Identification of three indolic compounds in a pigmented-melanoma cell-culture supernatant by gas chromatography-mass spectrometry.

In the supernatant of melanoma cell culture SK mel 25 three indolic compounds - 5-hydroxy-6-methoxyindole, 5-hydroxy-6-methoxyindolyl-2-carboxylic, and 6-hydroxy-5-methoxyindolyl-2-carboxylic acids - have been identified. The supernatants were extracted with ethyl acetate, derivatized, and analyzed by gas chromatography-mass spectrometry. The significance of this finding is discussed.

Capillary gas chromatographic profiling of urinary, plasma and erythrocyte sugars and polyols as their trimethylsilyl derivatives, preceded by a simple and rapid prepurification method.

A capillary gas chromatographic method for the profiling of trimethylsilylated mono- and disaccharides and polyols in urine, plasma and unwashed and washed erythrocytes is described. The prepurification method is based on the moderate inhibition of the derivatization experienced in the presence of physiological amounts of inorganic salts and the relative stability of the formed trimethylsilylethers towards treatment with water and dilute hydrochloric acid. Series to series quality control data for 31 sugars/polyols in a pooled urine are given. The method was used to establish age-dependent concentrations of 18 sugars/polyols in urines of 72 control persons on a free diet. Gas chromatographic profiles and quantitative data obtained from urines of pediatric patients with galactosemia treated with a diet low in lactose and galactose, type 1 hereditary tyrosinemia treated with a diet low in phenylalanine and tyrosine, and a neurological disorder with a high calorie gastric drip feeding, are presented and discussed. Examples of the profiling of sugars/polyols in the plasma, unwashed and washed erythrocytes of a healthy adult, and the plasma of a newborn with galactosemia prior to treatment, are given.

Hemoglobins S and C: reference values for glycohemoglobin in heterozygous, double-heterozygous and homozygous subjects, as established by 13 methods.

Glycohemoglobin (gly-Hb) reference ranges of non-diabetic adults with HbAA (n = 17), HbAS (n = 37), HbAC (n = 22), HbSC (n = 8), HbSS (n = 6) and HbCC (n = 3) were determined by 13 methods, based on affinity chromatography, HPLC, electrophoresis and immunoassay. Gly-Hb of subjects with HbAS and HbAC can be measured without major difficulties by most methods. Some give rise to absolute gly-Hb differences > or = 1% compared with subjects with HbAA. Measurement of HbA1c/total Hb cannot be recommended. Some HPLC and immunoassay methods cannot measure gly-Hb in subjects with HbSC, HbSS and HbCC, whereas others may suffer from interference. Most methods showed low gly-Hb, reflecting increased erythrocyte turnover. Use of special reference ranges requires previous knowledge of the condition (affinity chromatography and immunoassay) or separation of gly-Hb and its precursor Hb (HPLC and electrophoresis). Interpretation is, however, not recommended because of the numerous factors that determine erythrocyte turnover.

Identification of Thormählen-positive compound "B" in urine of patients with malignant melanoma.

The identification of the entire structure of the Thormählen-positive compound B from melanotic urine is described. The compound was separated from other Thormählen-positive compounds using DEAE-cellulose column chromatography. On the basis of differential enzymic hydrolysis followed by gas chromatography-mass spectrometry analysis and comparison with synthetically prepared compounds it is possible to conclude that the Thormählen-positive compound B is a mixture of O-sulphate 5-hydroxy-6-methoxyindole and 6-hydroxy-5-methoxyindole with predominance of the latter.

Glycohaemoglobin: comparison of 12 analytical methods, applied to lyophilized haemolysates by 101 laboratories in an external quality assurance programme.

