bluuebunny/crossref_metadata_2025 huggingface dataset:
Discrete automated chemistry system with tableted reagents.
Authors: Driscoll R C , Edwards R B , Liston M D , Love T W , Mazza J C , Trafton J E
Published: January 2020

An instrument/reagent system with tableted reagents has been developed for performing ultraviolet/visible photometric assays. Preformed disposable plastic cuvettes pass through a stationary track every 5 s, providing a maximum throughput of 698 tests/h. The photometric system consists of a single light source with a separate set of light guides for each of eight photometer stations; bichromatic readings are possible at any of seven wavelengths between 340 and 630 nm. Tablets for 30 different analytes have been developed, each of which contains all necessary components for a single determination. Tablets are dispensed from disposable dispensers into cuvettes under the direction of the system's microprocessor and are completely dissolved within 45 s by ultrasonic agitation. Sample volumes range from 2 to 20 microL. Test requests are selected on a cathode-ray tube display, and sample-container bar-code labels are automatically printed. Sample entry is totally random. The system is designed to provide reagent quality control and also serum and reagent blank corrections for each test. Examples of equilibrium, zero-order, and first-order kinetic assays are presented.
Rapid loss of lactate dehydrogenase isoenzyme activity in serum by cold-induced formation of immunoglobulin G-lactate dehydrogenase complex.
Authors: Wickus G G , Smith M J
Published: January 2020

Lactate dehydrogenase (LD; EC 1.1.1.27) activity in serum from a patient recovering from a myocardial infarction was extremely unstable when stored at 0 degree C. The activity of each LD isoenzyme except LD-1 decreased by at least 40% when serum was stored at 0 degree C for 4 h. The patient's erythrocyte LD activity had normal stability at lower temperatures, but LD from other sources, when added to the patient's serum, rapidly lost activity at 0 degree C. The patient's serum contained an immunoglobulin G that combined--at 0 degree C but not at 21 degrees C--primarily with LD isoenzymes containing one or more M subunits. Because this immunoglobulin-LD complex has no enzyme activity, we used 125I-labeled purified LD to study formation of the complex. NAD+ blocked the formation of immunoglobulin-LD complex but could not dissociate the complex and restore the LD activity.
Sex-dependent relation between ionized calcium in serum and blood pressure.
Authors: Fogh-Andersen N , Hedegaard L , Thode J , Siggaard-Andersen O
Published: January 2020

In an epidemiological study of 977 45-year-olds, both the mean concentration of ionized calcium in serum, [Ca2+]7.4, and arterial blood pressure were higher in men than in women. Among the men, there was a weak positive correlation between [Ca2+]7.4 and the systolic blood pressure, but no other significant correlation was found within each separate sex. Postmenopausal women had slightly increased [Ca2+]7.4 but normal blood pressure. Patients taking thiazide diuretics or beta-adrenergic blocking agents had normal [Ca2+]7.4.
Simultaneous on-line measurement of blood K+, Ca2+, Na+, and pH with a four-function ChemFET integrated-circuit sensor.
Authors: Sibbald A , Covington A K , Carter R F
Published: January 2020

On-line, simultaneous measurement of blood K+, Ca2+, Na+, and pH has been achieved by using a quadruple-function ChemFET (chemical-sensitive field-effect transistor) integrated-circuit sensor. Blood is withdrawn from the subject through an actively heparinized, dual-lumen cannula and passed into a flow-cell containing the sensor, in alternation with a calibration solution. A minicomputer controls the analysis system, logs data, and provides a continuous, graphic display of K+ and pH values.
Improved liquid-chromatographic determination of 3-methoxy-4-hydroxyphenylethyleneglycol in urine with electrochemical detection.
Authors: Shipe J R , Savory J , Wills M R
Published: January 2020

In this improved method for quantifying 3-methoxy-4-hydroxyphenylethyleneglycol (MHPG) in urine, after a multistep extraction of MHPG and internal standard (iso-MHPG) from 3.0 mL of urine, the compounds are separated on a C18 reversed-phase column and quantified by use of an electro-chemical detector. The isocratic chromatographic separation takes about 16 min. The mobile phase is phosphate buffer/acetonitrile (88/12 by vol), the flow rate 0.7 mL/min. Recycling the mobile phase and automating the sample injection make possible the unattended assay of more than 70 samples per day. The within-run precision of the method is excellent (CV 1.8%) at a mean concentration of 1.1 mg/L.
Enzyme-linked immunosorbent assay of retinol-binding protein in serum and urine.
Authors: Lucertini S , Valcavi P , Mutti A , Franchini I
Published: January 2020

This highly sensitive method for determining retinol-binding protein in human serum and urine is based on a double-antibody "sandwich"-type enzyme-linked immunosorbent assay. The assayable concentration range is 0.8-48 micrograms/L, the detection limit 0.2 micrograms/L. Within-assay coefficients of variation for 10 determinations at two different concentrations were 5.5 and 5.8%. The corresponding between-assay CVs were 7.9 and 9.2%. We saw no interference from any components of urine or serum. The mean urinary excretion by 30 healthy subjects, as determined by this method, was 101 micrograms/g of creatinine (SD 38.8). The concentration in serum averaged 43 mg/L (SD 12.1).
Sampling from a skewed population distribution as exemplified by estimation of the creatine kinase upper reference limit.
Authors: Miller W G , Chinchilli V M , Gruemer H D , Nance W E
Published: January 2020

