Showing papers in "Clinical Chemistry in 1974"

Enzymatic determination of total serum cholesterol.

Abstract: An enzymatic method is described for determination of total serum cholesterol by use of a single aqueous reagent. The method requires no prior treatment of sample and the calibration curve is linear to 600 mg/dl. Cholesterol esters are hydrolyzed to free cholesterol by cholesterol ester hydrolase (EC 3.1.1.13). The free cholesterol produced is oxidized by cholesterol oxidase to cholest-4-en-3-one with the simultaneous production of hydrogen peroxide, which oxidatively couples with 4-aminoantipyrine and phenol in the presence of peroxidase to yield a chromogen with maximum absorption at 500 nm. The method is reproducible, and the results correlate well with those obtained by automated Liebermann—Burchard procedures (AA-2 and SMA 12/60) and the method of Abell et al. The present method affords better specificity than those previously reported and has excellent precision.

Statistical Quality Control and Routine Data Processing for Radioimmunoassays and Immunoradiometric Assays

Abstract: Numerous methods are available for the graphical display of radioimmunoassay dose—response curves, for curve-fitting and dose interpolation, for statistical quality control, and for automation and computerization of data processing. The relative merits of these approaches are discussed. Minimal requirements for radioimmunoassay data-processing systems are presented. The features of an "ideal" system are discussed.

Measurement of Ceruloplasmin from Its Oxidase Activity in Serum by Use of o-Dianisidine Dihydrochloride

Abstract: We have developed a new method for determination of serum ceruloplasmin from its oxidase activity, in which o -dianisidine dihydrochloride is used as substrate. o -Dianisidine dihydrochloride is more stable than is the more widely used p -phenylenediamine substrate, and forms a stable product that is measured at 540 nm. Correlation was good between results of this method and those obtained with both a p -phenylenediamine substrate method ( r = 0.98) and a radial immunodiffusion procedure ( r = 0.97). There is little or no interference from reducing or colored components of serum. The coefficient of variation (day-to-day) for the method was 4.2%. A normal range of 62-140 U/ liter has been determined by the reported method.

Evaluation of Glucose-6-Phosphate Dehydrogenase from Leuconostoc mesenteroides in the Hexokinase Method for Determining Glucose in Serum

Abstract: The hexokinase (EC 2.7.1.1)/glucose-6-phosphate dehydrogenase (I) (EC 1.1.1.49) method of glucose analysis is highly accurate, precise, and sensitive when I from Leuconostoc mesenteroides and NAD+ are used. Coefficients of variation ranged from 0.68 to 8.9% for glucose standards between 0.56 and 27.7 mmol/liter. Recovery was 103 ± 10% for glucose standards added to sera of known glucose content. Although no substances investigated interfere with the test system when present at their normal or likely concentrations in serum, final reaction mixtures containing mannose (>0.28 mmol/liter), fructose (3.7 mmol/liter), or 2-deoxy-D-glucose (4.01 mmol/liter) inhibit the analysis. With all constituents lyophilized, the reagent mixture is stable for at least a year at 4 °C and for five days at 40 °C. Within-day reproducibility averaged 0.83%; day-to-day precision was 1.3%. We compared results of hexokinase/glucose procedures by using I from yeast (with NADP+) and from L. mesenteroides (with either NAD+ or NADP+ as coenzyme); there was complete correlation over a serum glucose range of 4 to 12 mmol/liter (72 to 217 mg/dl). Use of NAD+ with I from L. mesenteroides is advantageous because of the decreased cost and greater stability of the coenzyme.