Stable lyophilized ethylenediaminetetra-acetic acid (EDTA)-blood haemolysates were applied in an external quality assurance programme (SKZL, The Netherlands) for glycohaemoglobin assays in 101 laboratories using 12 methods. The mean intralaboratory day-to-day coefficient of variation (CV), calculated from the assay of 12 unidentified pairs over a period of 1 year, was 5.2% (range: 0.2-28.7). Forty-seven per cent of laboratories did not meet the criterion of CV < 5%, whereas 68% did not meet the clinically more desirable 3.3-3.6%. Linearity, as derived from the analysis of five combinations of two haemolysates with low and high glycohaemoglobin percentages over 6 months, was excellent (mean correlation coefficient 0.9953; range: 0.9188-0.9999). Analysis of two samples with high and low glycohaemoglobin percentages gave mean interlaboratory coefficients of variation of 10% for one method performed by several laboratories and 22% for all methods performed by all laboratories. It is concluded that the majority of laboratories do not meet the clinically desirable intralaboratory precision and that an unacceptably high interlaboratory precision exists.

Potential of descriptive linear discriminant analysis for studying clinical chemical and haematological data from persons with heterozygous sickle cell disease.

To study the potential of multivariate classification methods in order to obtain more insight into abnormal laboratory data from patients with sickle cell disease, we investigated standard haematological and clinical chemical variables of 18 controls and 37 apparently healthy persons with heterozygous sickle cell disease (Hb AS), all women, using both univariate and multivariate classification methods. In the univariate method, those with Hb AS showed decreased serum log aspartate aminotransferase (log AST) activity, mean corpuscular volume and mean corpuscular haemoglobin (MCH) and increased sodium concentration. The multivariate method identified sodium, potassium, urea, uric acid, log AST, alanine aminotransferase and MCH as the variables that produced maximal separation between persons with Hb As and controls. It increased the 'non-error rate' for classification of persons with Hb AS by 16.4% compared with classification based on the variable, MCH, that produced maximal separation by the univariate method. The frequency distribution of percentage Hb S in the Hb AS group proved bimodal with maximal separation at 37.0% Hb S. The subgroup with 37.0% or less (n = 16) was considered to have concomitant heterozygous alpha-thalassaemia-2. In the univariate method the subgroup characterized by greater than 37.0% Hb S (n = 21) had increased serum sodium and uric acid concentrations, perhaps related to sickle cell nephropathy, whereas the subgroup with less than or equal to 37% Hb S did not. The multivariate method added information to the univariate method by additionally identifying abnormalities in serum potassium and urea concentrations in the former subgroup.(ABSTRACT TRUNCATED AT 250 WORDS)

A new method for the determination of gamma-glutamyltransferase in serum.

A new, simple and sensitive method is described for assay of serum gamma-glutamyltransferase activity based on the hydrolysis of the substrate L-gamma-glutamyl-3-carboxy-4-nitranilide which offers the advantage of producing directly its own chromogen. The substrate is highly soluble and the method can therefore easily be adapted to any equipment for the automated assay of gamma-glutamyltransferase. The activities at measured optimum substrate concentration are slightly higher than with the Szasz ((1969), Clin. Chem. 15, 124-136) method in which L-glutamyl-p-nitroanilide is used as substrate.

Influence of hemoglobin variants and derivatives on glycohemoglobin determinations, as investigated by 102 laboratories using 16 methods.

Influences of hemoglobin (Hb) variants (HbSS, HbCC, beta-thalassemia, HbAE, HbAS, HbAC, hereditary persistent HbF) and Hb derivatives (carbamylated- and acetylated-Hbs, Schiff base, and those formed in stored blood) on results of glyco-Hb assays by 102 laboratories using 16 different methods were investigated. Affinity chromatography shows deviating results only with homozygous Hb S and C. Correct interpretation of results from patients with decreased erythrocyte half-lives requires previous knowledge on this condition. Measurements of HbA1c by HPLC and electrophoresis are obviously unsuitable for homozygous hemoglobinopathies; for heterozygous hemoglobinopathies and Hb synthesis variants, HbA1c should be expressed as percentage of HbA0 + HbA1c; abnormal Hbs are usually recognized; both carbamylated- and acetylated-Hbs interfere and Schiff base must be eliminated. Except for stored blood, all Hb variants and derivatives gave erroneous results with disposable ion-exchange columns. Dako's immunoassay is not affected by Hb derivatives; glycated Hb variants are not recognized as glyco-Hb and percentages are consequently too low. Glyco-Hb by the immunoassay of Bayer (performed by one laboratory) is not affected by Hb variants and derivatives.