Creatine kinase (EC 2.7.3.2) was measured in sera from 580 females, ages 1-77 years, and 550 males, ages 1-63 years. The distribution of results for male and female groups shows pronounced skewing toward higher values. The observed distribution of results could not be described by any of six mathematical formulas for skewed distributions, an indication of the unsuitability of such formulas to transform these data for parametric analysis. The range of 97.5 percentile estimates produced by six independent samples of 100, 200, and 400 observations randomly selected from a mathematical model defined by the adult female distribution showed progressive narrowing from the 150-380 U/L interval for the samples of 100 observations to 200-265 U/L for the samples of 400 observations; no further improvement was seen when 800 observations were used. The samples of 100 and 200 observations contained extreme value points that might appear as "outliers" but were shown to be valid members of the population distribution when larger sample sizes were collected.
Simultaneous determination of 25-hydroxy- and 1,25-dihydroxyvitamin D from a single sample by dual-cartridge extraction.
Authors: Kao P C , Heser D W
Published: January 2020

With this dual-cartridge system we extract 25-hydroxyvitamin D [25(OH)D] and 1,25-dihydroxyvitamin D [1,25(OH)2D] from a single serum sample by using a nonpolar octadecylsilanol silica cartridge to adsorb the vitamin D metabolites and other nonpolar substances; the polar substances wash through the cartridge. The eluted material is then applied to a second alkylamine cartridge, which adsorbs the relatively polar hydroxylated metabolites; the less-polar substances are washed from the second cartridge. Elution from the second cartridge purifies and also separates 25(OH)D and 1,25(OH)2D with analytical recoveries near 90%. The monohydroxyl metabolites are determined by "high-performance" liquid chromatography (HPLC); the dihydroxyl metabolites are further purified by HPLC and determined by radioreceptor assay according to established procedures. Mean (+/- SD) winter normal values (34 subjects of both sexes; blood drawn in mid-April) were 18 +/- 5 micrograms/L for 25(OH)D and 25 +/- 7 ng/L for 1,25(OH)2D. In nine laboratory volunteers, the mean increase in the serum 25(OH)D3 value 5 h after ingestion of 50 micrograms of 25-hydroxycholecalciferol (Calderol) was 9 (SD 4) micrograms/L.
Use of monoclonal antibodies in an enzyme immunoassay for factor VIII-related antigen.
Authors: Bradley L A , Franco E L , Reisner H M
Published: January 2020

Two monoclonal antibodies (MAb 53, MAb D7) were produced, each having specificity for Factor VIII-related antigen (FVIIIR:Ag), but exhibiting no inhibitory effect on either procoagulant activity or the ability of von Willebrand factor to agglutinate platelets in the presence of the antibiotic ristocetin. For quantification of FVIIIR:Ag, we used the antibodies in a competitive enzyme-linked immunosorbent assay (ELISA). Binding of either of the MAb's to solid-phase antigen was inhibited by free FVIIIR:Ag in the test sample. Dose-response curves for the reference standards were consistently linear (r2 greater than 0.990) and reproducible. The normal range of FVIIIR:Ag detected in plasma (normal defined as 1000 units/L) was similar to that reported for polyclonal heterologous antibodies in similar ELISA or immunoradiometric (IRMA) systems, and the assay was sensitive to 10 units of FVIIIR:Ag per liter. Inter- and intra-assay precision was good, coefficients of variation being less than 11%. Studies on patients showed good correlations between values measured by MAb ELISAS and IRMA (polyclonal rabbit antibody) over FVIIIR:Ag concentrations ranging from less than 10 to 2700 units/L (r = 0.971, p less than 0.001 for MAb 53; r = 0.938, p less than 0.001 for MAb D7). Both ELISAS could be used to quantify FVIIIR:Ag in other mammalian species. The assay is inexpensive and simple, and all reagents required for it are commercially available.
Development of a reference material for alkaline phosphatase.
Authors: Duncan P H , McKneally S S , MacNeil M L , Fast D M , Bayse D D
Published: January 2020

In developing a Reference Material for alkaline phosphatase, we studied the stability, kinetic properties, and commutability of separate preparations of the purified enzyme from human liver, intestine, bone, and placenta. The Michaelis constants (Km) for the preparations from liver, bone, and intestine agreed well with the Km values we obtained for five human serum specimens, whereas that for the placental isoenzyme differed significantly. The first three isoenzymes exhibited nearly identical response-surface patterns, which closely paralleled those observed for 12 human serum specimens (commutability), but not that of the placental isoenzyme. Thus, we believe that a reference material could equally well consist of either the bone, intestinal, or liver isoenzyme. All four isoenzymes were satisfactorily stable in temperature-accelerated degradation studies. We chose the liver isoenzyme as an appropriate reference material because liver tissue is easier to obtain than bone or intestine and the isoenzyme is abundant in liver, is easy to extract, and is the one most commonly increased in human serum. This material is stable at -20 degrees C, is free of interfering and degradative enzymes and, being of human origin, is commutable with the enzyme in human serum.
Association of an oxygen-sensitive lactate dehydrogenase isoenzyme, LDk, with LD-6 in serum of critically ill patients.
Authors: Onorato V A , Manly K F , Vladutiu A O
Published: January 2020

We measured a highly unusual, oxygen-sensitive lactate dehydrogenase, LDk, in the serum of six patients whose serum showed a band for LD-6 on routine agarose gel electrophoresis for LD isoenzymes. All these patients showed very high serum LDk activity, greatly exceeding the high values previously described in serum of patients with various malignant tumors. In two of the patients, LDk activity was low both before LD-6 was found in and after it disappeared from the serum, evidencing a correlation with LD-6. All of the six patients, five of whom died in the hospital, had severe hypotension. We suggest that hypoxia is responsible for the appearance of LD-6 in serum and that LD-6 is found in association with high LDk activity in serum of critically ill patients.
Interpreting the profile of liver-function tests in pediatric liver transplants.
Authors: Simonsen R , Virji M A
Published: January 2020

