Showing papers in "Clinical Chemistry in 1982"

Serum triglycerides determined colorimetrically with an enzyme that produces hydrogen peroxide.

Abstract: In this direct colorimetric procedure, serum triglycerides are hydrolyzed by lipase, and the released glycerol is assayed in a reaction catalyzed by glycerol kinase and L-alpha-glycerol-phosphate oxidase in a system that generates hydrogen peroxide. The hydrogen peroxide is monitored in the presence of horseradish peroxidase with 3,5-dichloro-2-hydroxybenzenesulfonic acid/4-aminophenazone as the chromogenic system. The high absorbance of this chromogen system at 510 nm affords useful results with a sample/reagent volume ratio as low as 1:150, and a blank sample measurement is not needed. A single, stable working reagent is used; the reaction is complete in 15 min at room temperature. The standard curve is linear for triglyceride concentrations as great as 13.6 mmol/L. Average analytical recovery of triglycerides in human sera is 100.1%, and within-run and between-run precision studies showed CVs of less than or equal to 1.6 and less than or equal to 3.0%, respectively. The method is suitable for automation.

Rapid assay for amino acids in serum or urine by pre-column derivatization and reversed-phase liquid chromatography.

Abstract: This method for estimating clinically important amino acids in serum or urine within 40 min involves o-phthalaldehyde/2-mercaptoethanol derivatization and reversed-phase "high-pressure" liquid chromatography. Homocysteic acid is an internal standard, and homoserine and norvaline are reference peaks. For all the amino acids estimated, the between-run coefficients of variation ranged from 2.0 to 13.5%, and the mean analytical recoveries from both serum and urine samples was 101%. Peak areas vary linearly with concentration up to 1500 mumol/L for all the amino acids assayed. The limit of detection for each amino acid was estimated to be 38 fmol.

Alkaline phosphatase isoenzymes.

Abstract: The human alkaline phosphatases constitute a system of multiple molecular forms of enzymes in which heterogeneity is partly due to genetic factors and partly to posttranslational modifications. Recognition of the nature and occurrence of these multiple forms has made a significant contribution both to the understanding of changes in alkaline phosphatase values for serum in disease and to the use of alkaline phosphatase measurements in diagnosis. Many of the diagnostic advantages of alkaline phosphatase isoenzyme analysis can be obtained with the aid of qualitative methods such as zone electrophoresis. However, quantitative methods are needed to take full advantage of the potential benefits of isoenzyme analysis. Selective inactivation methods can be applied successfully to the quantitative analysis of bone and liver alkaline phosphatases in serum. However, the aim of future research should be to remove the limitations at present imposed on quantitative analysis by the close similarities of bone and liver alkaline phosphatases.

Determination of glycosylated hemoglobin by affinity chromatography: comparison with colorimetric and ion-exchange methods, and effects of common interferences.

Abstract: An affinity-chromatographic method for determination of glycosylated hemoglobin (Anal. Lett. 14: 649-661, 1981) is compared with the thiobarbituric acid colorimetric (I) (Clin. Chem. 27: 669-672, 1981) and the ion-exchange liquid-chromatographic (II) (Diabetes 29: 623-628, 1980) methods. A correlation of 0.98 was obtained for the affinity method vs II and 0.97 for affinity vs I (n = 51). The within-run CV was 1.9% for specimens from non-diabetic individuals and 1.0% for those from diabetics. The respective between-run CVs were 3.4% and 2.4%. Failure to remove "labile" glucose adducts by 5-h incubation of erythrocytes in isotonic saline (37 degrees C) contributed an average error of 13.1% for II, 5.4% for I, and 1.6% for the affinity method. Affinity chromatography gave a decrease of 0.1-0.2% glycosylated hemoglobin for each 1.0 degree C temperature increase between 18 and 27 degrees C. Varying the pH of the wash buffer used in the affinity procedure from 7.75 to 8.25 (pH 8.0 optimum) produced at net change of 0.5% in glycosylated hemoglobin with one diabetic specimen. Using the affinity method, we determined the reference interval for glycosylated hemoglobin in 124 apparently healthy individuals to be 5.3 to 7.5% (mean 6.36%, SD 0.55%). Rechromatography by II and isoelectric focusing analysis of the fractions obtained by the affinity separation revealed a substantial population of glycosylated hemoglobins not measured by II. The affinity method offers a rapid, simple, precise, and accurate alternative to methods currently in use and gives substantial freedom from many common interferences.

