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A technic of autoanalysis of the elastase activity and the capacity of elastase inhibition of the serum is described. This estimation depends on the hydrolysis by elastase of N-ter-butoxycarbonyl-L-nitrophenyl ester with liberates p.nitrophenol the colour of which is read at 405 nm. The means of this sample estimation are reported in a few cases of respiratory, dermatological and pancreatic disease.
Analysis of blood urea nitrogen (BUN) on 160 sera by Urograph and autoanalysis revealed excellent correlation if the results of the Urograph method were reported in one of three ranges: under 20 mg. %, between 20 and 50 mg. %, and over 50 mg. %. Because of the simplicity of the Urograph method, it is suggested that this procedure should be useful for night, weekend and emergency analyses, or possibly for mass screening of BUN levels where automated equipment is not available.
The authors propose a method of autoanalysis of protein in presence of Triton X-100. This method uses Lowry's method in presence of sodium dodecyl-sulphate which permits inhibition of the formation of precipitates between Triton X-100 and Folin's reagent.
This work studies the quality of the response of the Electra 600 for the determination of the prothrombin time (PT) and the activated cephalin time (ACT). The intra-serial precision was good for the PT and the ACT with normal or pathological plasmas (CV 2%). The precision was excellent for the PT (r = 0.98; Electra 600-fibrometer). The correlation coefficient varies between 0.85 and 0.95 for the ACT depending on the nature of the activator chosen. Lactescent plasmas having a protein concentration greater than 85 g/l should be treated manually for measurement of the PT. No interference was noted with other biological substances: eg glucose, bilirubin, hemoglobin. The increasing addition of heparin indicates a correct sensitivity of the response with Electra 600 both for short and long ACT. Autoanalysis renders the determinations of PT and ACT independant of the manipulator, that of the PT being much more rapid than with the manual method
The usefulness of R(-)-4-(3-isothiocyanatopyrrolidin-1-yl)-7-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole [R(-)-DBD-PyNCS], a fluorescent chiral tagging reagent, for the determination of racemic amines and amino acids, was studied. The reagent reacted with beta-blockers selected as representative secondary amines to produce corresponding fluorescent diastereomers (excitation at 460 nm and emission at 550 nm). The yields of the derivatization reaction were dependent on the stereostructure arround the NH group in beta-blockers. The resulting diastereomers were completely separated with single chromatographic run using linear gradient elutions by reversed-phase chromatography. R(-)-DBD-PyNCS was also applied to the determination of DL-amino acid, considered to be one of the primary amines, in human urine and foodstuffs. DL-amino acids tested equally reacted with the reagent, and the thiocarbamoyl derivatives were separated with an ODS column. The epimerization during the derivatization reaction was negligible judging from the resolution of opposite diastereomers on the chromatogram. The occurence of D-amino acids (D-Ala, D-Ser, D-Asp and/or D-Glu) was identified in the samples tested. The structures and the purities were elucidated with on-line HPLC-MS. The chiral reagent possessing an isothiocyanate group (-NCS) in the structure seems to be applicable to continuous sequential analysis of peptides containing D-amino acids. The thiocarbamoyl derivatives obtained from the reaction with DL-amino acids were converted to thiohydantoins via thiazolinones in acidic medium. The thiohydantoins produced from acidic, basic, neutral, hydroxyl and aromatic amino acids were completely separated with isocratic elutions using acidic mobile phase containing 0.1% TFA. The separations were sufficient for the identification of DL-amino acid in peptide sequences. Although the epimerization during the conversion reaction to thiohydantoins was not avoidable, the descrimination of D- and L-configuration was demonstrated with some commercially available peptides such as beta-lipotropin and [D-Ala2]-deltorphin II. The Edman degaradation method using R(-)-DBD-PyNCS was also adopted to autoanlaysis by gas-phase sequencer. The separation and the detection (UV 254 nm) conditions of the derivatives were used without any change from those for the Edman degradation method using PITC as the tagging reagent. The three DL-amino acid residues (Tyr, Ala and Gly) in [L-Ala2]-leucine-enkephalin and [D-Ala2]-leucine-enkephalin were perfectly identidied with the autoanalysis.
