bluuebunny/crossref_metadata_2025 huggingface dataset:
An evaluation of the Du Pont aca IV: does it meet medical needs?
Authors: Lott J A , Abbott L B , Koch D D
Published: January 2020

A new bench-top analyzer from Du Pont, the "aca IV," was evaluated. To do so, we performed 10 different tests during an eight-week test period. The instrument is very precise (CVs less than 2%), results agree well with those obtained with the older acas (II and III), linearity is good in the clinically important ranges, and there is very little drift or carryover. From a medical-needs viewpoint, we found the analyzer satisfactory for all tests except possibly for calcium, for which the medical-needs criteria are so strict that few laboratories can meet them. We reached the latter conclusions by using a statistical model, described here, and from the predictive value of an abnormal test for populations with various prevalences of disease.
The upper limit of normal for thyrotropin is 3 or 4 milli-int. units/L.
Authors: Durham A P
Published: January 2020

Recently Musto et al. (Clin Chem 30: 329-330, 1984) noted that, despite the clinical importance of accurate measurement of thyrotropin at low concentrations, the upper limit of normal is well below the stated upper limit of normal of most commercial tests. Here I amplify their statement.
Preparation of urine samples for liquid-chromatographic determination of catecholamines: bonded-phase phenylboronic acid, cation-exchange resin, and alumina adsorbents compared.
Authors: Wu A H , Gornet T G
Published: January 2020

We compared results for the liquid-chromatographic determination of free norepinephrine and epinephrine in urine after purifying the catechols by the following methods: (a) acid-washed alumina, (b) weak cation-exchange resin (WCX), (c) a combination of weak cation-exchange resin followed by alumina (WCX-alumina), and (d) commercially available phenylboronic acid adsorbent. We evaluated analytical specificity, sensitivity, recovery, and turnaround time. The WCX-alumina combination produced the most sensitive and specific chromatograms for urinary catecholamines; the other methods took less processing time. Neither WCX nor alumina alone was suitable for routine work because of chromatographic interferences in a significant proportion of urines. The phenylboronic acid method is adequately sensitive and specific for norepinephrine and epinephrine, and samples can be assayed faster. Thus it provides a compromise between the high analytical performance of the WCX-alumina method and the speed of the WCX and alumina methods.
Rhabdomyolysis: two pediatric case reports.
Authors: Goldsmith B M , Hicks J M
Published: January 2020

We report two pediatric cases of rhabdomyolysis. This disease involves the destruction of skeletal muscle, which can present with myalgia and a brown-pigmented urine. The first patient presented with acute renal failure, hypertension, and hyponatremia. The second patient was pyrexic, hypernatremic, and hypokalemic, and later developed hypertension. Evidence of rhabdomyolysis in both patients included dark, o-tolidine-positive urine, granular casts in the urinary sediment, and grossly increased activities of creatine kinase (greater than 60 000 U/L) in serum. An uncommonly recognized entity in the pediatric age group, rhabdomyolysis often presents as an acute disease with severe onset but can be diagnosed with relatively simple laboratory tests.
Light chain disease and massive proteinuria.
Authors: Lessard F , Bannon P , Lepage R , Joly J G
Published: January 2020

We describe the case of a 64-year-old man with lambda type light chain disease, in whom panhypogammaglobulinemia was associated with anemia, massive Bence Jones proteinuria (24.6 g/L), and renal failure. Lambda type light chains were present in the serum.
Hypergastrinemia--a risk factor for myocardial infarction?
Authors: Lindstedt G , Bengtsson C , Lapidus L , Nyström E
Published: January 2020

We determined gastrin and pepsinogen I in serum samples obtained in 1968-69 from 256 women (175 women 54 years old and 81 women 60 years old when sampled). The concentration of gastrin in serum was significantly (p less than 0.01) and positively correlated with the incidence of myocardial infarction during a 12-year follow-up, with age taken into account as a background variable in multivariate analysis. It was not correlated with overall mortality or with the 12-year incidences of angina pectoris, electrocardiographic changes indicating ischemic heart disease, or stroke. The correlation with myocardial infarction was independent of smoking, systolic blood pressure, indices of obesity, concentrations of blood glucose during fasting and of serum triglycerides and cholesterol, and of the presence of diabetes mellitus at screening or during follow-up. Serum gastrin was significantly (p less than 0.05) related to body mass index (positive age-specific relation) and to smoking (negative age-specific relation). These findings may provide a new aspect to analysis of risk factors for myocardial infarction.
Radioenzymic determination of homocysteine in plasma and urine.
Authors: Refsum H , Helland S , Ueland P M
Published: January 2020

Using a modification of the radioenzymic assay described previously (J Biol Chem 259: 2360-2364, 1984) we measured homocysteine in freshly prepared plasma and urine from volunteers. The concentration of free homocysteine--i.e., the amount measurable in plasma after deproteinization by strong acid--was 2.27 (SEM 0.11) mumol/L for 18 men and 1.95 (SEM 0.13) mumol/L for 16 women (p greater than 0.05, not significant). About 70% of the total homocysteine in human plasma was associated with plasma proteins, and was precipitated with strong acid. The concentration of protein-bound homocysteine in plasma was 6.51 (SEM 0.32) mumol/L for men and 7.29 (SEM 0.65) mumol/L for women, a significantly (p less than 0.01) different spread. Homocysteine was rapidly released from plasma proteins in the presence of a reducing agent, dithioerythritol. By gel filtration of plasma on a "high-performance" liquid-chromatographic column, albumin was shown to be the sole carrier of homocysteine in plasma. Because the fraction bound to protein as determined by this procedure equaled that obtained by precipitation of plasma proteins with acid, we conclude that homocysteine is bound to albumin in vivo. The concentration of homocysteine in urine ranged from 3.5 to 9.5 mumol/L, about 6 mumol of homocysteine being excreted per 24 h.
Immunoenzymometric assay for insulin involving column chromatography and insulin immobilized on Sepharose.
Authors: Yamamoto R , Kimura S , Hattori S , Matsuura A , Hayakawa T
Published: January 2020