Determination of N tau-methylhistamine in urine by gas chromatography using nitrogen-phosphorus detection.

The determination of N tau-methylhistamine in urine, using gas chromatography with nitrogen-phosphorus detection and the homologue N tau-ethylhistamine as internal standard, is described. A comparison between the present method and a previously described stable isotope dilution mass fragmentographic method resulted in a regression line of Y = 0.023 + 0.944X mumol/l with a correlation coefficient of 0.996. The 24-h excretions of 35 normal adults on a free diet ranged from 0.4 to 1.8 mumol. Patients with mastocytosis, chronic myelocytic leukemia, anaphylactic reactions and a patient after bronchial provocation showed above normal values.

Automated method for the determination of 5'-nucleotidase in serum by continuous flow analysis.

The manual method of Persijn & Van der Slik (J. Clin. Chem. Clin. Biochem. 6, 441-446 (1968): 7, 493-497 (1969); 8, 398-402 (1970) for the determination of serum 5'-nucleotidase has been adapted to the Auto-Analyzer II System. In the Auto-Analyzer II, the incubation temperature for the enzyme reactions is 37 degrees C, and the effective sample speed is 30/h, at a sample/wash ratio of 2:1. There is good agreement and correlation between the manual method and the Auto-Analyzer II method (equation of regression line: y = x + 0.6, correlation coefficient = 0.988; the normal range is 2.9-10.5 U/l for both methods). In routine use, within-run and between-day reproducibility are considerably smaller for the Auto-Analyzer II than for the manual method, especially when large numbers of samples are assayed.

Determination of serum nucleotidase with cytidine monophosphate as substrate. Part II: Improvement of the procedure.

Serum mucleotidase activity (EC 3.1.3.5) is measured by the amount of ammonia liberated from cytidine after incubation with cytidine deaminase (EC 3.5 4.5). Purification of the cytidine deaminase results in the abolition of interfering reactions, so that routine application is possible.

Evaluation of a reference material for glycated haemoglobin.

The use of lyophilized blood as a reference material for glycated haemoglobin was investigated with respect to IFCC criteria for calibrators and control materials. Ninety-two laboratories, using 11 methods, detected no changes in glycated haemoglobin content when the lyophilizate was stored for one year at 4 degrees C. Affinity chromatography, HPLC, electrophoresis and immunoassay detected no changes following 18 months storage at -84 and -20 degrees C. Samples for HPLC are stable at 4 degrees C for one year, and 5 years at -20 degrees C. For the other three methods, samples are stable for 5 years at 4 degrees C. At 4 degrees C, reconstituted samples are stable for 2 days (HPLC) and 7 days (other three). Lyophilization does not cause matrix effects and inhomogeneity, since mean glycated haemoglobin and reproducibility for lyophilized samples and whole blood were similar. The coefficient of variation for vial filling precision was 0.59%. We conclude that lyophilized blood samples can be used as calibrators and control materials. Their use as calibrators, following assignment of the HbA(1c) value by HPLC, may contribute, in the interim, to the standardized interpretation of long term diabetic control.

Effect of calibration on dispersion of glycohemoglobin values determined by 111 laboratories using 21 methods.

One hundred eleven laboratories, using 21 different methods based on five different principles, determined glycohemoglobin (GHb) percentages in two identical series of six lyophilized hemolysates and three similarly processed calibrators, distributed 3 months apart. To assign GHb percentages to calibrators, we used HbA1c results from nine participants who used the Bio-Rad Diamat high-performance liquid chromatographic method. Three-point calibration with assigned values improved mean intralaboratory variation (CV) from 6.6% to 3.5%. For samples with low (5.5%) and high (14.1%) GHb percentages, respectively, calibration decreased interlaboratory variation per method (from 10% to 4% and from 6% to 3%), inter-method variation (from 18% to 4% and from 16% to 3%), and overall interlaboratory variation (from 25% to 7% and from 15% to 4%). Without calibration, 71% of the laboratories did not meet the clinically desirable intralaboratory CV of 3.5%; calibration reduced this proportion to 39%. We conclude that, irrespective of the analytical method used, calibration greatly reduces all sources of GHb variation.