bluuebunny/crossref_metadata_2025 huggingface dataset:
Some considerations of methodology and standardization of apolipoprotein B immunoassays.
Authors: Rosseneu M , Vercaemst R , Steinberg K K , Cooper G R
Published: January 2020

Apolipoprotein B, the major protein component of very-low-density (VLDL) and low-density (LDL) lipoproteins in serum, is being widely measured in serum, to help assess risk of cardiovascular disease. Standardization has proven difficult because of the physical and immunochemical heterogeneity of LDL and VLDL, the masking of antigenic determinants of LDL and VLDL, the insolubility of apolipoprotein B, the instability of lipoprotein reference materials, and the multiplicity of analytical methods. Here we have examined the literature and collected current information from lipoprotein scientists to determine the problems--and the ways investigators have attempted to solve them--in preparation, storage, and use of standards and anti-apolipoprotein B sera, analytical procedures and reagents, and the nature of apolipoprotein B in serum. Standardization appears possible with development of stable reference materials that are applicable to the various analytical methods, controlled immunochemical reaction conditions, and establishment of a selected interim reference method for use as a point of reference.
2,6-Dimethyloctanedioic acid--a metabolite of phytanic acid in Refsum's disease.
Authors: Greter J , Lindstedt S , Steen G
Published: January 2020

The urine of two patients with Refsum's disease consistently contained 2,6-dimethyloctanedioic acid, a compound not normally found in human urine. In addition, their urines contained increased concentrations of 3-methylhexanedioic acid. These two compounds may be formed from phytanic acid by an initial omega-oxidation and subsequent beta-oxidations. It was calculated that this oxidation pathway may metabolize at least 30 mg of phytanic acid per day.
Kinetic assay of human pepsin with albumin-bromphenol blue as substrate.
Authors: Gray S P , Billings J A
Published: January 2020

A novel substrate, albumin complexed with bromphenol blue, has been developed for the assay of human gastric juice pepsin by a kinetic method in the Cobas centrifugal analyzer. The action of pepsin on the complex degrades the albumin and releases the dye. The change in the color of the substrate is a zero-order reaction. Human and porcine pepsin have different Km's with the new substrate. This kinetic method has a throughput of 28 tests in approximately 10 min and good precision (CV = 2.0%). Other advantages are analysis in homogeneous solution (thereby eliminating the need to separate substrate and products), lack of interference from bilirubin or phenol red, and the expression of pepsin activity in IUB enzyme units.
Effect of aspirin on determinations of glycosylated hemoglobin.
Authors: Nathan D M , Francis T B , Palmer J L
Published: January 2020

We investigated the in vivo and in vitro effects of aspirin on several clinical assays of glycosylated hemoglobin. Acetylation of hemoglobin falsely increased the glycosylated hemoglobin fraction measured by "high-performance" liquid chromatography and electrophoresis, but isoelectric focusing and colorimetric techniques differentiated between acetylated and glycosylated fractions. Aspirin ingestion may result in an apparent increase in glycosylated hemoglobin measured with common clinical assays.
Simultaneous very fast liquid-chromatographic analysis of ethosuximide, primidone, phenobarbital, phenytoin, and carbamazepine in serum.
Authors: Kabra P M , Nelson M A , Marton L J
Published: January 2020

We describe a sensitive, specific, and very fast liquid-chromatographic assay for simultaneously determining five anticonvulsants (ethosuximide, primidone, phenobarbital, phenytoin, and carbamazepine) by using commercially available 5- or 3-microns particle size reversed-phase columns and a microflow-cell-equipped ultraviolet detector. The anticonvulsant drugs are extracted from 200 microL of serum containing 50 mg of cyclopal per liter as an internal standard, by elution from a Bond-Elut (Analytichem International, Harbor City, CA 90710) column with 300 microL of methanol. A 5-microL aliquot of the eluate is applied to an analytical column and eluted with a mobile phase of acetonitrile/methanol/phosphate buffer, 20 mmol/L, pH 3.7 (13.5/35/51.5 by vol), at a flow rate of 3.0 mL/min and at 50 degrees C. Detection is at 210 or 195 nm. The chromatography is complete in less than 2.5 min with the 5-microns-particle column, and in less than 1.4 min with the 3-microns-particle column. The sensitivity of the method for all drugs is less than 1 mg/L. Analytical recovery of drugs added to serum ranged from 92 to 109% for concentrations up to 200 mg/L. Between-run precision (CV) ranged from 1.3 to 4.1%.
Radioimmunoassay of bile acids in tissue, bile, and urine.
Authors: Wildgrube H J , Stockhausen H , Metz P , Mauritz G , Mahdawi R
Published: January 2020

Two commercially available (Abbott Labs.) radioimmunoassays for determination of conjugated cholic acid and sulfoglycolithocholic acid in serum have been modified for bile acid measurements in alcoholic tissue extracts, bile, and urine. The specificity of both radioimmunoassays has been determined with regard to 27 free and conjugated bile acids. After filtration, bile acids can be measured in urine and bile without prior extraction. Tissue is homogenized and the bile acids are extracted into methanol. Urinary excretion by 64 healthy humans was 2.09 (SD 1.09) mumol of conjugated cholic acid and 8.44 (SD 8.03) mumol of sulfated glycolithocholic acid per 24 h. In liver from 10 patients with various noncholestatic liver disease, the mean concentration of conjugated cholic acid was 32.4 (SD 15.9) nmol/g wet weight. In the liver of 27 male Wistar rats, the concentration of conjugated cholic acid was 41.3 (SD 11.7) nmol/g of tissue, of sulfoglycolithocholic acid 5.1 (SD 2.3) nmol/g of tissue.
Suitability of control materials in the differential inhibition assay for human pancreatic and salivary amylase.
Authors: O'Donnell M D , McGeeney K F
Published: January 2020

