bluuebunny/crossref_metadata_2025 huggingface dataset:
Global approaches to quantitative analysis of gene-expression patterns observed by use of two-dimensional gel electrophoresis.
Authors: Anderson N L , Hofmann J P , Gemmell A , Taylor J
Published: January 2020

A major difficulty in the use of two-dimensional protein maps to identify and classify cell types is the problem of acquiring, selecting, and analyzing quantitative data on hundreds of protein spots. Here we use methods of multivariate statistics to analyze the differences among a panel of human cell lines, in some cases involving quantitative data on more than 250 proteins. Principal-component and cluster-analysis techniques show that the lines can be easily distinguished, even by using the subset of proteins present in all cells. A preliminary analysis of the protein changes brought about by phorbol ester-induced differentiation of the line U937 is included.
"Map" of proteins resolved from human chorionic villi by two-dimensional electrophoresis.
Authors: Trnka P L , Pergament E , Anderson N G
Published: January 2020

Two-dimensional electrophoresis was applied to specimens of human chorionic villi obtained during the first trimester of gestation, the object being to simultaneously map several hundred polypeptide gene products. Genetically normal specimens were homogenized in a urea-based denaturant and the supernates were electrophoresed with use of the "ISO-DALT" system. Four categories of proteins are distinguished on the map: previously identified proteins present in chorionic villi and other cell types; unidentified proteins present in chorionic villi and other cell types; proteins present in chorionic villi and amniotic fluid but not in other cell types; and proteins probably originating from the amnio-chorionic plate. The reference map for chorionic villi provided in this study may serve as the basis of determining whether genetic analyses conducted in the first trimester accurately represent the fetal genotype.
Protein changes in activated human platelets.
Authors: Giometti C S , Anderson N G
Published: January 2020

Using two-dimensional electrophoresis, we mapped both the total and the cytoskeletal proteins of human platelets before and after activation with thrombin or the calcium ionophore A23187. Activation resulted in increased abundance of the phosphorylated form of myosin light chains with an approximate molecular mass of 20 kDa, decreased abundance of two proteins with molecular masses of approximately 18 and 25 kDa, and, in the case of activation with thrombin, the appearance of a new chain of protein spots (named "Thromb:1"). The latter, found associated with isolated detergent-insoluble cytoskeletons, reacted with antibody to human fibrinogen and thus were identified as gamma-gamma dimers of fibrin. The total number of proteins associated with the cytoskeleton increased after activation with either thrombin or A23187, but we observed some differences in which proteins were bound, and for how long.
Concentrations of non-glucose polyols in serum and cerebrospinal fluid from apparently healthy adults and children.
Authors: Yoshioka S , Saitoh S , Seki S , Seki K
Published: January 2020

Six non-glucose polyols--mannose, fructose, 1-deoxyglucose, mannitol, glucitol, and inositol--were identified and evaluated in human serum and cerebrospinal fluid by gas-liquid chromatography and by gas-liquid chromatography/mass spectrometry. Concentrations of fructose, mannose, and inositol in the serum of healthy persons or children without metabolic diseases varied with age, as already reported for 1-deoxyglucose. Fructose, inositol, and glucitol concentrations in cerebrospinal fluid significantly exceeded those in serum. The method described here for determining polyols and for evaluating polyol patterns in serum, as well as the resulting data on children and healthy subjects, should be useful in investigations of the clinical and physiological significance of polyols.
Liquid-chromatographic determination of two antidepressants, trazodone and mianserin, in plasma.
Authors: Wong S H , Waugh S W , Draz M , Jain N
Published: January 2020

Plasma containing trazodone or mianserin was extracted. The organic phase containing trazodone was evaporated and the residue was reconstituted in dilute acid. Mianserin was back-extracted from the organic phase with dilute acid. Both drugs were chromatographed on mu Bondapak C18 columns, with phosphate/acetonitrile as the mobile phase. Peak-height ratios of drug/internal standard were linearly correlated with concentrations between 25 and 2000 micrograms/L for trazodone, and between 25 and 200 micrograms/L for mianserin, with respective between-run CVs of 4.7% and 7.6%. Detection limits were 5 ng. Of some common drugs and metabolites examined, nortriptyline co-elutes with the internal standard used in the trazodone assay, while flurazepam co-elutes with mianserin. Concentrations of trazodone in 26 patients ranged from 73 to 1678 micrograms/L. For two geriatric patients, concentrations were about 2000 micrograms/L. For two overdose patients, they were about 5000 micrograms/L. The concentration of mianserin was 27 micrograms/L for a volunteer treated with a single 40-mg oral dose.
Rapid radioimmunoassay for corticotropin in unextracted human plasma.
Authors: Nicholson W E , Davis D R , Sherrell B J , Orth D N
Published: January 2020

This RIA for corticotropin (ACTH) involves use of a commercially available antiserum and permits measurement of immunoreactive ACTH in unextracted plasma. The assay takes 2.5 days, detects as little as 5 pg/mL of plasma, and is specific: structurally and (or) biosynthetically related peptides do not cross react. Generally, data on dilutions of almost all human plasma specimens produce curves parallel to that for the ACTH reference standard. Values correlate well with those obtained by a well-established but more laborious RIA. We measured immunoreactive ACTH in normal subjects at various times of the day and after modulation of their pituitary-adrenal axis, and in patients with hypo- and hyper-secretion of ACTH. We conclude that the full range of immunoreactive ACTH values, which accurately reflect the status of the subject's pituitary-adrenal axis, can be quickly and easily determined in samples of unextracted human plasma.
Evaluation of a direct solid-phase radioimmunoassay for progesterone, useful for monitoring luteal function.
Authors: Kubasik N P , Hallauer G D , Brodows R G
Published: January 2020