Results of traditional laboratory tests of liver function were correlated with the clinical course in 26 pediatric patients after liver transplantation. On the basis of clinical outcome after transplantation, the patients were divided into two groups: (a) uncomplicated course with short hospital stay, and (b) post-transplantation course complicated by multiple clinical problems. The patterns of results for tests reflecting liver function--bilirubin (total and conjugated), aspartate (EC 2.6.1.1) and alanine (EC 2.6.1.2) aminotransferases, and gamma-glutamyltransferase (gamma GT, EC 2.3.2.2)--were consistent with the clinical findings in these patients. Values for alkaline phosphatase (EC 3.1.3.1), however, were only rarely increased, even when there was clinical evidence of biliary obstruction. Not only was serum gamma GT increased in obstructive jaundice, but this sometimes was the only test giving results outside the normal limits. We suggest that the persistent and marked increases of gamma GT observed in half of the patients may have resulted from immune-mediated damage to the transplanted liver.
Kits for enzyme determinations compared: relation between composition and quality.
Authors: Peters F P , Hafkenscheid J C
Published: January 2020

Kits of five different suppliers, composed according to the Dutch recommendations for determination of the enzyme activity of alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase, gamma-glutamyltransferase, alkaline phosphatase, and creatine kinase, were intercompared. Activity concentrations of the enzymes in human sera were measured under defined conditions, evaluated, and related to the actual composition of the kits. Concentrations of all kit components were determined by various analytical techniques. The overall results of the activity measurements and the composition of at least three kits inter-agree well. We found deviations as great as 10% in our analytical evaluation of the kits of the other two suppliers, which can partly be accounted for.
Urinary excretion of acetaminophen and its metabolites as studied by proton NMR spectroscopy.
Authors: Bales J R , Sadler P J , Nicholson J K , Timbrell J A
Published: January 2020

Acetaminophen and its glucuronide, sulfate, N-acetyl-L-cysteinyl, and L-cysteinyl metabolites can be rapidly detected by 1H NMR spectroscopy of intact, untreated human urine. Study of the time course of excretion of these metabolites in five clinically normal men after ingestion of the usual 1-g therapeutic dose of the drug showed that the mean 24-h excretion of the drug and these metabolites as determined by NMR was 77.3% of the dose. Respective relative proportions of the above metabolites were 49.9%, 37.6%, 3.0%, and 9.5% (L-cysteinyl plus free drug). Excretion of some other metabolites in urine, including creatinine, citrate, hippurate, and sarcosine was measured concurrently. Excretion of creatinine and sarcosine was closely correlated.
Improved determination of manganese in hair by use of a mini-autoclave and flameless atomic absorption spectrometry with Zeeman background correction: an evaluation in unexposed subjects.
Authors: Guillard O , Brugier J C , Piriou A , Ménard M , Gombert J , Reiss D
Published: January 2020

Motivated by the relatively wide range of values published hitherto, we offer a new, reliable method for determination of manganese in hair from unexposed human subjects. By using a mini-autoclave, we have developed a method that obviates loss of manganese during digestion and, thanks to use of a Teflon receptacle and rigorous methodology, contamination has been minimized. We used Zeeman flameless atomic absorption spectrometry, with the method of standard additions, for quantification. Within-run CVs for concentrations of Mn of 0.26 and 0.28 microgram/g of dry hair were 3.63 and 3.93%; the day-to-day CV for a Mn concentration of 0.29 microgram/g of dry hair was 5.84%. Mean (+/- SD) analytical recovery of Mn added to samples of hair was 104% (+/- 9.2%). The mean concentration of Mn found in the hair of 15 healthy unexposed subjects was 0.26 (+/- 0.05) microgram/g dry weight. This sensitive, reproducible procedure is suitable for use in analysis for traces of Mn in hair.
A mathematical model of the hemoglobin-oxygen dissociation curve of human blood and of the oxygen partial pressure as a function of temperature.
Authors: Siggaard-Andersen O , Wimberley P D , Göthgen I , Siggaard-Andersen M
Published: January 2020

A mathematical model is described giving the oxygen saturation fraction (s) as a function of the oxygen partial pressure (p): y - y0 = x - x0 + h X tanh [k X (x - x0)], where y = kn[s/(1-s)] and x = ln(p/kPa). The parameters are: y0 = 1.875; x0 = 1.946 + a + b; h = 3.5 + a; k = 0.5343; b = 0.055 X [T/(K - 310.15)]; a = 1.04 X (7.4 - pH) + 0.005 X Cbase/(mmol/L) + 0.07 X [[CDPG/(mmol/L)] - 5], where Cbase is the base excess of the blood and CDPG is the concentration of 2,3-diphosphoglycerate in the erythrocytes. The Hill slope, n = dy/dx, is given by n = 1 + h X k X [1 - tanh2[k X (x - x0)]]. n attains a maximum of 2.87 for x = x0, and n----1 for x----+/- infinity. The model gives a very good fit to the Severinghaus standard oxygen dissociation curve and the parameters may easily be fitted to other oxygen dissociation curves as well. Applications of the model are described including the solution of the inverse function (p as a function of s) by a Newton-Raphson iteration method. The po2-temperature coefficient is given by dlnp/dT = [A X alpha X p + CHb X n X S X (1 - s) X B]/[alpha X p + CHB X n X s X (1 - s)], where A = -dln alpha/dT approximately equal to 0.012 K-1; B = (lnp/T)s = 0.073 K-1 for y = y0; alpha = the solubility coefficient of O2 in blood = 0.0105 mmol X L-1 X kPa-1 at 37 degrees C; CHb = concentration of hemoglobin iron in the blood. Approximate equations currently in use do not take the variations of the po2-temperature coefficient with p50 and CHb into account.
Enzymic fluorometric assay of plasma ammonia with a centrifugal analyzer.
Authors: Rodger M R , Jenkins P
Published: January 2020

