bluuebunny/crossref_metadata_2025 huggingface dataset:
Immunoenzymometric assays for simultaneous quantitation and distribution of erythrocyte antigen.
Authors: Makler M T , Mlecko C R , Pesce A J
Published: January 2020

We describe an adaptation and modification of the enzyme-labeled immunosorbent assay (ELISA), which yields information on the quantity and distribution of surface antigens on erythrocytes. The distribution assay, named "ELADA" (for Enzyme-Linked Antigen Distribution Assay), involves use of an alkaline phosphatase-immunoglobulin conjugate with alpha-naphthol phosphate and a diazonium salt as substrate. The insoluble reaction product forms a complex on the cell surface at the location of the antigen. Antigens in aliquots from the same enzyme-immunoglobulin conjugate are quantified with the soluble substrate p-nitrophenyl phosphate. One can thus establish whether the distribution of erythrocyte antigens is random by examining the ELADA-stained cells with a scanning electron microscope. Combining the quantitative ELISA with measurements of cell numbers makes it possible to estimate the number of antibody molecules-and thus the number of antigenic sites per cell. By using various modified antisera, substituted naphthol phosphates with higher avidities, and reaction conditions designed to optimize the kinetic reaction, we have been able to resolve the enzyme product deposited on the antigenic site to within 20 nm. It is not always possible to decide whether each deposit represents a cluster of antigenic determinants or a single determinant; however, if the number of antibody sites is known from ELISA quantitation, information about the number of antibody sites per deposit can be ascertained.
Homogeneous fluorescent immunoassay with dry reagents.
Authors: Greenquist A C , Walter B , Li T M
Published: January 2020

We described an assay for theophylline in serum in which the reagents for the substrate-labeled fluorescent immunoassay procedure are dry reagents in a paper matrix. This assay format is both rapid and convenient. In one such paper strip assay, the competing drug conjugate is present in the diluting buffer (dilution-into-conjugate strip assay); in another, all necessary assay reagents are contained on the strip (integral strip assay). The latter strip can be prepared by using appropriate solvents in a "two-dip" procedure, which prevents premature reaction of the assay constituents. The dilution-into-conjugates strip assays yield results that compare well with those by the corresponding solution assay (r = 0.975, standard error of estimate = 1.67 mg/L) and by "high-performance" liquid chromatography (r = 0.980, standard error of estimate = 1.49 mg/L). The integral strip results were compared with those by an enzyme immunoassay for theophylline (r = 0.990, standard error of estimate = 2.07 mg/L). Both strip assays require only a single dilution step before analysis.
Measurements of serum colloid osmotic pressure are of limited usefulness.
Authors: Duncan A , Young D S
Published: January 2020

We examined the usefulness of serum colloid osmotic pressure measurement in patients with chronic rather than acutely occurring low serum protein concentrations. We used two oncometers, the IL 186 Weil Oncometer and the Wescor Model 4100; results from the two instruments were interchangeable. Values for the colloid osmotic pressure were compared with those for serum total protein (r = 0.783) and albumin concentrations (r = 0.882), which were similar to previously published values. Our day-to-day CV was 2.8%. In studying over 100 patients we found that the previously reported occurrence of pulmonary edema in almost all patients whose colloid osmotic pressure was less than 12.5 mmHg was not seen in the chronic hypoproteinemic patients. We noted only one fatality in our patients whose colloid osmotic pressure was less than 10.5 mmHg, a value found to be associated with fatality in one previous study of acutely ill patients. Factors such as ambulation, fasting, dehydration, and the nature of the blood sample can markedly affect the value for colloid osmotic pressure value, and this, coupled with the good correlation with the serum albumin in several studies, leads us to question the usefulness of measuring colloid osmotic pressure in a non-specialist hospital environment, either as an adjunct to the measurement of serum protein or albumin, or as an independent test.
Two serum pancreatic isoamylase determinations compared.
Authors: Rammeloo T , van Haard P M , Beunis M H
Published: January 2020

Serum pancreatic isoamylase activity was measured by a method involving inhibition of salivary isoamylase and by a well-known agarose electrophoretic method, modified by us. We saw changes in electrophoretic patterns of pancreatic isoamylase fractions after storage of serum samples for three weeks at 4-8 degrees C, but with the inhibition method no alterations in activities were found. Within-assay and between-assay imprecisions of both methods were about the same. Serum pancreatic amylase activities as measured by the inhibition method exceeded by about 10% those obtained by the electrophoretic method. The inhibition method seems to be a reasonable candidate for routine application, whereas the electrophoretic method is still time-consuming and requires special skill to perform. Some suggestions are given for improving the calibration of the inhibition method recommended by the manufacturer.
Effect of duration and temperature of storage on serum analyte stability: examination of 14 selected radioimmunoassay procedures.
Authors: Kubasik N P , Ricotta M , Hunter T , Sine H E
Published: January 2020

We determined appropriate temperatures for sample storage and the resulting stability of 14 analytes commonly radioimmunoassayed in the clinical laboratory. Serum specimens to be tested for concentrations of cholylglycine, cortisol, digoxin, ferritin, follitropin, immunoglobulin E, lutropin, prolactin, thyroxin (also blood-spot thyroxin), triiodothyronine, and triiodothyronine uptake could be stored for up to two weeks at room temperature, refrigerated, or frozen without any loss of analyte activity. Specimens for insulin testing require freezing or refrigeration, and specimens for gastrin testing should be stored at -70 degrees C for optimal results.
Fluorometry of selenium in serum or urine.
Authors: Lalonde L , Jean Y , Roberts K D , Chapdelaine A , Bleau G
Published: January 2020