We have evaluated a commercially available, direct, solid-phase radioimmunoassay kit for progesterone determination in serum or plasma. The assay is precise, within-run precision (CV) in the clinically significant ranges being 2.5 to 5.2%, between-run 5.5 to 5.8%. Mean analytical recovery of different concentrations of progesterone added to serum was 99.7% (range 95.3 to 102.7%). Fourteen closely related steroids showed no cross reactivity. The minimum detection limit was 0.5 microgram/L. Luteal-phase progesterone concentrations in serum were increased (greater than 3 micrograms/L) in 19 normal ovulatory menstrual cycles and decreased (less than 1.5 micrograms/L) in two nonovulatory cycles. We found this direct assay for progesterone to be analytically and clinically sound, and useful for assessing luteal-phase function.
Dual-channel continuous-flow system for determination of phenylalanine and galactose: application to newborn screening.
Authors: Hoffman G L , Laessig R H , Hassemer D J , Makowski E R
Published: January 2020

We describe a dual-channel AutoAnalyzer (Technicon) system for the simultaneous measurement of phenylalanine and galactose from blood specimens on filter-paper. Using a single 1/4-in.-diameter (6-mm) specimen, we measure both components fluorometrically at a rate of 70/h. Analytical recovery with the method and the linearity of measurements vs sample concentration are excellent through the ranges of interest, 0-200 mg/L for phenylalanine and 0-800 mg/L for galactose. Carryover at the critical values during screening, 40 mg/L for phenylalanine and 100 mg/L for galactose, is essentially zero. The dual-channel system provides the means to incorporate a low-incidence test, i.e., galactosemia (incidence 1/70 000), into an existing program for phenylalanine analysis, for which the higher rates (phenylketonuria, incidence 1/11 500) easily justify the cost of mass screening.
Simple method for preparing and quantifying very-low-density lipoprotein.
Authors: Kohlmeier M
Published: January 2020

A micromethod is described for isolating and quantifying human very-low-density lipoprotein (VLDL) from 0.1 mL of human serum, by which more than 100 samples can be processed in half a day with a standard ultracentrifuge. Replicate analysis of 10 normal and hyperlipemic samples gave median coefficients of variation of 11.7% for VLDL cholesterol, 5.1% for VLDL triglycerides. Results obtained by micro-ultracentrifugation and enzymic cholesterol measurement agreed closely with those measured with an electrophoretic method (Pearson's r = 0.98). Compared with the established method, on the other hand, lower values were found for both VLDL cholesterol (r = 0.89) and triglycerides (r = 0.97).
Effect of pH and NaCl on measurements of ionized calcium in matrices of serum and human albumin with a new calcium-selective electrode.
Authors: Rehfeld S J , Barkeley J , Loken H F
Published: January 2020

A neutral ligand ion-exchanger type of ion-selective electrode is used in the current version of the Orion Model SS-20 calcium analyzer. We evaluated it over the pH range 4.0 to 8.5, using albumin solutions containing 150 mmol/L NaCl and pooled patients' serum. Within this pH range response was nernstian, and the relationship between potential (in millivolts) and log [Ca2+] at each pH was linear. Variation in Ca2+ with increasing ionic strength (because of added NaCl) was not significantly different between standards and albumin solutions; it decreased in both. In contrast, the proportion of calcium in serum increased with increasing NaCl concentrations. Ca2+ was not bound in whole serum albumin at a pH less than about 3.9. We observed a substantial difference between the degree of Ca2+ binding in solutions of fatty-acid-free human albumin and serum.
A comment on thyrotropin measurement and evaluation.
Authors: Musto J D , Pizzolante J M , Chesarone V P
Published: January 2020

There is a need for improvement of thyrotropin measurements in the clinical laboratory, specifically in the lower portion of the normal range. We cite realistic examples of incorrect, misleading data generated with many thyrotropin kits currently in use and propose a practical laboratory protocol to evaluate the performance of a procedure for determining thyrotropin. The protocol integrates medical physiology with analytical measurement.
Chemical and clinical evaluation of the random access analyzer "RA-1000".
Authors: Schwartz M K , Statland B E , Coughlin J , Eisen C , Fleisher M , Ito R , Levine J B , Ng R , Pennachia G M , Rose H N
Published: January 2020

In the RA-1000, a random-access discrete analyzer, an inert fluorocarbon fluid is used to prevent interaction and carryover. Production-model instruments were evaluated in two laboratories with respect to determination of glucose, creatinine, total protein, inorganic phosphorus, cholesterol, alkaline phosphatase, lactate dehydrogenase, creatine kinase, gamma-glutamyltransferase, and aspartate and alanine aminotransferases. Within-run, among-run, and day-to-day (for 15 days) precision was assessed, and results were correlated with those obtained by the methods routinely in use in our departments. Precision was excellent, correlation acceptable.
Use of monoclonal antibodies in a radioimmunoassay for human transcortin.
Authors: Faict D , De Moor P
Published: January 2020