This practical assay for measuring insulin involves use of a 4 X 8 mm chromatographic column. Serum samples are incubated at 30 degrees C for 1 h with beta-D-galactosidase-labeled antibody to insulin, then passed through the 0.1-mL column containing insulin immobilized on Sepharose 4B. After the column is washed to remove the bound label, the buffer in the column is replaced with a solution of o-nitrophenyl-beta-D-galactoside. The column is then incubated at 30 degrees C for 1 h, the enzyme reaction is stopped by washing the column with sodium carbonate solution, and the absorbance of the eluate is measured at 420 nm. Results obtained by this method were compared with those by a radioimmunoassay and a solid-phase enzyme immunoassay.
Macroamylase immunoglobulins show high affinity for animal and human amylases.
Authors: Urdal P , Landaas S , Kierulf P , Strømme J H
Published: January 2020

We have examined the affinity shown by the immunoglobulin fraction from each of five sera containing macroamylase for amylases from different sources: human saliva or human, porcine, or ovine pancreas. High affinity constants, 0.4 X 10(10) to 7.2 X 10(10) L/mol, were found in competitive binding experiments with human or porcine pancreatic amylase. All but one serum yielded linear Scatchard plots, indicating that in most sera the amylase-binding immunoglobulins are homogeneous, possibly monoclonal. The immunoglobulin fractions from different sera differed in their specificity: two of them bound all four types of amylases, whereas two bound only one type. Three of the five immunoglobulin fractions showed considerably higher affinity towards one or both of the animal amylases than towards the human ones, and may be primarily directed against some animal amylase.
Fluorometric assay for phospholipase A2 in serum.
Authors: Thuren T , Virtanen J A , Lalla M , Kinnunen P K
Published: January 2020

In this fluorometric assay for phospholipase A2 (EC 3.1.1.4) in serum we use the fluorescent phospholipid analog 1-octacosanyl-2-(pyren-1-yl)hexanoyl-sn-glycero-3-phosphatid yl monomethyl ester as substrate. The optimized conditions are: 28 mumol of the substrate per liter of Tris buffer (20 mmol/L, pH 7.4). The hydrolytic reaction is allowed to proceed for 30 min at 37 degrees C. The fluorescent reaction product, (pyren-1-yl)hexanoic acid, is then separated from the unreacted substrate by liquid-liquid phase partition. The concentration of the liberated fatty acid analog is determined fluorometrically. The detection limit is approximately 6 pmol min-1 mL-1. Forty duplicate samples can be assayed in about 2 h. The activity of this enzyme in serum of 20 healthy volunteers averaged 69 pmol min-1 mL-1. For seven patients with clinically diagnosed pancreatitis the average activity was 1092 pmol min-1 mL-1.
Evaluation of a benchtop capillary gas chromatograph-mass spectrometer for clinical toxicology.
Authors: Deutsch D G , Bergert R J
Published: January 2020

We evaluated the Hewlett-Packard 5995B benchtop capillary gas chromatograph-mass spectrometer (GC-MS) for its ability to identify drugs commonly detected and (or) measured in the clinical toxicology laboratory. Initial experiments indicated that the instrument as originally configured, with an isolation valve between the gas chromatograph and mass spectrometer, was unsatisfactory for the identification of hypnotics-sedatives. However, with the capillary inserted directly into the ion source, we could detect 10 ng of these drugs on a total-ion chromatogram. The software programs cause the instrument to be highly automated. In terms of ease of operation and speed it was found suitable for use in a routine clinical laboratory. Chromatography of urine extracts on the capillary gas chromatograph-mass spectrometer yielded excellent resolution of parent compounds and metabolites (e.g., diphenhydramine together with approximately four metabolites and propoxyphene with four metabolites). However, the manufacturer's computer program used to evaluate the quality of the match between the experimental mass spectra and the 375 drug reference spectra was only moderately successful in identifying unknown compounds. The ability of this capillary GC-MS to identify most compounds with a high degree of confidence will be increased by enlarging the library to include more drugs and metabolites and by using a more reliable computerized matching program.
A radioimmunoassay for 18-hydroxycortisol in plasma and urine.
Authors: Corrie J E , Edwards C R , Budd P S
Published: January 2020

Increased excretion of 18-hydroxycortisol has been proposed as a specific biochemical marker for differential diagnosis of primary aldosteronism. We describe the development of a direct RIA with an 125I label that permits measurement of the steroid in less than or equal to 0.5 microL of urine or less than or equal to 25 microL of plasma. For control subjects, the mean concentrations of 18-hydroxycortisol in urine and plasma were 310 (SD 178) nmol/24 h (n = 32) and 2.27 (SD 0.80) nmol/L (n = 37), respectively; patients with Conn's adenoma or glucocorticoid-remediable aldosteronism had values for urine in the range 1084 to 6534 nmol/24 h and concentrations in plasma ranging from 6.49 to 31.20 nmol/L. Patients with idiopathic zona glomerulosa hyperplasia had values for urine and plasma ranging from 353 to 734 nmol/24 h and from 0.26 to 6.60 nmol/L, respectively. Concentrations of 18-hydroxycortisol in urine clearly discriminate patients with idiopathic hyperplasia from those with other forms of primary aldosteronism, but further work is required to assess the diagnostic accuracy of determinations in plasma.
Evaluation of five kits for detecting choriogonadotropin in urine.
Authors: Sheehan C , Carreiro E
Published: January 2020