The activities of serum malate dehydrogenase (MDH) and its mitochondrial isoenzyme (MDHm) were studied in sera of patients with liver disease. They proved to be more useful than those of aspartate aminotransferase (AST) and its mitochondrial isoenzyme for detection of hepatocellular carcinoma and acute circulatory failure, and for estimation of the severity of acute hepatitis. The N/T value measuring system, which is adaptable for autoanalysis and allows simultaneous determination of activities depending on NAD and thionicotinamide adenine dinucleotide (thio-NAD), yields both the total activity of MDH and the N/T value which was correlated significantly with MDHm/MDH (r = 0.748). Assay of MDH and its mitochondrial isoenzyme in association with the N/T value measuring system seems to be more useful and less time consuming for estimation of the severity of liver diseases than that of AST and its mitochondrial isoenzyme.
1. The amino acid composition of total proteins in six stages of the life cycle of Schistosoma mansoni was determined by routine autoanalysis of acid hydrolysates. Aspartate, glutamate and glycine were consistently the most abundant protein amino acids in all stages. 2. Incorporation of each of the protein amino acids into adult and egg proteins was determined using 72 hr cultures in complex media. Incorporation rates varied widely and there was no correlation between abundance in protein and the rate of incorporation. 3. Only five amino acids were interconverted to other amino acids which were themselves incorporated into worm and egg proteins (ala, arg, asp, gly, ser); of these only two (glu from ala and pro from arg) appeared to be of quantitative significance. Exogenous glucose yielded only three protein amino acids (ala, asp, glu). 4. The data are considered in the light of differences in egg and adult protein synthesis and with particular regard to potential chemotherapy at this level.
An automated chromatographic detection system for the simultaneous determination of riboflavin phosphate, caffeine, nicotinamide and pyridoxine hydrochloride in a multivitamin pharmaceutical preparation was constructed. Hydrolytic pretreatment of riboflavin phosphate to riboflavin was carried out using a pre-column enzyme reactor, in which immobilized sweet potato acid phosphatase was packed, and then enzymatically hydrolyzed riboflavin and other ingredients in the pharmaceutical preparation were concentrated in an ODS trap column. The concentrated riboflavin and other ingredients were back-eluted from the trap column using a mobile phase containing 1-decanesulfonate as an ion-pair reagent, and then subsequently chromatographed on an ODS analytical column. It was necessary to wash the ODS trap column with aqueous acetonitrile to remove 1-decanesulfonate in the trap column, which is advantageous to concentrate the riboflavin and other ingredients for the subsequent analysis. The calibration curves for riboflavin phosphate and other ingredients were linear over the concentration ranges tested, and correlation coefficients for standard curves were 0.9999 for all four ingredients. Analytical recoveries of the four ingredients at different levels of concentration added to the ordinary pharmaceutical preparation were also in the range of 99.1-101.2%. The present method was superior to the ordinary manual and batch-wise enzymatic methods in being harmless to the environment, rapid and accurate under continuous autoanalysis.
BACKGROUND: Reports about side effects from cromolyn sodium (DSCG) are few and only a minority of them indicate the possibility of an immediate type I hypersensitivity reaction. METHODS: We report an 8-year-old boy with a history of seasonal rhinoconjunctivitis and asthma. The patient reported two immediate episodes of acute asthma following inhalation of a cromolyn sodium (DSCG) capsule with an interval of 6 months. The latter occasion was also associated with conjunctivitis and generalized urticaria, requiring emergency treatment. RESULTS: An end-point prick test elicited a 4-mm wheal at 10 mg/mL with DSCG solution for inhalation and DSCG eye drops. An end-point intradermal skin test with DSCG solution for inhalation elicited a 12-mm mean diameter wheal at 10 mg/mL. During the intradermal test, the patient developed conjunctivitis and wheezing with a FEV1 fall of 27% from baseline. Controlled conjunctival and bronchial challenge tests were positive. The bronchial challenge test was repeated 3 years later showing a FEV1 fall of 22% five minutes after inhalation of 1 mg/mL DSCG during one minute. A leukocyte histamine release test performed by an autoanalysis fluorometric procedure, with several dilutions of DSCG, was negative. CONCLUSIONS: This case of DSCG anaphylaxis satisfies the criteria for an IgE-mediated reaction. We believe that thought should be given more frequently to this mechanism when patients report adverse reactions to DSCG.
