bluuebunny/crossref_metadata_2025 huggingface dataset:
Estradiol-17 beta determined in plasma by gas chromatography-mass spectrometry with selected ion monitoring of mixed silyl ether-perfluoroacyl ester derivatives and use of various stable-isotope-labeled internal standards.
Authors: Dehennin L
Published: January 2020

A highly specific method is described for measuring estradiol-17 beta (E2) in plasma by gas chromatography-mass spectrometry (GC-MS) associated with stable isotope dilution. A mixed derivative, E2-3-trimethylsilyl ether-17-heptafluorobutyrate (E2-3-TMS-17-HFB), was found to have excellent analytical properties. The specificity of the derivatization procedure exploits a unique feature of estrogens: the selective exchange of a phenolic perfluoroacyl ester for a trialkylsilyl ether. No significant differences in E2 concentration could be ascribed to the use of 2H- or 13C-labeled analogs, thus ruling out interferences from possible isotope exchange commonly attributed to deuterated compounds. Precision is closely similar to that for methods in which the more common E2-3, 17-bis(trimethylsilyl) ether and E2-3, 17-bis(heptafluorobutyrate) derivatives are used. Sensitivity and specificity of the mixed 3-TMS-17-HFB derivative allow adequate determinations of E2, even in plasma from males, in 2-mL samples. Interlaboratory mean concentrations of E2 obtained by routine immunoassays were consistently higher than the target values estimated by GC-MS, particularly at concentrations less than 100 pmol/L.
Dependence of the thyroxin/thyroxin-binding globulin (TBG) ratio and the free thyroxin index on TBG concentrations.
Authors: Nelson J C , Tomei R T
Published: January 2020

We studied the dependence of the free thyroxin (T4) index and the ratio of T4 to thyroxin-binding globulin (TBG) on TBG concentrations, using sera from cases of congenital TBG deficiency and congenital TBG excess. Two such sera with similar concentrations of albumin, transthyretin, and free T4 were mixed to provide test samples with TBG concentrations covering the range of clinical interest without changes in the other T4-binding proteins. Total T4, free T4, TBG, triiodothyronine (T3) uptake, and the free fraction of T3 in serum were measured, and we calculated the free T4 indices, T4/TBG ratios, and the free fraction of T4. A 100-fold variation in TBG concentration was associated with a 10-fold variation in total T4, a fourfold variation in T3 uptake, and a 10-fold variation in the T4/TBG ratio. As TBG concentrations increased, the T4/TBG ratio decreased and the free T4 index increased. The free T4 index did not parallel the T4/TBG ratio, and neither the T4/TBG ratio nor the free T4 index reflected the concentrations of free T4 in serum.
Solid-phase enzymoimmunoassay of estrone in saliva.
Authors: Folan J , Gosling J P , Finn M F , Fottrell P F
Published: January 2020

In this solid-phase enzymoimmunoassay for estrone in saliva, microtiter plates are used after extraction of the sample with diethyl ether. No chromatographic step is involved. The detection limit of the assay is 450 fg per well (33 pmol/L). Intra- and interassay coefficients of variation for the assay of low, medium, and high concentrations of estrone in saliva were respectively 4.2, 12.7; 5.2, 8.7; and 2.7, 5.8%. Using this assay, we found a highly significant correlation (P less than 0.001) between estrone concentrations in time-matched serum and saliva samples. The analytical procedure is rapid and relatively simple. One person can assay 50-60 saliva samples during a normal working day. We conclude that the assay is very suitable, even in small laboratories, for saliva estrone measurements, which, in facilitating serial sampling, enables dynamic observations of estrone concentrations and ovarian activity to be more easily made.
High-yield purification of the complex-forming glycoprotein in urine from normal and abnormal subjects.
Authors: Wakui H , Kobayashi R , Itoh H , Imai H , Nakamoto Y , Miura A B
Published: January 2020

Ordinary methods for purification of complex-forming glycoprotein (protein HC) or alpha 1-microglobulin, a protein closely related to protein HC, require either large volumes of urine or special collection of urine specimens from patients with tubular proteinuria. Here we describe a fast, efficient procedure for isolating protein HC in high yield from urines from healthy and diseased subjects, with use of Cibacron blue, hydroxylapatite, and gel chromatography. Using this procedure, we also obtained a considerable amount of polymeric protein HC from the urine of a patient with chronic renal failure. The pattern of charge heterogeneity and immunoreactivity with anti-protein HC differed between the polymeric and monomeric forms of protein HC. We also observed a variability in charge heterogeneity of protein HC among patients with renal disorders. These results demonstrate that this purification method is useful for further studies to elucidate the biochemical properties of protein HC and its clinical significance in renal disorders.
Liquid-chromatographic assay for free and transthyretin-bound retinol-binding protein in serum from normal humans.
Authors: Burri B J , Kutnink M A
Published: January 2020