We report an evaluation of the performance characteristics, accuracy, and sensitivity of five test kits designed to detect human choriogonadotropin (hCG) in urine: "Sensitex" (latex agglutination inhibition), "beta-Neocept" (hemagglutination inhibition), "Duoclone" (direct agglutination), "Tandem-Visual HCG" (enzyme immunoassay), and "Sensichrome" (enzyme immunoassay). For comparison, all specimens and dilutions were also quantified by a radioimmunoassay. The accuracy of these kits was excellent and the manufacturers' stated sensitivity was confirmed. We conclude that the enzyme immunoassay method is the most sensitive procedure for qualitatively detecting hCG.
Concentrations of protease and anti-protease in serum of patients with pancreatic cancer.
Authors: Buamah P K , Skillen A W
Published: January 2020

We measured the concentrations of trypsin, elastase, and three anti-proteases-alpha 1-macroglobulin, alpha 1-antitrypsin, and alpha 1-antichymotrypsin-in serum from 10 patients with pancreatic carcinoma. All 10 showed increased elastase and decreased alpha 2-macroglobulin concentrations, nine had increased alpha 1-antichymotrypsin, and eight had increases in alpha 1-antitrypsin and trypsin. Serial studies during chemotherapy of one patient showed that the protease concentrations decreased during treatment but the concentrations of the anti-proteases remained abnormal.
A rapid, sensitive enzyme-linked immunoassay for human thyrotropin.
Authors: Tseng Y C , Burman K D , Baker J R , Wartofsky L
Published: January 2020

In this enzyme-linked immunoassay for human thyrotropin (TSH) in unextracted serum we use 96-well immunoenzymometric assay plates, first coated with polyclonal antibody to TSH, then incubated with the serum samples and reacted with mouse monoclonal antibody to human TSH. After incubation with alkaline phosphatase-labeled antibody against mouse IgG, disodium p-nitrophenyl phosphate is added and the color change is measured spectrophotometrically. Assay sensitivity is 0.1 milli-int. unit/L. Cross reactivity with lutropin, follitropin, or choriogonadotropin was negligible. TSH concentrations ranged from 0.4 to 4.1 milli-int. units/L in 43 normal subjects (mean 2.0, SD 1.0), and were uniformly less than 0.3 milli-int. unit/L in 23 patients with hyperthyroidism. Features which make this assay advantageous to the clinical laboratory include ease of set-up, ability to assay many samples at a time, high sensitivity, rapid turnaround time (8 h), and absence of requirements for radioactive materials.
Single-reagent polarization fluoroimmunoassay for amphetamine in urine.
Authors: Colbert D L , Gallacher G , Mainwaring-Burton R W
Published: January 2020

This simple polarization fluoroimmunoassay for detection of amphetamine in urine involves the use of a single reagent: suitably diluted antiserum plus fluorescein-labeled amphetamine as tracer. To this, one adds 10 microL of urine, incubates for a few minutes at room temperature, and measures the fluorescence polarization. By using an antiserum obtained against an immunogen conjugated via the para position of the drug's phenyl ring, we have attained greater specificity than that of most existing immunoassays. As little as 1 mg of amphetamine per liter was easily detected. Results for 266 patients' urines agreed completely with those by gas-liquid chromatography. When a nonseparation enzymoimmunoassay (EMIT-d.a.u., Syva) was applied to 62 specimens chosen without conscious bias, three results were falsely negative, three falsely positive.
Liquid-chromatographic determination of propisomide and its mono-N-dealkylated metabolite in plasma and urine.
Authors: Houin G , Jeanniot J P , Ledudal P , Barré J , Tillement J P
Published: January 2020

We describe a "high-performance" liquid-chromatographic assay for simultaneously determining propisomide and its mono-N-dealkylated metabolite in plasma and urine. After extraction with dichloromethane at alkaline pH, the unchanged drug, its metabolite, and the internal standard are separated by liquid chromatography on a reversed-phase column and the absorbance of the eluate is measured at 254 nm. Selectivity, sensitivity, and reproducibility are excellent. Results are similar to those by gas chromatography for propisomide but, in addition, the metabolite can be simultaneously measured in the same clinical sample. We also report results by this method for blood and plasma samples from a volunteer receiving a single 200-mg dose of propisomide.
Volatile organic compounds in exhaled air from patients with lung cancer.
Authors: Gordon S M , Szidon J P , Krotoszynski B K , Gibbons R D , O'Neill H J
Published: January 2020

Using a specially developed breath collection technique and computer-assisted gas chromatography/mass spectrometry (GC/MS), we have identified in the exhaled air of lung cancer patients several volatile organic compounds that appear to be associated with the disease. The GC/MS profiles of 12 samples from lung cancer patients and 17 control samples were analyzed by using general computerized statistical procedures to distinguish lung cancer patients from controls. The selected volatile compounds had sufficient diagnostic power in the GC/MS profiles to allow almost complete differentiation between the two groups in a limited patient population.
Rapid quantitative, specific measurement of pancreatic amylase in serum with use of a monoclonal antibody.
Authors: Mifflin T E , Benjamin D C , Bruns D E
Published: January 2020

