bluuebunny/crossref_metadata_2025 huggingface dataset:
Serum ferritin compared with other indices of iron status in children and teenagers undergoing maintenance hemodialysis.
Authors: Ellis D
Published: January 2020

To determine how best to assess iron status, I studied 12 young renal patients (ages 5.5 to 20 years) undergoing regular hemodialysis treatments. Iron balance was estimated by monitoring iron loss ascribable to blood loss during dialysis and diagnostic testing, and iron intake in the form of oral and intravenous iron supplements and blood transfusions. Traditional methods of evaluating iron status--measurement of hemoglobin, erythrocyte indices, reticulocyte count, iron, and transferrin--were compared with measurement of serum ferritin. The serum ferritin measurements provided superior information. In three cases this method was superior to visual assessment of bone marrow stained for iron.
Double-antibody method and the protein-A-bearing Staphylococcus aureus cells method compared for separating bound and free antigen in radioimmunoassay.
Authors: Gupta R K , Morton D L
Published: January 2020

We compared the protein-A-bearing Staphylococcus aureus immunoadsorbent to the double-antibody precipitation method for separating bound and free radiolabeled antigen in a radioimmunoassay. With human albumin (antigen) and rabbit anti-human albumin (antibody) as a model, our results indicate that formalin-fixed, heat-killed S. aureus cells could be substituted for the double-antibody precipitation method. Ease of preparation and high adsorption capacity of protein-A-bearing S. aureus for most mammalian IgG make this method economical and time saving.
Simultaneous measurements of acetaminophen and salicylate in plasma by liquid chromatography.
Authors: Miceli J N , Aravind M K , Cohen S N , Done A K
Published: January 2020

We describe a "highly-performance" liquid chromatographic method in which 8-chlorotheophylline is used as an internal standard to determine acetaminophen and salicylate simultaneously in plasma samples. Therapeutic as well as toxic concentrations can be determined on samples as small as 50 micro L, which makes the method particularly useful for determinations on samples from young children. The procedure requires a simple extraction of the drugs, and the chromatography is completed in 6 min.
Calibration and monitoring of spectrometers and spectrophotometers.
Authors: Frings C S , Broussard L A
Published: January 2020

We have delineated some of the factors affecting the performance of spectrometers and spectrophotometers in the clinical laboratory and have presented some of the methods for verifying that these instruments are functioning properly. At a minimum, every laboratory should perform periodic inspections of spectrometric functions to check wavelength calibration, linearity of detector response, and stray radiation. Only through such an inspection program can a laboratory ensure that these instruments are not contributing to inaccurate analytical results.
The use of linear models and matrix least squares in clinical chemistry.
Authors: Deming S N , Morgan S L
Published: January 2020

We present a unified approach to the use of linear models and matrix least squares with the intention of providing a better understanding of the techniques themselves and of the statistics that arise from these techniques as they are used in clinical chemistry. Emphasis is placed on the importance of appropriate experimental designs and adequately precise measurement processes for efficiently obtaining the desired information.
Microencapsulated antibodies in radioimmunoassay--I. Determination of digoxin.
Authors: Halpern E P , Bordens R W
Published: January 2020

We describe the application of the microencapsulated-antibody technique to the radioimmunoassay of digoxin in serum. Droplets of emulsified rabbit antibody are microencapsulated in a semipermeable nylon membrane by an interfacial polymerization technique. The antibody microcapsules are incubated with 125I-labeled digoxin and unlabeled digoxin for 15 min at 37 degrees C, then free and bound digoxin are separated by centrifugation. Subtherapeutic, therapeutic, and toxic concentrations of digoxin in sera can be determined, with use of a standard curve prepared by use of known amounts of digoxin. With this technique we obtained an intra-laboratory correlation coefficient of 0.945 for 100 patients' sera and one of 0.940 for interlaboratory results for 21 sera (10 laboratories) when compared to a routine clinical laboratory radioimmunoassay for digoxin. Icterus, lipemia, hemoglobin, or disproteinemia had no effect on the analytical recovery of digoxin. The standard curve was linear to 6 microgram/L; the sensitivity was 0.25 microgram/L.
Preparation and stability of a liquid creatine kinase isoenzyme control from rabbit serum.
Authors: Lum G
Published: January 2020

The MB isoenzyme of creatine kinase (CK) may be prepared in vitro from rabbit serum containing only the MM and BB isoenzymes, by means of a hybridization technique. The MM and BB dimers dissociate in 4 mol/L urea, which allows random recombination of M and B monomers. A liquid CK-isoenzyme control can be made from mixtures of rabbit sera obtained after hybridization and stabilized with glycerol and 25 mmol of 2-mercaptoethanol per liter. A liquid control stored at 4 degrees C showed good stability over a three-month period, declining to a mean residual activity of CK of approximately 90% after three weeks and a mean residual activity of MM, MB, and BB of 80--85% after six weeks. At 25 degrees C, CK activity of the liquid control declined to 75--80% after the fourth week. CK-BB at 25 degrees C was the least stable isoenzyme, declining to 75% after the third week and reaching 60% of activity after 12 weeks. CK-MB and CK-MM showed approximately 10--15% less stability at 25 degrees C than at 4 degrees C.
Factors affecting the concentration of triacylglycerols (triglycerides in plasma: reference values for adults.
Authors: Steinmetz J , Panek E , Siest G , Gueguen R
Published: January 2020