We quantified vitamin A-transporting retinol-binding protein (RBP) in serum or plasma by size-exclusion "high-performance" liquid chromatography, using a TSK 2000 column and fluorescent detection of the bound retinol. Serum or plasma samples filtered through a 0.20-microns (pore size) Millex filter were applied directly to the column. The pH 7.0 mobile phase contained sodium phosphate, disodium EDTA, and mercaptoethanol. Two peaks with RBP immunological activity were eluted: the smaller peak containing at least 86% of the vitamin A, which was identified as transthyretin-bound RBP; the larger peak containing a small amount (less than 14%) of a highly fluorescent vitamin A-containing protein, identified as free RBP. Both free and transthyretin-bound RBP can be quantified by this method.
Pyroglutamic acidemia in an adult patient.
Authors: Creer M H , Lau B W , Jones J D , Chan K M
Published: January 2020

Pyroglutamic acidemia, a rare metabolic disorder, usually appears in infancy. It is characterized by retardation, ataxia, hemolytic anemia, and chronic acidosis and is caused by a marked deficiency of glutathione synthetase (EC 6.3.2.3) activity. This disease is inherited as an autosomal recessive trait, but the clinical condition is also detected in heterozygotes. We report an unusual case of high-anion-gap metabolic acidosis in a 52-year-old woman who was admitted with neurological complaints and breathing problems but without the characteristic clinical features of congenital glutathione synthetase deficiency. The etiology of the acidosis could not be attributed to ketoacidosis, lactic acidosis, or ingestion of methanol, salicylate, or ethylene glycol. Analysis of the patient's plasma and urine for organic acids revealed the presence of high concentrations of pyroglutamate (5-oxoproline), which remained high throughout her hospitalization.
Standardization of methods for measuring plasminogen activator inhibitor activity in human plasma.
Authors: Chandler W L , Loo S C , Nguyen S V , Schmer G , Stratton J R
Published: January 2020

We have standardized the measurement of plasminogen activator inhibitor type 1 (PAI-1) activity in plasma. One-chain tissue-type plasminogen activator (t-PA; EC 3.4.21.31; final activity, 5 int. units/mL) was incubated with plasma (final dilutions 1:4 to 1:40) in phosphate buffer (pH 7.4, ionic strength = 0.15) for 15 min at 37 degrees C, followed by acidification and measurement of residual t-PA activity by an amidolytic method. The PAI-1 activity assay was 98% specific for PAI-1 activity in samples from both pregnancy and nonpregnancy, and varied linearly with added plasma volume when the percent inhibition of t-PA was between 8% and 50%. For the standardized method, analytical recovery was 93 +/- 5%, the detection limit was 1.6 arbitrary units per milliliter (1 arb. unit of PAI-1 activity = inhibition of 1 int. unit of t-PA activity), and total imprecision was 10.2 (SD 0.7) arb. units/mL (CV = 7%, n = 20). The average PAI-1 activity in 10 healthy individuals drawn between 0800 and 1000 hours was 23.9 +/- 15.4 arb. units/mL. Compared with the standardized assay, two of three previously described assays underestimated PAI-1 activity in plasma by 77% and 85%, respectively.
Enzymatic determination of potassium in serum.
Authors: Berry M N , Mazzachi R D , Pejakovic M , Peake M J
Published: January 2020

This is a kinetic assay for measuring K+ in serum, based on the activation of pyruvate kinase (EC 2.7.1.40) by K+. We eliminated interference from Na+ and NH4+ ions, which also activate this enzyme, by including Na+-binding and NH4+-consuming reagents in the reaction mixture. The assay was developed with and evaluated in the Cobas Fara centrifugal analyzer (and has been used in other kinetic analyzers). Within-run and between-run CVs were less than 1.4% and less than 1.6%, respectively. The reaction rate per millimole of K+ per liter (0.05 delta A/min) was more than double that of the reagent blank (0.02 delta A/min). Results correlated well with those by flame photometry, and interference from bilirubin, hemoglobin, lipids, heparin, and other cations was negligible. This method, in conjunction with a previous method we have reported in which beta-galactosidase is used for measuring Na+ in serum, offers a practical alternative to the use of ion-selective electrodes and flame photometry for measuring these clinically important monovalent cations in high-throughput or "stat" biochemical analyzers.
A simplified and rapid test for acetylator phenotyping by use of the peak height ratio of two urinary caffeine metabolites.
Authors: el-Yazigi A , Chaleby K , Martin C R
Published: January 2020