In this rapid quantitative assay for pancreatic alpha-amylase (EC 3.2.1.1) in serum, we precipitate salivary amylases by 10-min incubation with monoclonal anti-salivary amylase antibody immobilized on particles of polyvinylidene fluoride. We then centrifuge the serum mixture and measure the pancreatic amylase activity remaining in the supernate by a kinetic method. The assay requires 50 microL of serum and the standard curve is linear to at least 1300 U of pancreatic amylase per liter of serum. CVs were 1.3% within-run, 6-8% day-to-day. Apparent analytical recovery of pancreatic amylase activity added to serum was 101% +/- 2%. Addition of purified salivary amylase, 356 U/L, to sera gave a value for apparent pancreatic amylase of less than 4 U/L, or 1% of the added salivary amylase activity. This assay correlated well with an electrophoretic method (slope, 0.97-0.99; intercept, 0.5 to -4 U/L; correlation coefficient, 0.946-0.990; and standard error of the estimate 3-5 U/L). Estimated normal reference intervals with maltotetraose as substrate were: total amylase, 39-118 U/L; pancreatic amylase, 11-50 U/L; and salivary amylase, 18-79 U/L.
Two radioassays for serum vitamin B12 and folate determination compared in a reference interval study.
Authors: Christenson R H , Dent G A , Tuszynski A
Published: January 2020

For 154 subjects, we verified that vitamin B12 and folate status was normal, using as criteria the average polymorphonuclear lobe count, mean corpuscular volume, and hemoglobin concentration. We then used blood from these subjects to compare values obtained with two radioassay kits, each designed for simultaneous vitamin B12 and folate determination. Although regression analysis showed reasonable correlation between the folate (r = 0.87) and vitamin B12 (r = 0.94) kits, we observed significant differences in the overall mean values for vitamin B12 (p less than 0.01) and folate (p less than 0.001) as measured with the kits in this population. Radioassay standard-curve data for the folate assays were similar, but these data indicated greater sensitivity at low concentrations for one vitamin B12 kit than the other. Using reference intervals recommended in the kit inserts, we found that the vitamin B12 status for 9% of these subjects would have been misclassified by one kit, 2% by the other.
Concentrations of lipids, lipoprotein, and apolipoproteins in serum of Zimbabwean Africans.
Authors: Gomo Z A
Published: January 2020

The subjects in this study were volunteers from a Zimbabwean population: 794 men and 705 women, ages between 20 and 65 years. They were receiving no medication and had no disease that could influence lipid metabolism. For determination of high-density lipoprotein cholesterol and apolipoproteins, they were screened for the known risk factors for coronary heart disease, to exclude factors known to influence those analytes. The results showed a significant sex- and age-dependence. The means and ranges for cholesterol, apolipoprotein B, and triglycerides were lower than those found in European populations. The high-density lipoprotein cholesterol and apolipoprotein A concentrations, on the other hand, were higher than in the European populations. This study established the reference ranges of the analytes studied and suggests that the prevalence of coronary heart disease may be low in Zimbabwean Africans.
Coupling aminohexyl-FAD to proteins with dimethyladipimidate.
Authors: Schroeder H R , Dean C L , Johnson P K , Morris D L , Hurtle R L
Published: January 2020

We used an efficient method having general applicability to couple N6-aminohexyl-flavin adenine dinucleotide (AHFAD) to several proteins for use in an apoenzyme reactivation immunoassay system (ARIS). AHFAD is first activated with 40-fold molar excess of dimethyladipimidate, excess imidate is removed rapidly by gel filtration, the activated product is incubated with the protein, and the conjugate formed is purified. This labeling technique permits incorporation of a controlled amount of amino-label into a protein, and eliminates the possibility of self-crosslinking, which would reduce the immunoreactivity of the conjugate. Here we demonstrate the utility of such a conjugate in a totally automated ARIS assay for thyroxin-binding globulin (TBG). After a competitive protein-binding reaction, apoglucose oxidase is added to combine with free TBG-AHFAD conjugate and produce active glucose oxidase, which is measured colorimetrically in a peroxidase-linked reaction. The assay covers the clinically significant range for TBG from 0 to 60 mg/L and has a throughput of 60 reactions in 75 min. Comparison with an RIA method (x) by regression analysis yielded the equation y = 0.890x + 1.217 (r = 0.975, n = 47, Syx = 1.906 mg/L).
Subtyping lymphocytes in peripheral blood by immunoperoxidase labeling and light scatter/absorption flow cytometry.
Authors: Kim Y R , Martin G , Paseltiner L , Ansley H , Ornstein L , Kanter R J
Published: January 2020

Lymphocyte subpopulations in a whole-blood sample can be detected by adapting mouse monoclonal antibodies (MAbs) and peroxidase (EC 1.11.1.7) labeling to a flow cytometer equipped with a tungsten-halogen light source and scatter/absorption optics (Technicon H6000). In the optimized cytochemical conditions each cell population generates a distinct, well-separated cluster, for accurate "thresholding" of the surface-antigen negative and positive lymphocyte populations in the presence of other leukocytes. After reaction with MAb, the erythrocytes are lysed, and the lymphocytes and other leukocytes are fixed. Biotinylated anti-mouse IgG, used as a bridge, amplifies the response from the avidin-peroxidase label. Granulocytes and monocytes, which have high endogenous peroxidase activity, and the labeled lymphocytes are stained in a specific amount of hydrogen peroxide plus 4-chloro-1-naphthol in 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid buffer. Accuracy and precision are equivalent to those of flow cytometers that measure immunofluorescence (e.g., Ortho Spectrum III), as demonstrated with OKT3, OKT4, OKT8, OKT11, and Leu 12 MAbs.
Gene probes: application to prenatal and postnatal diagnosis of genetic disease.
Authors: Kazazian H H
Published: January 2020