This method for rapid plasma ammonia determination, supplied in the form of a commercially available kit, is based on a combination of enzymic reaction and fluorometry. Concentrations up to 350 mumol/L can be measured. The detection limit is 15 mumol/L. Intra-assay coefficients of variation for 30 and 100 mumol/L concentrations were 5.0% and 3.0%, respectively. The corresponding interassay CVs were 8.0% and 3.8%. Assay of 16 samples takes about 15 min. Only 25 microL of sample is required, which makes this assay suitable for pediatric use and small-animal research.
Determination of volatile alcohols and acetone in serum by non-polar capillary gas chromatography after direct sample injection.
Authors: Smith N B
Published: January 2020

In this method for detection and quantification of volatile alcohols by capillary gas chromatography, the serum sample is deproteinized, then directly injected into the gas chromatograph with 1-propanol as the internal standard. The capillary column is a 30-m bonded methylsilicone-coated, fused-silica column. With helium as the carrier gas, the injector inlet is set at a split ratio of 1/30 and the average linear velocity in the column is 25 cm/s. Injector and flame-ionization detector temperatures are 280 degrees C, oven temperature 35 degrees C. Chromatography time is less than 3 min.
Silver-stained phenotyping of alpha 1-antitrypsin in dried blood and serum specimens.
Authors: Massi G , Marano G , Patalano F , Auconi P
Published: January 2020

In this technique for determining the electrophoretic phenotype of alpha 1-antitrypsin in dried blood or serum specimens, the adsorbed material is eluted with a concentrated solution of dithiothreitol, focused on polyacrylamide thin-layer gel, and made visible with silver stain. With this staining technique all normal and pathological alpha 1-antitrypsin phenotypes can be detected. The procedure is relatively simple, inexpensive, and suitable for use in large-scale screening for alpha 1-antitrypsin deficiency in selected populations.
Enzyme immunoassay of free thyroxin in serum.
Authors: Ito M , Miyai K , Doi K , Mizuta H , Amino N
Published: January 2020

We describe a double-antibody enzyme immunoassay for free thyroxin (FT4) in serum with use of beta-D-galactosidase conjugated to thyroxin. The method is uninfluenced by thyroxin-binding globulin or albumin. Values for FT4 so determined correlated well with those determined by radioimmunoassay (r = 0.98) and equilibrium dialysis (r = 0.89). The mean variability (CV) within and between assays was 7.4% and 7.6%, respectively. The measurable range of FT4 in serum was 2.8 to 109 ng/L. The FT4 concentrations in serum as determined by this method were 8.4 to 15.5 ng/L for 26 normal adult subjects; 26 to greater than 109 ng/L for 10 patients with hyperthyroidism; less than 2.8 to 8.0 ng/L for seven patients with hypothyroidism; 7.3 to 15.8 ng/L for eight pregnant women; and 12.2 and 13.5 ng/L for two patients with low concentrations of thyroxin-binding globulin.
Single-reagent polarization fluoroimmunoassay for barbiturates in urine.
Authors: Colbert D L , Smith D S , Landon J , Sidki A M
Published: January 2020

We describe a rapid polarization fluoroimmunoassay for screening for the presence of barbiturates in urine. The single reagent is prepared by pre-mixing antiserum with a fluorescein-labeled barbiturate derivative as tracer. Use of a mixture of two sheep antisera, raised against different barbiturate immunogens, results in an assay with a broad cross-reactivity spectrum for most common barbiturates. One adds urine to the pre-mixed reagent, incubates the solution for a few minutes at room temperature, and measures the fluorescence polarization. The tracer/antiserum reagent is stable for at least six months at 4 degrees C. Results compare favorably with two other commonly used screening techniques for barbiturates, thin-layer chromatography and the EMIT-stTM (Syva) system. Although designed as a qualitative screen, some drugs can be quantified by means of a standard curve of the relevant barbiturate.
Stabilized analysis of antidepressant drugs by solvent-recycled liquid chromatography: procedure and proposed resolution mechanisms for chromatography.
Authors: Lensmeyer G L , Evenson M A
Published: January 2020

In this efficient extraction and isocratic liquid-chromatographic procedure for measuring eight antidepressant drugs in serum (amoxapine, 8-hydroxyamoxapine, doxepin, desmethyldoxepin, imipramine, desipramine, amitriptyline, and nortriptyline), they are extracted from serum as a group by use of disposable solid-phase cyanopropyl columns; the eluate is injected directly onto a Zorbax cyanopropyl analytical column. This sample preparation circumvents potential problems of drug instability associated with the usual evaporating/concentrating techniques. Recycling the acetic acid/acetonitrile/n-butylamine mobile phase not only maintains a stable, cost-effective system, it also prolongs column life. Standard curves for all eight drugs are linear to 1000 micrograms/L; the detection limit is 8-10 micrograms/L. For all drugs and metabolites, within-run CVs were 0.9 to 2.2%, between-run CVs 2.1 to 3.8%. Analytical recovery of the solid-phase extraction step was 85 to 100% (mean = 94.5%) for all analytes. Standards and controls, stored frozen in drug-free serum, are stable for at least six months. We also report a study of separation mechanisms.
Determination of enzyme activity in biological fluids by means of electrochemical oxidation of NADH at a modified glassy carbon electrode.
Authors: Bartalits L , Nagy G , Pungor E
Published: January 2020