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.
Macro creatine kinase: determination and differentiation of two types by their activation energies.
Authors: Stein W , Bohner J , Steinhart R , Eggstein M
Published: January 2020

Determination of the MB isoenzyme of creatine kinase in patients with acute myocardial infarction may be disturbed by the presence of macro creatine kinase. The relative molecular mass of this form of creatine kinase in human serum is at least threefold that of the ordinary enzyme, and it is more thermostable. Here we describe our method for determination of macro creatine kinases and an easy-to-perform test for differentiating two forms of macro creatine kinase, based on their distinct activation energies. The activation energies of serum enzymes are mostly in the range of 40-65 kJ/mol of substrate. Unlike normal cytoplasmatic creatine kinases and IgG-linked CK-BB (macro creatine kinase type 1) a second form of macro creatine kinase (macro creatine kinase type 2) shows activation energies greater than 80 kJ/mol of substrate. The exact composition of macro creatine kinase type 2 is still unknown, but there is good reason to believe that it is of mitochondrial origin.
Glucose determinations in plasma and serum: potential error related to increased hematocrit.
Authors: Sidebottom R A , Williams P R , Kanarek K S
Published: January 2020

This study reports the effect of increased hematocrit (packed cell volume) on the rate of decrease in plasma glucose during 6 h in blood specimens from seven newborns (cord blood) and heparin-treated blood specimens from eight adults. Mean hematocrits ranged frm 0.43 to 0.75 for the adults and from 0.51 to 0.81 for the infants. The decrease in glucose was related to time and to hematocrit. For example, in adults with a mean hematocrit of 0.43, glucose decreased from 890 +/- 40 mg/L to 570 +/- 30 mg/L (mean +/- SEM) at 6 h, whereas in adults with a mean hematocrit of 0.75, glucose decreased from 890 +/- 40 to 200 +/- 30 mg/L. In infants with a mean hematocrit of 0.51, glucose decreased from 1010 +/- 20 to 480 +/- 70 mg/L at 6 h; infants with a mean hematocrit of 0.81 had blood glucose decrease from 1010 +/- 20 to 50 +/- 30 mg/L. These results indicate that the incidence of hypoglycemia in infants with polycythemia may be overestimated, and emphasize the need for prompt handling of blood samples collected from newborns for glucose determinations.
Enzymic determination of citrate in serum and urine, with use of the Worthington "ultrafree" device.
Authors: Tompkins D , Toffaletti J
Published: January 2020

We describe an enzymic method for conveniently measuring citrate in serum or urine. Interfering enzyme proteins are removed by a disposable ultrafilter ("Ultrafree"; Worthington Diagnostics), obviating the need for hazardous protein precipitants. A 50 mmol/L Tris buffer adequately controls pH, no lactate dehydrogenase is necessary in the reagents, and the reaction of citrate catalyzed by citrate lyase (EC 4.1.3.6) is complete in 2-3 min. Within-run and day-to-day coefficients of variation were 7.5% and 5.4%, respectively. Serum citrate concentrations for 20 apparently healthy persons ranged from 0.08 to 0.17 mmol/L (mean 0.12, SD 0.03). Urinary citrate excretion by six normal volunteers ranged from 2.2 to 4.4 mmol/24 h. We observed no detectable changes in citrate in whole blood stored at room temperature for 90 min or longer. Overall, the method is faster and less hazardous than other methods for citrate that require protein precipitation.
Effect of sample instability on glycohemoglobin (HbA1) measured by cation-exchange chromatography.
Authors: Simon M , Hoover J D
Published: January 2020

We compared results for glycohemoglobin obtained from fresh whole blood or separated erythrocyte samples with results obtained after storage. We judged the storage condition to be acceptable ("stable") if the glycohemoglobin results after storage were within the 95 confidence interval (+/- SD) of the results obtained for the specimens on the day of venipuncture. Hemolysates can validly be stored for five months at -70 degrees C. Whole-blood samples stored at 4 degrees C remain stable for four days; whole blood treated with heparin or EDTA (but not oxalate) is stable for seven days. At 30 degrees C, whole blood or erythrocytes from some donors are stable for one day, but after two days and seven days, results are frequently higher. We confirmed previous findings that the separated erythrocytes can be stored at -20 degrees C for at least seven days. In addition, we compared the elution profiles for stored samples showing increased values.
Quantitation of urinary normetanephrine and metanephrine by reversed-phase extraction and mass-fragmentographic analysis.
Authors: Canfell C , Binder S R , Khayam-Bashi H
Published: January 2020

