
An endogenous beta-glucuronidase that hydrolyses the chromogenic substrate 5-bromo-4-chloro-3-indolyl-beta-D-glucuronide (X-gluc) in Aspergillus niger is reported. The activity was induced when the fungus was grown in media containing xylan, but was either very low, or absent, when grown on glucose. Endogenous beta-glucuronidase was primarily located in newly formed hyphae, and was apparent at pH values between 3 and 6. Hydrolysis of X-gluc was sensitive to the inhibitor D-saccharic acid 1,4-lactone and was irreversibly inactivated by heating. The bacterial uid A beta-glucuronidase reporter gene was strongly expressed in the hyphae of transformed A. niger but, in contrast to the endogenous activity, the enzyme was also active at pH 7-8.5. Histochemical localization of uidA expression in A. niger, without interference from the endogenous beta-glucuronidase activity, was achieved by staining at this pH.
In this note the genetic characterization of the peptide degrading system of Propionibacterium freudenreichii was addressed. Genomic fragments of P. freudenreichii subsp. freudenreichii LMG 16415 were cloned in Escherichia coli XL1 Blue, and those leading to an increase in peptidase-like activity using chromogenic substrates aminoacyl-beta-naphtylamides (aminoacyl-betaNA) were isolated and sequenced. This strategy allowed the identification of partial gene regions of P. freudenreichii LMG 16415 with significant similarity to proteins directly or indirectly involved in peptide and amino acid metabolism, i.e., an oligopeptide transporter, a D-amino acid oxidase, a muropeptidase, and an ABC transporter involved in osmoregulation similar to glycine betaine transporters.
The effects of 2,3-butanedione monoxime (BDM) were examined using rat ventricular myocytes loaded with Indo-1 to measure the intracellular Ca concentration ([Ca2+]i). BDM (10 mM) produced a transient increase of the systolic Ca transient with no steady-state effect on its magnitude. This transient increase was more marked when BDM was applied after having decreased the external Ca concentration from 1 to 0.1 mM. There was a transient increase of resting [Ca2+]i in both quiescent and electrically stimulated cells. Prior application of BDM decreased the rise of [Ca2+]i produced by caffeine. In voltage-clamped cells the rise of [Ca2+]i produced by BDM was accompanied by a transient inward current attributed to the electrogenic Na-Ca exchange. The amount of Ca lost from the cell upon application of 10 mM BDM could be estimated either from the integral of the BDM-evoked current or from the reduction of the integral of a caffeine-evoked current and corresponded to about 50% of the sarcoplasmic reticulum (s.r.) Ca content. The decrease of s.r. Ca content and the transient potentiation of the systolic Ca transient suggest that BDM acts by stimulating Ca-induced Ca release. These effects must be allowed for when using BDM.
Inhibition of protein synthesis by cycloheximide during vetch seed germination, did not prevent globulin breakdown as indicated by a decrease in vicilin- and legumin-specific immunosignals on Western blots. Protein bodies isolated from embryo axes and cotyledons of dry vetch (Vicia sativa L.) seeds using a non-aqueous method were found to be free of cytoplasmic and organellar contaminations. Lysates of these purified protein bodies were capable of degrading globulins; this process was blocked by the cysteine proteinase (CPR) inhibitor iodoacetic acid. Protein bodies contained the papain-like CPR2 and CPR4, and the legumain-like CPR VsPB2. In vitro assays showed that albumin extracts from protein bodies degraded oligopeptide substrates in the PepTag-Assay and degraded the legumain substrate N-benzoyl-asparaginyl-p-nitroanilide. We conclude that, during germination, globulin mobilization is initiated by stored CPRs in protein bodies of embryonic axes as well as cotyledons, and that de-novo-formed proteolytic enzymes mainly mediate bulk degradation of stored globulin in cotyledons after germination.