We describe the production of monoclonal antibodies to human transcortin and their use in a radioimmunoassay (RIA). A high-affinity antibody (Ka = 4 X 10(10) L/mol) made possible a sensitive RIA for transcortin (detection limit = 0.23 ng per tube), whereas use of an antibody of moderate affinity (Ka = 5 X 10(8) L/mol) was more suitable for the routine measurement of transcortin in serum, only a 25-fold dilution of the sample being required instead of 1500-fold. The correlation was good between both RIAs (r = 0.959) and between each of the RIAs and radial immunodiffusion (r = 0.955 and 0.976 for the methods with high- and low-affinity antibody, respectively). Although monoclonal antibodies were used in the RIAs and polyclonal ones in the radial immunodiffusion procedure, similar values were obtained by all techniques.
Reference intervals for serum lipids, lipoproteins, and apoproteins in the elderly.
Authors: Alvarez C , Orejas A , Gonzalez S , Diaz R , Colomo L F
Published: January 2020

We measured total cholesterol, high-density-lipoprotein cholesterol, low-density-lipoprotein cholesterol, triglycerides, apoprotein A, and apoprotein B in serum. The subjects were a volunteer group of 145 white people (50 men and 95 women) ranging in age between 65 and 95 years, who were not receiving medical treatment and had no disease that could influence lipid metabolism. Before further categorization, we saw no significant sex-related difference in any of these lipid constituents. The mean age of the group was about 80 years, and we compared results for those older and younger. For the women, the only significant difference was a decrease in low-density-lipoprotein cholesterol for those older than 80 years. In men over 80 there was a significant decrease in triglycerides and in apoprotein B and an increase in high-density-lipoprotein cholesterol. The only sex-related difference for persons under and over 80 was in values for high-density-lipoprotein cholesterol, which were higher for men over 80, whereas triglycerides were higher for women over 80.
Determination of sodium and potassium with ion-selective electrodes.
Authors: Fogh-Andersen N , Wimberley P D , Thode J , Siggaard-Andersen O
Published: January 2020

We compared different sample-handling techniques for measurement of Na+ and K+ with ion-selective electrodes (ISE). Imprecision was less for venous blood (with a minimum of heparin) than for plasma, serum, or capillary blood. The results for K+ were higher for serum than for whole blood, and higher for whole blood than for plasma. The latter difference was apparently due to release of K+ during the analysis. Values were more stable for whole blood stored at 20 degrees C than at 4 degrees C or 37 degrees C. The molality of Na+ in the plasma of mixed whole blood changed by -10.5 mmol/kg per unit change in blood pH. This could be explained by the different H+ buffering capacities of plasma and erythrocyte fluid, because when the pH is changed, the concentration of small anions in erythrocytes changes more than it does in plasma, with a consequent osmotic movement of water across the erythrocyte membrane. When we took into account the residual liquid-junction potential and the mass concentration of water in each of 65 patients' sera, the molality determined for Na+ was 1% lower and that of K+ 3% lower by ISE than by flame photometry--differences that may be related to ionic binding or to a lower molal activity coefficient in serum than in the calibrator.
New rapid kit for determining calcium in serum and urine evaluated.
Authors: Zerwekh J E , Nicar M J
Published: January 2020

We describe the performance of a colorimetric reagent kit (Diagnostic Systems Laboratories, Inc., Webster, TX 77598) for measuring calcium directly in urine, serum, and ultrafiltered serum, and compare the results with those obtained with atomic absorption spectrophotometry. The CV for within-run precision was 2.8 and 2.1% for urine and whole serum, respectively (n = 10 each). Between-run precision for urine, whole serum, and ultrafiltered serum was 1.9, 1.6, and 2.2%, respectively (n = 8 to 10). Analytical recovery of added calcium from three different urine and serum specimens, to which three different concentrations of calcium had been added, was 101.9 (SD 0.3%) for urine and 100.9 (SD 0.2%) for serum. Assay of 30 urine specimens, 15 ultrafiltered serums, and 20 whole serums by both the kit and atomic absorption spectrophotometry demonstrated correlation coefficients of 0.993, 0.828, and 0.751, respectively. Mg2+, hemolysis, or lipemia does not interfere. Compared with atomic absorption spectrophotometry, the calcium kit procedure is rapid and simple.
Collection and handling of 24-hour urine specimens for measurement of analytes related to renal calculi.
Authors: Ng R H , Menon M , Ladenson J H
Published: January 2020

Analysis for calcium, magnesium, phosphorus, oxalate, uric acid, and creatinine in 24-h urine collections is often needed for the differential diagnosis of patients with renal calculi. Considerable attention has been given to improving the methods of analysis, but improper sample collection and processing can cause significant errors for calcium and oxalate in urine samples not treated with acid and for uric acid in urine samples not treated with base. The errors are related to the concentration of the analyte, the interval the sample is stored before analysis, and the original pH of the urine sample. We describe here a system of sequential acidification (to pH 1.5) and alkalinization (to pH 9) of 24-h urine samples, followed by heating at 56 degrees C for 10 min. This procedure allows accurate analysis for all the above analytes in the same 24-h collection of urine. We validated the sample-treatment protocol for 80 24-h urine collections.
Sodium dodecyl sulfate polyacrylamide gel electrophoresis of serum after protein denaturation in the presence or absence of 2-mercaptoethanol.
Authors: Marshall T
Published: January 2020