A defined substrate method was developed to simultaneously enumerate total coliforms and Escherichia coli from drinking waters without the need for confirmatory or completed tests. It is a new method based on technology that uses a hydrolyzable substrate as a specific indicator-nutrient for the target microbes. No equipment other than a 35 degrees C incubator and long-wavelength (366-nm) light is necessary. To perform the test, one only has to add water to the powdered ingredients in a tube or flask. If total coliforms are present in the water sample, the solution will change from its normal colorless state (no target microbes present) to yellow. The specific presence of E. coli will cause the same tube to fluoresce under a longwave (366-nm) UV lamp. The test, called Autoanalysis Colilert (AC), was compared with Standard Methods for the Examination of Water and Wastewater 10-tube multiple tube fermentation (MTF) in a national evaluation. Five utilities, representing six U.S. Environmental Protection Agency regions, participated. All water samples came from distribution systems. Split samples from a wide variety of water sources were analyzed for the MPN-versus-MPN comparison. A total of 1,086 tubes were positive by MTF, and 1,279 were positive by AC. There was no statistical difference between MTF and AC. Species identifications from positive tubes confirmed the sensitivity of the AC. A national evaluation of the AC test showed that it: (i) was as sensitive as Standard Methods MTF, (ii) specifically enumerated 1 total coliform per 100 ml, in a maximum of 24 h, (iii) simultaneously enumerated 1 E. coli per 100 ml in the same analysis, (iv) was not subject to false-positive or false-negative results by heterotrophic bacteria, (v) did not require confirmatory tests, (vi) grew injured coliforms, (vii) was easy to inoculate, and (viii) was very easy to interpret.
A defined substrate method was applied to drinking water to simultaneously enumerate total coliforms and total Escherichia coli directly from samples. After incubation at 35 degrees C for 24 h, the development of yellow in an initially colorless solution was specific for total coliforms; fluorescence at 366 nm in the same tube(s) or vessel demonstrated the presence of E. coli. No confirmatory or completed steps were necessary. Known as autoanalysis colilert (AC), this method was constituted as a presence-absence test and compared with the methods described in Standard Methods (SM) in the P-A format. Seven water utilities representing a wide geological and hydrological spectrum participated in the evaluation. A total of 702 split drinking water samples were analyzed. Of these, 358 were negative in both tests (SM- and AC-); 302 were positive (SM+ and AC+); and 42 were mixed (SM+ and AC-, 20; AC+ and SM-, 22). The overall agreement rate was 94%. Comparison of the SM and AC results by nonparametric statistics demonstrated no differences. Heterotrophic plate count bacteria exerted no discernible effect on the AC test. By subculture, each time the AC test was yellow, a total coliform was present; when the test was fluorescent, E. coli was isolated.
The new United States Drinking Water Regulations state that water systems must analyze for Escherichia coli or fecal coliforms on any routine or repeat sample that is positive for total coliforms. The proposed methods for the detection of E. coli are based on beta-glucuronidase activity, using the fluorogenic substrate 4-methylumbelliferyl beta-D-glucuronide (MUG). This study was conducted to determine whether beta-glucuronidase negative E. coli were present in significant numbers in environmental waters. Two hundred and forty E. coli cultures were isolated from 12 water samples collected from different environmental sources. beta-glucuronidase activity was determined using lauryl tryptose broth with MUG, EC broth with MUG, and the Autoanalysis Colilert (AC) procedure. The isolates were also evaluated by the standard EC broth gas fermentation method for fecal coliforms. The results confirm that assaying for the enzyme beta-glucuronidase utilizing the MUG substrate is an accurate method for the detection of E. coli in environmental waters.
An appropriate procedure for analysis of cystine in the hair has been established, and this procedure was applied to the hair of Japanese women. The recovery of authentic cystine added to a hair sample was 85-90% in 6N HCl hydrolysis, and 96% in acid hydrolysis after performic acid oxidation. The 18-hr acid hydrolysis, but not 4-hr one, was sufficient to digest the hair. In colorimetry by using phosphotungstate, Brown's reagent gave a stable color development. The cystine content by amino acid autoanalysis was significantly correlated with that by the phosphotungstate colorimetry by the modified method of Kassel et al. or of Shinohara. The cystine content in women's hair, which was collected from specimens of different individuals cut during the period from 1910s to 1980, indicated a wide variation ranging from 0.654 to 1.607 mmol half-cystine per g of hair after washing with 0.5% sodium laurylsulfate.