This amperometric technique for the determination of enzyme activity is based on detecting a decrease in the concentration of the NADH co-factor of the enzyme reaction. A glassy carbon electrode, modified by adsorption of Mg2+ and NADH, is used to measure the anodic peak current that corresponds to the oxidation of NADH. We found no significant difference between the enzyme activity of lactate dehydrogenase (E.C.1.1.1.27) preparations as measured by the above amperometric technique and by a spectrophotometric method.
Precipitation of apo E-containing lipoproteins by precipitation reagents for apolipoprotein B.
Authors: Gibson J C , Rubinstein A , Brown W V
Published: January 2020

We measured the solubility of apolipoprotein E (apo E) after precipitation, with heparin-Mn2+ or dextran sulfate-Mg2+, of lipoproteins containing apo B. Data from 46 randomly selected subjects suggest that apo E is readily precipitated by dextran sulfate-Mg2+, but that heparin-Mn2+ preferentially precipitates apo E associated with apo B-containing lipoproteins while leaving the apo E-containing fraction of high-density lipoproteins (HDL) in solution. In a more detailed analysis of three subjects, we measured the lipoprotein association of apo E by column chromatography on agarose beads, before and after its precipitation from plasma. This study confirmed the preferential solubility of apo E associated with HDL lipoproteins. Using plasma from two normolipidemic subjects, we maintained the heparin concentration at 1.30 g/L and varied the manganese concentration from 9.2 to 184 mmol/L. A 46 mmol/L concentration best separated apo E-containing HDL from apo B-containing lipoproteins. Thus, at these final concentrations, heparin-Mn2+ appears to precipitate the apo E associated with apo B-containing lipoproteins, leaving soluble most of the apo E associated with lipoproteins of HDL size.
An automated, affinity-column-mediated, enzyme-linked immunometric assay for digoxin on the Du Pont aca discrete clinical analyzer.
Authors: Leflar C C , Freytag J W , Powell L M , Strahan J C , Wadsley J J , Tyler C A , Miller W K
Published: January 2020

We describe an automated assay for digoxin that requires a 200-microL sample of serum. Total analysis time is 18 min. The method is extremely precise, with within-run CVs of 2.6, 1.6, and 4.9%, respectively, at 0.5, 1.5, and 3.5 micrograms of digoxin per liter (n = 20). The lower limit of detection is 0.2 micrograms of digoxin per liter. For patients' samples, the correlation with RIA (x) is excellent (r = 0.95; y = 0.95x - 0.14; standard error = 0.17 micrograms/mL). We saw no interferences in samples having high concentrations of rheumatoid factors, lipid, bilirubin, or hemoglobin. Cross reactivity with digoxin analogs and steroidal compounds is similar to that observed by RIA.
Improved assay for alcohol dehydrogenase activity in serum by centrifugal analysis.
Authors: Kato S , Ishii H , Kano S , Hagihara S , Todoroki T , Nagata S , Takahashi H , Nagasaka M , Sato J , Tsuchiya M
Published: January 2020

We describe an improved method for determination of alcohol dehydrogenase (EC 1.1.1.1) activity in 60 microL of human serum, based on conversion of ethanol to acetaldehyde with simultaneous reduction of NAD+ in glycine NaOH buffer (pH 9.0) at 37 degrees C in a centrifugal analyzer. The final concentration of NAD+ was 10 mmol/L and ethanol was 20 mmol/L. The dilution curve was linear with enzyme activity up to 200 U/L, and results by this method correlated well with those by a manual method (N Engl J Med 279: 241-248, 1968). Within-run precision (CV) was 0.9 to 8.2% over the range of 4.5 to 88.1 U/L, and day-to-day precision was 5.4 to 5.6%. In sera from 198 healthy individuals, mean alcohol dehydrogenase activity was 1.6 (SD 1.2, range 0-5) U/L. To evaluate the clinical utility of determining alcohol dehydrogenase, we measured the activity of alanine aminotransferase and alcohol dehydrogenase in sera from 470 patients with various diseases in our hospital, and found that results for the two enzymes did not correlate well.
Automated kinetic assay for lactate dehydrogenase isoenzyme 1 by centrifugal analysis after alkaline inactivation.
Authors: Eckfeldt J H , Kershaw M J , Lewis L A
Published: January 2020

Lactate dehydrogenase isoenzyme 1 (LD1, EC 1.1.1.27) has been used widely as a marker for myocardial infarction, and many analytical methods for it have been developed, most of which are relatively labor intensive. On the basis of a recent report by Takizawa et al. (Clin Chem 29: 1941-1945, 1983) describing the marked stability of LD1 in buffered alkaline solutions, and with use of a centrifugal analyzer, we have developed a fully automated assay for LD1. Results with this method are precise (between-day SD = 2.1 U/L), vary linearly with LD1 activity to 600 U/L, and correlate well with LD1 as determined by immunological (Roche Isomune-LD) and agarose electrophoretic methods (r = 0.985 and 0.986, respectively). Furthermore, the method is easy and convenient, and should provide a substantial savings in labor and reagent costs to laboratories currently determining LD1 by electrophoretic or immunologic methods.
Effect of protein concentration on the determination of digoxin in serum by fluorescence polarization immunoassay.
Authors: Porter W H , Haver V M , Bush B A
Published: January 2020