We investigated the behavior of 26 quality-control sera with the inhibitor method for differential amylase (EC 3.2.1.1) assay. We also studied the sensitivity to the wheat-derived inhibitor of pancreatic amylases from 10 different animals in comparison with human pancreatic and salivary amylase. The results indicate that only control materials containing human amylases can be measured accurately. The animal amylases (bovine, equine, porcine) used in many quality control sera are relatively insensitive to the inhibitor as compared with human pancreatic and salivary amylase.
Liquid-chromatographic profiles of urinary porphyrins.
Authors: Schreiber W E , Raisys V A , Labbé R F
Published: January 2020

Information on changes in the urinary excretion pattern of porphyrins can be especially useful in the diagnosis of disorders of porphyrin metabolism. Most clinical laboratory procedures are designed for assay of uroporphyrin and coproporphyrin only, and in many cases even these are not cleanly separated. Hence, we developed a "high-performance" liquid-chromatographic procedure to separate and quantify all five urinary porphyrins--that is, those with four through eight carboxyl groups. Before chromatography, the porphyrins are isolated from other urinary components by two simple, rapid pretreatment steps, then injected into the chromatograph in nonesterified form. They are separated and eluted with a step gradient of methanol/phosphate buffer, pH 3.0, in which the methanol content is first 650, then 850 mL/L. As little as 1 ng of eluted porphyrins can be measured fluorometrically. Analytical recovery of coproporphyrin is virtually 100% and of uroporphyrin 75-80%. CVs are about 10% for coproporphyrin at 70 micrograms/L and 20-40% for uroporphyrin at 8 micrograms/L.
Alpha-fetoprotein concentrations in maternal serum: relation to race and body weight.
Authors: Crandall B F , Lebherz T B , Schroth P C , Matsumoto M
Published: January 2020

We confirmed the relation between maternal weight and serum alpha-fetoprotein concentration and have shown a further relation--to ethnic origin. Of these two, maternal weight is the more closely related. Oriental, white, and Hispanic women showed no significant differences in serum AFP concentrations when corrections for maternal weight were applied. Black women showed consistently higher values, by an average of 10% at each week of gestation. These corrections only affected values falling just above or below the 95th centile cutoff. We conclude that corrections for maternal weight and race should be applied when values for alpha-fetoprotein in maternal serum are being interpreted.
Sensitivity and specificity of the hydrogen breath-analysis test for detecting malabsorption of physiological doses of lactose.
Authors: Rosado J L , Solomons N W
Published: January 2020

We examined the changes in sensitivity and specificity that would occur with alterations in the sample-collection schedule and (or) cutoff criterion for the increase in hydrogen concentration in breath after administration of doses of lactose in the dietary range. In a breath-analysis test to classify individuals as lactose-absorbers or lactose-malabsorbers, 41 subjects drank 360 mL of intact cow's milk, containing 18 g of lactose, and breath samples were collected and analyzed at 30-min intervals for 5 h. An increase in H2 concentration of greater than or equal to 20 microL/L above basal values at any of the 10 intervals was diagnostic of malabsorption. Increases of greater than or equal to 18 or greater than or equal to 15 microL/L were only 85% as specific in classifying the same individuals. Reduction in the number of samples tested per subject uniformly reduced the sensitivity. However, a simplified procedure suitable for field studies (in which four samples--at 0, 2, 3, and 4 h--are collected and analyzed with greater than or equal to 20 microL/L as the cutoff value) gives 80% sensitivity and 100% specificity, as compared with the 11-sample procedure.
The "eyetone" blood glucose reflectance colorimeter evaluated for in vitro and in vivo accuracy and clinical efficacy.
Authors: Hay W W , Osberg I M
Published: January 2020

We evaluated the performance of a blood glucose reflectance colorimeter ("Eyetone," Ames Co.) for accuracy and precision with use of "Dextrostix" (Ames Co.) glucose oxidase reagent strips for blood samples with known and unknown concentrations of glucose covering the usual range of neonatal blood glucose (200-800 mg/L). The meter was calibrated and tested by research nurses and one clinical chemist. Five unknowns were tested for accuracy and precision (56-92 determinations per unknown) and compared with Beckman Astra values (plasma and calculated whole blood). Eyetone/Dextrostix values differed (gave lower values) from the calculated whole-blood values only at concentrations less than 300 mg/L. On 258 clinical specimens from newborn infants, Eyetone/Dextrostix values were not different from calculated whole-blood values (p less than 0.05, r = 0.80). Operator training to develop a consistent procedure was the most critical factor in achieving accurate and precise results.
An evaluation of four serum tests for pregnancy.
Authors: Ryder K W , Munsick R A , Oei T O , Young P C , Blackford H F
Published: January 2020