The effects of prolonged administration of anticonvulsants were analyzed on different biochemical parameters related to phosphocalcium metabolism in 98 children aged 1 to 14, not affected by other chronic pathology, and to whom no Vitamin D nor other vitamin-mineral complexes had been administered. We take as reference a group of normal children studied during the same period. Determinations were accomplished with the usual chemical controls in our laboratory (autoanalysis of continuous flow, radioimmunoassay and colorimetric measurements) and the following mean levels were obtained for treated children: Calcium: 9.2 +/- 0.4 mg/dl, phosphorous: 3.5 +/- 1.9 mg/dl, alkaline phosphatase: 252 +/- 72 U/L, 25-Hydroxycholecalciferol: 35.6 +/- 18 ng/ml, Osteocalcine: 5.4 +/- 3.6 ng/ml, and parathyroid hormone: 0.4 +/- 0.1 ng/ml. These values differed significantly from those found in the control group for phosphorous, lower in children under treatment (p = 0.000), and the 25-Hydroxycholecalciferol was likewise lower (p = 0.000). In other way the mean levels of alkaline phosphatase were higher in treated children (p = 0.000). No significant differences were obtained for mean levels of calcium, parathyroid hormone and osteocalcine. These differences are maintained when distributing patients among different age groups. Solar radiation received by treated children during the months preceding the extraction, did not produce significant differences on 25-Hydroxycholecalciferol, with mean values that were similar at the end of summer (34 +/- 9 ng/ml) and the end of winter (36.6 +/- 18 ng/ml).
The level of diabetes education has been evaluated by means of a test questionnaire with 62 items in a group of 57 insulin-dependent Diabetes Mellitus (type I) patients who were cared for at a Hospital Diabetes Unit for a least six months prior the study. We observed that only 60% of our patients had an acceptable diabetes education. Our educational programme seems to be efficient for patients between 20 and 40 years old with a High School or University degree, although we think that the educational method should be changed for older or less educated patients. Moreover, we have analyzed the possible influence of diabetes education on the glycemic control, both globally, and in specific areas. We observed a significantly lower MBG in the patient group with a higher level of diabetes knowledge. The MBG and HbA1c values correlate with training in blood glucose autoanalysis.
Forty-six patients presenting typical or non-typical angina pectoris were all submitted to analysis of effort angina, traditional or automatic, and to analysis of the amplitude of the R wave and to an effort myocardial scan using thallium 201 (taken as reference substance) during maximal muscle exercise. The following conclusions may be drawn from this study. 1) Autoanalysis of the ECG improves considerably the sensitivity without changing the specificity of the method (92 and 82 p. cent for 79 and 82 p. cent). Analysis of the variations in amplitude of the R wave gave no further information. It should be reserved for patients whose effort ECG was difficult to interpret: e.g. left or right bundle branch block, digitalis effect, very abnormal resting ECG, or average age female population.
In medical practice, laboratory tests, especially chemical tests, has been used as an important supplementary tool for diagnosis. However, recent "clinical chemistry" as a science is stagnant and wandering. Since the clinical importance of "clinical chemistry" has been clarified in various fields and deeply permeated into daily practice, "clinical chemistry" as a science has come up against a brick wall. With such a background, some researchers attempted to find a way in the technical field or accuracy management field, i.e., "analytic chemistry". In this field, certain results have been obtained, and the analytic accuracy has been so improved as to reflect in vivo changes at extremely high accuracy. However, this "analytical chemistry" is, so to speak, a field of emergency refuge of "clinical chemistry" and not its original state. Many clinical chemical items can be analyzed using automatic analyzers, and academic interest is lost due to autoanalysis as a black box. In present status, we should develop a new "clinical chemistry". The borders between examination areas have become unclear, and immunological laboratory techniques have been introduced into clinical chemistry. We should estimate new approach using these new techniques and establish their clinical significance. In medical practice, not only diagnostic imaging test, such as ultrasound imaging CT, MRI for evaluation of organic in vivo changes but also clinical test including chemical test for sensitive evaluation of functional changes is indispensable. These two examination methods should be combined in medical care with "patients" placed in its central position.
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