Human serum proteins were evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis after protein denaturation in the presence or absence of 2-mercaptoethanol. Both electrophoretic techniques give characteristic and reproducible banding patterns and achieve a high degree of resolution within the limits of a one-dimensional separation. The major protein bands have been identified, and the merits of the two techniques are compared. Protein denaturation in the absence of 2-mercaptoethanol gives more discrete bands for the purpose of protein identification by maintaining the disulfide-bridge-dependent polypeptide associations characteristic of many serum proteins. However, simultaneous use of both electrophoretic techniques enhances identification by exploiting the response of an individual protein to mercaptoethanol treatment. The clinical potential of this approach for detecting protein disorders is discussed.
Glycated hemoglobin in uremic patients as measured by affinity and ion-exchange chromatography.
Authors: Bannon P , Lessard F , Lepage R , Joly J G , Dufresne L
Published: January 2020

We estimated glycated ("glycosylated") hemoglobin in erythrocytes of hemodialyzed uremic patients by the aminophenylboronic acid affinity-chromatographic method and by an ion-exchange chromatographic method. As expected, apparent HbA1 concentrations were above normal in the uremic patients, owing to interference by the carbamylated hemoglobin. However, there was no significant difference between values for uremic and normal subjects when glycated hemoglobin was measured by the affinity method. Evidently it is unaffected by the presence of hemoglobin species modified by reactants not displaying a cis-1,2-diol group, such as carbamylated hemoglobin. For this reason it is preferable to the ion-exchange chromatographic method for accurate measurement of glycated hemoglobin.
"Nonspecific" increases in plasma immunoreactive calcitonin in healthy individuals: discrimination from medullary thyroid carcinoma by a new extraction technique.
Authors: Body J J , Heath H
Published: January 2020

Occasional seemingly healthy individuals have above-normal concentrations of calcitonin-like immunoreactivity in their plasma, which can lead to mistaken diagnosis of thyroidal or other cancer. We measured immunoreactive calcitonin (CT) before and after extracting the plasma on columns of silica (to improve sensitivity and specificity of the assay for monomeric calcitonin) in five "healthy high-CT" men (I), five patients with known medullary thyroid carcinoma (II), and 30 normal controls (III). Median (and range) values (pg/mL = ng/L) for whole-plasma immunoreactive CT in these groups were, respectively, 379 (157-526), 429 (174-563), and 33 (less than 25-92). Dose-dilution curves for plasma samples from group I did not parallel the standard curve, in contrast to samples from the other two groups. Values for extractable CT from plasma from groups I and III, however, were indistinguishable, but remained significantly increased in group II. Infusions of Ca, 2 mg/kg body wt. in 5 min, produced the expected (normal) increases in extractable CT in group I. The occasional factor (or factors) in plasma of healthy persons that interferes in assays for CT is eliminated by the silica extraction method, and in this way such cases can be distinguished from cases of medullary thyroid carcinoma.
Mechanism of platelet interference with measurement of lactate dehydrogenase activity in plasma.
Authors: Peake M J , Pejakovic M , Alderman M J , Penberthy L A , Walmsley R N
Published: January 2020

Platelets reportedly inhibit lactate dehydrogenase activity in plasma under reaction conditions of low osmolality. We describe observations inconsistent with these reports, and we attribute this "inhibition" to optical interference by platelets during the course of a reaction. We conclude that when platelet lysis is prevented and the optical interference of platelets corrected, platelet-rich plasma, platelet-poor plasma, and serum show essentially the same lactate dehydrogenase activity. Furthermore, platelet contamination can cause unexpected problems when lactate dehydrogenase is assayed with centrifugal analyzers. Results can be high or low, depending on the volume of diluent pipetted with the sample, and extreme within-run variations in activity are possible. When plasma is used instead of serum for routine analyses, regular checks for platelet contamination should be performed as a quality-control procedure, especially by laboratories separating plasma with bench-top centrifuges. Platelets can also interfere optically with assay of other enzymes and metabolites.
Changes in fatty acids in phospholipids of the bronchoalveolar fluid in bacterial pneumonia and in adult respiratory distress syndrome.
Authors: Baughman R P , Stein E , MacGee J , Rashkin M , Sahebjami H
Published: January 2020

Fatty acids of the phospholipid fraction of bronchoalveolar lavage fluid from patients with bacterial pneumonia or with the adult respiratory distress syndrome were chromatographed and the patterns compared with those for a control group. In the control group, palmitic acid (16:0) was the predominant fatty acid, accounting for 58.0% (SD 8.25%) of the total fatty acid, a proportion significantly higher (p less than 0.001) than in the distress-syndrome group (42.1%, SD 4.88%) or the acute pneumonia group (32.1%, SD 1.73%). There was a greater proportion of oleic acid (18:1) in the disease groups; thus the ratio of palmitic to oleic acid was useful in distinguishing these three groups. No patient with a palmitic/oleic acid ratio greater than 2.45 had evidence of parenchymal inflammation. Of those with a ratio less than 1.3, 89% had acute bacterial pneumonia.
Automated prothrombin-time test with use of a chromogenic peptide substrate and a centrifugal analyzer.
Authors: Becker U , Jering H , Bartl K , Jilek F
Published: January 2020

We modified a test for prothrombin time, to automate its performance by centrifugal analysis. The sample is activated with calcium-thromboplastin in the presence of a thrombin-specific chromogenic peptide substrate, Tos-Gly-Pro-Arg-p-nitroaniline (Chromozym TH). The endpoint of the reaction is set as the time after onset until a defined amount of substrate has been cleaved, as reflected in a fixed change in absorbance. The assay is as sensitive as clotting-type methods are to the clotting factors of the extrinsic pathway--except for fibrinogen, which can be estimated from the maximum absorbance difference. Substrate concentration has little effect on the result, and varying it does not significantly change the sensitivity to factor X, which indicates little interference with the enzymes of the coagulation cascade. The method was applied to the Cobas Bio centrifugal analyzer. An external calculator must be used to convert absorbance readings into reaction times, which can be expressed as percent-of-normal values or as "ratios" by use of reagent-specific factors. CVs were less than 2% within run, less than 6% day to day. Overall correlation with the clotting test (r = 0.95) was good. Using the Cobas Bio, we achieved a throughput rate of 200 samples per hour.
Simpler liquid-chromatographic screening for organic acid disorders.
Authors: Bennett M J , Bradey C E
Published: January 2020