We evaluated four pregnancy tests (Biocept-G, Beta-CG, Preg/Stat, and HCG-Beta Screen), using sera from 59 nonpregnant subjects and 77 patients with serum human choriogonadotropin beta-subunit (beta-hCG) concentrations ranging from 4 to 100 000 int. units/L. The results obtained for each test were compared with the results predicted on the basis of the sample's beta-hCG concentration and the beta-hCG concentration the manufacturer claimed necessary for a positive result (the test's analytical sensitivity). Biocept-G had the best sensitivity (100%), specificity (98.9%), and accuracy (99.2%). Beta-CG had the poorest sensitivity (86.4%), Preg/Stat the poorest specificity (87.5%), and accuracy (92.6%). We confirmed the manufacturer's claimed analytical sensitivity (200 int. units/L) for the Biocept-G procedure, but our calculated analytical sensitivity for the other tests was significantly different from that claimed by their manufacturers. Best results were obtained with Biocept-G, but with its analytical sensitivity of 200 int. units/L, samples from early pregnancy will give negative results. None of the pregnancy tests evaluated here will establish the presence or absence of early pregnancy with certainty.
Immunonephelometry of specific proteins in human seminal plasma.
Authors: Lizana J , Blad E
Published: January 2020

Using immunonephelometry, we measured albumin, transferrin, alpha 1-antitrypsin, orosomucoid, hemopexin, IgG, IgA, and secretory IgA in seminal plasma of men who were attending a fertility clinic. Seminal plasma, obtained by centrifugation of semen, is a turbid liquid producing high blank values that were not diminished by ultracentrifugation, filtration, or treatment with protamine, dextran sulfate, or various lipidophilic gels. Pretreatment of seminal plasma with polyethylene glycol 6000 (final concentration, 40 g/L) considerably decreased the blank values without appreciably changing the relative concentration of the eight proteins studied. Our investigations illustrate the feasibility of this application of immunonephelometry. This approach can be a valuable diagnostic tool in the laboratory evaluation of the male partner in infertile unions.
Specificity of human beta-choriogonadotropin assays for the hormone and for an immunoreactive fragment present in urine during normal pregnancy.
Authors: Schroeder H R , Halter C M
Published: January 2020

We evaluated four high-purity commercial preparations of human choriogonadotropin (hCG; Boehringer-Mannheim, Calbiochem, Cambridge, and Radioassay Systems) by two radioimmunoassays and one radioreceptor procedure. Their specific activities were less than half that of the first International Reference Preparation of hCG for immunoassay. In addition, a fragment that was immunoreactive in radioimmunoassays for hCG with antisera to the beta-subunit conformational determinant was isolated from crude hCG (Roussel Corp.). The fragment adsorbed to concanavalin A and exhibited a relative molecular mass of 12 000 by gel filtration. In a study with 94 urines from women three to 24 weeks pregnant, the fragment contributed more than 70% of the immunoreactivity detected by the above radioimmunoassays for hCG in 70% of the samples. The fragment was present in urine throughout pregnancy, but was not detected in the serum of any of seven pregnant women tested.
Liquid-chromatographic profiling of endogenous fluorescent substances in sera and urine of uremic and normal subjects.
Authors: Mabuchi H , Nakahashi H
Published: January 2020

Endogenous fluorescent substances increase in serum during uremia. We have used "high-performance" liquid chromatography to profile these fluorescent substances in both uremic and normal body fluids. The fluorescence excitation and emission maxima we used were 322 and 415 nm, respectively. Of the numerous fluorescent substances found in uremic body fluids and in normal urine, some were also detectable in normal serum, but at relatively weak fluorescence intensities.
Immunoadsorption to improve gas chromatography/high-resolution mass spectrometry of estradiol-17 beta in plasma.
Authors: Gaskell S J , Brownsey B G
Published: January 2020

We describe a new, highly selective procedure for the determination of estradiol-17 beta in plasma. Samples are extracted with a micro-cellulose-coupled antiserum to estradiol-17 beta. Conversion of the extracted steroid to the bis(trimethylsilyl) ether is followed by gas chromatography/high-resolution mass spectrometry with selected ion monitoring. Precise quantification is achieved through the use of [2H3]estradiol-17 beta as internal standard.
Rapid quantification of C-reactive protein by centrifugal analysis.
Authors: Melamies L
Published: January 2020

I describe a rapid, simple immunoturbidimetric method for determining C-reactive protein in serum. With the Instrumentation Laboratory Multistat III microcentrifugal analyzer, quantitative results are obtained automatically after a few minutes of reaction time. Within-run and between-run coefficients of variation ranged from 2.5 to 12.7% at C-reactive protein concentrations of 55 to 69 and 14 to 25 mg/L, respectively, normal values being less than 10 mg/L. Comparison with the commercially available radial immunodiffusion method (y) yields the regression equation y = 1.011x - 2.112 (r = 0.979, n = 100).
Determination of urinary oxalate with commercially available oxalate oxidase.
Authors: Buttery J E , Ludvigsen N , Braiotta E A , Pannall P R
Published: January 2020

For this direct colorimetry of urinary oxalate, commercially available oxalate oxidase (EC 1.2.3.4) is used. The urine is first diluted, to diminish the effect of interfering substances. Analytical recovery of oxalate from urines with five different oxalate concentrations (0.4 to 2.0 mmol/L) ranged from 92 to 109% (mean 99%). The within-day and between-day precision (CV) of the method for a wide range of oxalate concentrations averaged better than 10%. There is good correlation (r = 0.977) between this enzymatic method (y) and the chemical method of Hodgkinson and Williams (x) [Clin Chim Acta 36: 127-132, 1972], the regression equation being y = 1.014x + 0.061. Urines with added ascorbate give falsely increased results. The proposed method is inexpensive and simple to perform.
Improved separation and quantification of the "middle molecule" b4-2 in uremia.
Authors: Faguer P , Man N K , Cueille G , Di Giulio S , Funck-Brentano J L
Published: January 2020