We studied factors influencing concentrations of triglycerides in the blood of a presumably healthy population of about 9000 persons. Using multi-dimensional analysis, we show, in addition to various related to sex and age, the relation between triglycerides and uric acid, overweight, arterial blood pressure, use of oral contraceptives (in women), consumption of alcohol and tobacco, and lack of physical exercise. These factors were classified as a function of their discriminative power by the statistical method of segmentation. We thus chose the following factors as criteria for exclusion from a reference population for the values of triglycerides in blood: in men 20 to 39 years old, overweight more than 90%; in men 40 to 59 years old, overweight of more than 115%, consumption of more than half a liter of wine per day or of more than 11 cigarettes, use of medicines, and a blood uric acid exceeding 420 mumol/L; in women 20 to 39 years old, contraceptives and overweight; in women 40 to 59 years old, overweight or more than 130% and the intake of medicines. At the 97.5th percentile of the reference population as compared with a non-selected population, the values were 80% less for men 20 to 39 years old, 95% less for men 40 to 59 years old, and only 26 to 30% less for women 20 to 59 years old.
Heterogeneity of chloramine T- and lactoperoxidase-radioiodinated human calcitonin.
Authors: Dermody W C , Levy A G , Davis P E , Plowman J K
Published: January 2020

Radioiodination reportedly damages peptides, but the nature of the damage has not been adequately examined. Utilizing isoelectric focusing, we examined the products of Chloramine T- and lactoperoxidase-directed radioiodinations of human calcitonin. Initially, the reaction products were purified by adsorption onto and elution from microfine silica (QUSO-G32). Radioiodination of the calcitonin by Chloramine T and lactoperoxidase produced a heterogeneous population of 125I-labeled peptides exhibiting apparent isoelectric points that were more acidic than that of unlabeled synthetic calcitonin. Variation in the products among radioiodinations and the inability of QUSO-G32 to resolve the components of the reaction mixture prompted our examination of alternative purification procedures. Anion-exchange chromatography on QAE-Sephadex effectively separated [125I]diiodotyrosine containing calcitonin from free iodine and [125I]iodolactoperoxidase. Our data indicate that: (a) radioiodination of human calcitonin by Chloramine T and lactoperoxidase induced alteration in the peptide as evidenced by isoelectric point, (b) specific [125I]iodopeptides vary in incidence and relative abundance among radioiodinations, (c) identification of the labeled amino acid in [125I]iodopeptides cannot ensure intergrity of the molecule, and (d) isoelectric focusing provides a method of comparing the products of peptide radioiodinations among laboratories.
Separation of bound from free hormone in radioimmunoassay of lutropin and follitropin.
Authors: Peterson M A , Swerdloff R S
Published: January 2020

A new technique in which polyethylene glycol is added to double-antibody assays of lutropin and follitropin eliminates the need for an incubation period during the separatory phase. After the antigen-first-antibody equilibrium reaction, the second antibody and polyethylene glycol (30 g/L final dilution) are added, the tubes are immediately centrifuged, each supernate is aspirated, and the precipitated antibody-bound hormone is counted. With this combined polyethylene glycol-second-antibody separatory method, the incubation time is shorter than the usual double-antibody separation procedure, but avoids the problem of high nonspecific precipitation, which occurs with polyethylene glycol alone. In addition, the combined separation technique allows the use of sera containing low titers of second antibody and use of smaller volumes of sera containing high titers of second antibodies, thus conserving second-antibody reagent.
X-ray fluorescence and potentiometry compared for determining iodine content of thyroid glands.
Authors: Puttemans F , Deconinck F , Jonckheer M , Vandeputte M , Massart D L
Published: January 2020

We compare results for iodine quantitation by x-ray fluorescence of excised thyroid glands with results of the quantitation by means of an original, newly developed chemical method, a Schöniger combustion technique, in which iodie is quantitated potentiometrically. Subsequently, we establish the accuracy and clinical suitability of the former technique in quantifying the intrathyroidal iodine.
Performance assessment of the GammafloTM automated radioimmunoassay system by assaying for digoxin.
Authors: Valdes R , Savory G , Bruns D , Renoe B , Savory J , Wills M R
Published: January 2020

We report an evaluation of the GammafloTM automated continuous-flow radioimmunoassay instrument in which we used a digoxin assay to assess system performance. System operation was based on combined continuous-flow and column-chromatographic techniques. No drift or carryover was detectable in 180 within-assay consecutive determinations performed at a rate of 42 determinations per hour (5 h of continuous operation). Within-assay and between-assay precision were less than 6% (coefficient of variation). The automated method correlated well (r = 0.960 and 0.952, respectively) with two established manual digoxin radioimmunoassay procedures. The data suggest this automated system offers a valid alternative to manual radioimmunoassay procedures in terms of overall precision, simplicity of operation, and sample throughout capacity.
A liquid chromatography-assisted assay for angiotensin-converting enzyme (peptidyl dipeptidase) in serum.
Authors: Chiknas S G
Published: January 2020