Zinc in plasma, neutrophils, lymphocytes, and erythrocytes as determined by flameless atomic absorption spectrophotometry.

Abstract: Zinc is determined in neutrophils and lymphocytes (isolated from whole blood on discontinuous gradients of Ficoll-Hypaque) and in microliter quantities of plasma and erythrocytes by flameless atomic absorption spectrophotometry with greater sensitivity than with conventional flame atomic absorption spectrophotometry. Before analysis, neutrophils and lymphocytes are digested with nitric acid and diluted with de-ionized water. Plasma and erythrocytes required no digestion, only dilution. Overall CVs were 4.0, 3.0, 5.0, and 4.6% for neutrophils, lymphocytes, erythrocytes, and plasma, respectively. Matrix effects were fully compensated for by use of standard solutions that simulated the sample matrix. Results for plasma and erythrocytes agreed with those obtained by the conventional technique.

Determination of selenium concentration and glutathione peroxidase activity in plasma and erythrocytes.

Abstract: We determined selenium concentrations and activities of the selenoenzyme, glutathione peroxidase (EC 1.11.1.9), in the plasma and erythrocytes of 38 apparently healthy women. We determined selenium concentrations directly by polarized Zeeman-effect flameless atomic absorption spectroscopy. Within-run precision studies for the assays gave CVs of 5.6% for a mean erythrocyte selenium concentration of 149.9 (SD 8.3) microgram/L (n = 10) and 6.4% for a mean plasma selenium concentration of 97.3 (SD 6.2) microgram/L (n = 12). For the women, mean selenium concentrations were 141.4 (SD 14.3) microgram/L of erythrocytes [0.49 (SD 0.07) microgram/g of hemoglobin and 96.3 (SD 14.2) microgram/L of plasma. Glutathione peroxidase activities were measured by a modification of the method of Paglia and Valentine (J. Lab. Clin. Med. 70: 158--169, 1967). Within-run precision studies for the glutathione peroxidase assays gave CVs of 12.8% for mean erythrocyte glutathione peroxidase activity of 77.2 (SD 9.9) U/g of hemoglobin (n = 13), and 8.1% for mean plasma activity of 312.5 (SD 25.2) U/L (n = 11). Mean enzyme activity was 78.7 (SD 12.9) U/g of hemoglobin for erythrocytes and 424 (SD 40) U/L for plasma. Erythrocyte selenium concentrations and glutathione peroxidase activities were positively, but poorly, correlated (r = 0.41, p less than 0.01).

The Human Protein Index.

Abstract: The feasibility of constructing a human protein index and the data base associated with it is discussed. The index would be used to explore molecular anatomy and pathology of human cells. At present, the index is closely tied to the only technique that provides the requisite resolution: two-dimensional electrophoresis. Mapping of human cells, tissues and body fluids together with the mapping of micro-biopsy samples of tissues from individuals with many different diseases should create a systematic description of disease at the protein level. (JMT)

Enzyme immunoassay for factor VIII-related antigen.

Abstract: I describe a simple enzyme-linked immunosorbent assay (ELISA) for the quantitation of Factor VIII-related antigen in plasma with use of commercially available peroxidase-labeled antiserum and solid-phase support. Regression analysis of 85 plasma samples analyzed by this technique (y) and by a commonly used electroimmunoassay (Anal. Biochem. 15: 45-52, 1966) (x) gave the equation y = 0.223 + 0.77x (r = 0.973). The present method was also compared with enzyme immunoassay in which a phosphatase-labeled antiserum prepared in our laboratory was used; the correlation between the two assays was very good. The simplicity and specificity of the ELISA technique should make it a useful alternative to the more difficult and time-consuming Laurell method.

Reference intervals for serum IgG, IgA, IgM, C3, and C4 as determined by rate nephelometry.

Abstract: We report reference intervals for IgG, IgA, IgM, C3, and C4 for a population of 750 well children and 120 healthy adults. Ranges were established by rate nephelometry (previous studies have been based on immunodiffusion). Our results generally agree with previously established immunoglobulin ranges, except for some disagreement as to ages when adult values are attained.

On the use and computation of likelihood ratios in clinical chemistry.