The plasma of uremic patients usually contains high concentrations of the so-called middle molecules (molecular mass, 300 to 1500 Da), which exert various toxic effects. Among these numerous substances, only one, named peak b4-2, has been correlated with uremic neuropathy. We describe our improvement of a two-stage chromatographic method, gel permeation followed by anion-exchange chromatography (J. Chromatogr. 146: 55-65, 1978), for separation and quantification of b4-2 in body fluids. In analyzing more than 300 samples from 43 uremic patients and 12 healthy subjects, we found a linear correlation between peak area at 254 nm and b4-2 concentration in the range 0.8 to 32 mg/L. The coefficient of variation, including data acquired during seven changes of columns, was 9%. Analysis time (80 min) was shorter than required with other methods. Our results confirm previous data for urinary b4-2 excretion by healthy subjects and for b4-2 removal rate in uremic patients undergoing hemodialysis or hemofiltration. Patients treated with continuous ambulatory peritoneal dialysis have a higher b4-2 excretion than do healthy subjects, suggesting a higher production of this solute in uremic patients.
Solid-phase radioimmunoassay of fragment D of human fibrinogen by use of a low-affinity monoclonal antibody.
Authors: Kennel S J , Lankford P K
Published: January 2020

Increased concentrations of plasmin-catalyzed degradation products of fibrin and fibrinogen in body fluids have been correlated with many disease states. We have developed monoclonal antibodies that react with one of these products, fragment D. Antibodies binding fragment D but not fibrinogen were found to have low affinity constants (Ka = 10(7) L/mol). A double monoclonal-antibody assay was developed in which a specific antibody attached to Sepharose beads is used to concentrate fragment D selectively from a test sample. Fragment D bound to the washed beads was then quantified with a high affinity (Ka = 10(10) L/mol) monoclonal antibody that reacts equally well with fragment D and fibrinogen. This solid-phase assay is shown to be specific and about 10-fold more sensitive than a standard liquid-phase competition radioimmunoassay in which the same antibody was used.
Interlaboratory study of the proficiency of cholinesterase phenotyping.
Authors: Evans R T , Wardell J , Rapier C
Published: January 2020

We report the results of a proficiency survey into the accuracy of cholinesterase (EC 3.1.1.8) phenotyping undertaken by 12 laboratories over a three-year period, during which 21 samples of nine different genotypes were analyzed. Specimens from patients who were homozygous for the atypical variant AA or from UA and AF heterozygotes were identified without difficulty, but the less-well-recognized combinations AJ and AK were detected by only two and three laboratories, respectively. Some degree of difficulty was also experienced in differentiating among UU, UF, and FS specimens. Clinically significant ascription errors occurred on 11 occasions. We recommend that detailed studies of cholinesterase status be undertaken only by laboratories experienced in the assay, those likely to be able to produce accurate results and offer appropriate advice.
Creatine kinase response surfaces explored by use of factorial experiments and simplex maximization.
Authors: Fast D M , Sampson E J , Whitner V S , Ali M
Published: January 2020

We conducted a five-component, five-level response-surface experiment to optimize the pH and the concentrations of magnesium, creatine phosphate, adenosine diphosphate, and buffer in an assay for creatine kinase. Under optimal conditions, creatine kinase activity was about 5% greater than that obtained with a previously reported assay (Clin Chem 23: 1569, 1977). We also applied a simplex maximization algorithm to the response-surface equation to locate areas of maximum sensitivity. Reaction conditions for two such areas were found, each yielding approximately 11% more activity than with the previously reported method.
Clinical evaluation of the liquid-chromatographic determination of urinary free cortisol.
Authors: Nakamura J , Yakata M
Published: January 2020

We recently reported (Clin. Chem. 28: 1497-1500, 1982) a liquid-chromatographic method for quantifying free cortisol in urine. We have since evaluated the clinical utility of our method by assaying cortisol in urine from normal subjects, patients, and subjects undergoing endocrine tests. We found that, in contrast with plasma cortisol, urinary cortisol is not bound to protein. It shows some correlation with 17-hydroxycorticosteroids in urine, but is independent of creatinine excretion. The amount of cortisol excreted daily by a particular individual was found to be fairly constant during nine or 10 days. Normal values determined for 203 apparently healthy individuals were 35.8 (SD 18.7) micrograms/day, with no significant sex-related differences but a tendency for a gradual decrease of cortisol excretion with age. We also report urinary cortisol excretion by patients with pituitary-adrenal disorders and some other diseases, and the pattern of response to dexamethasone and metyrapone administration.
External quality assessment of assays for cortisol in plasma: use of target data obtained by gas chromatography/mass spectrometry.
Authors: Gaskell S J , Collins C J , Thorne G C , Groom G V
Published: January 2020

We describe procedures for measuring cortisol in plasma and serum by isotope dilution and mass spectrometry. A method that incorporated solvent extraction, derivatization, and gas chromatography/high-resolution mass spectrometry provided data of good precision; interassay CVs were generally 3 to 4% for the concentration range of 100-650 nmol/L. Replacing solvent extraction with extraction on a column of Lipidex 1000 or extraction by immunoadsorption yielded data in excellent agreement with the first method. Plasma and serum pools were analyzed to provide target data for use in the U.K. National External Quality Assessment Scheme for cortisol assays. Routine laboratory assays, as judged by comparison with mass-spectrometric data, were generally positively biased except for analysis of a charcoal-stripped plasma supplemented with cortisol. The results emphasize the importance of using unadulterated plasma or serum pools in assessments of steroid assay procedures.
Osmometry of CO2 in gas samples.
Authors: Oh M S , Kee C S , Uribarri J , Carroll H J
Published: January 2020