Hydrolyzed urine with added ring-trideuterated normetanephrine and metanephrine is applied to wet C18-reversed-phase minicolumns. The "metanephrines" are eluted, dried, derivatized with pentafluoropropionic anhydride, and analyzed with the gas chromatograph-mass spectrometer. Ions for the nondeuterated and trideuterated compounds are monitored at ml z 458 and 461, respectively. For both normetanephrine and metanephrine, the standard curve is linear over the range 10-2000 micrograms/L and the procedure has adequate precision both within-run (CV less than 3%) and between-day (CV less than 7%). Alkaline pH in the extraction is important for optimal analytical recovery. We have examined the potential value of untimed urine specimens for screening purposes and compared 24-h urine concentrations of these analyses in normotensive and hypertensive persons.
Liquid-chromatographic determination of ethylene glycol in plasma.
Authors: Gupta R N , Eng F , Gupta M L
Published: January 2020

In this novel procedure for determining ethylene glycol in plasma by liquid chromatography, benzoyl esters of ethylene glycol and of benzyl alcohol (used as the internal standard) are prepared directly in plasma. The benzoyl esters, highly ultraviolet-absorbing chromogens, are ideal compounds for analysis by reversed-phase liquid chromatography with methanol/water as the mobile phase. The benzoyl derivative of ethylene glycol is well separated from the derivative of the internal standard and from plasma constituents. The standard curve is linear to 400 mg of ethylene glycol per liter. As little as 10 mg of ethylene glycol per liter of plasma can be measured. Other commonly ingested alcohols do not interfere.
Electrophoretic determination of isoamylases in serum with commercially available reagents.
Authors: Leclerc P , Forest J C
Published: January 2020

We describe and evaluate a method for determining isoamylases in serum. With this method, which involves conventional electrophoretic apparatus and commercially available reagents, seven isoenzymes can be resolved. The electrophoresis is performed in agarose gel, and isoenzymes are detected with a water-insoluble cross-linked starch polymer linked to a blue dye. The isoenzymes are quantified by densitometry. Within-assay imprecision (CV) was less than 7%, between-assay CV less than 11%. This method has the sensitivity to detect the major isoamylases (P2 and S2) in the serum, even at low total amylase activity. Reference intervals for P-type isoenzymes were 2-52 U/L and for S-type 2-85 U/L. P2S2 is the most common pattern found in normal individuals, then (in decreasing order) P1P2S2, P2S2S3, and P2 patterns. Patterns found in serum from cases of acute pancreatitis, mumps, macroamylasemia, and pelvic inflammatory disease are illustrated.
Characterization of uremic "middle molecular" fractions by gas chromatography, mass spectrometry, isotachophoresis, and liquid chromatography.
Authors: Schoots A C , Mikkers F E , Claessens H A , De Smet R , Van Landschoot N , Ringoir S M
Published: January 2020

Uremic ultrafiltrates (and normal serum, for comparison) were fractionated by means of gel filtration. The collected fractions were further investigated by combined analytical techniques: "high-performance" liquid chromatography, gas chromatography, mass spectrometry, and isotachophoresis. Ultrafiltrate fractions in the so-called middle molecular mass region (Mr 500-2000) contained a considerable amount of substances of low molecular mass, such as carbohydrates, organic acids, amino acids, and ultraviolet absorbing solutes. Ultraviolet absorbance in the "middle molecular mass region" of the gel chromatogram is mainly due to the presence of these rather low-molecular-mass solutes. Therefore this signal is not a quantitative measure of molecules with a "middle" molecular mass.
Gas-chromatographic determination of ethylene glycol in serum.
Authors: Porter W H , Auansakul A
Published: January 2020

We describe a new gas-chromatographic procedure for ethylene glycol in serum, determined as the cyclic phenylboronate ester. The method is rapid, requires only 100 microL of serum, and either an OV-1 or an OV-17 column is used. The linear dynamic range extends to at least 5000 mg/L, with a lower limit of detection of 10 mg/L. Analytical recovery of ethylene glycol from serum averaged 97% (95-101%) over the concentration range of 250-5000 mg/L. The method demonstrates good precision, with CVs less than 2.5%, over the concentration range 500-2000 mg/L.
More sensitive flameless atomic absorption analysis of vanadium in tissue and serum.
Authors: Stroop S D , Helinek G , Greene H L
Published: January 2020

We describe a sensitive method for the determination of vanadium in biological substances by atomic absorption spectroscopy. By using background correction and pyrolytically coated graphite tubes with flameless apparatus, this vanadium assay has a sensitivity of 65 pg and a detection limit of 30 pg, approximately sixfold more sensitive than previously reported assays involving this technique. The vanadium concentrations have been determined in several biological materials, including bovine liver and human serum. To investigate signal interferences, we have determined vanadium concentrations in matrix solutions containing several elements at physiologically relevant concentrations. Evaluated by the method of standard additions, the vanadium signals are linear with concentration over a range of 3 to 100 micrograms/L. Tissue vanadium concentrations are similar to those previously reported. Serum vanadium concentrations can be determined directly, and we found values ranging from 2.22 to 3.94 micrograms/L in human serum. The improved sensitivity permits the use of sample sizes substantially smaller than previously required.
Alkaline phosphatase isoenzymes.
Authors: Moss D W
Published: January 2020

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.
Gas-chromatographic assay for heme oxygenase activity.
Authors: Sunderman F W , Downs J R , Reid M C , Bibeau L M
Published: January 2020