I describe modification of the spectrophotometric assay described by Cushman and Cheung [biochem. Pharmacol. 20, 1637 (1971)] for serum angiotensin-converting enzyme, with use of "high-pressure" liquid chromatography to measure the hippuric acid end product. After reaction of 10 microL of untreated serum with the angiotensin-converting enzyme substrate analog hippuryl-L-histidyl-L-leucine, the hippuric acid produced is extracted into ethyl acetate and quantitated, relative to an added internal standard, by liquid chromatography. Total chromatographic running time is 3 min per sample, with a within-run CV of 4.3% and a day-to-day CV of 6.6%, for aliquots of plasma supplemented with 1 mmol of hippuric acid per liter. The measurements are linear for hippuric acid in amounts up to 20 nmol per assay. The presence of large quantities of lipid in the serum did not affect the accuracy of the determination.
Human corticotropin (ACTH) radioimmunoassay with synthetic 1--24 ACTH.
Authors: Kao P C , Jiang N S , Carpenter P C
Published: January 2020

A corticotropin antiserum was obtained from rabbits immunized with synthetic 1--24 corticotropin conjugated with bovine serum albumin. The antiserum did not cross react with synthetic alpha-melanotropin or with synthetic beta-endorphin and had a cross reactivity of 0.23% with human beta-lipotropin. We developed a radioimmunoassay with the antiserum obtained, in which we used polyethylene glycol in conjunction with a second precipitating antibody for fast (15-min) separation of antibody-bound and free corticotropin. The assay had a sensitivity of 16 ng/L and was validated on patients with various pituitary and adrenal diseases. From 103 normal subjects, the median value for corticotropin in specimens collected during the morning was 34 ng/L of plasma; the upper 95% confidence limit of the normal range was 98 ng/L.
Radial and linear thin-layer chromatographic prodedures compared for screening urines to detect oligosaccharidoses.
Authors: McLaren J , Ng W G
Published: January 2020

We describe a circular (radial) thin-layer chromatographic procedure for separating urinary oligosaccharides. Results were better than those obtained by a single linear development. Bands and specific patterns were finely resolved for various known oligosaccharidoses. The procedure provides a simple means of screening for these disorders.
Improved liquid-chromatographic method for determination of serum cortisol.
Authors: Kabra P M , Tsai L L , Marton L J
Published: January 2020

We describe a specific and precise method for measuring concentrations of cortisol in serum or plasma by liquid chromatography. Cortisol, together with an internal standard, equilenin, is extracted from 1 mL of serum or plasma and analyzed isocratically on a reversed-phase column with a mobile phase of acetonitrile/phosphate buffer (30/70, by vol.), at a flow rate of 2.0 mL/min. The eluted cortisol is detected by its absorption at 254 nm and quantitated by peak height measurements. Each analysis requires no longer than 15 min at the optimum column temperature of 50 degrees C. The lower limit of detection for cortisol is about 2 ng/sample for a standard solution; sensitivity is routinely 5 micrograms/L of serum. Analytical recoveries exceeded 95%, with good day-to-day precision (coefficients of variation between 4 and 7%). Of more than 50 drugs and steroids tested for possible interference, only the steroids cortisone, prednisone, and prednisolone may interfere with the analysis of cortisol.
Measurement of Erythrocyte 2,3-diphosphoglycerate with a centrifugal analyzer.
Authors: Hickey T M , Uddin D E , Kiesow L A
Published: January 2020

Intra-erythrocytic concentration of 2,3-diphosphoglycerate is a major determinant of the oxygen affinity of hemoglobin. We report here the adaptation of its assay to a centrifugal analyzer, with use of a commercially available reagent. Results are calculated by using the reagent-blank-corrected absorbance change at 340 nm between 120 and 300 s for the samples and a 2.5 mmol/L standard. Under these conditions the standard curve is linear to 5 mmol/L. The compound in a 101-fold aqueous hemolysate is stable for several weeks at either -4 or -70 degrees C. Assay sensitivity and precision are excellent and results agree well with those by the corresponding manual method.
D-Mannose in human serum, measured as its aldononitrile acetate derivative.
Authors: Monson T P , Wilkinson K P
Published: January 2020

We report a procedure for determining D-mannose in serum. After removal of proteins and lipids, the carbohydrate-containing fraction is treated to form the aldononitrile acetate derivative and analyzed by gas--liquid chromatography with a flame-ionization detector. D-Mannose and D-glucose had retention times of 31 and 34 min, respectively. Day-to-day reproducibilities (CV) of between 2 and 12% for mannose were attainable for quantities of 100--900 ng. Related hexoses and derivatives do not interfere. The sensitivity was such that 10 mg/L could be detected in a 0.1-mL sample of serum. This method may be of use in diagnosing invasive Candida infection.
Evaluation and application of magnetizable charcoal for separation in radioimmunoassays.
Authors: Al-Dujaili E A , Forrest G C , Edwards C R , Landon J
Published: January 2020

We compared conventional charcoal separation of antibody-bound and free antigen with a novel method involving magnetizable particles containing charcoal. The magnetizable charcoal separation method was as effective as the conventional method for a wide range of radioimmunoassays, including those for several steroids and small polypeptide hormones. In a detailed comparison of conventional vs. magnetizable charcoal for radioimmunoassay of aldosterone, the magnetizable charcoal had several advantages: it did not require prior coating with dextran or protein, varied less with time and temperature, required no centrifugation, and took less time for separation. The correlation of results obtained with magnetizable and conventional charcoal separation was excellent (n = 89, r = 0.98, p less than 0.001), and the coefficient of variation for the separation with magnetizable charcoal was 0.5%.
Creatine kinase isoenzyme--antibody reactions in immuno-inhibition and immunonephelometry.
Authors: Morin L G
Published: January 2020