Measurement of solute concentrations in biologic fluids by an osmometric technique has been described previously. Here, we describe an osmometric technique for measuring CO2 in gas samples. A solution of NaOH is injected into a graduated syringe containing the gas sample and CO2 is trapped in the reaction: 2NaOH + CO2 leads to Na2CO3 + H2O. The decrease in osmolality of the NaOH solution allows estimation of pCO2 in the original gas sample.
Variations in amylase isoenzymes and lipase during acute pancreatitis, and in other disorders causing hyperamylasemia.
Authors: Leclerc P , Forest J C
Published: January 2020

We compare the clinical value of assay of amylase (EC 3.2.1.1) isoenzymes with that of lipase (EC 3.1.1.3) in serum from patients with proven acute pancreatitis or with hyperamylasemia from other causes. In the former group we measured amylase, lipase, and isoamylases daily. Lipase and P(pancreas)-type isoamylases reached the highest mean values on the first day of an attack of acute pancreatitis (day one). Lipase declined rapidly, and by day four its mean activity was about the same as that of amylase and lower than that of the P-type isoamylases. Great inter-individual variations were found among patients with a similar clinical course. Of the 85 samples analyzed, amylase activity for 36 declined to within reference limits, but 18 of the 36 had high lipase activity, 18 had high P-type isoamylases activity, and 31 had P3 isoenzyme, which is not detectable in normal sera. Determination of isoamylases is a more sensitive index to acute pancreatitis than lipase assay and may be particularly useful when pancreatitis is suspected despite a normal total amylase activity. In the group of patients with hyperamylasemia from other origins, three had macroamylasemia, one had mumps, one had abdominal trauma without pancreatic injury, and one had pelvic inflammatory disease. The specific pattern of macroamylase on electrophoresis permitted a precise diagnosis of macroamylasemia; normal lipase had only ruled out pancreatitis. In the three other cases, lipase and isoamylases excluded pancreatic involvement.
125I-labeled radioimmunoassay kits for progesterone evaluated for use in an vitro fertilization program.
Authors: Blight L F , White G H
Published: January 2020

We have evaluated two commercially available 125I radioimmunoassay kits (Diagnostic Products Corp., DPC; and Radioassay Systems Laboratories, RSL) for measurement of serum or plasma progesterone, to determine their suitability for use in in vitro fertilization programs. Both kits were suitably rapid for program requirements. Results by both were linear with concentration up to 60 nmol/L, and both had acceptable lower detection limits of 0.3 nmol/L. Kit-determined progesterone concentrations (y) for 100 patients' samples correlated well with results by our existing 3H radioimmunoassay method (y = 1.11x + 0.2, r = 0.965 for the DPC kit; y = 1.01x + 1.4, r = 0.974 for the RSL kit). Mean analytical recovery for the RSL kit was 116%, that for the DPC kit, 202%. Within-batch precision, expressed as the mean CV for three concentrations of progesterone, was 6.5% for the RSL kit, and 16.4% for the DPC kit; between-day CV was 8.1% for the RSL kit, 17.7% for the DPC kit. We conclude that the RSL kit provides a rapid, precise, and accurate assay for serum progesterone, suitable for use in a fertilization program, but do not recommend the DPC kit for either this purpose or the more general purpose of tracking menstrual cycles.
Pseudocholinesterase/high-density lipoprotein cholesterol ratio in serum of normal persons and of hyperlipoproteinemics.
Authors: Jain R , Kutty K M , Huang S N , Kean K
Published: January 2020

The proposed complementary risk factor, pseudocholinesterase/high-density lipoprotein cholesterol ratio, was significantly higher in patients with type IIb and IV hyperlipoproteinemias then in controls. In contrast, the established risk factor, total cholesterol/high-density lipoprotein cholesterol ratio, was significantly higher in patients with type IIa and IV hyperlipoproteinemias. Discriminant analysis indicated that prediction of risk for coronary heart disease on the basis of lipoprotein phenotypes can be improved by about 20% when both the above factors are assessed concurrently. On the basis of earlier studies in humans and animals, we also suggest that the proposed risk factor may provide a better understanding of events leading to enhanced risk for coronary heart disease as a consequence of nutrition and of abnormal metabolism of lipids and lipoproteins.
Reflectance digital matrix photometry.
Authors: Neeley W E , Zettner A
Published: January 2020

We have designed and constructed a digital matrix photometer for quantitative measurement of reflected light of small chromophoric areas or colored spots. The areas are divided conceptually into small subunits in which the reflected light is measured. This is done through stepwise scanning with a photodiode array. The sum of the individual reflectance measurements is related to the total amount of an analyte in the spot. We have found that this technique makes possible accurate and precise quantitation of light-absorbing substances in small areas of different sizes, shapes, and internal irregularity measuring as little as 1 mm across.
Simultaneous determination of phenytoin and phenobarbital in serum or plasma by substrate-labeled fluorescent immunoassay.
Authors: Dean K J , Thompson S G , Burd J F , Buckler R T
Published: January 2020