Gene probes can now be used to detect a variety of mutations that produce single-gene disorders. In present clinical practice, restriction endonuclease analysis is used for the prenatal diagnosis of sickle cell anemia, alpha-thalassemia, and beta-thalassemia. Direct detection of the mutation is possible in alpha-thalassemia, where a deletion has usually occurred, and in sickle cell anemia, where the mutation alters the recognition sequence of the restriction endonuclease, Mst II. Indirect detection of beta-thalassemia is based on using normal variations in DNA (DNA polymorphisms) to track normal and affected beta-globin genes in families. This latter kind of analysis is also useful in detecting the phenylalanine hydroxylase genes affected in phenylketonuria and will often be used in disorders where the mutations are unknown. In cases where the mutation is known, direct analysis by use of oligonucleotide probes is a new and important advance. An example of this type of gene detection in a family with classical hemophilia is presented. In addition, with chorion villus biopsy, detection of these inherited diseases is feasible by the 12th week of pregnancy.
Hemoglobin interference from in vivo hemolysis.
Authors: Blank D W , Kroll M H , Ruddel M E , Elin R J
Published: January 2020

Laboratory values for specimens from a case of intravascular hemolysis showed that hemoglobin was significantly increased and thus could interfere with the determination of other analytes. We studied this problem by adding increasing amounts of purified hemoglobin (to a maximum concentration of 19.3 mg/L) to aliquots of pooled serum samples. The hemoglobin significantly interfered with the determination of only five analytes: albumin, aspartate aminotransferase, direct bilirubin, and total protein on the SMAC, and creatinine on the Astra. We propose that for cases of proven intravascular hemolysis, values for only the analytes not affected by hemoglobin should be reported. We find lactate dehydrogenase activity useful in assessing the components of in vivo hemolysis; the differences between serum and plasma values for potassium, lactate dehydrogenase, and hemoglobin are related to in vitro hemolysis. Criteria for specimen collection and assessment of type of hemolysis are proposed.
Immunochemical approach to the question of inactive subunits in cases of lactate dehydrogenase B subunit deficiency.
Authors: Maekawa M , Sudo K , Kanno T
Published: January 2020

If there were enzymatically inactive B subunits in a solution containing lactate dehydrogenase (LD) B4 prepared from the hemolysates of heterozygous individuals with LD-B subunit deficiency, the inactive subunits would compete with normal subunits for antibodies to the B4 subunit. Using the activity of normal LD-B4 as an index, we could ascertain the proportion of free antigen (LD-B4) and antibody-antigen complexes (LD-B4-anti-LD-B4) and calculate the apparent equilibrium constant, Keq. Doing so, we found the value for Keq in LD-B subunit deficiency to be smaller than that for normal controls. Evidently the expected competitive reaction takes place. We conclude that heterozygous individuals with LD-B subunit deficiency do indeed produce variant (enzymatically inactive) B subunits of LD.
Immunoradiometric assay of sex-hormone binding globulin with use of two different monoclonal antibodies.
Authors: Gershagen S , Fernlund P
Published: January 2020

Two monoclonal antibodies (MK1 and MK2) reacting with human sex-hormone binding globulin (SHBG) were obtained from mice hybridomas. The dissociation constants for the binding of SHBG to MK1 and MK2 were 7.5 and 75 pmol/L, respectively. MK1 was coupled to polyacrylamide beads with a yield of 37%, resulting in 15 mg of antibody per gram of beads. The maximal binding of SHBG by the MK1-beads was 16% of the theoretical capacity. The amount of 125l-labeled MK2 bound to MK1-beads was related to the amount of SHBG present. The system has been used for the immunoradiometric assay (IRMA) of SHBG in serum, and has been standardized with purified SHBG. Assay sensitivity is 3 micrograms/L; intra- and inter-assay (total variation) CVs were 5% and 10%, respectively. Values obtained with the assay for 100 patients' sera agreed well with those obtained with a conventional radioimmunoassay, and SHBG in a patient's serum subjected to gel chromatography eluted as a symmetrical peak with the expected retention when the effluent was analyzed with the present assay. Analytical recovery of SHBG added to serum from a man, a woman, and a pregnant woman ranged between 93% and 107%. The mean (and SD) concentrations of SHBG in sera from healthy women and men were 3.7 +/- 1.1 and 1.8 +/- 0.9 mg/L, respectively.
A radioreceptor assay in which iodocyanopindolol is used to determine propranolol and its active metabolites in unextracted serum or plasma.
Authors: Elkins R P , Kelly J F , Rosenberg B J
Published: January 2020

This rapid, sensitive, simple radioreceptor assay (RRA) for l-propranolol and its active metabolites in unextracted samples requires 5 microL of sample, a beta-adrenergic antagonist, 125-l-labeled (-)cyanopindolol (125ICYP), and turkey erythrocyte membrane receptors (Kd = 40 pmol/L). Equal volumes (100 microL) of diluted sample and 125ICYP are incubated with 500 microL of erythrocyte membranes for 30 min. Cold isotonic saline (2.5 mL) is added, and the mixture is centrifuged. The concentration of drug that inhibited receptor binding by 50% (IC50) was l-propranolol, 1.5 micrograms/L; d-propranolol, 243 micrograms/L; and 4-hydroxypropranolol, 8.8 micrograms/L. Analytical recovery of propranolol added to serum was 93 to 120%; results for 138 clinical samples tested by this method and by liquid chromatography correlated well (r = 0.96). Results correlated strongly (r = 0.98) for 23 clinical samples tested by RRA and analyzed for both propranolol and 4-hydroxypropranolol by liquid chromatography. The sensitivity of this RRA was 0.3 micrograms/L, and both intra- and interassay CVs were less than 12%. Modifications of this method could permit testing of other nonselective beta-blockers.
Effects of temperature on measurement of aspartate aminotransferase and alanine aminotransferase in commercial control sera.
Authors: Hafkenscheid J C , Kohler B E
Published: January 2020