We have developed an improved assay for microsomal heme oxygenase activity, based on the enzymic release of CO from the alpha-methene bridge of hemin and the quantitation of CO by gas chromatography. The within-run coefficient of variation (CV) of heme oxygenase assays in microsomes from rat tissues (liver, kidney) averaged 8%; the between-run CV averaged 15%. The detection limit for heme oxygenase activity was approximately 1 nmol/h per milligram of microsomal protein. Gas-chromatographic assays of heme oxygenase activities in rat tissues correlated well (r = 0.94) with results by a spectrophotometric assay based on bilirubin production. In untreated rats, heme oxygenase activity averaged 7 +/- 3 nmol/h per milligram of protein (n = 36) in kidney microsomes and 14 +/- 5 nmol/h per milligram of protein (n = 17) in liver microsomes. Heme oxygenase activity was increased 10-fold in kidney microsomes and threefold in liver microsomes from rats killed 17 h after subcutaneous injection of NiCl2 (0.5 mmol/kg body wt). These findings illustrate the efficacy of the gas-chromatographic assay for measuring xenobiotic effects on heme oxygenase activity.
High-density lipoprotein subfractions as measured by differential polyanionic precipitation and rate zonal ultracentrifugation.
Authors: Simpson H S , Ballantyne F C , Packard C J , Morgan H G , Shepherd J
Published: January 2020

Recent interest in the putative protective role of high-density lipoprotein (HDL) and its subfractions against atherosclerosis has highlighted the need for a rapid, simple subfractionation procedure. Here we compared HDL subfractionation by two recently developed polyanionic-precipitation methods with the values obtained by rate zonal ultracentrifugation. A similar result for total HDL cholesterol was obtained by all three methods. However, HDL2 cholesterol as measured by the precipitation procedures was significantly higher than the zonal value, and HDL3 was lower. This reflects the different underlying principles involved in the separations and highlights the need for a clearer understanding of the functional roles of the HDL fractions.
Measurement of prostatic acid phosphatase in serum and bone marrow: radioimmunoassay and enzymic measurement compared.
Authors: Huber P R , Scholer A , Linder E , Hagmaier V , Vogt H , Christen P , Eppenberger U , Rutishauser G
Published: January 2020

We quantitated the concentrations of prostatic acid phosphatases (EC 3.1.3.2) in serum and bone-marrow aspirates with three commercial radioimmunoassay kits, and the catalytic activities with a thymolphthalein monophosphate-based enzyme test. The enzyme's immunological activity in serum was compared with its catalytic activity for its potential as a detector of early prostatic cancer and its performance as an early marker of metastatic activity in bone. Neither measurement is useful for detecting early stages of prostatic cancer. The spread of carcinoma to lymph nodes or to bone is detected with greater frequency by radioimmunoassay than by the enzymic test. Radioimmunoassay also detected metastasis to the bone more frequently than did physical methods. Analytical and clinical performance of the four methods is described.
Five kits for estimating free thyroxin concentration in serum evaluated, and correlated with other indices to thyroid status.
Authors: Wilke T J
Published: January 2020

I evaluated Amerlex (Amersham International), Coat-A-Count (Diagnostic Products Corporation), Gammacoat (Clinical Assays), Immophase (Corning Medical), and Liquisol (Damon Diagnostics) radioimmunoassay kits for determinations of free thyroxin concentration in sera. Kit results for free thyroxin and the free thyroxin index and thyroxin/thyroglobulin ratio were compared for patients with thyroid disease and for subjects with normal and altered thyroglobulin binding capacity. Values for free thyroxin, free thyroxin index, and thyroxin/thyroglobulin clearly distinguished hyperthyroid and hypothyroid patients from euthyroid subjects. Each free thyroxin kit showed that fewer euthyroid patients with increased or decreased thyroglobulin binding capacity had values outside of normal limits than was indicated by kits for either free thyroxin index or thyroxin/thyroglobulin. The free thyroxin kits not only provided a better overall index of thyroid status in euthyroid patients who had alterations in thyroglobulin binding capacity than either of the indirect assessments of free thyroxin, but also supplied similar diagnostic information in thyroid disease.
Predicting recurrence in patients with breast cancer from cumulative laboratory results: a new technique for the application of time series analysis.
Authors: Winkel P , Bentzon M W , Statland B E , Mouridsen H , Sheike O
Published: January 2020

We followed the cases of 26 consecutive postmenopausal patients operated on for primary breast cancer. Serum specimens were obtained each month for 1.5 years and stored at -80 degrees C until assayed for carcinoembryonic antigen (CEA) and other quantities. Ten patients developed recurrence, while 16 qualified as controls (no clinical recurrence for at least 1.7 years after the last venipuncture). Using the homeostatic autoregressive time series model, modified by us to be particularly sensitive to sustained deviations from the mean, we detected four recurrences by CEA without having any falsely positive alarms. Group-based reference limits and application of the unmodified homeostatic model were less effective (fewer detected and shorter lead time). Simulation studies, involving use of a mathematical model relating CEA concentration to tumor growth and using parameters estimated from patient data, verified this and indicated that at least five stable baseline values are needed to detect 100% of recurrences before they are detected by the group-based limit.
Determination of fluoxetine and norfluoxetine in plasma by gas chromatography with electron-capture detection.
Authors: Nash J F , Bopp R J , Carmichael R H , Farid K Z , Lemberger L
Published: January 2020

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.
Evaluation of a new commercial solid-phase direct radioimmunoassay for unconjugated estriol in pregnancy plasma.
Authors: France J T , Knox B S , Fisher P R
Published: January 2020