This assay system for simultaneously determining phenytoin and phenobarbital in serum and plasma is based on the substrate-labeled fluorescent immunoassay technique. A beta-galactosylcoumarin derivative of phenobarbital and a 4-methylcoumarin phosphodiester derivative of phenytoin are used as substrate labels for Escherichia coli beta-galactosidase and Crotalus atrox phosphodiesterase I, respectively. The smallest measurable concentrations are about 1.6 mg/L for phenytoin, 2.7 mg/L for phenobarbital. Within-run coefficients of variation are about 5% for phenytoin and 2% for phenobarbital, about 6% for both between-runs. Results for phenytoin and phenobarbital in serum and plasma correlate well with those determined by the Ames TDA (r = 0.944 and 0.986, respectively) and Syva's EMIT (r = 0.977 and 0.969, respectively) assays.
Direct spectrophotometric assay for angiotensin-converting enzyme in serum.
Authors: Ronca-Testoni S
Published: January 2020

The appropriate conditions for determining angiotensin-converting enzyme in human serum with use of 2-furanacryloyl-L-phenylalanylglycylglycine as substrate are described. The method, a modification of that reported by Holmquist et al. (Anal. Biochem. 95:540-548, 1979) for purified rabbit lung enzyme, is based on the blue shift of the absorption spectrum that occurs upon hydrolysis of the substrate into furanacryloyl-L-phenylalanine and glycylglycine. The Km value for the substrate is 0.31 mmol/L. Some kinetic properties determined by this method are similar to those previously reported for the purified serum enzyme. Results by the present assay and Cushman's modified method correlate closely (r = 0.995). The normal reference interval for 42 adult donors was 43-137 U/L (mean 90 U/L, SD 23 U/L). The within-run and between-run CVs ranged from 3.0 to 4.1%. Its rapidity, simplicity, sensitivity, and precision make the proposed method very suitable for routine work, clinical investigation, and kinetic and structural studies of the serum enzyme.
Kinetic measurement of total amylase and isoamylase activities with a centrifugal analyzer.
Authors: de Rijke D , Kreutzer H J
Published: January 2020

We evaluated a new kinetic assay for alpha-amylase (Phadebas IsoAmylase Test), using modified starch as the substrate and a CentrifiChem 400 centrifugal analyzer system. We determined isoamylase activities by using a selective inhibitor. Results were compared with those obtained with the chromogenic Phadebas dyed-starch technique. The method for total amylase appeared to be rapid and precise (CV = 4%) and results correlated well with the chromogenic method. Samples with activities up to 3000 arb. units/L can be analyzed without dilution. Glucose and pyruvate interfere with the assay, but hemoglobin and bilirubin do not. Pancreatic (P) and salivary (S) isoamylase activity can be determined with acceptable precision (CVP = 8%, CVS = 10%) by an automated routine procedure with commercially available reagents.
Interference from renal dialysis patients' specimens in a direct method for serum iron.
Authors: Bunting P S , Aggarwal M
Published: January 2020

In a direct-measuring, sample-blank procedure for serum iron we obtained spuriously high results for renal hemodialysis patients. These spurious results were found to be the result of protein precipitating out of solution owing to anticoagulation of these patients with heparin during hemodialysis. Two methods are described for circumventing this interference: adjusting the pH of the buffer and adding sodium dodecyl sulfate (lauryl sulfate). We prefer the latter procedure for reasons which are discussed, and optimized conditions are described for it. The effects on results by this latter method of bilirubin, hemolysis, and turbidity are described. The method is also evaluated for precision and accuracy, and it is recommended for routine use. A significant "protein effect" (due to viscosity) is noted, which has implications for calibration of the American Monitor KDA instrument.
Boehringer immunoinhibition procedure for creatine kinase-MB evaluated and compared with column ion-exchange chromatography.
Authors: ter Welle H F , Baartscheer T , Fiolet J W
Published: January 2020

In determination of creatine kinase isoenzyme MB (CK-MB), the Boehringer immunoinhibition method gives a high and variable blank activity as compared with column-chromatography. Thus a correction must be applied. Furthermore, a second correction of 1% of total creatine kinase activity is necessary to compensate for nonspecific creatine phosphate-dependent activity. As a consequence, two immunoinhibition determinations--one for CK-MB and one for blank activity--and a determination of total creatine kinase are required. Use of the manufacturer's diagnostic criteria, on the basis of which suspected myocardial infarction is confirmed or eliminated, leads to a high frequency of false-negative conclusions.
Bioluminescent assay for total bile acids in serum with use of bacterial luciferase.
Authors: Styrélius I , Thore A , Björkhem I
Published: January 2020

This simple, rapid, sensitive kinetic bioluminescent method for the assay of bile acids in serum involves use of 3 alpha-hydroxysteroid dehydrogenase (EC 1.1.1.50) in combination with a new, commercially available NADH Monitoring Reagent (LKB-Wallac) containing a low activity of NADH:FMN-oxidoreductase and a high activity of bacterial luciferase. Interfering dehydrogenases in serum are inactivated in the test tube with trichloroacetic acid before the assay. The standard curve is linear for concentrations of bile acids up to about 300 mumol/L. With a sample volume of 20 microL, the detection limit is about 0.2 mumol/L. The within-run precision (CV) is about 10%, both at high and low concentrations of bile acids in serum. Correlation is good (r = 0.996) between results by this method and an enzymatic method based on spectrophotometry. However, the latter method is considerably less sensitive, and it is less precise at low concentrations of bile acids.
Determination of nifedipine in serum or plasma by reversed-phase liquid chromatography.
Authors: Bach P R
Published: January 2020