We describe an isocratic cation-exchange chromatographic technique, with ultraviolet detection, for determination of urinary organic acids. The method is useful for screening urines from neonates and infants suspected of having an organic aciduria, prior to confirmatory analysis by gas chromatography-mass spectrometry. Advantages of this technique over conventional gas chromatography include ease of sample preparation and the simultaneous detection of volatile fatty acids; limitations include its lack of an identification system like that of mass spectrometry, the presence of nonultraviolet-absorbing fatty acids, and the relatively long retention times of phenolic compounds. Chromatograms for patients with methylmalonic aciduria, propionic aciduria, isovaleric aciduria, beta-ketothiolase deficiency, lactic aciduria, and glutaric aciduria type II are compared with those for a normal infant.
Prevention of interference by dextran with biuret-type assay of serum proteins.
Authors: Flack C P , Woollen J W
Published: January 2020

In assay of serum proteins by use of the biuret reaction, dextran can cause turbidity by formation of an insoluble complex of dextran with copper and tartrate (or EDTA) in strongly alkaline solution. Whether or not the turbidity occurs depends on the tartrate concentration: turbidity is maximal at about 10 g/L, absent at 20 g/L or more, and only slight and delayed at 4 g/L. Two biuret reagents, containing respectively 5.6 and 22.5 g of tartrate per liter, obviate the interference, but the former is suitable only when a short (5 min) incubation is used. Both reagents show linear calibration curves and yield virtually identical results.
Specific affinity-chromatographic measurement of glycated hemoglobins in uremic patients.
Authors: Bruns D E , Lobo P I , Savory J , Wills M R
Published: January 2020

The clinical utility of glycated hemoglobin measurements in renal failure is controversial, given numerous earlier studies showing no correlation between glycated hemoglobin and other indicators of blood glucose control in uremic subjects. This problem is attributable, in part, to analytical interferences from carbamylated hemoglobins. We report use of a specific affinity method to measure glycated hemoglobins in a group of uremic patients, diabetic and nondiabetic, undergoing treatment by continuous ambulatory peritoneal dialysis. Concentrations of glycated hemoglobins correlated significantly with values for fasting plasma glucose (r = 0.52, n = 17, p less than 0.05) in these patients and with mean glucose measurements in five diabetic patients who used home glucose monitoring (r = 0.91, p less than 0.05). Contrary to studies with ion-exchange chromatography, our measurements of glycated hemoglobin showed no positive correlation with concentrations of urea in serum. In a separate group of patients, we found that hemodialysis sessions produced no acute effect on glycated hemoglobin. Measurements of glycated hemoglobins by analytically specific methods may thus better reflect long-term control of blood glucose in renal dialysis.
Determination of steroid hormones in a human-serum reference material by isotope dilution--mass spectrometry: a candidate definitive method for cortisol.
Authors: Patterson D G , Patterson M B , Culbreth P H , Fast D M , Holler J S , Sampson E J , Bayse D D
Published: January 2020

We report a method, based on isotope dilution--mass spectrometry, for determining cortisol in a pooled specimen of human serum. Isotopically labeled cortisol is added to 5.0 mL of serum so that the molar concentrations of labeled cortisol and unlabeled cortisol are approximately equal. The specimen and two calibration standards are extracted with dichloromethane, and the extracted cortisol is converted to the methoxime-trimethylsilyl ether derivative. Samples and standards are analyzed by gas chromatography--mass spectrometry by monitoring the peak areas for m/z 605 and 608. The cortisol concentration is calculated by linear interpolation between the two bracketing standards. Variances of data collected during six weeks showed that the overall coefficient of variation (CV) was 0.69% (n = 32); the within-vial CV, 0.63%; the among-vial CV, 0.22%; and the among-day CV, 0.15% (means = 3.973 nmol/vial). Method specificity was demonstrated by liquid chromatographic as well as C8 mini-column cleanup of samples before derivation, by alternative ion monitoring at m/z 636 and 639, and by negative-ion chemical ionization at m/z 459 and 462. Derivatives of all observed degradation products of cortisol under basic, neutral, and acidic conditions did not interfere.
Simultaneous determination of lidocaine and its principal metabolites by liquid chromatography on silica gel, with aqueous eluent.
Authors: Kushida K , Oka K , Suganuma T , Ishizaki T
Published: January 2020

We describe the simultaneous determination of lidocaine and its pharmacologically active metabolites, monoethylglycinexylidide and glycinexylidide, in plasma by "high-performance" liquid-chromatography. By use of a bare ( unbonded ) silica gel with aqueous eluents, separations of organic amines such as lidocaine and its metabolites, which are very difficult and have a poor peak symmetry on bonded reversed-phase packings, were easily accomplished with a good peak symmetry. The method is sufficiently precise, sensitive, and specific. Analytical recoveries of all compounds were greater than 90%; CVs for reproducibility were less than 5% for all compounds; the lower detection limits were 0.1 mg/L or less. This method can be used to monitor the concentrations of these compounds in plasma and to prevent the concentration-related side-effect(s).
Measurement of prostate-specific antigen by radioimmunoassay.
Authors: Liedtke R J , Batjer J D
Published: January 2020