Effects of Intra-and Interindividual Variation on the Appropriate Use of Normal Ranges

Abstract: Normal ranges based on the distribution of single samples from a large number of individuals reflect both intra- and interindividual variation. If the average ratio of these two sources of variation is small, then, assuming gaussian distributions, the conventional normal range will usually include a larger than expected proportion of an individual9s distribution of values. When the average ratio exceeds 1.4, the normal range will include a proportion either larger or smaller than expected, depending on whether the individual9s variability is less than or greater than average intra-individual variation. Investigation of multivariate normal regions in certain cases where calculations are feasible produced similar results. With these numerical guidelines, data from recent blood-chemistry studies indicate that conventional normal ranges are likely to be less sensitive than desired to significant changes in an individual9s biochemical state. This analysis supports the continued development and use of cumulative (in time) systems for reporting laboratory test results for individuals.

Single-Antibody Technique for Radioimmunoassay of Cortisol in Unextracted Serum or Plasma

Abstract: A radioimmunoassay procedure for cortisol is described that requires only 5 µl of serum or plasma. Native cortisol-binding proteins are denatured by heating 5 µl of sample in 0.2 ml of glutamate buffer in a boiling water bath for 15 min. After a 2-h incubation of heat-treated serum or plasma with [H3]cortisol and cortisol antibody, "free" and "bound" cortisol are separated by use of hemoglobin-coated charcoal. The method is easy and sensitive, and results compare favorably with those of a well-accepted acid-fluorescence method. In addition, we show that serum and heparinized plasma yield the same results by this method, and therefore either can be assayed by this procedure.

Separation of tissue and serum creatine kinase isoenzymes by ion-exchange column chromatography.

Abstract: I describe a simple, rapid anion-exchange column chromatographic technique for separating the creatine kinase (CK) isoenzymes in human serum and tissue. Extracts of CK-rich tissues (skeletal muscle, cardiac muscle, and brain) were used to determine optimum conditions for separating CK isoenzymes MM, MB, and BB. Samples, layered on mini-columns (0.5 x 6.0 cm) of DEAE-Sephadex A-50, were eluted stepwise with Tris-buffered sodium chloride (100, 200, and 300 mmol/Iiter). Column effluents were assayed by the Rosalki CK method. Distribution of total activity among the eluted fractions was tissue-specific and reproducible. Evaluation of sera from 71 patients with myocardial infarction and other diseases associated with elevated CK activity revealed isoenzyme patterns that resembled those of either cardiac muscle or skeletal muscle. Cardiac pattern (presence of MB isoenzyme) and clinical documentation of myocardial infarction were 100% correlated in the 35 patients so studied.

Determination of Unbound Bilirubin in the Serum of Newborns

Abstract: An enzymatic assay is described for non-albuminbound bilirubin in the serum of newborn infants. Unbound bilirubin is oxidized to colorless compounds by ethyl hydroperoxide in the presence of horseradish peroxidase (EC 1.11.1.7), while albumin-bound bilirubin is protected from oxidation. Because the equilibrium between albumin and bilirubin occurs rapidly, the oxidation step is rate limiting, and the initial oxidation velocity of total bilirubin is proportional to the unbound bilirubin concentration. By titrating serum with bilirubin in vitro, the association constant and binding capacity of high-affinity sites for albumin binding can be determined. Normal human serum albumin tightly binds 1 mole of bilirubin per mole of albumin (binding constant, 2-4 x 108 liter/mol). Although weaker secondary binding occurs, the unbound bilirubin fraction increases rapidly after the high-affinity binding sites are saturated. Compromised newborns may have a decreased apparent binding capacity and (or) binding affinity. The method can be used to assess the risk of a jaundiced infant for bilirubin encephalopathy.

Simple Enzymatic Determination of Polysaccharide (Glycogen) Content of Animal Tissues

Abstract: For determination of (chiefly) glycogen content, tissue homogenates in citrate buffer are mixed with exo-1,4-α-glucosidase (EC 3.2.1.3). Liberated glucose is estimated with a Beckman Glucose Analyzer. This method was checked with commercial glycogen (from oysters) and with fish (carp) tissues. Comparative measurements were made with the classic Good-Kramer-Somogyi method [ J. Biol. Chem. 100, 485 (1933)]. The described method, suitable for small samples of tissue, such as those obtained by biopsy, seems to be simple and accurate, but gives values 5 to 10% higher than those given by the classic KOH-ethanol method.