Therapeutic concentrations of nifedipine in serum or plasma were measured by reversed-phase liquid chromatography, with detection by ultraviolet absorbance at 235 nm. In the procedure a disposable reversed-phase extraction column is used. A 1-mL sample is required. The method is sensitive to 3 micrograms of nifedipine per liter and the standard curve is linear to at least 400 micrograms/L. Coefficients of variation at 100 micrograms/L were 2.2% within-run, 2.8% between-run. The method has been used to determine nifedipine in patients involved in a test of its efficacy in treating muscular dystrophy.
Liquid-chromatographic determination of indole-3-acetic acid and 5-hydroxyindole-3-acetic acid in human plasma.
Authors: Martínez E , Artigas F , Suñol C , Tusell J M , Gelpí E
Published: January 2020

We describe a "high-performance" reversed-phase liquid-chromatographic method for determination of indole-3-acetic acid (I) and 5-hydroxyindole-3-acetic acid (II) in human plasma. I is eluted at 1.0 mL/min with a mixture of 1-pentanesulfonic acid (pH 3.1), methanol, and water. It is detected by fluorometry. A mixture of citric acid/sodium phosphate solution (pH 4.8) and methanol, at 1.5 mL/min, is used to elute II, which is detected with an electrochemical cell. Platelet-poor plasma samples were pretreated with HCl, perchloric acid, and trichloroacetic acid for protein precipitation. Best results were obtained with the last (protein precipitation is incomplete with HCl, while recoveries of I are concentration dependent with perchloric acid). Analytical recoveries were 58% (SD 3.1%, CV 5.3%, n = 12) and 79% (SD 3.3%, CV 5.3%, n = 9) for I and II, respectively. Concentrations of I and II in plasma ranged from 0.61 to 3.32 (mean 1.54, SD 0.59, n = 15) mumol/L and from 33.0 to 102.6 (mean 51.8, SD 20.1, n = 16) nmol/L, respectively.
Immunonephelometry of apolipoprotein A-II in hyperlipoproteinemic serum.
Authors: Heuck C C , Erbe I , Frech K , Lohse P , Münscher G
Published: January 2020

Immunonephelometry of apolipoprotein A-II (apo A-II) in serum requires that factors responsible for nonspecific increases of light scattering be eliminated. Complete delipidation is not necessary for immunonephelometry of apo A-II; in fact, light scattering of immunocomplexes from delipidated HDL is greater than from isolated apo A-II. Therefore, using the isolated peptide as a standard does not suffice for measurements in serum. The concentration of protein influences the development of light scattering and must be considered in standardizing this measurement.
A nonlinear regression-kinetic method for quantification of serum triglycerides.
Authors: Hamilton S D , Skoug J W , Pardue H L
Published: January 2020

We report a new kinetic approach to the quantification of triglycerides (triacylglycerols) in human sera. The new approach combines a commercially available enzyme-reagent system with a multiple-linear-regression data-processing method. Values of absorbance collected at 3-s intervals between 20 and 305 s are fitted to a pseudo-first-order model to compute the total change in absorbance expected if the reaction were to proceed to equilibrium. The computed absorbance change varies linearly with triglyceride concentration between 3 and 20 mumol/L (after 250-fold dilution of sample). The pooled relative standard deviation was 7.2% for 23 serum samples with triglyceride concentrations between 3 and 20 mumol/L. Comparison of kinetic (y) and equilibrium (x) absorbance changes for the 23 sera gave a least-squares equation of y = 0.994x + 0.00066, confirming good agreement between the methods. The temperature coefficient is less than 0.2% per degree Celsius.
Sample preparation with ion-exchange resin before liquid-chromatographic determination of plasma catecholamines.
Authors: Frayn K N , Maycock P F
Published: January 2020

Estimation of plasma catecholamine concentrations by "high-performance" liquid chromatography, although increasingly used, still presents difficulties. We describe a method for clean-up of plasma samples on cation-exchange resin before they are extracted with alumina. This procedure eliminates many of the interfering compounds often present in plasma extracts prepared with the alumina treatment alone. The method can be adapted to use commercially available disposable cation-exchange columns.
Free erythrocyte protoporphyrin and zinc protoporphyrin measurements compared as primary screening methods for detection of lead poisoning.
Authors: Kaul B , Slavin G , Davidow B
Published: January 2020

We compared two commonly used primary screening methods (FEP and ZnP) for detecting Pb poisoning in children, used according to three major protocols. The results showed that FEP and ZnP values were comparable for only 36% of the children examined; 54% had FEP greater than ZnP and 10% had ZnP greater than FEP. One would identify approximately twice as many children with Pb greater than 300 micrograms/L by an FEP (500 micrograms/L cutoff) as with a ZnP (400 micrograms/L cutoff) screen. With a cutoff of 350, as compared with 500 micrograms/L for ZnP, one would perform 33% additional confirmatory blood Pb determinations with a 3% increased detection of Pb intoxication. If the number of false-negative Pb specimens is to be minimized, the cutoff "action level" for hematofluorometers should be lowered from the currently recommended 500 micrograms/L of whole blood to 350 micrograms/L. Our long experience and that of other laboratories leads us to recommend a revision of Centers for Disease Control risk categories and cutoff values, depending on whether FEP or ZnP is measured in combination with a patient's blood Pb concentration. Our finding of above-normal values for ZnP and FEP with Pb concentrations less than 300 micrograms/L indicates that iron deficiency constitutes an equally important public-health problem for children in New York City.
Cathodally migrating creatine kinase in sera of patients treated with theophylline, and its diagnostic implications.
Authors: Delahunty T J
Published: January 2020