We describe the performance of an RIA for the measurement of prostate-specific antigen (PA). Between-assay precision (CV) for control sera with various analyte concentrations was as follows: mean = 1.67 micrograms/L, CV = 7.1%; mean = 4.47 micrograms/L, CV = 5.6%; mean = 7.15 micrograms/L, CV = 5.5% (n = 19 each). Analytical recovery of PA (nine concentrations ranging from 2.3 to 21.1 micrograms/L) added to a serum pool averaged 101.8% (range 96.1 to 116.1%). Sensitivity (detection limit) of the RIA was 0.25 micrograms/L. Cross reactivity of prostatic acid phosphatase (PAP) in this assay was less than 0.022%. The mean percent B/B0 for 74 specimens from women was 98.9%, not statistically different from that for the zero standard. The normal reference interval for men was 0-2.7 micrograms/L ( 99th percentile), as established by assay of specimens from 276 apparently normal men. Measurement of PA and prostatic acid phosphatase in 205 consecutive serum specimens from patients with clinical evidence of prostate disease similarly placed patients into normal (98) or abnormal groups (54) in 152 cases. However, in 49 cases only the concentration of PA in serum was abnormal. Sequential measurement of both tissue markers in specimens from several patients who were undergoing therapy for prostate cancer appeared to provide supplemental information regarding treatment success.
A "sandwich" solid-phase enzyme immunoassay for lutropin in urine.
Authors: Rathnam P , Saxena B B
Published: January 2020

In this "sandwich" technique of enzyme immunoassay for lutropin in urine, a highly purified lutropin-specific anti-gamma-globulin is conjugated with alkaline phosphatase. The conjugate is then extensively purified to remove free enzyme and free antibody molecules. The solid phase consists of anti-lutropin immune globulin coupled to polystyrene tubes or beads. The specific antibody on the solid phase binds the lutropin in the urine sample or in the standard solution, which in turn binds to the specific antibody conjugated to the enzyme. The amount of bound enzyme, which is determined colorimetrically, is thus directly proportional to the amount of lutropin in the sample. We optimized assay conditions and report them here. The sensitivity of the assay is equal to that of radioimmunoassay and almost 10-fold that of the hemagglutination inhibition assay. The test can be used to detect the pre-ovulatory surge of lutropin in women.
Particle-counting immunoassay of human somatotropin.
Authors: Castracane C E , Cambiaso C L , Retegui L A , Gilbert I , Ketelslegers J M , Masson P L
Published: January 2020

Human somatotropin was assayed by a novel automated nonradioisotopic technique, "particle-counting immunoassay," that requires a 45-min incubation and only 60 microL of 10-fold diluted sample. The principle of the assay is agglutination of antibody-coated latex particle by somatotropin, the reaction being measured by the (instrumented) counting of residual non-agglutinated particles. The dynamic range in serum extends from 0.2 to 40 micrograms/L. The between-assay CV was 12% for a concentration of 3.3 micrograms/L and 8.5% for 31.9 micrograms/L. The coefficient of correlation (r) with radioimmunoassay was 0.97. Curves for various dilutions of the macromolecular and monomeric forms of the hormone were not parallel.
Total polyamines and their non-alpha-amino acid metabolites simultaneously determined in urine by capillary gas chromatography, with nitrogen-phosphorus detector; and some clinical applications.
Authors: Muskiet F A , van den Berg G A , Kingma A W , Fremouw-Ottevangers D C , Halie M R
Published: January 2020

A capillary gas-chromatographic method with nitrogen-phosphorus detection is used here to simultaneously determine 1,3-diaminopropane, putrescine, cadaverine, spermidine, spermine, isoputreanine , putreanine , and N-(3-aminopropyl)-N'-(2-carboxyethyl)-1,4- diaminobutane in urine. After acid hydrolysis the compounds are isolated by adsorption onto silica gel and converted into their methyl-heptafluorobutyryl derivatives. We give quality-control data and age-dependent values for urinary excretion of these analytes by 76 apparently healthy controls. Circadian rhythmicity in the excretion of spermidine and (especially) isoputreanine was established and is discussed in the light of its implications for monitoring therapy of cancer. Investigation of menstrual-cycle-dependent diurnal variation in one normal woman showed no distinct, consistent fluctuations. We applied the method to monitor (by use of 24-h urine specimens) an uncomplicated, normally progressing pregnancy, a patient with metastatic melanoma being treated with cytostatic drugs, and (in more detail) the treatment of a patient with high-grade non-Hodgkin lymphoma.
Activities of some enzymes in serum after therapy with intracoronary streptokinase in acute myocardial infarction.
Authors: Kwong T C , Fitzpatrick P G , Rothbard R L
Published: January 2020