Criteria for Judging Precision and Accuracy in Method Development and Evaluation

Abstract: We describe an approach for formulating criteria that can be used to judge whether an analytical method has acceptable precision and accuracy. We derive criteria for several experiments that are commonly used in method-evaluation studies: precision or replicates, recovery, interference, and comparison of patient values between the new method and a proven method. These criteria are based on the medical usefulness of the test results, thus the acceptability of the method is judged with respect to the clinical requirements.

Automated method for determining serum thiocyanate, to distinguish smokers from nonsmokers.

Abstract: A chemical test to distinguish smokers and nonsmokers is important in many epidemiologic studies. We have developed an automated method for determining serum thiocyanate by use of the reaction between thiocyanate and ferric ions to form a colored complex. Our data show that this method distinguishes cigarette smokers and nonsmokers: of 197 healthy individuals studied, 1.8% false-positive and 6.7% false-negative results were obtained when 85 µmol of SCN- per liter was the critical value used to distinguish the two populations.

Plasma Cortisol Determination: Radioimmunoassay and Competitive Protein Binding Compared

Abstract: A simple radioligand assay has been developed for measuring cortisol in plasma and urine. The antiserum was produced in rabbits by using cortisol-21-hemisuccinyl-bovine serum albumin as the antigen. The standard curve was linear from 0-1000 pg. A small aliquot of ethanol-precipitated plasma or urine is directly assayed. Interassay variability of 56 separate assays of a normal plasma pool during six months was: mean, 224 µg/liter; range, 210-238 µg/liter; CV, 3.2%. In assay of multiple samples obtained between 0800 and 1800 hours from normal young men, radioimmunoassay (RlA) gave consistently lower values than did competitive protein binding (CPB). The relationship, as calculated from results for 41 single samples, is expressed by the equation RIA = 0.713 CPB + 0.59, suggesting that the normal range for RIA should be 35-180 µg/liter rather than the 50-250 µg/liter usually cited. Although there is crossreactivity with 11-deoxycortisol, this assay is clearly superior to CPB: it is more specific, has a wider range (<8-400 µg/liter), and requires less technician time.

Specific Gravity of Blood and Plasma at 4 and 37 °C

Abstract: The specific gravity (relative density) of human whole blood and plasma from 25 healthy volunteers was determined gravimetrically. For whole blood it was found to be 1.0621 (95% confidence interval: 1.0652-1.0590) at 4 °C and 1.0506 (95% confidence interval: 1.0537-1.0475) at 37 °C. Plasma specific gravity was 1.0310 (95% confidence interval: 1.0324-1.0296) at 4 °C and 1.0205 (95% confidence interval: 1.0216-1.0193) at 37 °C. All of these values are referred to the density of water at 4 °C. We show the relationship between these values and those given in the literature for measurements at 25 °C. There was a small increase in whole blood specific gravity with increasing hematocrit, but it was not statistically significant over the 40-56 hematocrit range studied.

Detection and Measurement of Total Bilirubin in Serum, with Use of Surfactants as Solubilizing Agents

Abstract: We describe a new method for measuring total bilirubin in serum. Nonionic, cationic, or anionic surfactants can be used as solubilizing agents to promote the diazo coupling of indirect-reacting bilirubin. A representative surfactant—azobilirubin system is illustrated, in which absorbance is maximum at 560 nm and linear to a concentration of 200 mg of bilirubin per liter. At pH 2.5 and in the presence of Du ponol (an anionic surfactant), bilirubin is completely coupled within 6 mm at 37 °C. All ingredients required for an assay are combined in two dry reagents, which are stable for a year at room temperature. Sensitivity is comparable to that of procedures routinely used in clinical laboratories; precision, linearity, and stability of the reagents in solution are excellent. Results for fresh sera correlated well with those obtained by the Jendrassik—Grof method. Hemolysis is minimized and turbidity eliminated.