The catalytic activity concentrations of aspartate aminotransferase (EC 2.6.1.1) and alanine aminotransferase (EC 2.6.1.2) in several commercial control sera were determined at three different temperatures. Although these sera were of various biological origins, the temperature-conversion factors calculated are nearly identical to those for native human sera. The proportional increase of the activity concentration caused by adding pyridoxal-5'-phosphate to the reaction mixture is independent of the reaction temperature. Arrhenius plots and some thermodynamic activation parameters of both enzymes are independent of the presence of the coenzyme pyridoxal-5'-phosphate.
Liquid-chromatographic determination of urinary glycosaminoglycans for differential diagnosis of genetic mucopolysaccharidoses.
Authors: Kodama C , Ototani N , Isemura M , Aikawa J , Yosizawa Z
Published: January 2020

Glycosaminoglycans in urine from patients with various mucopolysaccharidoses were digested with chondroitin ABC lyase (EC 4.2.2.4) or chondroitin AC lyase (EC 4.2.2.5), then converted into fluorescent pyridylamino derivatives and analyzed by "high-performance" liquid chromatography. Chromatograms of the chondroitin ABC lyase digests of samples from nine patients with Hunter's syndrome all showed a major peak for unsaturated disaccharide-4-sulfate, derived from dermatan sulfate, and another specific but unidentified peak (peak x). All samples from patients with Hurler's and Scheie's syndromes contained another specific component (peak y), besides the disaccharide-4-sulfate. A sample from a patient with Maroteaux-Lamy syndrome showed peaks for N-acetylgalactosamine-4-sulfate and the disaccharide-4-sulfate, but neither peak x nor peak y. A sample from a patient with Morquio's disease showed a much higher ratio of the 6-sulfate to 4-sulfate than in other diseases, and N-acetylgalactosamine-6-sulfate was detected. We tentatively identified peaks x and y as the pyridylamino derivatives of 2-O-sulfo-L-idosyluronic acid 4-O-sulfo-N-acetylgalactosamine and L-idosyluronic acid 4-O-sulfo-N-acetylgalactosamine, respectively. This method may be useful in differential diagnosis.
Creatine kinase isoenzyme MM variants in skeletal muscle and plasma from marathon runners.
Authors: Apple F S , Rogers M A , Ivy J L
Published: January 2020

We investigated the patterns of variants of creatine kinase isoenzyme MM (CK-MM) in gastrocnemius muscle and plasma sampled from male and female long-distance runners before and after a marathon race. The proportions of CK-MM variants MM1 (pI 6.90) and MM2 (pI 6.62), identified in the skeletal muscle from both sexes, did not differ significantly from those in skeletal muscle from nonrunning controls or from heart muscle. CK-MM1 was the major (84-85% of total CK-MM) variant form. Patterns of CK-MM in plasma collected from male runners 24, 48, 72, and 96 h after the race were similar to those for female runners, but we detected two new additional variants, which we designate MM1B (pI 6.76) and MM2B (pI 6.49). For both sexes the total CK-MM activities in plasma were significantly (p less than 0.05) greater after the race, but the women's total CK-MM activities were significantly (p less than 0.05) less than the men's. The rates of disappearance of MM1, MM2, and MM3 from plasma after the race differed significantly (p less than 0.05) between men and women, MM1 clearing the fastest. Determination of the CK-MM variants in plasma after strenuous exercise may be of help in assessing CK release from injured skeletal muscle.
Effect of streptokinase on fibrinogen and related proteins in plasma.
Authors: Bowie L J , Kinney R , Vye M , Eckert B , Kwaan H C
Published: January 2020

We investigated the effect of streptokinase on determination of fibrinogen and related clottable proteins in an effort to assess the relative reliability of these determinations in monitoring patients being treated with streptokinase. Citrated plasma was incubated with and without streptokinase, then assayed for fibrinogen (I), fibrin(ogen) degradation products (II), total clottable protein (III), and plasminogen (IV). Values for I decreased rapidly. Values for III and IV generally paralleled those for I, values for III lacked adequate sensitivity. II, low initially, increased to 80 mg/L in 1-2 h. Soybean trypsin inhibitor effectively stabilized all these constituents (except IV) in streptokinase-treated plasma, and we recommend that this (or some other such) inhibitor of fibrinolysis be used routinely when specimens are collected for these determinations. Data are presented that suggest that transient polymers are formed, the clinical significance of which is not known.
Reference intervals for calcium, phosphate, and alkaline phosphatase as derived on the basis of multichannel-analyzer profiles.
Authors: Sinton T J , Cowley D M , Bryant S J
Published: January 2020

In an attempt to demonstrate a rapid and economical approach to deriving reference intervals, we analyzed data on plasma calcium and phosphate from a multichannel analyzer for more than 20 000 subjects and data on alkaline phosphatase from more than 10 000 subjects. Subjects were selected by the criterion, that their results for constituents other than the one of interest were within current reference intervals. Thus we have been able to include older patients who have diseases that accompany old age, but that do not affect test results. The mean concentrations of calcium and phosphate decreased with increasing age in both sexes, except for an abrupt increase for women about the time of reaching menopause. Similarly, the mean alkaline phosphatase activity increased with age in both sexes, reflecting a skewed frequency distribution. Here also, there was an abrupt increase in the modal value for women near menopause.
Enzyme-amplified immunoassays: a new ultrasensitive assay of thyrotropin evaluated.
Authors: Clark P M , Price C P
Published: January 2020