We evaluated a new radioimmunoassay kit for unconjugated estriol in pregnancy plasma. The overall mean intra-assay precision (CV), as determined from replicate analyses of three plasma pools with different estriol concentrations, was 5%; the overall mean inter-assay precision was 7.7%. The assay system had acceptable linearity, with a correlation coefficient of 0.97 between results for 24 plasma samples assayed at 10 and 20 microL. Analytical recovery of estriol added to plasma to give three concentrations averaged 98.6%. Estriol values were generally higher with the kit than with our conventional charcoal-separation RIA method. The regression line equation was y = 1.11x + 1.0, the correlation coefficient 0.97. In plasma from 28 normal pregnant women, sampled serially during the last trimester, the mean unconjugated estriol concentration in plasma increased steadily from 29 nmol/L at 28 weeks of gestation to 42 nmol/L at 34 weeks, and then more rapidly to 93 nmol/L at term. This kit provides a rapid, technically simple, and reliable assay method, offering advantages to clinical laboratories with a high estriol workload.
Evaluation of determination of glucose in urine with some commercially available dipsticks and tablets.
Authors: Bandi Z L , Myers J L , Bee D E , James G P
Published: January 2020

Four commercial products for urine glucose determination were evaluated and compared with a quantitative hexokinase procedure. We examined precision, sensitivity, and analytical recovery of glucose from glucose-supplemented urine samples and comparison of methods, using patients' samples. Only "Chemstrip uG" (Bio-Dynamics Inc.) could differentiate between 0.3 g/L (upper limit of normal) and 0.6 g/L urine glucose concentrations. "Tes-Tape" (Lilly) and "Diastix" (Ames) gave positive readings at 0.3 g/L; "Clinitest" (Ames) detected glucose only over 1 g/L. Analytical recovery of glucose was best, for all four products, between 1 and 2.5 g/L; Chemstrip uG was the most nearly accurate among the four. Between 5 and 20 g/L glucose concentrations, Tes-Tape, Diastix, and Clinitest tended to give falsely low results; the use of Chemstrip uG resulted in overestimates of concentration at 20 g of glucose per liter. Only Chemistrip uG and Clinitest (two-drop method) had linear ranges extending to 50 g/L; Chemstrip uG had better precision and accuracy at this concentration. Of the four products, Chemstrip uG had the lowest within-technologist and technologist-to-technologist random analytical errors. In method comparison on patients' samples, Chemstrip uG was significantly stronger in its association with the quantitative hexokinase method than was Diastix, Clinitest, or Tes-Tape.
Evaluation of a modified alcohol dehydrogenase assay for the determination of ethanol in blood.
Authors: Poklis A , Mackell M A
Published: January 2020

We evaluated a new alcohol dehydrogenase (EC 1.1.1.1) enzymic assay (ADH-glycine, Sigma Chemical Co.) for the determination of ethanol in blood. This assay differs from the manufacturer's previous assay (ADH-pyrophosphate) in that glycine replaces pyrophosphate as the buffer and hydrazine replaces semicarbazide as the trapping agent. The standard curve for the assay was linear over blood ethanol concentrations of 0.50-5.00 g/L. The reaction time of the assay was 10 min. At 1.00 g/L within-run and between-run CVs were 3.96% (n = 20) and 4.01% (n = 20), respectively. Mean analytical recovery of ethanol added to whole blood at 0.50-5.00 g/L was 99.7% (SD 2.6%). We performed 100 consecutive clinical and forensic determinations by the ADH-glycine assay, the ADH-pyrophosphate assay, and gas chromatography. Correlation coefficients of the results by least-square linear regression were 0.995 for ADH-pyrophosphate vs ADH-glycine, and 0.990 for gas chromatography vs ADH-glycine. The major advantage of the ADH-glycine assay over the ADH-pyrophosphate assay is the shorter reaction time, 10 min vs 30 min.
Simultaneous liquid-chromatographic determination of carbamazepine and its epoxide metabolite in plasma.
Authors: Sawchuk R J , Cartier L L
Published: January 2020

We describe a liquid-chromatographic method for the simultaneous quantification of carbamazepine and its 10,11-epoxide metabolite in plasma. The method is used routinely in the analysis of carbamazepine and its epoxide in 0.5 mL of plasma at concentrations of 1 to 20 and 0.2 to 5 mg/L, respectively. The use of peak-height ratios as a measure of detector response appeared to provide better precision and accuracy than peak-area ratios.
Liquid-chromatographic determination of amitryptyline and its metabolites in serum, with adsorption onto glass minimized.
Authors: Edelbroek P M , de Haas E J , de Wolff F A
Published: January 2020

To study correlations between the concentrations, in serum, of amitriptyline and its most important metabolites with clinical response in patients, we developed a "high-performance" liquid-chromatographic method for routine determination of amitriptyline, nortriptyline, total 10-hydroxy-amitriptyline, desmethylnortriptyline, and E(trans)- and Z(cis)-10-hydroxynortriptyline. These compounds are extracted from 1 mL of alkalinized serum into hexane/isoamyl alcohol (99/1 by vol). Perazine is the internal standard. To minimize irreversible adsorption of the drugs onto the glassware, 5 micrograms of maprotiline is added to the organic phase just before evaporation. After a 10-min resolution on a silica column eluted with acetonitrile/methanol/NH4OH (1 mol/L), absorbance is measured at 240 nm. Only chlorimipramine, doxepin, procainamide, and N-acetylprocainamide may interfere with assay of the compounds that probably are therapeutically relevant: amitriptyline, nortriptyline, and E-10-hydroxynortriptyline. Uremia, lipemia, and icterus also do not affect the analysis.
Semiautomated system for simultaneous assays of serum vitamin B12 and folic acid in serum evaluated.
Authors: Chen I W , Silberstein E B , Maxon H R , Volle C P , Sohnlein B H
Published: January 2020