Abstract: The clinical relevance of likelihood ratios (L-values) for revising the physician's diagnostic probabilities has been recognized. However, the calculation of L-values, particularly in the case of quantitative or mixed quantitative-binary test results, raises problems that have not yet been addressed. Based on a very general assumption that yields a simple functional form for the likelihood ratio, a method is developed that allows such calculations regardless of the nature and the number of clinical laboratory tests to be interpreted simultaneously. Hence the notion of predictive value (posterior probability) is extended from binary or dichotomized tests to quantitative tests, and from univariate to multivariate clinical laboratory results. The simplicity and flexibility of this approach eliminates difficulties in computation arising from the addition of new data to an existing data base. It is hoped that this method will now allow L-values to be reported along with the original test results in daily laboratory practice.

Radioimmunoassay for somatomedin C: comparison with radioreceptor assay in patients with growth-hormone disorders, hypothyroidism, and renal failure.

Abstract: We raised an antiserum (Tr4) in rabbits against a basic somatomedin C-like peptide preparation. Using high-immunoreactivity somatomedin C tracer, we compared the performance of radioimmunoassays in which we used the Tr4 antiserum and a well-characterized somatomedin C antiserum distributed by the National Pituitary Agency (NPA) with that of the human placental-membrane somatomedin radioreceptor assay (RRA). In their cross reactivity toward various somatomedin-like and unrelated peptides, the two radioimmunoassay methods were almost identical, although NPA antiserum, with about fourfold higher titer than Tr4 antiserum, showed a slightly greater sensitivity for most peptides tested. Radioimmunoassay of acid-ethanol-extracted plasma samples from normal persons and acromegalic, hypopituitary, hypothyroid, and renal-failure patients revealed no analytical differences between the antisera (for 122 samples, r = 0.979 between methods). Somatomedin values for acromegalic and hypopituitary samples showed no overlap with normals. Values for hypothyroid and pre-dialysis renal-failure samples were significantly lower than normal. By comparison, the RRA showed greater cross reactivity toward some somatomedin-like peptides and gave significantly lower values than radioimmunoassay for acromegalic and hypothyroid plasma extracts, and significantly higher value for hypopituitary and renal-failure samples. We conclude that the radioimmunoassay methods clearly are of greater diagnostic value than RRA for clinical somatomedin measurement.

Up-dated catalogue of HeLa cell proteins: percentages and characteristics of the major cell polypeptides labeled with a mixture of 16 14C-labeled amino acids.

Abstract: A total of 1357 polypeptides [946 acidic (isoelectric focusing) and 411 basic (nonequilibrium pH-gradient electrophoresis)] from human HeLa cells have been separated and catalogued with use of high-resolution two-dimensional gel electrophoresis. Of these polypeptides, 1266 were detected by labeling cells with [35S]methionine, while the rest were revealed by silver staining or by labeling with a mixture of 16 14C-labeled amino acids. For convenience, all these polypeptides have been numbered and are indicated in a large fold-out protein map. The percentages of some of the major 14C-labeled proteins have been determined, and for some we list a few characteristics such as: variation during the cell cycle; cellular distribution in cytoplasts and karyoplasts; presence in Triton- and salt-extracted cytoskeletons; and phosphorylation and sensitivity to neoplastic transformation.

Liquid-chromatographic assay for retinol (vitamin A) and retinol analogs in therapeutic trials.

Abstract: A "high-performance" liquid-chromatographic separation of retinoids (retinol, isotretinoin, all-trans retinoic acid, retinal, etretinate, and retinyl acetate) in serum is described. The separation was used in developing a quantitative assay for retinol (vitamin A) and two therapeutic analogs, isotretinoin (13-cis-retinoic acid) and etretinate (Ro 10-9359). The procedure requires 1 mL of serum. Overall analytical recovery for retinol, isotretinoin, and etretinate from serum was 100% (SD 7%). The between-day coefficient of variation for specimens with concentrations ranging from 0.70 to 0.95 mg/L was less than 4%. Normal reference intervals for serum retinol in men and women are 0.61 to 1.33 and 0.44 to 1.19 mg/L, respectively.

Six methods for urinary protein compared.