I have raised, in rabbits, antibodies against MM and BB isoenzymes of creatine kinase. The antibodies produced were homogeneous by Ouchterlony double immunodiffusion and did not cross react with their opposite antigens. Both antisera, however, appeared to be mixtures of antibodies, displaying different equivalences for activation, inactivation, and, possibly, precipitation. Inactivation studies indicated the presence of antibodies effective against dimer only and antibodies effective against monomer or dimer. Both antisera cross reacted with MB and displayed antibodies that appeared to block only half of the activity as well as antibodies that blocked all of the activity. The antisera produced were useful for measuring MB by both immuno-inhibition and immunonephelometry, but neither appears to be advantageous over current electrophoresis or ion-exchange methods. A comparison of decay of MB in patients between activity and mass measurements indicated that activity decay is about 12-fold faster than mass decay.
Creatine kinase BB: a new tumor-associated marker.
Authors: Silverman L M , Dermer G B , Zweig M H , Van Steirteghem A C , Tökés Z A
Published: January 2020

Creatine kinase isoenzyme BB (CK-BB) is found in the serum of patients with various types of cancer. Using both radioimmunoassay and agarose electrophoresis, we found abnormal amounts of this isoenzyme in the serum of 15 of 17 patients with untreated prostatic carcinoma. Among patients with other types of adenocarcinomas and metastatic disease, serum CK-BB was increased in most of those who were unresponsive to therapy. In benign or malignant prostate tissue and in malignant pleural effusions, cytoplasmic CK-BB as determined by immunoperoxidase staining was found in epithelial cells. Prostatic fluid had high concentrations of CK-BB, as did malignant, but not benign, pleural fluid supernates. We conclude that glandular epithelial cells contain CK-BB, which is released in benign and malignant states and may appear in higher concentrations in the circulation in malignant states. These conclusions are consistent with predictions we have made from a model experimental system concerning characteristics of possible tumor markers. The observations indicate a role for CK-BB as a tumor marker, particularly for adenocarcinoma of the prostate.
Separation of five isoenzymes of serum lactate dehydrogenase by discontinuous gradient elution from a miniature ion-exchange column.
Authors: Hsu M Y , Kohler M M , Barolia L , Bondar R J
Published: January 2020

We describe a simple, fast chromatographic technique for quantitatively separating the five isoenzymes of lactate dehydrogenase (LD; EC 1.1.1.27) in serum. A 250-microL serum sample is applied to a 6.0 x 0.7 cm column of QAE-Sephadex A-50 and eluted stepwise with five different buffers. The isoenzyme fractions, assayed by the method of Wroblewski and LaDue [Proc. Soc. Exp. Biol, Med. 90, 210 (1955)], are stable at room temperature for 24 h. For all five isoenzymes the average within-day coefficient of variation is 4.3%; the day-to-day CV for 20 days is 6.3%. Of some common potentially interfering substances tested, only sodium fluoride (238 mmol/L) was found to do so, by slowing the elution from the column and making the fractions turbid. The expected range in international (IUB) united and percent for each isoenzyme was determined from data on 73 men and 70 women. From these data we calculate, by a percentile estimate of a nongaussian distribution, a normal range of LD-1/LD-2 ratio of 0.55--0.87 for men and 0.52--0.91 for women. The LD isoenzyme patterns in both normal and above-normal samples of 147 sera, as evaluated by the present column method and by electrophoresis, correlated well.
An improved micromethod for theophylline determination by reversed-phase liquid chromatography.
Authors: Butrimovitz G P , Raisys V A
Published: January 2020

We describe an improved micro-scale method for determining serum theophylline by reversed-phase liquid chromatography. Samples are deproteinized with two volumes of methanol containing beta-hydroxyethyltheophylline as an internal standard. The supernate is chromatographed with a methanol/sodium acetate buffer (15/85 by vol) mobile phase, and the amount of theophylline is calculated from the ratio between peak heights for theophylline and the internal standard at 273 nm. We also monitor for chemical interferences at 254 nm. We found no interferences from ampicillin, methicillin, cephalosporin, and cephalothin. None of the drugs tested or metabolites and compounds related to theophylline have been found to interfere. The method is rapid, accurate, sensitive, and specific for theophylline.
Homogeneous enzyme immunoassay for thyroxine compared with a radioimmunoassay technique.
Authors: Gustafsson J E
Published: January 2020

A homogeneous enzyme immunoassay (EMIT) for serum thyroxine determination has been evaluated. It was also compared to our routine method, a radioimmunoassay with use of a commercial kit, Tetra-Tab RIA. Within-day precision was very similar for the two methods, the CV ranging from about 2.5% at 150 nmol of thyroxine per liter to about 5.7% at 25 nmol/L. The two methods were compared by running 100 patients' serum samples. The resulting linear regression equation was: y = 1.088x - 17.9, with a correlation coefficient r = 0.979. The deviation from the theoretical line, y = x, wash shown to be primarily caused by the calibration of the methods, shown by running the EMIT calibrators in the radioimmunoassay method and vice versa. The choice of thyroxine method in the individual laboratory will then depend on considerations other than methodological factors.
Hyperxanthinemia interferes with serum uric acid determinations by the uricase method.
Authors: Hande K R , Perini F , Putterman G , Elin R
Published: January 2020