We evaluated performance characteristics of a semiautomated radioassay system (ARIA II, Becton Dickinson Immunodiagnostics) for simultaneous measurements of vitamin B12 and folic acid in serum. The system requires off-line boiling of samples, which are then processed on-line. The automated process includes dispensing of boiled samples, tracers, and binders (purified intrinsic factor and milk binder); incubation, separation of bound and free ligands; elution; counting radioactivity of 57Co and 125I; and data reduction. Results were compared with those obtained by a manual method (Diagnostic Products Corp.). Although the nonspecific binding of the ARIA II is more than double that of the manual system, the precision values are comparable (within- and between-assay CVs are less than 8.5%). Values obtained with these two systems correlated well except that folic acid concentrations obtained with ARIA II tended to be lower at high folic acid concentrations. We observed no significant total carryover or drift in the semiautomated system.
Five creatine kinase isoenzymes in serum of a patient with severe heart disease.
Authors: Csako G , Papadopoulos N M , Jett G K , McIntosh C L
Published: January 2020

Abnormal creatine kinase (CK) isoenzyme patterns were observed in the serum of a 64-year-old woman with severe heart disease. Agarose electrophoresis revealed the presence of all the usual CK isoenzymes (MM, MB, and BB) plus an extra band between MM and MB. Total serum CK activity was within the normal range. Within 2 h after the patient suffered cardiorespiratory arrest, a fifth CK isoenzyme appeared, cathodal to MM. After cardiac valve replacement, the patient's serum showed a high activity of CK, but the isoenzyme pattern showed only MM and, transiently, an MB band. With return of the serum CK activity to normal, the CK isoenzymes pattern also became normal, virtually ruling out genetic variant(s). The abnormal CK isoenzyme patterns might have been the consequence of severe hypoxemia in the patient, thus such patients may represent an ominous prognostic sign. The association of the abnormal pattern upon admission with rapid deterioration of the condition of the patient suggests prompt attention for the prevention of complications.
A calibration protocol for serum-based secondary standards.
Authors: Miller W G , Rhodes D J , Moore C J
Published: January 2020

In this procedure, tentative calibration values are assigned to secondary standards by analysis of that material with the routinely used instrument calibrated by use of the former lot of material. Aliquots of serum from patients are then analyzed by the routine method and an appropriate calibration method in which primary standards are used. If the patient-sera results by both methods are the same, the assigned calibration value is assumed to be correct; if not, the calibration value of the secondary-standard material is adjusted to produce agreement in patient-sera results between the two methods. Thus, calibration of the routine method is linked via the calibration method to primary standards as the criterion of accuracy. This approach corrects for matrix interference of serum-based secondary standards. All that is expected for the secondary standard to obtain an accurate result for patient sera is to produce an analytical signal (irrespective of the origin of the signal) that is equivalent to the calibration value. Differences were significant between the secondary-standard calibration value we determined by this procedure and that assigned by the manufacturer for bilirubin, calcium, phosphorus, chloride, CO2, creatinine, glucose, sodium, uric acid, and commonly measured enzymes.
Performance of a new rate-nephelometric assay for rheumatoid factor, and its correlation with tube-titer results for human sera and synovial fluid.
Authors: Painter P C , Lyon J M , Evans J H , Powers W W , Whittaker R L , Decker M J
Published: January 2020

We evaluated a rate-nephelometric assay for rheumatoid factor (RF), as developed for use with the Beckman Immunochemistry System (ICS). Within- run precision (CV) for low-, mid-, and high-dilution samples (60-400 kilo-int. units/L) was 3.5, 1.5, and 1.6%; between-run precision was 3.4%. Analytical linearity was excellent. Biological interference resulted in some degree of nonlinearity in more than 70% of the patients' samples tested. Sensitivities were 96.5 and 94.1% and specificities were 98.5 and 95.3% for the ICS RF and Wampole slide methods, respectively, for a clinically defined population of 170 patients. Results for 100 ICS RF-positive samples correlated well with concomitantly measured Calbiochem-Behring tube-titers. Weekly measurement of Calbiochem-Behring. Hyland Diagnostics, and ICL Scientific tube-titer values for RF along with the ICS RF values on samples from the same patients indicated stable ICS RF values but showed at least +/- 1 tube dilution variances both within and between the tube-titer methods. Therefore, it may not be appropriate to compare a precise new method with a relatively imprecise comparison method. Treatment of RF-positive samples with dithiothreitol, which disrupts the pentameric character of immunoglobulin M, rendered the samples negative by repeat ICS RF assay and confirmed the method's specificity for pentameric immunoglobulin M-RF. Serum RF concentration as determined by ICS paralleled changes in clinical symptoms in a patient treated with both effective and relatively ineffective regimens, which suggests a useful role for the assay in monitoring efficacy of clinical treatment.
Enzymic determination of free and esterified cholesterol in serum by microcalorimetry.
Authors: Rehak N N , Young D S
Published: January 2020