Abstract: Six assays for protein in urine were compared for linearity, ease of standardization, precision, comparability of assay values, technical ease of assay, and current cost. The assays investigated were three dye-binding techniques, a recent turbidimetric technique, the trichloroacetic acid--biuret reaction, and a tannic acid protein precipitation reaction with ferric chloride. All assays suffered from standardization problems, although the biuret method showed the best analytical recovery of albumin and gamma-globulin. The tannic acid/ferric chloride method is dependent on sample pH. The turbidimetric assay exhibited the greatest imprecision; i.e., CVs were 19.5% at a protein concentration of 0.13 g/L and 6.0% at a protein concentration of 1.3 g/L. On the basis of all the factors assessed, we conclude that the Pesce/Strande Ponceau-S and the Bio-Rad Coomassie Brilliant Blue dye-binding techniques offer certain advantages over the other assays studied.

Free amino acid content of lymphocytes nd granulocytes compared.

Abstract: For extracting free amino acids from human leukocytes, we find that disruption of the cells by ultrasonication is more reliable than freezing and thawing. The amount of free amino acids extracted by the latter method depends on the temperature and duration of thawing. We extracted free amino acids from leukocytes of healthy men by ultrasonication and compared their concentrations in lymphocytes and granulocytes. The amino acid content of granulocytes significantly (p less than 0.001) exceeded that of lymphocytes. Of the amino acids extracted from granulocytes, 76% was taurine; for lymphocytes taurine, aspartic acid, and glutamic acid were predominant, respectively composing 44%, 17%, and 26% of the total. Taurine is proposed as an index of cell disruption.

Immunoturbidimetry of albumin and immunoglobulin G in urine.

Abstract: I describe a rapid, sensitive immunoturbidimetric assay for measuring urinary albumin and immunoglobulin G with use of an automated spectrophotometer. Diluted urine samples and polyethylene glycol in phosphate-buffered saline are pipetted into the cuvettes of the spectrophotometer. The initial absorbances of the samples are measured at 340 nm; antiserum to albumin or to immunoglobulin G is added to each tube, and after 2 min at 37 degrees C the absorbance of the mixtures is read at 340 nm. The initial blank absorbances of the samples are subtracted from the final absorbances automatically. The change in absorbance is linear with concentration in the range of 5-400 mg/L for albumin and 3-1000 mg/L for IgG. The lower limit of the determination is 5 mg/L for albumin, 3 mg/L for IgG. Linear correlations were observed between the concentrations of albumin and immunoglobulin G determined by this method (x) and those determined by radial immunodiffusion (y). The regression equation for albumin was y = 0.84x + 0.03 (r = 0.99, n = 87), and for IgG y = 0.94x + 0.02 (r = 0.98, n = 87).

Specific spectrophotometry of ascorbic acid in serum or plasma by use of ascorbate oxidase.

Abstract: We describe a specific enzymatic spectrophotometric method for ascorbic acid in serum or plasma. Samples are analyzed indirectly by measuring the absorbance at 593 nm of a reaction product, a complex of ferrous ion and 2,4,6-tris(2-pyridyl)-s-triazine (Fe2+-TPTZ). This product is formed by reduction of the corresponding ferric ion complex (Fe3+-TPTZ), which is nonspecifically reduced by various biological reducing agents under acidic conditions. Ascorbic acid is specifically quantified by pretreating one of a pair of replicate samples with ascorbate oxidase (EC 1.10.3.3), to oxidize the ascorbic acid, then reacting both samples with Fe3+-TPTZ and measuring the difference between the absorbances at 593 nm of the treated and untreated samples. This difference is linearly related to ascorbic acid concentrations from 10 to 100 mg/L. Ten repeat determinations of a serum pool with added ascorbic acid yielded a CV of 2.8% and a mean of 47.2 mg/L. The correlation (r) between the proposed method and the dinitrophenylhydrazine method was 0.93 for 32 samples analyzed by both methods. The present method is specific for ascorbic acid and requires no deproteinization.

Improved direct determination of alpha- and gamma-tocopherols in plasma and platelets by liquid chromatography, with fluorescence detection.

Abstract: Tocopherols extracted from plasma with methanol or from platelets with chloroform/methanol were injected in methanol on a reversed-phase (C18) "high-performance" liquid-chromatographic column and eluted with water/methanol (2/98, by vol) at a flow rate of 1.4 mL/min. A "high-performance" spectrophotofluorometer was used for detection. Analytical recoveries ranged from 89 to 106%. The response was linear to at least 0.3 micrograms of either tocopherol (alpha- or gamma-) applied to the column, and the limit of detection was 0.1 ng. The method was used to measure tocopherols in plasma and platelets from human subjects, and some values are presented.

Isolation and preliminary characterization of a fraction of bilirubin in serum that is firmly bound to protein.