Serum xanthine concentrations as high as 148 mg/L were noted after treatment of a patient with Burkitt's lymphoma who was receiving allopurinol. These markedly above-normal values for xanthine led to spuriously low values for serum uric acid as measured by the uricase method. Rapid tumor lysis in patients who are receiving allopurinol may lead to marked hyperxanthinemia, which in turn may obscure hyperuricemia in such patients when the uricase method is used for uric acid analysis. In such situations, uric acid concentrations should be measured by the phosphotungstate colorimetric assay.
New developments in analysis of isoenzymes separated by "high-performance" liquid chromatography.
Authors: Schlabach T D , Fulton J A , Mockridge P B , Toren E C
Published: January 2020

We have developed two enzyme analyzers for use in "high-performance" liquid chromatography. In both systems two detectors are used, placed after the column effluent has been combined with assay reagent. In one system, an absorbance detector is placed before and after a post-column reaction coil. Peaks observed at one detector are subtracted from those at the other, to produce a two-point measurement of enzyme activity. The linear dynamic range was 17--1700 U/L for lactate dehydrogenase (EC 1.1.1.27). In the other system, two reaction coils were used and a single fluorescence detector was placed at the end of each coil. These coils were kept at different temperatures, and an automated switching valve diverted equal amounts of column effluent and reagent into both coils. The fluorescence readings were then subtracted to produce a differential measurement of enzyme activity. The linear dynamic range was 20--1000 U/L. We used both systems to chromatographically analyze lactate dehydrogenase isoenzymes, and could separately determine both the distribution and activity of sample isoenzymes.
High-performance liquid chromatography for ultraviolet-absorbing constituents in human urine.
Authors: Miyagi H , Miura J , Takata Y , Ganno S
Published: January 2020

Several liquid-chromatographic systems involving anion-exchange resins and linear acetate gradient elution have been reported previously for the analysis of ultraviolet-absorbing constituents in human urine. We describe a high-performance liquid-chromatographic system on which a macroreticular anion-exchange resin, and stepwise elution are used. Separations were more rapid with ammonium chloride/acetonitrile stepwise elution than with acetate stepwise elution, and urine constituents could be detected at 225 nm with the former system. Analytical conditions were convenient and the method has proven to be useful for routine clinical analysis.
Properties and uses of immobilized light-emitting enzyme systems from Beneckea harveyi.
Authors: Jablonski E , DeLuca M
Published: January 2020

Bacterial luciferase and NADH:FMN oxidoreductase have been immobilized onto arylamine glass beads. These immobilized enzymes can detect as little as 0.2 pmol of NADH per assay sample. Glucose-6-phosphate dehydrogenase has been co-immobilized with these enzymes, and with this system it is possible to quantitate 1 pmol of glucose 6-phosphate. By co-immobilizing a fourth enzyme, hexokinase, onto the glass beads, the system can reproducibly detect 20 pmol of glucose per liter. These immobilized enzyme systems are potentially superior to soluble enzymes by being reusable and much more stable. We compared the light-emitting properties of the immobilized enzyme systems with that of an equivalent mixture of the soluble enzymes. The most striking difference was the apparently more efficient conversion of NADH or glucose 6-phosphate to light by the immobilized enzymes. We used hydroxysteroid dehydrogenase in developing a soluble coupled system for the assay of androsterone and testosterone. The lower limit of detection was 100 pmol.
Flow-injection analysis with chemiluminescence detection.
Authors: Rule G , Seitz W R
Published: January 2020

In "flow-injection analysis," the sample is inserted into a stream of reagent by use of a sample-injection valve. Mixing occurs downstream from the valve in a coil of tubing. With chemiluminescence detection this coil is positioned in front of a photomultiplier. We have evaluated the system for detection of hydrogen peroxide, using the luminol reaction with cupric ion as a catalyst. The effects of flow rate, sample volume, and reaction kinetics on the magnitude, duration, and repeatability of the chemiluminescent response have been evaluated. Precisions of 1 to 2% relative standard deviation on replicate measurements are readily achievable. A sample throughput of six samples per minute is possible with very little peak overlap. This detection system can be coupled to any process in which peroxide is generated.
Mechanism of interference by hemoglobin in the determination of total bilirubin. I. Method of Malloy-Evelyn.
Authors: Shull B C , Lees H , Li P K
Published: January 2020

Oxyhemoglobin is the species of hemoglobin in erythrocyte hemolysates that inhibits the diazo reaction. Ferric hemoglobin derivatives and species with relatively low molecular mass do not interfere. Conversion of oxyhemoglobin to acid hematin under assay reaction conditions is associated with rapid destruction of bilirubin, which accounts for the diazo reaction error. The most probable mechanism for this destruction of bilirubin is an oxidative reaction involving H2O2, formed in the oxidation of hemoglobin, and acid hematin acting as a pseudoperoxidase. We could find no evidence for other mechanisms of interference such as spectral error or azobilirubin destruction. Addition of potassium iodide, 4.0 mmol/L final concentration in the reaction mixture, eliminates interference from hemoglobin added to give concentrations as great as 10 g/L. It also eliminated the effects of hemolysis in the method of Ertingshausen et al. (Clin. Chem. 19: 1366, 1973), in which ethylene glycol is used as the accelerator.
"High-pressure" liquid chromatography of sulfisoxazole and N4-acetylsulfisoxazole in body fluids.
Authors: Jung D , Oie S
Published: January 2020