2-Amino-2-methyl-1-propanol as the Alkalizing Agent in an Improved Continuous-Flow Cresolphthalein Complexone Procedure for Calcium in Serum

Abstract: We describe an improved cresolphthalein complexone procedure for calcium in which diethylamine is replaced by a nontoxic amino alcohol. Advantages of the procedure are increased sensitivity, excellent baseline stability, a more optimum reaction environment (pH 10.0) that eliminates blanking problems, and freedom from interference by magnesium. Values obtained by this method are not significantly different ( P < 0.05) from those obtained by atomic absorption spectrophotometry.

Measurement of IgG and Albumin Content of Cerebrospinal Fluid, and its Interpretation

Abstract: A high correlation ( r = 0.85) normally exists between the concentration of albumin and of IgG in cerebrospinal fluid (CSF). The correlation is still better ( r = 0.96) if the concentrations of the two proteins are also measured in plasma and the CSF/ plasma ratios compared for albumin and IgG. The scatter diagram for these ratios in persons without disease of the nervous system is most useful as a reference when differentiating between local IgG production in the subarachnoid space and an increase in CSF protein for other reasons. The analysis can be performed on less than 0.1 ml of CSF, in contrast to agarose-gel electrophoresis, for which 5-10 ml is necessary.

Creatinine Metabolism in Humans with Decreased Renal Function: Creatinine Deficit

Abstract: This study was designed to test the hypothesis that, in a patient with decreased renal function and an increased plasma creatinine concentration, a significant quantity of creatinine is excreted into the gut, as is true of urea and uric acid, and is metabolized by gut flora. [ Methyl -14C] creatinine was given intravenously to five patients and [ methyl -14C] creatinine or [ carbonyl -14C] creatinine was given orally to five patients. Extracts of excreta, plasma, and urine were subjected to ion-exchange chromatography, with monitoring for 14C and for ninhydrin-positive material. Respiratory gases, collected in acid and base, were assayed for radioactivity. Blood specimens were obtained at intervals to furnish data on decay of labeled creatinine in the body pool. The data show that there is a true creatinine deficit (15.9 to 65.7% of the creatinine formed is metabolized or excreted via extrarenal routes) in patients with decreased renal function. In these patients, creatinine is metabolized to CO2 and methylamine, presumably by the microflora of the gut. A significant portion of the carbonyllabeled creatinine appeared in plasma in an unidentified compound.

Differentiation of Electrophoretically Similar Hemoglobins—Such as S, D, G, and P; or A2, C, E, and O—by Electrophoresis of the Globin Chains

Abstract: Electrophoresis of the globin chains of hemoglobin on cellulose acetate in both acidic and alkaline buffers (pH about 6 and 9) containing urea and 2-mercaptoethanol is a simple, rapid means of characterizing hemoglobins. Erythrocyte hemolysate is electrophoresed in the presence of a large amount of mercaptoethanol, which liberates heme from globin, and keeps it in solution during its rapid electrophoretic removal. Each globin chain migrates at a characteristic rate, which varies with the pH and composition of the buffer. The combined data permit differentiation, with a high degree of specificity, of some similarly charged hemoglobins. They may also be useful in assessing the effect of secondary and tertiary structure on molecular charge.

Screening Cord Bloods for Detection of Sickle Cell Disease in Jamaica

Abstract: A cord-blood screening program, designed primarily for detecting sickle cell disease, has been in operation for seven months (8000 samples) at a large maternity unit in Kingston, Jamaica. We describe techniques of cord-blood collection and electrophoretic investigation on both cellulose acetate and agar gel. These methods appear to give rapid, valid results at minimal expense and are well adapted to screening large populations.

Principles of competitive binding assays (saturation analyses). II. Sequential saturation.

