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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Abstract: Acetaminophen is a commonly used analgesic, available without prescription. Several of its metabolites have heretofore been isolated from physiologic fluids and analytically characterized. In general, the separation methods are complicated, usually requiring extensive sample pretreatment, and do not measure the individual conjugated metabolites. High-resolution anionexchange separation of urinary samples from subjects receiving acetaminophen reveals eight chromatographic peaks, representing seven metabolites and the free drug itself. Metabolites separated include 2-methoxyacetaminophen, its glucuronide and sulfate conjugates, the sulfate conjugate of 2-hydroxyacetaminophen, the glucuronide and sulfate conjugates of acetaminophen, S -(5-acetamido-2-hydroxyphenyl)cysteine, and S -(5-acetamido-2-glucuronosidophenyl)cysteine. Urinary and serum concentrations of the drug and its seven metabolites were determined by high-resolution liquid chromatography as a function of time after two clinically normal men ingested 1950 mg of the drug. Concentrations in urine and serum are compared, and estimated urinary excretion rates are reported for all metabolites except S -(5-acetamido-2-hydroxyphenyl)cysteine. Serum concentrations of the glucuronide were higher than concentrations of the free drug 2 h after the drug was ingested, indicating that solvent-extraction procedures for serum will yield low estimates of total drug unless hydrolysis precedes the extraction step.

Abstract: Measurement of blood pH, p o2 and p co2 also involves calculation of two or more derived quantities and correction of the measured values in cases where the body temperature of the patient differs from the temperature of measurement. References to the pertinent calculations and the temperature corrections are scattered through the literature of several medical specialties, and much new information has been gathered in recent years that directly affects these calculations. This review explains each of the derived quantities and correction factors most used in this field and also provides the best available data for the calculations, in a form that can readily be adapted to electronic data processing.

Abstract: I describe a method for measuring insulin antibodies in insulin-treated subjects. The antibodies are characterized by maximum binding capacity and affinity constant. Endogenous and therapeutic insulin interferes in insulin antibody assay, and it is removed by dextran—charcoal treatment at pH 3.5. Endogenous total and free insulin may be measured by a simple extension of the method.

Abstract: Serum triglycerides can be enzymatically determined by hydrolysis of triglycerides to glycerol and free fatty acids [ Clin. Chem. 19, 476 (1973)]. Enzyme integral (end-point) and fixed-time rate analyses of serum triglycerides by this procedure have been investigated with a centrifugal analyzer. We determined the precision of blank determinations, the contribution of normal serum alkaline phosphatase to total values, and the amount of endogenous glycerol in fasting serum samples and blood collection tubes. We also made a statistical analysis of enzymatic triglyceride values obtained for a healthy, fasting (12-16 h) population of men and women. The relative merits of both procedures are discussed.

Abstract: Determination of serum creatinine by the Jaffe reaction may give erroneous results because of interfering noncreatinine chromogens. Studies of the Jaffe reaction in alkaline picrate media of various pH demonstrated that creatinine, proteins, and certain chromogens have characteristic properties as regards the appearance and intensity of the color reaction. Thus a method for determining "true" creatinine has been developed in which simple spectrophotometric measurements are made at 500 nm in two alkaline picrate reagents, buffered at pH 9.65 and 11.50. Results agree well with those obtained by the use of an ion-exchange method. The proposed method is easy and rapid and offers the advantages of increased specificity and direct analysis of serum and urine.

Abstract: A radioimmunoassay is described for parathyroid hormone in human serum, in which commercially available reagents are used almost exclusively. This assay can be done by any laboratory with experience in radloimmunoassay. Thirty-two of thirty-three patients with surgically proven primary hyperparathyroidism had detectable concentrations of parathyroid hormone in their serum, and concentrations of the hormone exceeded the normal range in 24 of them. Significant positive correlations were found between pre-operative serum calcium, pre-operative serum parathyroid hormone, and the weight of parathyroid tissue removed at operation. These three parameters were also significantly correlated with severity of the skeletal changes as assessed by semiquantitative histological methods.

Abstract: We describe a modification of the fluorometric method of Haussler and Hajdu [ Arzneim. Forsch. 14, 704 and 709 (1964)] for assay of furosemide in either serum or urine. A 1-ml sample, acidified to pH 2, is extracted with 5 ml of diethyl ether; 4 ml of the ether is back-extracted into 1 ml of phosphate buffer (pH 7.0, 0.1 mol/liter), and finally acidified with 1 ml of dilute HCl (0.6 mol/liter). A procedure for estimating blanks in urine was derived to correct for dilution caused by diuresis. Internal standards are used, and the "effective" extraction ratio is used to correct for the effects of quenching and extraction differences. In equilibrium, 93% of the drug is bound to serum proteins; 65% is tightly bound. Erythrocytes contain less than 5% of the drug. Quantum yield of fluorescence at pH 1 is 0.0496 for furosemide and is 0.0163 for 4-chloro-5-sulfamoylanthranilic acid. Furosemide fluorescence diminishes with increasing pH, while that of 4-chloro-5-sulfamoylanthranilic acid (a degradation product) increases.