We describe a simple, rapid chromatographic method for separating and quantitatively determining sulfisoxazole and its N4-acetyl metabolite in plasma and urine. A 100-micro L sample of plasma or urine is combined with 200 micro L of a solution containing 12 mg/L of the internal standard, N4-acetylsulfamethoxazole, in absolute methanol and centrifuged to obtain a clear supernatant solution. This solution is then eluted through a 10-micron microparticulate column with a mobile phase of 32/68 (by vol) methanol/sodium acetate buffer (0.01 mol/L, pH 4.7), at a flow rate of 1.2 mL/min. The eluted sompounds are detected by their absorption at 254 nm. We calculated concentration from the peak-height ratios of sulfisoxazole or N4-acetylsulfisoxazole to N4-acetylsulfamethoxazole. The peak-height ratio was linear with concentration in the range 0.05--200 mg/L for both drug and metabolite in plasma and urine. Because this assay can be completed within 30 min of obtaining a blood or urine sample, it should be a valuable tool in clinical drug monitoring and pharmacokinetic studies.
Spectrophotometric screening method for acetaminophen in serum and plasma.
Authors: Liu T Z , Oka K H
Published: January 2020

We describe a simple, economical procedure for rapidly detecting acetaminophen in serum or plasma. The method is based upon the reduction by the drug of ferric 2,4,6-tris(2-pyridyl)-s-triazine, at an acidic pH, to ferrous 2,4,6-tris(2-pyridyl)-S-triazine complex, which absorbs maximally at 593 nm. Absorbance and acetaminophen concentration are linearly related from 25 to 400 mg/L, and so therapeutic and toxic concentrations can be measured. The method is accurate; day-to-day CV's for two pooled control specimens (103 and 227 mg/L) were 4.4 and 6.6%. Correlation studies, with an established nitration method and with the free-radical diphenylpicrylhydrazyl dye method, showed correlation coefficients of 0.985 and 0.915, respectively. Of 25 commonly used drugs tested, only levodopa, oxyphenylbutazone, and phenylephrine interfere significantly. Interference from salicylate, salicylamide, and phenylbutazone was insignificant.
Metastable creatine kinase MM and complexes of creatine kinase BB and immunoglobulin G, "atypical" isoenzymes with similar electrophoretic mobility.
Authors: Homburger H A , Wold L , Jacob G L , O'Brien J
Published: January 2020

Metastable creatine kinase MM isoenzyme was isolated and partially purified from homogenates of myocardium and skeletal muscle by gradient elution on carboxymethyl cellulose. This variant isoenzyme migrated between the MM and MB isoenzymes on agarose electrophoresis, accounted for 3.5% of the total creatine kinase activity in each tissue, was not a macromolecule, and had stable electrophoretic mobility only in borate buffer (0.02 mol/L). By comparison, the creatine kinase isoenzymes with similar "atypical" electrophoretic mobility in serum specimens were complexes of the BB isoenzyme and immunoglobulin G. These complexes were measured by a radioimmunoassay specific for the creatine kinase B-subunit and eluted predominantly with the MB isoenzyme in a commercial anion-exchange reagent system.
A fully automated, continuous-flow radioimmunoassay for methotrexate.
Authors: Kamel R , Landon J , Forrest G C
Published: January 2020

We describe a fully automated continuous-flow radioimmunoassay for methotrexate. [125I]Histamine-labeled methotrexate was used as tracer. Anti-methotrexate serum was coupled to a magnetizable solid-phase and the bound and free fractions were separated with an electromagnetic field. The assay is precise (CV less than 2.5%) and rapid (30 samples per hour), incubation volume is small (about 160 micro L), and incubation brief (10 min). The accurate timing inherent in the system obviates the need to attain equilibrium, so that assay of each sample takes only 15 min. The assay is sensitive (1--100 microgram/L). There is no significant carryover between samples of high and low concentration. Results by the automated method correlated well with those by both a manual assay in which the same reagents and separation technique are used (r - 0.99) and a competitive protein-binding assay (r = 0.96).
Bilirubin binding by primary sites of human serum albumin, with suppression of secondary-site interference by use of a high salt concentration.
Authors: Rutkowski R B
Published: January 2020

A high (2.0 mol/L) NaCl concentration apparently suppresses the secondary-site binding of bilirubin by human serum albumin. Thus if an excess of bilirubin is added to human serum albumin or to neonatal serum in buffer containing 0.1 mol of tris(hydroxymethyl)aminomethane and 2.0 mol of NaCl per liter (pH 7.5), and the bilirubin not bound to albumin is removed by treatment with calcium carbonate, the bilirubin binding reserve of primary binding sites can be estimated from direct measurement of spectral absorbance at 468 nm. Hemolysis and conjugated bilirubin apparently do not interfere. In a comparison study with serum samples from neonates the method gave results that generally agreed with those obtained by a commercially available Sephadex G-25 column procedure. In serum samples from adults the calculated unbound unconjugated bilirubin (free bilirubin) values derived by using binding reserves determined by the proposed method correlated well with the free bilirubin concentrations measured by a peroxidase method, but were only about one-half the amounts obtained by the peroxidase method.
Monitoring clorazepate dipotassium as desmethyldiazepam in plasma by electron-capture gas--liquid chromatography.
Authors: Haidukewych D , Rodin E A , Davenport R
Published: January 2020