Determination of digoxin by fluorescence polarization immunoassay (FPIA) with the Abbott "TDx" is significantly influenced by the concentration of total serum protein. Each 10 g/L increase in serum protein results in an 8% decrease in measured digoxin. Studies with [3H]digoxin confirmed that digoxin binds to the protein pellet during the trichloroacetic acid precipitation step before the immunoassay. Serum protein, or equal concentrations of albumin or gamma-globulin, exert an equivalent effect on the apparent digoxin value. Because the total protein concentration of the assay calibrators is low (50 g/L) compared with its reference interval in serum (60-80 g/L), results by FPIA may be expected to be low by an average of 16% (range, 8-24%). Digoxin results by FPIA will be most nearly accurate when the calibrators include a total protein concentration of about 70 g/L. Patients' specimens with abnormally high or low protein content will give falsely high or low results for digoxin.
Performance of the ASTRA enzyme systems evaluated.
Authors: Janssen J W , Verdegaal M C , Visser R W
Published: January 2020

We evaluated the ASTRA enzyme systems for the kinetic measurement of some commonly measured enzymes. The comparison studies with the SMAC and the LKB 8600 show that results by the ASTRA enzyme systems correlate well with those by our routine methods. Studies with several control sera yielded excellent results for both within-run and between-day variation. The response of the photometer is linear up to 3.0 A. For three enzymes the range of linearity exceeded the limits proposed by the supplier, but for lactate dehydrogenase it did not extend as far as proposed. Reagents are stable for at least one week at 16-24 degrees C.
Nephelometry of acute-phase glycoproteins by binding to concanavalin A.
Authors: Pitkänen E M , Palosuo T , Aho K , Putus-Tikkanen T , von Essen R
Published: January 2020

Nephelometry of serum acute-phase glycoproteins by binding to concanavalin A (con-A) was compared with assays for haptoglobin and alpha 1-acid glycoprotein, and for C-reactive protein. The cutoff points for positive reactions were determined on the basis of results for a random sample (n = 130) from a middle-aged population. The sensitivity of the con-A binding assay compared favorably with that of individual acute-phase glycoproteins in a follow-up cohort of 198 patients with inflammatory joint diseases. Unlike the case in many individual acute-phase glycoprotein assays, the distribution of con-A binding values in healthy subjects is remarkably symmetrical, allowing an easy distinction between abnormal and normal values.
Interference of endogenous lactoperoxidase antibodies in a solid-phase immunosorbent radioassay for antibodies to protein hormones.
Authors: Ericsson U B , Larsson I
Published: January 2020

Interference of endogenous antibodies to lactoperoxidase (EC 1.11.1.7) has been demonstrated in a solid-phase immunosorbent radioassay for the detection of antibodies to protein hormones. On enzymic iodination with lactoperoxidase, a little of the 125I is incorporated in the enzyme itself by self-iodination. Because the 125I-labeled lactoperoxidase was not removed from the tracer by chromatography on Sephadex G-50, G-100, and G-200 columns, endogenous antibodies to the enzyme, which were present in 25 of 28 sera from apparently healthy individuals, were detected in conjunction with the measurement of antibodies to growth hormone (somatotropin), prolactin, and thyroglobulin, thus causing false-positive results. Contaminating radioactive lactoperoxidase can be removed by adsorption chromatography on cellulose or by liquid chromatography, and can be avoided by performing the iodination with immobilized lactoperoxidase or with Chloramine-T.
Standardized automated assay for functional alpha 1-antitrypsin.
Authors: Mullins R E , Miller R L , Hunter R L , Bennett B
Published: January 2020

We describe an enzymic assay for the functional activity of alpha 1-antitrypsin, in which tosyl-Gly-Pro-Lys-p-nitroanilide acetate (Chromozym PL) is the trypsin substrate. For the indicating reaction, the concentration of substrate is about double the Km of its reaction with trypsin. This assay is an improvement over reactions involving N-alpha-benzoyl-DL-arginine p-nitroanilide as substrate, the solubility of which in water is less than the Km of its reaction with trypsin. The assay is standardized in terms of active sites of trypsin inhibited per liter of serum, with p-nitrophenyl-p'-guanidinobenzoate as the active site titrant. The results agreed well with those by an immunonephelometric assay for alpha 1-antitrypsin (r = 0.953). The within-run and run-to-run precision was approximately 3%. The results of the assay varied linearly with alpha 1-antitrypsin concentration from 0 to 60 mumol/L of serum. The minimum sample size was 50 microL, and analysis of 24 samples took less than 20 min.
Abbott radiative energy attenuation method for quantifying ethanol evaluated and compared with gas-liquid chromatography and the Du Pont aca.
Authors: Cary P L , Whitter P D , Johnson C A
Published: January 2020

Quantification of ethanol by a radiative energy attenuation (REA) technique was evaluated and compared with results by gas-liquid chromatography (GLC) and by the Du Pont aca. Within-assay CVs were less than 5.5%. Between-assay CVs ranged from 1.9% to 6.0% for serum and blood controls at concentrations of 0.5, 1.0, and 2.5 g/L. We observed no cross reactivity with methanol, isopropanol, or acetone, and analytical recovery of ethanol from serum averaged 101%. For the three-method comparison we performed parallel determinations of 156 blood, 92 serum, and 54 urine samples containing a wide range of ethanol concentrations. Linear regression analysis of the REA results vs those of GLC or aca yielded the following: for serum, REA = 1.03 GLC -0.03 (r = 0.998), REA = 1.13 aca -0.04 (r = 0.999); for blood, REA = 0.97 GLC + 0.05 (r = 0.994), REA = 0.99 aca + 0.06 (r = 0.996); and for urine, REA = 1.01 GLC -0.03 (r = 0.998). We also discuss the clinical and forensic use of the REA method for ethanol.
Lactate production by aerobic bacteria grown in cerebrospinal fluid.
Authors: Kiechle F L , Kamela M A , Starnes R W
Published: January 2020