Abstract: The principles of sequential saturation as a form of competitive binding assays are discussed in detail and differentiated from those of equilibrium techniques. The rationale of its applicability in practice and its potential to increase sensitivity under certain assay conditions are demonstrated with three binding systems involving digoxin, insulin, and folates. The advantages and disadvantages of the sequential saturation technique are outlined.

Mechanisms of the Liebermann-Burchard and Zak color reactions for cholesterol.

Abstract: Correlation of SO2 and Fe2+ measurements with new spectral data indicates that the Liebermann-Burchard (L-B) and Zak color reactions for cholesterol have similar oxidative mechanisms, each yielding, as oxidation products, a homologous series of conjugated cholestapolyenes. These studies further suggest that the colored species observed in these two systems are enylic carbonium ions formed by protonation of the parent polyenes. Thus, the red (λmax, 563 nm) product typically measured in the Zak reaction is evidently a cholestatetraenylic cation, and the blue-green product in the L-B reaction (λmax, near 620 nm) is evidently the pentaenylic cation. The effects of rate of carbonium ion formation and sulfuric acid concentration on sensitivity and color stability are discussed. A solvent extraction procedure is described for specifically converting cholesterol to 3,5-cholestadiene. Incorporating this step into the typical L-B method can increase the L-B sensitivity for cholesterol by several fold.

Polymer Enhancement of Automated Immunological Nephelometric Analysis, as Illustrated by Determination of Urinary Albumin

Abstract: We have developed a new automated immunonephelometric method for determination of albumin in urine, by exploiting the polymer-enhancing effect on the immunological reaction. The original continuous-flow manifold has been simplified and the reaction time shortened to about 3 min. The antiserum was diluted 100-fold in a solution (100 g/liter) of polyethylene glycol (av mol wt, 6000) and mixed with the prediluted sample in the continuous-flow system. The method is highly sensitive and is unaffected by high blank and (or) high absorbance values of the specimens. Day-to-day variation was 3.8-4.3% (CV). Accuracy, as estimated from recovery experiments, was also good. A comparative study of 176 urines with the single radial immunodiffusion technique showed a correlation coefficient of 0.994. We therefore suggest the new method for routine use for determination of urinary albumin, because it is fast, accurate, precise, and sensitive, and requires only small amounts of antiserum.

Factors Contributing to Intra-Individual Variation of Serum Constituents: 4. Effects of Posture and Tourniquet Application on Variation of Serum Constituents in Healthy Subjects

Abstract: We studied the effects on 18 serum constituents of posture and prolonged tourniquet application. The subjects were 11 healthy men, ages 20-25 years. The assays were performed on the AutoChemist Multi-Channel Analyzer (AutoChem Instrument AB, Lidingo, Sweden). To compensate for the within-hour variation in these constituents, we drew blood samples at 1100 h and 1130 h on several days. The 1100-h sample was taken after the subjects had been sitting erect for 60 min. The 1130-h sample followed different posture regimens: Control day: sitting for 15 min; experimental days: after ( a ) being supine for 30 min, ( b ) standing for 30 min, and ( c ) sitting erect for 30 min. The 1130-h/ 1100-h ratios for the three experimental days were compared with those for the control day. Significant differences ( P <.05) were found for serum potassium, calcium, total protein, albumin, aspartate aminotransferase, and acid phosphatase under condition a ; for phosphate ion, total protein, total lipid, cholesterol, and alkaline phosphatase under condition b ; and for aspartate aminotransferase under condition c . The effect of a 3-minute tourniquet application was similarly studied. The ratio of the "prolonged tourniquet application day" differed significantly from the control day with regard to serum potassium, total protein, iron, total lipid, cholesterol, aspartate aminotransferase, and bilirubin. Significance of posture and tourniquet time in blood-sampling and their effect on total intra-individual variation are discussed.