Sera from 30 patients, selected only on the basis of having serum theophylline concentrations greater than or equal to 18 mg/L, were assayed for total creatine kinase (CK), isoenzyme CK-MB, and cathodally migrating CK (mCK-2). CK-MB was measured by ion-exchange chromatography. mCK-2 was detected by electrophoresis on agarose gel, with fluorometric scanning. The mCK-2 variant was found in 37% of these 30 patients. None of the patients in the mCK-2 group had CK-MB values exceeding the cutoff value associated with myocardial infarction, so extensive cardiac ischemia was considered unlikely. A review of the patients' records supported this conclusion, and no particular association with any specific disease was found among the mCK-2 group. Investigations with use of goat anti-CK-MM and anti-theophylline indicated that neither CK-MM nor theophylline were present in the mCK-2. Results of specific radioimmunoassay for CK-B subunit were negative. The mobility of the mCK-2 peak was unresponsive to 2-mercaptoethanol, suggesting that CK of mitochondrial origin was not present. These results provide insight into the clinical relevance of mCK-2 and some information concerning its composition.
Improved turbidimetric assay for lysozyme in urine.
Authors: Houser M T
Published: January 2020

I describe an improved turbidimetric assay for lysozyme in urine. This method is simple, reproducible, linear over a 100-fold concentration range, sensitive to concentrations as low as 10 micrograms/L, and only commercially available products are required. Evaluation of urinary lysozyme concentration with this assay demonstrated a within-run and between-run CV of 9.9% and 4.8%, respectively, for normal urine, minimal chemical interference, and a mean analytical recovery of 104.8%. There were differences in the rate of clearing and in the linearity of the assay with use of three commercial preparations of Micrococcus lysodeikticus, and significant differences in results when different lysozyme standards were used. Reference values were established by use of data on 169 healthy subjects, ages six months to 61 years. Clinical efficacy of the assay was demonstrated in children with renal disorders, especially those associated with chronic renal failure or tubular dysfunction.
Microsampling on the Technicon SMAC system.
Authors: Walmsley T A , Fowler R T , Abernethy M H
Published: January 2020

The sample volume needed for a Technicon SMAC continuous-flow analyzer has been reduced for routine operation. Two options are available: 141 microL for a 17-test profile, or 224 microL, which allows the direct-sampling assays for creatinine and iron to be included. The sample decrease is achieved by the sequential dialysis of creatinine and iron, an increased sample dilution from sixfold to ninefold, the strict minimization of diluted sample stream wastage, and development of more sensitive methods for glucose and alkaline phosphatase to allow greater use of the diluted sample stream. A glycine-containing diluent increases the sensitivity of the iron method by 25% and prevents the protein precipitation that plagues the continuous-flow analysis for iron in plasma. No deterioration in performance of the analyzer has been detected during nine months of routine operation at the reduced sample size. Added advantages are the decreased consumption of calibration materials and an increased ability to do repeat tests.
High creatine kinase MB isoenzyme activity associated with a rhabdomyosarcoma.
Authors: Hickman P E , Messina S , Trotter J M , Masarei J R
Published: January 2020

A 20-year-old man was found to have high plasma creatine kinase (EC 2.7.3.2) activity in association with a rhabdomyosarcoma of the prostate. There was a very high proportion of the MB isoenzyme. Clinical findings and changes in creatine kinase activity with time showed that this increase was not ascribable to myocardial disease. We believe that the high proportion of CK-MB, confirmed by a combination of electrophoretic and immunoinhibition studies, originated from the tumor.
Novel liposome composition for a rapid colorimetric test for systemic lupus erythematosus.
Authors: Janoff A S , Carpenter-Green S , Weiner A L , Seibold J , Weissmann G , Ostro M J
Published: January 2020

This test for systemic lupus erythematosus utilizes a novel liposome composition entrapping the cation-responsive red dye Arsenazo III. In dilutions of normal sera the liposome membranes undergo a rearrangement when divalent cations are added, resulting in the release of the encapsulated dye and the rapid formation of a stable blue cation-dye complex. Microliter amounts of sera from patients with active lupus stabilize the liposome preparation such that the vesicles remain intact in the presence of the added divalent cations and thus maintain their red color for extended periods. The assay requires 1-min incubations in sera at room temperature and can be performed with standard microtiter plates, allowing the screening of large numbers of serum samples in a short time. Moreover, the unique absorption spectra of the complexed and uncomplexed dye allow for quantification of results.
An internally referenced test strip immunoassay for morphine.
Authors: Litman D J , Lee R H , Jeong H J , Tom H K , Stiso S N , Sizto N C , Ullman E F
Published: January 2020

We describe an internally referenced immunochemical test-strip for use in the rapid detection of morphine. The method is based on the "enzyme-channelling" immunoassay technique, and a glucose oxidase-horseradish peroxidase enzyme pair is used to immunospecifically generate an insoluble, colored reaction product on the test-strip surface. Test strips are composed of two active surfaces, each of which contain co-immobilized glucose oxidase and antibody. The indicator pad contains antibody directed against the drug, and the color that develops on its surface is inhibited by the presence of drug in the sample. The reference pad contains anti-peroxidase and is used to set the assay detection limit and normalize for variations in temperature, timing, and sample interference. The 10-min assay protocol involves incubating the strip in sample, then incubating it in a developer solution containing glucose, a peroxidase chromogenic substrate, and a peroxidase conjugate of the analyte. The ratio of the color formed on the indicator pad to that formed on the reference pad is used to score the test as positive or negative for drug at a predetermined concentration.