The source of the abnormally high concentration of lactate in the cerebrospinal fluid in patients with bacterial meningitis is not known. It may represent a bacterial metabolite. To determine whether cerebrospinal fluid possesses appropriate substrates to support the growth of pathogenic aerobic bacteria, three Gram-positive and nine Gram-negative bacteria were separately inoculated in pooled normal cerebrospinal fluid. After incubation for 24 h, all Gram-positive bacteria increased lactate, eight Gram-negative bacteria decreased lactate, and one Gram-negative bacteria failed to significantly change the lactate concentration. We conclude that lactate produced in cerebrospinal fluid in patients with aerobic bacterial meningitis is not necessarily a bacterial metabolite.
Two-dimensional electrophoresis of proteins from cultured human retinal-pigment epithelial cells: internal references, cataloging, and glycoproteins.
Authors: Haley J E , Gouras P
Published: January 2020

Two-dimensional gel electrophoresis of acidic and basic [35S]methionine-labeled polypeptides derived from primary cultures of human retinal-pigment epithelial cells revealed about 850 proteins. By co-electrophoresis with highly purified, evolutionally conserved proteins, alpha-actinin, calmodulin, cytosol retinal-binding protein, alpha- and beta-tubulin, and vinculin (mass: 130 000 Da) were tentatively identified in the fluorograms. Quantification of greater than 100 of the excised radioactive spots by liquid scintillation counting revealed an estimated overall gel/gel and donor/donor variation of 40% (SEM, 21%), the latter for data on three to four donors 57 to 81 years old. Therefore, for a difference from normal to be significant (p less than or equal to 0.01), it would, on average, have to exceed 88% of the control mean for that protein. Putative glycoproteins were independently radiolabeled, with tritiated sugars as precursors. Glucosamine was incorporated most rapidly and with the highest specific activity. It labeled about 170 polypeptides. Fucose and N-acetylmannosamine, respectively, labeled 74 and 27 polypeptides. The glycoprotein label was maximal in about 16 very acidic proteins with apparent molecular masses between 50 000 and 150 000 Da. Parallel use of both a sugar and an amino acid label facilitates identification of proteins in two-dimensional gels.
Quantitative analysis and pattern recognition of two-dimensional electrophoresis gels.
Authors: Ridder G , VonBargen E , Burgard D , Pickrum H , Williams E
Published: January 2020

We describe a system, both hardware and software, that provides quantitative analysis and data reduction of two-dimensional electrophoresis gels. Image-analysis techniques are used to determine spot intensities and to match spot patterns among many gels. A pattern-recognition program is used to extract the useful information contained in the spot lists. The application of this technology to a study of supernates from bacterial cultures is described.
A metrological study of autoradiographs from two-dimensional gel electrophoresis.
Authors: Valiron O , Lefkovits I , Garderet P , Steinberg C
Published: January 2020

Samples prepared from a single batch of labeled cells were subjected to two-dimensional gel electrophoresis and autoradiography. Three factors were varied: total quantity of protein, quantity of labeled protein, and exposure time. The mean background absorbance of the film remained identical (about 0.5 A) for all the treated series, whatever the exposure time and whether or not there were unlabeled proteins in the sample. Hence any spot with a peak A of the same order of magnitude can be seen. The standard deviation was about 0.05 A. Thus, the measurement precision is 2.5% of full scale for digitalization over 0 to 2 A. We derived experimental calibration curves, which are neither linear nor logarithmic because of the film response and which can be used on randomly chosen spots.
An approach to molecular analysis of T-cell clones by two-dimensional gel electrophoresis: is there interclonal and intraclonal heterogeneity?
Authors: Kettman J , Lefkovits I
Published: January 2020

To understand the regulation of immunity one must understand the processes and the consequences of the activation of the lymphoid cell, its clonal proliferation, and the ultimate differentiation of the progeny into the effector elements that constitute the immune system. By use of polyclonal mitogens, individual T-cell precursors can be activated for study of their clonal growth and generation of T effector cells. We used two-dimensional gel electrophoresis to study the gene products expressed by such T-cell clones. On comparing the radiofluorograms of such gels for a large set of T-cell clones we find that many gene products are expressed in common, as expected, but that another large set of products varies from one clone to the other and might be designated as markers of T-cell subsets or stages of differentiation. By subdividing the growing clone and analyzing the family of daughter clones, we find considerable variation in gene expression during clonal expansion.
Identification of coordinate pairs of polypeptides: a technique for screening of putative precursor product pairs in 2D gels.
Authors: Lemkin P F , Sonderegger P , Lipkin L E
Published: January 2020

Two-dimensional electrophoresis, combined with computerized quantification of individual proteins, provides a sensitive and accurate approach to detection of small differences in the protein pattern. As an extension of "GELLAB," a data base system for the analysis of two-dimensional electrophoretic gels, we report here on a screening algorithm for detection of certain post-translational modification events. It is based on the assumption that, if there is a post-translational modification of a protein in the transition between two functional states of a biological system, in many cases the sum of the protein concentration of a precursor-product pair in one is equivalent to that in the other. In addition to the identification of candidate pairs with a precursor-product relationship, cues to the nature of the post-translational modification that might relate them might also be derived from comparisons of the isoelectric point and apparent molecular mass. This subset of the GELLAB system may be thought of as a screening tool in the search for and investigation of post-translational protein-processing events so that the more time-consuming structural investigations to follow can be performed on a smaller set of proteins.
Quality control and technical outcome of ISO-DALT two-dimensional electrophoresis in a clinical laboratory setting.
Authors: Daufeldt J A , Harrison H H
Published: January 2020