Manganese, Copper, and Zinc Concentrations in Serum and Packed Blood Cells during Acute Hepatitis, Chronic Hepatitis, and Posthepatitic Cirrhosis

Abstract: Manganese, copper, and zinc concentrations were determined in serum and packed blood cells of normal controls, patients with acute and chronic (persistent or aggressive) hepatitis, and cases of postnecrotic cirrhosis. During the active phase of acute hepatitis, serum manganese concentrations are invariably increased; the difference between the mean value and the normal is highly significant, P < 0.001. The mean serum copper is also significantly increased ( P < 0.01). The concentrations become normal during the subsiding phase. In chronic aggressive hepatitis and posthepatitic cirrhosis, the mean serum manganese concentration is increased, P < 0.001, whereas the serum zinc concentration is frequently decreased. There is a highly significant ( P < 0.001) positive correlation between serum manganese concentration and the activity in serum of aminotransferases, in subjects with acute or chronic hepatitis or postnecrotic cirrhosis.

Standardization of Serum Ceruloplasmin Concentrations in International Enzyme Units with o-Dianisidine Dihydrochloride as Substrate

Abstract: We describe a method for calculating the absorptivity (in terms of substrate consumed) of the colored solution obtained when o -dianisidine dihydrochloride is oxidized by ceruloplasmin. By oxidizing o -dianisidine dihydrochloride with known amounts of hydrogen peroxide we could determine that the molar reacting ratio of o -dianisidine to hydrogen peroxide is 2:1. So calculated, absorptivity is 9.6 ml µmol -1 cm -1 , the figure used to estimate ceruloplasmin oxidase activity in terms of International Units.

Optimized Determination of γ-Glutamyltransferase by Reaction-Rate Analysis

Abstract: We describe a method for measuring γ-glutamyltransferase (EC 2.3.2.2) activity in serum, which can be used with automated enzyme analyzers (such as the LKB 8600 Reaction Rate Analyzer) that require enzyme reactions to be initiated with substrate. The method also permits optimal determination conditions to be obtained at 37 °C. The enzymatic reaction is commenced by adding γ-glutamyl- p -nitroanilide dissolved in dilute hydrochloric acid to samples pre-incubated with tris(hydroxymethyl)aminomethane—glycylglycine buffer. The p -nitroaniline liberated is continously monitored at 37 °C at 405 nm. The pH of the pre-incubation buffer is such that the optimal pH for the enzyme reaction results from addition of the acid substrate solution.

Relative Affinity of Hemoglobin S and Hemoglobin A for Carbon Monoxide and Oxygen

Abstract: The relative affinity constants of hemolysates from individuals with hemoglobins A, S, or AS have been measured at 37 and 26 ° C. Observed values of all hemoglobin types were the same at both temperatures, K 37 = 230, K 26 = 296. Control measurements on whole blood containing Hb A gave values of K 37 = 222. The small but significant difference between K 37 values measured in whole blood and in hemolysates may be a result of the greater increase of MetHb in the hemolysates during the in vitro equilibration.

Electrochemical Detection of Selected Organic Components in the Eluate from High-Performance Liquid-Chromatography

Abstract: High-performance liquid chromatography can be combined with hydrodynamic thin-layer electrochemistry for determination of trace amounts of organic constituents in complex samples. With small and inexpensive analyzers based on these two techniques, as little as 1 pg of an electrochemically active component can be detected in a few minutes. Because many of the important low-molecular-weight organic constituents of body fluids— both endogenous metabolites and drugs—undergo electrochemical reactions, it seems reasonable to presume that useful assays might be developed by using the above methodology. Beginning to explore this presumption, we illustrate how uric acid, ascorbic acid, catecholamines, and related tyrosine metabolites might be measured in urine and serum. In some cases, liquid chromatography with electrochemical detection provides better sensitivity, selectivity, and speed than traditional methods, while minimizing the need for analytical reagents. We describe the basic approach and progress to date and suggest future applications.