Abstract: We have isolated from pathological sera a bilirubin fraction (delta) that is very tightly, if not covalently, bound to protein, most likely albumin. This delta fraction absorbed at a lambda max of 433 nm in the visible spectrum, between the lambda max of unconjugated (alpha) and that of conjugated (Bc) bilirubin when measured in solutions containing albumin. However, unlike the other bilirubin species, this fraction could not be separated from the proteins in serum by exhaustive ultrafiltration in the presence of caffeine/benzoate solution. In the Jendrassik-Grof diazo procedure for bilirubin analysis, the delta fraction gave a large direct reaction (76-89% of the total reaction). Yet, when relatively hydrophobic azo dyes were formed by reaction of the delta fraction with the diazonium salt of dichloroaniline, only 50% of the dyes were extractable from aqueous solution. On chromatography the rest remained associated with protein. Of the extractable dye, more than 70% was accounted for by two liquid-chromatographic peaks with retentions identical with those of azo dyes formed from unconjugated bilirubin. This delta fraction was not appreciably separated from protein by treatment with strong acid or base, or by prolonged digestion with various enzymes. Finally, in a highly denaturing solvent (urea/mercaptoethanol), this fraction was not dialyzable through a membrane with a 12 000-dalton cutoff.

beta 2-Microglobulin instability in pathological urine.

Abstract: Urinary excretion of beta 2-microglobulin (beta 2M) is a widely used test of renal proximal tubular function. beta 2M is known to be unstable in normal urine with a pH less than 5.5. The results of a study of beta 2M excretion in patients receiving gentamicin suggested that beta 2M might be more unstable in pathological urine. A series of experiments was designed to define the extent, variability, and mechanism of its instability in pathological urine. It was unstable in pathological urine with a pH of 6.0 at 37 degrees C and pH 5.5 at room temperature, but this varied in urines from different patients. The instability was abolished by heating the urine to 80 degrees C. These results suggest that the most likely explanation for the instability is enzymic degradation. Because enzymuria will vary among patients, investigators should test the stability of beta 2M in urine from the patients they wish to study before trying to define conditions for urine collection.

Two-dimensional gel electrophoresis of serum specimens from a normal population.

Abstract: We examined sera from a normal population by two-dimensional gel electrophoresis, to establish the normal pattern of serum proteins and to investigate genetic polymorphisms. With such information in hand, specimens from patients with certain diseases may be readily evaluated. Towards this goal, we optimized the ISO-DALT system (Proc. Natl, Acad, Sci, USA 74: 5421--5425, 1977) for routine phenotyping of alpha 1-antitrypsin, haptoglobin, GC-globulin, alpha 2-HS-glycoprotein, and transferrin, as well as a previously unknown polymorphic protein. We examined the effects of aging the specimens for 2 h at room temperature (no changes) or at -20 degrees C for several months (small changes), as well as serum/plasma differences and the effect of protease inhibitors. Silver-stain methods were modified to allow simultaneous staining of 10 gels, with reasonably good reproducibility of stain intensity. We quantitated silver-stained gels by densitometry of photographic transparencies. Very small samples suffice with this stain (0.5 microL of serum or plasma), allowing the use of "finger-stick" methods instead of venipuncture, yet the patterns are better resolved and easier to read than those for 10-microL specimens processed on gels stained with Coomassie Blue. Our techniques for rapidly removing albumin and IgG allow the investigator to examine areas on the gel that ordinarily are obscured. The region of haptoglobin has been examined by using serum from an ahaptoglobinemic donor. Finally, we present an expanded "normal" map illustrating the composite protein pattern.

Use of glycerol-cryoprotected Lactobacillus casei for microbiological assay of folic acid.

Abstract: A simple procedure for preparing glycerol-cryoprotected Lactobacillus casei cultures has been developed. L. casei grown in medium supplemented with low concentrations of folic acid (0.3 micrograms/L) is diluted with an equal volume of glycerol (800 mL/L) and stored at -20 degrees C. Growth response of the glycerol-cryoprotected L. casei to low concentrations of folic acid exceeded that of cultures maintained by monthly agar stab transfer. Also, growth for the zero-folate blanks was considerably less for the cryoprotected cultures. Assay of folate in several rat tissues correlated well (r = 0.999) with the standard microbiological assay. The growth rate of the culture depends on the inoculum size, and a heavy inoculum of cryoprotected L. casei may be used to complete the assay after only an overnight incubation.