We describe a procedure for determing clorazepate dipotassium as its decarboxylated, pharmacologically active metabolite, desmethyldiazepam, in 100 microL of plasma, with use of electron-capture gas--liquid chromatography and with methylnitrazepam as the internal standard. The procedure is a one-tube, one-step extraction without derivative formation and is accurate, reproducible, and rapid. The sensitivity limit is 20 micrograms/L. Within-run and between-run CV's (concentration, 3.5 mg/L) were 2.9 and 3.5%, respectively. Within-run CV's for 1.5 and 1.0 mg/L concentrations were 3.9 and 4.3%, respectively. For a 1.0 mg/kg per day dose of clorazepate dipotassium, the mean steady-state concentration of desmethyldiazepam in plasma was 1.037 mg/L.
New perspectives in coagulation testing.
Authors: Fareed J , Messmore H L , Bermes E W
Published: January 2020

The field of coagulation testing has undergone some major technological and conceptual developments, which are briefly reviewed here. The assessment of coagulation parameters is no longer restricted to the study of clot formation and its dissolution. The understanding of the biochemical nature of coagulation processes, coupled with the development of new therapeutic agents in the treatment of hemostatic disorders, has brought about the development of fast, reliable, and clearly defined laboratory test procedures to evaluate the components of this system. The introduction of methods involving synthetic substrates has been very significant because many of the coagulation parameters can now be measured with a spectrophotometer or fluorometer, by methods that lend themselves to the automation found in most large clinical chemistry laboratories. In our laboratory, we use automated synthetic-substrate methods for antithrombin-III, plasminogen, and prothrombin, and are developing the synthetic-substrate assay equivalent of clot-based prothrombin time and partial thromboplastin. Immunological methods such as laser/rate nephelometry, enzyme-linked immunoassays, electroimmunodiffusion, and radioimmunoassays have been utilized to evaluate coagulation proteins. The relation of functional and immunological properties of these proteins to their physiological function is being studied. In coming years the testing of coagulation function will undergo some major changes and will require input from clinical chemists and other laboratory scientists to facilitate the technology transfer and proper standardization of new methods.
Use of electrochemical sensors for on-line monitoring of ionized calcium, potassium, and glucose in whole blood of living dogs.
Authors: Fogt E J , Eddy A R , Clemens A H , Fox J , Heath H
Published: January 2020

We report the development and evaluation of electrochemical sensors for continuous on-line measurement of whole-blood ionized calcium, potassium, and glucose in living animals. Ionized calcium and potassium are measured with ion-selective electrodes, by using a unique differential approach. A polarographic sensor coupled with a membrane with bound glucose oxidase is used in the glucose analyzer. The modular concept of instrument design permits one mainframe, computer, and pump system to house any analyzer module or a combination of modules. A small volume of venous blood (about 2 mL/h) is continuously withdrawn from the subject through a double-lumen catheter and pumped through the analyzer(s). Results of in vitro correlation studies and studies in normal dogs (including responses of ionized calcium, potassium, and glucose to various stresses) support the validity of the measurements.
Automated multiple flow-injection analysis in clinical chemistry: determination of total protein with Biuret reagent.
Authors: Shideler C E , Stewart K K , Crump J , Wills M R , Savory J , Renoe B W
Published: January 2020

We have examined the feasibility of the automated multiple flow-injection technique for application to clinical chemistry by adapting to this system the biuret method for the determination of total protein. Samples were discretely and rapidly introduced into a continuously flowing, nonsegmented reagent stream by means of an automatic sampler and high-pressure injection valve. Pumps operating at 1380-2070 kPa (200-300 psi) were utilized to introduce the biuret reagent and saline diluent into the system separately at flow rates of 72 and 47 microL/s, respectively. Use of 20-microL sample and a 3.0-s reaction-delay coil was adequately sensitive for analysis for total protein by this method. Samples were analyzed at a rate of 150/h with no detectable between-sample carryover. Within-run precision studies yielded relative standard deviations of 2.5% and less. Total protein values obtained by this method correlated well with those obtained by centrifugal analyzer and bubble-segmented continuous-flow biuret methods.
Monitoring serum valproic acid by gas chromatography with electron-capture detection.
Authors: Chan S C
Published: January 2020

I have developed a procedure to monitor, by gas chromatography with electron-capture detection, the concentration of valproic acid in serum. The valproic acid is derivatized into its phenacyl ester. The assay is very sensitive, measuring as little as 100 pg of valproic acid easily and accurately. Solvent extraction and concentration of the drug are not necessary; instead, one only has to precipitate the serum proteins. n-Octanoic acid is used as the internal standard. The peak-height ratio of phenacyl valproate to phenacyl octanoate is linearly related to concentration up to 200 mg of valproic acid per liter.
Kinetic immunoturbidimetric measurement of thyroxine binding globulin.
Authors: Spencer K , Price C P
Published: January 2020

We describe the extension of kinetic immunoturbidimetry to measurement of low-concentration proteins in serum, specifically thyroxine binding globulin. Method performance is superior to that of currently available direct assays for this protein by radioimmunoassay or the Laurell "rocket" techniques. The assay is rapid (1-min measurement period), economical, and results compare well with those by established techniques. The technique has wide applicability to instruments with kinetic measuring ability and could provide a useful support to other non-isotopic thyroid-function tests. The assay further emphasizes the sensitivity and usefulness of kinetic immunoturbidimetry for measuring low concentrations of proteins.
Monitoring 2-ethyl-2-phenylmalonamide in serum by gas-liquid chromatography: application to retrospective study in epilepsy patients dosed with primidone.
Authors: Haidukewych D , Rodin E A
Published: January 2020