Concentrations of free and esterified cholesterol in serum can be determined simultaneously by measuring, with a batch-type microcalorimeter, the heat released during the coupled cholesterol esterase/cholesterol oxidase/catalase enzymic reaction. To differentiate the two forms of cholesterol, we used kinetic calorimetry: the rate of heat output due to enzymic hydrolysis of esterified cholesterol (the rate-determining reaction) was subtracted from the measured heat, the difference being the heat released during the enzymic oxidation of free cholesterol (the fast reaction). Results obtained by the kinetic calorimetric method agreed with those obtained by separate sequential end-point calorimetric determinations of free and total cholesterol. We also compared the kinetic calorimetric method with the cholesterol method of Abell and Kendall and a continuous-flow modification of the Liebermann-Burchard method (Technicon SMAC). De-biased linear-regression analysis of the data indicates acceptable agreement between the calorimetric and the Abell-Kendall methods (y = 0.98x + 11.5). The correlation between results by calorimetric and SMAC methods shows a significant proportional error (y = 1.17x - 159.4). Bilirubin (up to 200 mg/L) does not interfere with the calorimetry.
Liquid-chromatographic estimation of saturated phospholipid palmitate in amniotic fluid compared with a thin-layer chromatographic method for acetone-precipitated lecithin.
Authors: Sax S M , Moore J J , Oley A , Amenta J S , Silverman J A
Published: January 2020

The "high-performance" liquid-chromatographic method we report for measuring palmitate derived from saturated phospholipids in amniotic fluid is sensitive and precise and possesses a wide dynamic range. Palmitate from nonsurfactant sources is largely excluded. The procedure involves oxidation of the chloroform/methanol-extracted lipids with osmium tetroxide, precipitation of the resulting phosphatides with cold acetone, mild alkaline hydrolysis, derivatization of the liberated fatty acids to phenacyl esters, and reversed-phase chromatography. The method is no less convenient or more time-consuming than measurement of the lecithin/sphingomyelin ratio by thin-layer chromatography. The clinical usefulness of the procedure is indicated by results of a correlative study of 26 amniotic fluids evaluated by "lung profile" tests in a second laboratory. A value for dipalmitoyl lecithin of 13 mg/L or greater indicates fetal lung maturity.
Fluorescence polarization immunoassay IV. Determination of phenytoin and phenobarbital in human serum and plasma.
Authors: Lu-Steffes M , Pittluck G W , Jolley M E , Panas H N , Olive D L , Wang C H , Nystrom D D , Keegan C L , Davis T P , Stroupe S D
Published: January 2020

Fluorescence polarization immunoassays for phenytoin and phenobarbital in human serum or plasma are described and shown to be clinically useful. No sample pretreatment, extraction, or phase separation is required. A determination can be made with less than 20 microL of sample in 15 min, with incubation at ambient temperature. Abnormal concentrations of protein, lipid, hemoglobin, or bilirubin do not interfere. In addition, drug metabolites and commonly co-administered drugs do not affect the assay results at clinically significant concentrations. Analytical recoveries of each of the two anticonvulsant drugs from serum averaged 101%. Between-assay CVs were less than 6.5%, with sensitivities of 0.5 mg/L. Comparison of this method with "high-performance" liquid chromatography, homogeneous enzyme immunoassay, and radioimmunoassay for phenytoin determinations yielded correlations of 0.99, 0.98, and 0.99, respectively. Similar comparison studies for phenobarbital yielded correlation coefficients of 0.99, 0.98, and 0.99, respectively.
Liquid-chromatographic measurement of phenylalanine and tyrosine in serum.
Authors: Spierto F W , Whitfield W , Apetz M , Hannon W H
Published: January 2020

With phenylalanine ammonia-lyase (EC 4.3.1.5) we converted phenylalanine (Phe) and tyrosine (Tyr) to transcinnamic acid and p-coumaric acid, respectively. These were separated by "high-performance" liquid chromatography and detected at 280 nm. We measured the Phe and Tyr content of human serum by adding 100 mU of the enzyme to a 20-microL serum aliquot, mixing for 2 h at 24 degrees C, then stopping the reaction with 1 mL of cold methanol. Precipitated proteins were removed by centrifugation and the separated clear supernates were stored at -20 degrees C. For chromatographic separation, detection, and quantification, we used a system equipped with a C-18 reversed-phase column, a variable-wavelength spectrophotometer, a printer-plotter, and a microcomputer. The mobile phase was a mixture of dilute aqueous (50 g/L) acetic acid and CH3CN (80/20, by vol). CVs for specimens containing 100 mg of Phe or Tyr per liter varied from 5 to 10%. Analytical recoveries were near 100%.
Pitfalls in the American Monitor kit methods for determination of total and "direct" bilirubin.
Authors: Doumas B T , Perry B W , Jendrzejczak B , Katona V
Published: January 2020