Enzyme kinetics for creatine kinase (CK), CK-MB, aspartate aminotransferase (AST), and lactate dehydrogenase (LD) in serum were followed in 14 patients who had suffered acute myocardial infarction and who were given intracoronary streptokinase shortly (mean 4.9 h, SD 2.6 h) after onset of symptoms. In the 10 patients for whom thrombolysis was successful, CK activity peaked earlier (12.8 vs 21.6 h) and at higher values (3548 vs 2436 U/L) than in the four patients for whom the treatment was unsuccessful. The mean maximum rate of increase in CK was threefold greater in the former group (574 vs 169 U/L per hour), but the total amount of CK released into the circulation and the fractional disappearance rates were similar for both groups. The profiles for AST and CK-MB for successfully treated patients closely resembled those for CK. LD, however, peaked significantly later than CK (25.7 vs 12.8 h). Early peaking of CK or CK-MB after nonsurgical reperfusion can be potentially useful as a noninvasive in vitro index to the success of therapy of myocardial infarction with thrombolytic agents.
Fluoroimmunoassays for procainamide and N-acetylprocainamide compared with a liquid-chromatographic method.
Authors: MacKichan J J , Coyle J D , Shields B J , Boudoulas H , Lima J J
Published: January 2020

We measured procainamide and its active metabolite, N-acetylprocainamide (NAPA), in 80 sera from 37 patients by a new fluorimmunoassay procedure and an established "high-performance" liquid-chromatographic method. Additive and proportional differences between the methods were 0.07 mg/L and 9%, respectively, for procainamide and 0.62 mg/L and 16% for NAPA. Between-day CVs by the chromatographic and immunoassay methods, respectively, were 3.9% and 2.2% for procainamide at a concentration of 6 mg/L, and 5.1% and 1.2% for NAPA (14 mg/L). We applied a modification of the fluoroimmunoassay for determination of procainamide concentrations, using sera obtained during a pharmacokinetic study, and demonstrated excellent agreement with the chromatographic method.
Two direct radioimmunoassays for 17 beta-estradiol evaluated for use in monitoring in vitro fertilization.
Authors: Haning R V , Meier S M , Boehnlein L M , Gerrity M , Shapiro S S
Published: January 2020

Two direct 125I radioimmunoassays for 17 beta-estradiol were evaluated by comparing results with those obtained by a comparison RIA involving extraction and chromatography (x) and with ultrasound parameters of ovarian activity. The correlation coefficients (R2) for the Immuchem Covalent-Coat Solid Phase 125I results (y1) and the Pantex Immuno-direct results (y2) with the comparison method results were 0.56 and 0.96, respectively, after log transformation of data (N = 42). Similar differences were observed in the correlations with the ultrasound parameters. We also evaluated the untransformed data by linear regression and obtained: y1 = 254 + 0. 410x (R2 = 0.65) and y2 = 48.2 + 0. 749x (R2 = 0.96). Between- and within-assay variations for a serum pool containing 17 beta-estradiol at 1278 pg/mL were respectively 9.0% and 9.4% for Immuchem , 3.3% and 6.7% for Pantex , and 7.3% and 9.4% for the comparison method; for the 267 pg/mL pool, these were 21.8% and 16.6% for Immuchem , 4.7% and 4.6% for Pantex , and 21.2% and 13.1% for the comparison method. Differences in the results obtained with the Pantex method and the comparison method were not clinically significant for monitoring superovulation for in vitro fertilization; the performance of the Immuchem method was less satisfactory.
Design of a microcomputer-based densitometer system: application to serum protein electrophoresis.
Authors: Neeley W E
Published: January 2020

We describe how a microcomputer (Apple II Plus) can be interfaced with a commercial densitometer to provide control functions, do automated data processing, and print final reports. The amplified densitometer photodetector signal is digitized, stored, and plotted on the cathode-ray tube to provide a real-time graphical display of data acquisition. The software was designed to identify automatically the five main serum protein fractions and to compute final results. Algorithms were designed to provide medically relevant comments for most of the common abnormal protein patterns, and the comments section was designed so that it could be modified to use specific comments composed by the user. A final report, printed in duplicate, contains information such as the patient's name, identifying numbers, numerical results, normal values, medically relevant comments, and a digitized high-resolution plot of the electrophoretic pattern. The densitometer was one that already had been in use for several years; the microcomputer system, software, and interfaces cost less than $2200.
Simplified determination of carboxyhemoglobin.
Authors: Beutler E , West C
Published: January 2020

This spectrophotometric method for estimating carboxyhemoglobin is based on the production of a two-pigment mixture by reducing oxyhemoglobin and methemoglobin with sodium hydrosulfite . Absorbances of the pigment are measured at 420 and 432 nm. Protecting the solutions against exposure to air is unnecessary. Values are stable and reproducible when the solutions are buffered at pH 6.85.
Microscale ultrafiltration technique for determining free drug in 50-microL serum samples.
Authors: Wittfoht W , Duwe K , Kuhnz W , Nau H
Published: January 2020

This ultrafiltration technique allows determination of free drug in 50 microL of serum. We ultrafiltered sera containing the following drugs--valproic acid (and its major metabolites), phenobarbital, diazepam, indomethacin, phenytoin, furosemide, and chloramphenicol--using both the commercially available micropartition system (MPS-1, Amicon), which requires a 200-microL sample, and our modified micro system, which requires only 50 microL. The value for the free fraction of each drug obtained in the two experiments agreed well. The smaller sample requirement makes the micro method particularly suited for pediatric samples and studies on small laboratory animals.
A unifying reference system for clinical enzymology: aspartate aminotransferase and the International Clinical Enzyme Scale.
Authors: Bowers G N , McComb R B
Published: January 2020