In this paper, we present initial quality-control evaluations for a prototype clinical laboratory, recommendations for clinical and technical data record-keeping, and laboratory protocols designed to minimize operator-dependent analytical error in a multi-user environment. These evaluations and recommendations are based on qualitative and semiquantitative two-dimensional electrophoresis studies of the College of American Pathologists' Reference Preparation for Serum Proteins (RPSP). We include the schematic map of RPSP and manually derived estimates of analytical precision for within-run and run-to-run performance with the standard 18 X 18 cm ISO-DALT system.
Improved record keeping and photography of silver-stained two-dimensional electrophoretograms by way of "XRD images".
Authors: Harrison H H
Published: January 2020

Kodak X-Ray Duplication film provides a relatively simple and inexpensive medium for producing full-size positive-image transparencies ("XRD images") from silver-stained two-dimensional polyacrylamide gel electrophoretograms. In these XRD images, the spot or band pattern of a polychromatic silver-stained gel is represented as black against a clear background, regardless of the color of the spot in the gel itself. Thus, XRD images may be overlapped with transillumination for visual gel-to-gel comparisons, may be easily subjected to scanning densitometry, and may have the exposure adjusted to provide the proper background for black-and-white photography. I describe here the details of the XRD method and the design of a new apparatus for producing XRD images in a wet-gel "contact-print" format, which improves the method.
Proteins in normal, irradiated, and postmortem human brain quantitatively compared by using two-dimensional gel electrophoresis.
Authors: Narayan R K , Heydorn W E , Creed G J , Kornblith P L , Jacobowitz D M
Published: January 2020

Using a combination of two-dimensional gel electrophoresis (2DE), silver staining, and computerized densitometry, we studied protein patterns in human cerebral cortex: normal fresh-frozen, fresh-frozen but previously irradiated, and post-mortem. The relative molecular mass of the resolved proteins ranged from 14 400 to 100 000, the isoelectric points from 4.75 to 7.0. The pattern of proteins (six of them identified) was essentially the same for all three groups. However, computerized densitometry demonstrated significant alterations in the density of several spots in the irradiated and postmortem groups as compared with the normal controls. Irradiated cortex showed statistically significant changes in only six spots (three increased and three decreased in density); postmortem material showed 20 altered spots (16 diminished and four increased). Evidently normal human cerebral cortex has a consistent protein pattern on 2DE, which is quantitatively (but not qualitatively) altered in irradiated and postmortem material. These findings provide a point of reference against which proteins from abnormal brain material can be compared, both qualitatively and quantitatively.
Two-dimensional gel electrophoresis used in neurobiological studies of proteins in discrete areas of the rat brain.
Authors: Jacobowitz D M , Heydorn W E
Published: January 2020

Using two-dimensional gel electrophoresis, we studied proteins in the rat brain. The relative amounts of individual proteins differ in discrete areas of the brain, and the concentrations of three different proteins can be altered by chronic administration of desmethylimipramine or reserpine. Brain proteins can be radiolabeled in vitro by incubating samples of fresh tissue with [35S]methionine. We identified several proteins by using immunoblotting and comigration. Finally, we developed a possible animal model for studying proteins related to Alzheimer's disease by depleting the cholinergic innervation to the cortex and the hippocampus.
Gamma heavy chain disease studied by two-dimensional electrophoresis and immuno-blotting techniques.
Authors: Blangarin P , Deviller P , Kindbeiter K , Madjar J J
Published: January 2020

We used two-dimensional polyacrylamide gel electrophoresis and immunoblotting techniques to study serum proteins from a patient with a monoclonal gammopathy. Two-dimensional electrophoresis was optimized for serum proteins with two main goals: (a) to allow the resolution of many serum proteins in both directions, with penetration of the maximum number of proteins in the first dimension; and (b) to obtain the best reproducibility from one experiment to another, within the limits of the current technique. These analyses, combined with immunoblotting, permitted us to characterize a gamma heavy chain disease protein of 34 000-Da molecular mass. Moreover, two-dimensional mapping of the patient's serum proteins allowed demonstration of the microheterogeneity of this monoclonal component.
Systematic characterization of human prostatic fluid proteins with two-dimensional electrophoresis.
Authors: Tsai Y C , Harrison H H , Lee C , Daufeldt J A , Oliver L , Grayhack J T
Published: January 2020

We present a systematic analysis of human prostatic fluid with two-dimensional gel electrophoresis (the ISO-DALT system) and a characterization of normal and disease-related protein patterns. A reference map for prostatic fluid proteins was established by analysis of pooled prostatic fluids from 80 men (age less than or equal to 50 years) without prostatic lesions. Proteins in prostatic fluid that share immunogenicity with serum proteins were identified by use of antibody to whole human-serum protein in an affinity-column fractionation of a reference pool and differential analysis of the absorbed (serum components) and unabsorbed (non-serum components) fractions. Individual prostatic fluids from 30 patients (eight with prostatic cancer, 10 with prostatitis and benign prostatic hyperplasia, six with benign prostatic hyperplasia alone, and six with asymptomatic chronic prostatitis) were scored qualitatively with respect to the presence or absence of 57 major prostatic fluid proteins. Statistically significant, disease-correlated alterations were observed for at least eight of the proteins so scored.