We evaluated the American Monitor Corporation kit for total and direct-reacting bilirubin and found that it suffers serious deficiencies, which lead to inaccurate and imprecise results. The main problem with the total-bilirubin procedure is that the short reaction time (2 min) is inadequate for completion of the reaction. The poor precision of the direct-bilirubin method is due to the short reaction time and the inability of the "stabilizer" (hydroxylamine sulfate) to completely destroy the diazo reagent. Depending on when Fehling's reagent is added, the reaction time may vary from 2 min to 7 min. Values for direct bilirubin at 7 min exceed those obtained at 2 min by 17 to 29%. The short reaction time makes color development temperature dependent, an additional source of imprecision. The suboptimal concentration of the diazo reagent results in underestimation of direct-reacting bilirubin. We recommend changes that improve both precision and accuracy of the kit procedures.
Kinetic method having a linear range for substrate concentrations that exceed Michaelis-Menten constants.
Authors: Hamilton S D , Pardue H L
Published: January 2020

We describe a new data-processing method for the kinetic quantification of substrates of enzyme-catalyzed reactions. Nonlinear regression is used to fit data for absorbance (A) and rate (dA/dt) vs time to the rate form of the Michaelis-Menten equation. Fitting parameters are the maximum velocity (Vmax), the Michaelis constant (Km), and the total absorbance change (delta A infinity) that would be observed if the reaction were monitored to completion. The method is evaluated with use of the uricase-catalyzed oxidation of uric acid, monitored at 293 nm, as a model reaction. Results for aqueous solutions demonstrate linear calibration plots for concentrations from well below to 3.5-fold the Michaelis constant, a zero temperature coefficient (36-38 degrees C), and near zero dependence on inhibitor (xanthine) concentrations that reduce the initial rate of 28% of its uninhibited value. Relative standard deviations (RSDs) vary from about 2 to 15%, depending on the data range (95-65% completion) used to process the data. For an 80% reaction data range, the pooled RSD was 6%. The sensitivity for uric acid is 1.15 x 10(4) L mol-1 cm-1 and the detection limit (95% confidence level) is 1.2 x 10(-6) mol/L.
Sodium measurements in multiple myeloma: two techniques compared.
Authors: Ladenson J H , Apple F S , Aguanno J J , Koch D D
Published: January 2020

Sodium was determined by flame photometry and by direct potentiometry in 56 serum or plasma samples from 24 patients with multiple myeloma or macroglobulinemia. We observed differences between the two techniques as large as 17 mmol/L (12%). The flame-photometric values decreased relative to the direct-potentiometric values as protein increased or water content decreased. Moreover, the two sodium measurements could not be interconverted simply on the basis of correcting for protein or water content. There was significantly lower residual variance (p less than 0.005) when the direct-potentiometric sodium values were compared with the osmolality (corrected for the influence of glucose and urea nitrogen) than when the flame-photometric values for sodium were so compared. We conclude that direct potentiometric measurements of sodium in patients with multiple myeloma gives clinically relevant results but flame photometry does not. Clearly, the method by which sodium is measured in patients with multiple myeloma must be considered if results are to be interpreted correctly.
Biliprotein in adult icteric serum--demonstrated by extension of the alkaline methanolysis procedure.
Authors: Wu T W , Sullivan S S
Published: January 2020

We confirmed that the alkaline methanolysis procedure of Blanckaert (Biochem. J. 185: 115-128, 1980) converts the sugar conjugates of bilirubin (Bc) into their corresponding methyl and dimethyl esters, which can be extracted into chloroform along with underivatized unconjugated bilirubin (Bu). By this procedure, we accounted for Bu nearly quantitatively, but only 76-83% of total Bc. By pretreating samples containing Bu and (or) Bc with a caffeine/benzoate reagent, we improved the analytical recovery of Bc to 85-93% without affecting the Bu. When the method (+ caffeine/benzoate) was applied to adult icteric serum, a variable fraction (20-75%) of the original total bilirubin (based on diazo reactivity) remained with the protein pellet, which is routinely discarded in the original methanolysis procedure. In this pellet we demonstrated the occurrence of a strongly protein-bonded bilirubin fraction (biliprotein) similar to the recently described "delta" fraction (Clin. Chem. 28: 629-637, 1982). The analytical and clinical implications of our findings are discussed.
Optimizing the o-phenylenediamine assay for horseradish peroxidase: effects of phosphate and pH, substrate and enzyme concentrations, and stopping reagents.
Authors: Bovaird J H , Ngo T T , Lenhoff H M
Published: January 2020

Horseradish peroxidase, assayed with o-phenylenediamine, is irreversibly inactivated when incubated in phosphate buffer, 100 mmol/L, at pH 5. The inactivation depends on both duration and incubation and phosphate concentration. Phosphate was the most potent inactivator and citrate the least potent of a series of buffers tested. The inactivation is not attributable to ionic strength per se or to Na+ or K+. The observed inactivation did not occur at high concentrations (2500 nmol/L, 0.1 g/L) of enzyme; however, this "protective" effect could not be reproduced by adding bovine serum albumin or a surfactant (Tween 20) to lower concentrations of enzyme. The inactivation was independent of commercial source of the enzyme or the kind of chromogenic assay used. On the basis of this information, we optimized the assay so that it gave eightfold greater absorbance values than those reported by others. The improved assay was sensitive to as little as 0.4 pmol/L (16 ng/L) of peroxidase, and was linear over the range of 0.4 to 5 pmol/L (16-200 ng/L).