A review of methodology for determining aspartate aminotransferase (ASAT; EC 2.6.1.1), including recent national and international recommendations, indicates that standardization of methodology alone will not bring interlaboratory compatibility of ASAT results. We propose that an additional component to standardization is needed, namely, enzyme reference materials. Furthermore, we suggest that stable, well-defined ASAT materials from human sources are currently available. These primary reference materials and the state-of-the-art IFCC Reference Method for ASAT provide the basis for a unifying reference system for ASAT. Given such a reference system, we propose a practical way to promote compatibility of currently incompatible numerical results for ASAT through the use of one ASAT scale of units, the "International Clinical Enzyme Scale." This scale-unification concept would permit all current methods, instruments, and temperature choices to be used for ASAT determinations in the daily working laboratory. We present illustrative examples and demonstrate the unique ability of this concept to promote compatibility of the ASAT results from numerous laboratories using many different ASAT methods.
Identification and quantification of hemoglobins A2 and Barts with an enzyme-labeled immunosorbent assay.
Authors: Garver F A , Singh H , Moscoso H , Kestler D P , McGuire B S
Published: January 2020

The presence and quantity of hemoglobins Barts and A2 in hemolysates from normal donors and individuals with alpha- and beta-thalassemia trait, respectively, were determined with an enzyme-labeled immunosorbent assay (ELISA). This technique requires the incorporation of monospecific antisera capable of specifically reacting only with these hemoglobins, e.g., with the delta chain of Hb A2 and gamma 4 chains of Hb Barts. By the ELISA, the mean percentage of Hb Barts in hemolysates from normal persons and persons with alpha-thalassemia was 0.25 (SD 0.07) and 6.1 (SD 0.40), respectively. Corresponding values for Hb A2 in hemolysates from normals and persons with beta-thalassemia were 3.1 (SD 0.22) and 5.9 (SD 0.21), respectively. The results obtained by the ELISA procedure were in good agreement with those determined by radioimmunoassay or microcolumn chromatography. The ELISA technique is more sensitive and specific than biochemical assays currently used to measure these hemoglobins and can detect 250 ng of Hb Barts in 100 micrograms of hemoglobin or 50 ng of Hb A2 in 5 micrograms of hemoglobin.
Citramalic acid in cerebrospinal fluid of patients with bacterial meningitis.
Authors: Perlman S , Carr S A
Published: January 2020

Cerebrospinal fluid (CSF) from uninfected patients and from patients with bacterial and viral meningitis was analyzed by gas-liquid chromatography, with use of a flame ionization detector, and by gas chromatography-mass spectrometry. The resulting profiles were consistent and reproducible. Hydroxy acids were the compounds found in greatest abundance in both normal and infected CSF. Control experiments to establish the sensitivity and efficiency of the extraction and derivatization methods are also presented. Constituents of CSF from patients with bacterial meningitis differed quantitatively and qualitatively from those of CSF from uninfected patients or patients with nonbacterial infections. CSF from seven of eight patients with bacterial meningitis contained citramalic acid, a compound not previously identified in either normal or infected CSF. The implications of these findings are discussed.
A new monoclonal-antibody two-site solid-phase immunoradiometric assay for human thyrotropin evaluated.
Authors: Pekary A E , Hershman J M
Published: January 2020

We compared results with a commercial solid-phase two-site immunoradiometric assay kit for human thyrotropin in which monoclonal antibodies are used (TANDEM-R TSH; Hybritech Inc.) with those by our radioimmunoassay (J Clin Endocrinol Metab 41:676, 1975), which is optimized for measurement of low concentrations of thyrotropin. In the immunoradiometric assay a specific antibody to the beta subunit of human thyrotropin is immobilized on a polystyrene bead, and a radiolabeled monoclonal antibody directed against the alpha subunit provides a measure of bead-immobilized hormone. The mean thyrotropin concentrations in 70 euthyroid serum samples were similar in the two assays. Values for hypothyroid patients were clearly higher in both assays than values for euthyroid individuals. In commercial assays the major source of error in measurement of thyrotropin response to thyroliberin in terms of the increment over the basal concentration of thyrotropin has been systematic errors in the measurement of those basal concentrations. With the present assay, however, basal values are obtained with good precision and accuracy: CV = 2.9% at 6 milli-int. units/L (n = 48).
A commercially available S-type amylase inhibitor evaluated for determination of amylase isoenzymes in serum.
Authors: Tietz N W , Shuey D F
Published: January 2020

On evaluation, an S-type alpha-amylase inhibitor from Sigma shows essentially the same performance characteristics as the inhibitor from wheat germ prepared according to O'Donnell and McGeeney (Biochim Biophys Acta 422:159, 1976). This inhibitor is now commercially available; thus any clinical laboratory can perform isoamylase determinations with a simple, fast procedure that is also suitable for emergency situations.
A kit for citrate in foodstuffs adapted for assay of serum and urine.
Authors: Warty V S , Busch R P , Virji M A
Published: January 2020

A kit method for rapid analysis of citrate in foodstuffs, based on use of citrate lyase (EC 4.1.3.6), has been adapted and evaluated for use with clinical samples. Membrane filters removed serum proteins, which interfere with the analysis, and reagent and sample volumes were decreased from those in the manufacturer's protocol, thereby decreasing the cost of the assay. Using gravimetrically prepared citrate standards, we determined that assay results varied linearly with concentration up to sixfold the upper reference limit for serum citrate. Intra- and interassay variation were within acceptable limits (CVs less than 3% and less than 8.5%, respectively). Kit reagents were stable for four weeks at -20 degrees C. Assay results were unaffected by hemolysis or other biochemical interferences. The method evidently provides a rapid, convenient microassay for citrate in clinical samples.