An enzyme-amplified immunoassay for thyrotropin has been evaluated. The lower limit of detection of the assay (mean + 3 SD of the zero standard) was 0.037 milli-int.unit/L and the precision was good, giving a working range of 0.13-24.0 milli-int.units/L. The accuracy of the assay was good, as judged from analytical-recovery experiments, analysis of external quality-assessment samples and comparison with in-house assays. No significant interferences or cross reactivities were identified. Assay of a 25-microL serum sample on the microtiter plate takes 3 h. Values for healthy subjects ranged from 0.4 to 4.0 milli-int.units/L. For 32 thyrotoxic patients thyrotropin concentrations were clearly suppressed and completely distinct from normal values. Patients taking triiodothyronine also showed lower concentrations. The enzyme-amplified immunoassay may thus be a suitable "front line" test for assessing thyroid function.
Enzyme-linked immunochemical measurement of estrogen receptor in gynecologic tumors, and an overview of steroid receptors in ovarian carcinoma.
Authors: Holt J A , Bolanos J
Published: January 2020

We tested whether a newly available enzyme-linked immunoassay (EIA) validly measures estrogen receptor (ER) in gynecologic tumors. We first documented that ER so measured agreed with results by established radioligand-based assays [dextran-coated charcoal (DCC) and hydroxylapatite (HAP)] for in-house breast carcinomas and for proficiency testing specimens. Then, for gynecologic tumors, we found strong correlations between results for ER as measured by the two methods; e.g., for 27 ovarian carcinomas, r greater than or equal to 0.86. The same was true for ER measured in nine specimens of ovarian carcinoma from women who had undergone chemotherapy: r greater than or equal to 0.94. Radioinert estradiol or serum had no discernible effect on EIA measurements of ER, whereas our DCC assay was rendered uninterpretable. Evidently the EIA validly measures ER in steroidogenic tissues, including ovarian, and also in breast and uterine carcinomas. Clinical management of the latter is now based in part on results of steroid receptor assays. For ovarian carcinomas, ER assay can be helpful for determining the probable primary site of adenocarcinomas of unknown origin, and it is providing a rational basis for development of new diagnostic and therapeutic strategies.
Radial partition immunoassay applied to automated quantification of human choriogonadotropin with use of two monoclonal antibodies.
Authors: Rugg J A , Rigl C T , Leung K , Lamar S L , Welsh M , LeBlanc M , Evans S A
Published: January 2020

We describe a novel application of radial partition immunoassay to quantification of human choriogonadotropin (hCG). In this "sandwich"-type assay, two monoclonal antibodies, specific for different sites on the intact molecule are used. The solid phase consists of tabs of glass-fiber filter paper containing a pre-immobilized antibody specific for the alpha subunit of hCG. The patient's sample is first applied directly to the central "reaction zone" of the tab, allowing hCG to bind to the solid-phase antibody. Then a buffered solution containing enzyme-labeled Fab' fragments of a monoclonal antibody specific for the beta subunit of hCG is applied, initiating "sandwich" formation. Finally, a wash buffer containing a fluorogenic substrate is applied, eluting unbound conjugate to the tab periphery. Bound enzyme conjugate is quantified by measuring the rate of increase in fluorescence. Rates are converted to clinical units by comparison with a stored calibration curve. Elapsed time from sample application to results is less than 8 min. Specific performance characteristics of this assay are reported.
Turbidimetric determination of prothrombin time by clotting in a centrifugal analyzer.
Authors: Gogstad G O , Dahl K H , Christophersen A , Bjerke A
Published: January 2020

Two thromboplastin reagents ("Thrombotest" and "Normotest Automated") were used in evaluation of an automated method for determination of prothrombin time based on turbidimetric measurement of clot formation in a centrifugal analyzer. We used 60 plasma samples from patients with various diseases or being treated with oral anticoagulant and 16 normal plasma samples. Prothrombin times were calculated by a computer connected to the analyzer, a reading being made at either a certain per cent increase in total absorbance or a fixed absorbance increase. Both correlated well with the manual method (r = 0.98-0.99). The reading points best fitting the manually obtained data were estimated by minimizing the residual sum of squares in regression analyses performed at various absorbance increases. The per cent reading was better in this respect. Normotest Automated could be nearly perfectly related to the manual method, whereas Thrombotest showed a (negligibly) small deviation. Reproducibility was good within run (CV less than or equal to 3.2%) as well as between batch of the reagents, as assessed from variation in INR (CV less than or equal to 4.9%). We conclude that turbidimetry of clot formation may be validly used in automation of the prothrombin-time test. The equipment needed and the total time per analysis are about as for chromogenic substrate methods, but reagent cost is considerably lower.
Two-monoclonal-antibody sandwich-type assay for thyrotropin, with use of an avidin-biotin separation technique.
Authors: Odell W D , Griffin J , Zahradnik R
Published: January 2020