We describe a procedure for determining 2-ethyl-2-phenylmalonamide (I) in serum of epilepsy patients dosed with primidone (II) for seizure control, by extracting the sample with chloroform under basic conditions, with use of an internal standard, 2-ethyl-2-(p-tolyl)malonamide, and without derivative formation. The sensitivity limit is 1.0 mg/L. Within-run CVs for 5, 10, and 30 mg/L concentrations were 3.5, 2.5, and 0.7%, respectively. For a 1.0 mg/kg body wt per day dose of II in patients co-medicated with phenytoin (Group A), the mean steady-state concentrations of I, II, and phenobarbital (III) were 0.72, 0.62, and 2.24 mg/L, respectively. For patients co-medicated with carbamazepine and phenytoin (Group B), I, II, and III concentrations were 0.68, 0.44, and 2.12 mg/L, respectively. Between these groups, only for II were the concentrations statistically different (t = 2.762; p < 0.01). For Group A no correlation was found between II and III. For Groups A and B, the coefficients of correlation between I and III were 0.626 and 0.826, respectively.
Comparability of results by simplified methods for measuring serum lipoprotein cholesterol in a pediatric population.
Authors: Srinivasan S R , Foster T A , Berenson G S
Published: January 2020

We compared serum lipoprotein cholesterol concentrations in a randomly selected subsample (n = 406) population of children (ages 5 to 17 years), as measured by the heparin-Ca2+ and the heparin-Mn2+ precipitation methods. Statistical analysis showed significant differences between results by these methods, especially in values for VLDL- and HDL-cholesterol (mean difference: VLDL-cholesterol, -61 mg/L; HDL-cholesterol, + 108 mg/L). Lipoprotein electrophoretic data indicated that estimating VLDL-cholesterol from serum triglyceride concentrations invariably resulted in higher values for children with an extremely faint pre-beta-lipoprotein band. Among the HDL-subfractions, 14 to 20% of HDL2 was precipitated by heparin-Mn2+ despite the presence of 80 g of albumin per liter. Heparin affinity chromatography of HDL2 in the presence of Ca2+ and Mn2+ showed an interaction of apolipoprotein E-containing HDL2 subfraction only in conditions involving Mn2+. The heparin-Ca2+ procedure precipitated the equivalent of 2 g of beta- plus pre-beta-lipoprotein cholesterol per liter from serum, with no detectable apolipoprotein B in the supernate. We conclude that these precipitations are affected by cation specificity and by differences in lipoprotein makeup of a given individual or population group.
Spectrophotometric micro method for measurement of dialyzable calcium by use of cresolphthalein complexone and continuous-flow analysis.
Authors: Toffaletti J , Kirvan K
Published: January 2020

We adapted a method for dialyzable calcium from fluorometric detection by use of calcein to a more specific spectrophotometric determination with cresolphthalein complexone. The reagents are available commercially and perform satisfactorily with respect to noise level, drift, stability, and sensitivity. Construction of the continuous-flow manifold with commercially available components (injection blocks, coils, and dialyzer) and the use of a 9-mm pathlength flow cell in an AutoAnalyzer I colorimeter have permitted a sampling rate of 70/h, and decreased the volume of serum required to 130 microL. A comparison of 71 sera analyzed by the present and the calcein method gave means of 1.39 (SD 0.14) and 1.39 (SD 0.13) mmol/L, respectively. The regression equation was: present method = 1.016 calcein--0.022 mmol/L (r = 0.97). The CV for the new method, as determined from 46 randomized duplicate analyses, was < 1%. The reference interval, as evaluated from results for 93 different individuals, was 1.26-1.43 mmol/L. We conclude that our methods is an improvement with respect to noise level, drift, specificity, detectability, and more general availability of instrumentation. Moreover, the smaller sample volume required makes possible the routine measurement of dialyzable calcium in pediatric samples.
Radioimmunoassay of human thyroglobulin with use of "thyroglobulin-free" plasma prepared by ultracentrifugation as diluent.
Authors: Ikekubo K , Jutton J , Schneider A B
Published: January 2020

We describe the preparation and evaluation of a new diluent for use in preparing standard curves for the radioimmunoassay of human thyroglobulin (TG). This diluent, "TG-free human plasma, allows measurement of TG in lower concentrations than with previous assay systems because the components of the standards are more similar to those of the samples. TG-free plasma was prepared by a modification of the differential centrifugation method of Edelhoch (J. Biol. Chem. 235:1326, 1960). Standard curves prepared by use of TG-free plasma and plasma obtained from thyroidectomized patients were identical. In our assay, plasma from patients who had undergone total thyroidectomy produced no displacement when 25 to 100 microL of plasma was assayed. Twenty-five normal subjects all had detectable TG concentrations, ranging from 4.0 to 25.7 microgram/L. Only 27 of 2000 determinations performed by this new method had undetectable values and 26 of these were from patients with total or near-total thyroidectomies. TG concentration determined by this assay may be more sensitive for finding recurrent thyroid malignancy.
Rapid sodium dodecyl sulfat/polyacrylamide gel electrophoresis of urinary proteins.
Authors: Cachera C , Mizon C , Fruchart J C , Mizon J , Tacquet A
Published: January 2020

We describe a procedure for the convenient separation of proteins by sodium dodecyl sulfate/polyacrylamide gel electrophoresis of urine from cases of renal disease. A precipitation method that requires no special apparatus was used to concentrate the urinary proteins; for electrophoretic separation we used a commercially supplied polyacrylamide/cellulose gel slab. This method seems to be valuable for investigation of proteinuria; we recommend it for routine use.