Simultaneous measurement of plasma apolipoproteins A-I and B by electroimmunoassay.

Abstract: We describe a simplified electroimmunoassay for quantification of human apolipoproteins A-I and B on prepared plates. A solution of agarose at 55 degrees C, containing hydroxyethylcellulose and antibodies, is poured onto a plastic film and allowed to gel. Wells are punched in the gels and the plates are dried for storage. Before use, they are rehydrated and buffered. We use succinylated Sudan Black to prestain lipoprotein fractions of plasma. After electrophoresing samples of plasma or standards for 3 h at 4 degrees C at 12.5 V/cm, we measure the peak heights and read the results from a standard curve prepared by using calibrated sera of known apolipoprotein B and A-I content as secondary standard. The within- and between-assay coefficients of variation were less than 4% in all cases. Results correlated well with those obtained by classic electroimmunodiffusion. Subjects with confirmed atherosclerotic lesions had significantly (p less than 10(-9)) lower ratios of apolipoprotein A-I to apolipoprotein B, compared with ratios in controls.

Two-dimensional gel electrophoresis of serum specimens from patients with monoclonal gammopathies.

Abstract: We modified the ISO-DALT two-dimensional gel electrophoresis system to allow the routine examination of serum specimens from patients with monoclonal gammopathies. This system, MC-Iso 1, is characterized by a broad pH gradient for resolving the basic immunoglobulin heavy and light chains. The increased resolution of basic proteins may be explained on theoretical grounds by an increase in voltage in this region of the cell. Ancillary techniques, such as those for albumin removal and pI assignment through use of charge standards, have also been implemented. The locations of immunoglobulin heavy chains have been confirmed by examination of over 250 serum samples as well as by "electro-blotting," with use of specific antisera. IgG subclass may also be predicted by location, but not with perfect accuracy. Differentiation of kappa and lambda light chains by relative mobility has been examined; the predictive value for correct identification of kappa chains is 83%, that for lambda chains 69%. Several unknown proteins have been observed in macroglobulinemia, related to mu heavy chain. Finally, we have determined that there is excellent correlation between non-denaturing isoelectric focusing and our system for pI assignment of light chains. This has importance due to reports of the potential importance of light-chain pI in the development of renal disease in patients with monoclonal gammopathies.

Determination of fluoxetine and norfluoxetine in plasma by gas chromatography with electron-capture detection.

Abstract: This gas-chromatographic method for assay of fluoxetine and norfluoxetine in human plasma involves extraction of the drugs and use of a 63Ni electron-capture detector. The linear range of detection is 25 to 800 micrograms/L for each drug. Overall precision (CV) in the concentration range of 10 to 100 micrograms/L for both drugs was approximately 10%. Accuracy (relative error) in the same concentration range was approximately +10%. None of the commonly prescribed antidepressants or tranquilizers that we tested interfere with the assay.

Fluorometry of selenium in serum or urine.

Abstract: This fluorometric procedure for determining selenium in human serum or urine is sensitive (requiring only 0.4 mL of sample), accurate, simple, and can be performed on several samples concurrently. Using this technique, we found a mean selenium concentration in the serum of normal Canadian men of 142.9 (SD 16.1) micrograms/L. The mean urinary excretion rate was 124.5 (SD 76.0) micrograms/day.

Mechanism of action of human pancreatic and salivary alpha-amylase on alpha-4-nitrophenyl maltoheptaoside substrate.

Abstract: We evaluated the enzymic mechanism by which alpha-4-nitrophenyl maltoheptaoside serves as a substrate for serum amylase (EC 3.2.1.1). Because polymeric substrates possess many potential sites of cleavage, the expression of enzymic activity as the number of micromoles of substrate transformed under defined conditions by an enzyme must be changed to "one microequivalent of group transformed." Therefore, we measured the activity of the isoenzymes of alpha-amylase with regard to suitable oligosaccharide substrates, which allowed us to express the catalytic activity in IUB units (U). By "high-performance" liquid chromatography we investigated the mechanism of human pancreatic and salivary alpha-amylase action, both alone and in combination with alpha-glucosidase (EC 3.2.1.20). On the basis of these results, we can describe exactly the entire reaction sequence and determine the stoichiometric coefficient of 4-nitrophenol within 0.02 mol/L (SD) produced under the assay conditions.