We have developed a sensitive, specific, noncompetitive, sandwich-type radioimmunoassay for human thyrotropin (hTSH), which can be performed in 30 min. The assay involves two monoclonal antibodies, selected for high affinity and specificity and also for reaction against antigenic sites on hTSH that are distal from each other. One of these antibodies is labeled with 125I; the other is conjugated covalently to biotin. Polystyrene beads were also conjugated covalently to biotin. After conjugation, the beads were incubated with avidin. These beads represent a rapid, simple method for separating hTSH-bound antibody from free antibody. The biotin-antibody-hTSH-125I-labeled antibody complexes bind to the beads and hTSH concentration is directly related to counts per minute. This assay can detect hTSH at a concentration of 0.06 milli-unit/L in serum.
Abnormal composition of apoproteins C-I, C-II, and C-III in plasma and very-low-density lipoproteins of non-insulin-dependent diabetic Chinese.
Authors: Pan X R , Cheung M C , Walden C E , Hu S X , Bierman E L , Albers J J
Published: January 2020

Apoprotein (apo) C-I, C-II, and C-III were measured in plasma from 35 white American non-diabetic subjects. The mean concentrations (mg/L) in plasma from normolipidemic and hypertriglyceridemic subjects, respectively, were apo C-I, 78 and 311; apo C-II, 37 and 200; and apo C-III, 98 and 529. These concentrations were strongly correlated with the concentrations of plasma triglyceride (TG). We also measured these apoproteins in 23 Chinese non-insulin-dependent diabetic patients (13 men, 10 women) and in eight male and 10 female non-diabetic Chinese controls. The male diabetics had significantly lower concentrations of apo C-I in plasma, and lower apo C-I/TG, apo C-II/TG, apo C-III/TG, and apo C-I/C-III ratios than did the controls. The female diabetics had significantly higher apo C-II concentrations than the controls. The median value for plasma glucose during fasting was 1400 mg/L in the male diabetic subjects, similar to the 1370 mg/L value in female diabetics. Therefore, the higher concentrations of very-low-density lipoprotein in male diabetics may not reflect hyperglycemia per se.
Separation of an inhibitor of erythropoiesis in "middle molecules" from hemodialysate from patients with chronic renal failure.
Authors: Saito A , Suzuki I , Chung T G , Okamoto T , Hotta T
Published: January 2020

A "middle molecule" inhibitor of erythropoiesis in patients with chronic renal failure was separated from a large amount of hemodialysate. Hemodialysate was passed through Amberlite XAD-4 resin, Sephadex G-50 gel, and DEAE-Sephadex A-25, followed by reversed-phase liquid chromatography. The erythroid colony assay (CFU-E) was used to detect the inhibitory effect of the sample solution. Certain fractions from the DEAE-Sephadex A-25 column showed a dose-related inhibition of CFU-E formation as great as that of standard spermine. The inhibitory effect of these fractions decreased to the control value after proteolytic digestion. The inhibitor was eluted from the liquid-chromatographic column by a solvent gradient containing 390 to 425 mL of methanol per liter. These results suggest there is an inhibitor of CFU-E with a relative molecular mass of 1000 to 10 000 and an active site composed of peptide. This technique may prove useful for separation of the inhibitor of erythropoiesis in uremic body fluid.
Minimizing between-kit variability in immunoassay for carcinoembryonic antigen by use of a common standard.
Authors: Zucchelli G C , Pilo A , Chiesa M R , Masini S , Baccini C
Published: January 2020

Between-laboratory agreement of CEA determinations collected in a multicenter study has been substantially improved (CV decreased from 60.4 to 21.6%) by converting the results from mass concentration units (ng/mL) of local kit standards to int. units/L of the international standard first IRP 73/601. Conversion factors for the kits most used were computed, during the same survey, from results of two analytical-recovery experiments in which control samples, supplemented with the international standard, were analyzed by participating laboratories (n = 96).
Liquid-chromatographic assay of urinary vanillylmandelic acid and homovanillic acid, with clean-up by on-column injection of acetonitrile or methanol.
Authors: Kodama K , Yamanaka K , Nakata T , Aoyama M
Published: January 2020

To separately measure vanillylmandelic acid (VMA) and homovanillic acid (HVA) in urine, the sample is diluted 10-fold with 50 mmol/L phosphoric acid, then exactly 5 microL is injected directly onto a 50 X 4.6 mm column of Nucleosil 3C18. Samples can be injected at 5-min intervals because all peaks after these compounds of interest are washed away. VMA is eluted from the column after 2.8 min with 50 mmol/L phosphate buffer, pH 2.2, and measured by electrochemical detection. All peaks eluting after VMA are washed away together, by injection of 50 microL of acetonitrile onto the column. HVA is eluted from the column after 3.3 min with a 100/10 (by vol) mixture of the phosphate buffer and acetonitrile. All later-eluting peaks are washed away together, by injection of 50 microL of methanol onto the column. Analytical recoveries of VMA and HVA were 98.5% and 100.6%, respectively; the CVs for various concentrations of either in urine were about 3%.
Immunoradiometric assay with use of magnetizable particles: measurement of thyrotropin in blood spots, to screen for neonatal hypothyroidism.
Authors: Waite K V , Maberly G F , Ma G , Eastman C J
Published: January 2020

We adapted a commercial immunoradiometric assay (IRMA) to measure thyrotropin in filter-paper blood spots. Two 3-mm blood spots are used for each standard and sample. These are incubated for 2 h with radiolabeled antibody and for 30 min with magnetic antibody, followed by a 10-min separation procedure. Assay sensitivity is 6 milli-int. units/L. Coefficients of variation (precision profile of the standard curve) ranged from 4.3 to 9.6%. The coefficient of correlation (r) between thyrotropin concentrations in the blood spots and in serum was 0.93. Pre-elution of the blood spots is necessary for short incubation time. Short incubation time, little need for specialized equipment, the high precision and sensitivity characteristic of IRMA, and ease of collection, transport, and storage of the blood-spot samples make this assay suitable for neonatal hypothyroid screening.