We describe a simplified liquid-chromatographic test in which acetylator phenotype is determined by measuring the peak height ratio of two urinary caffeine metabolites, 5-acetylamino-6-formylamino-3-methyluracil and 1-methylxanthine. We applied this test to determine the acetylator phenotypes of 52 subjects who regularly drink coffee, tea, or caffeinated beverages. Also, we determined the acetylator phenotypes of these subjects according to a well-established sulfasalazine test, which yielded identical results. We established the reproducibility of the described test by determining the acetylator phenotypes of 10 additional subjects on two different days separated by a period of two to five weeks. Of the 52 subjects examined by both tests, 40 (76.9%) were classified as slow acetylators, which agrees well with the percentage reported elsewhere for 297 similar subjects from the Saudi population.
Concurrent measurement of flecainide acetate and propranolol by normal-phase high-performance liquid chromatography.
Authors: Rognerud C L , Ou C N
Published: January 2020

This simple, isocratic, normal-phase liquid-chromatographic method concurrently measures flecainide acetate and propranolol in 100 microL of serum within 8 min. The chromatographic system consists of a Waters "Resolve" column packed with 5-microns silica spheres and a mobile phase of ammonium sulfate (10 mmol/L, pH 6.8)/methanol (22/78 by vol), pumped at 0.9 mL/min and monitored by a fluorometer (excitation at 225 nm and emission at 340 nm). After 100 microL of serum is mixed with 200 microL of the internal standard solution [N-(2-piperidylmethyl)-2,3-bis(2,2,2-trifluoroethoxy)-benzamide HCl, 2500 micrograms/L] and 200 microL of 0.2 mol/L sodium carbonate, the sample is extracted into butanol/hexane (20/80 by vol). The organic layer is separated and evaporated, and the residue is redissolved in 200 microL of methanol; 50 microL of this is injected onto the column. Relative recovery was 100% over the assay range of 25-2000 micrograms/L for flecainide and 10-2000 micrograms/L for propranolol. Within-run CVs were less than 2% for flecainide and less than 5% for propranolol; day-to-day CVs ranged from 5.0% to 6.5% for flecainide and from 3% to 12% for propranolol.
Lactate dehydrogenase isoenzyme patterns in serum of patients with metastatic liver disease.
Authors: Rotenberg Z , Weinberger I , Davidson E , Fuchs J , Harell D , Agmon J
Published: January 2020

Total lactate dehydrogenase (LD, EC 1.1.1.27) and LD isoenzymes were determined in serum of 170 patients with metastatic liver disease, 35 of whom had multiple metastatic sites. Overall, values of LD were above normal for 78% of the 170 patients. Half of the patients had an isomorphic pattern of LD isoenzymes (i.e., relative increase in all five isoenzymes); the other half had an increased LD-4,5 pattern, mostly a solitary increase in LD-5 only. Of those patients with normal LD values, 92% had the increased LD-4,5 pattern, whereas 70% of patients with LD values greater than 350 U/L had an isomorphic pattern of LD isoenzymes. All 35 patients with multiple metastatic sites had LD activity greater than 350 U/L; in the majority of them (74%) it was greater than 500 U/L; in 31 (89%), the increase was isomorphic. The diagnostic efficiency of the combined LD greater than 225 U/L (upper limit of normal) and increased LD-4,5 test results was much better than that of LD greater than 225 U/L alone (93% vs 74%). We conclude that serum LD and LD isoenzymes should be determined in every patient with suspected liver metastatic disease. The isomorphic pattern of LD isoenzymes is apparently associated with higher values for total LD and was common among the patients with multiple metastatic sites.
Status of drugs-of-abuse testing in urine under blind conditions: an AACC study.
Authors: Frings C S , Battaglia D J , White R M
Published: January 2020

We report results of a blind study designed to determine the accuracy of drugs-of-abuse testing in urine as done in 31 laboratories across the United States. The drugs studied were amphetamines, cannabinoids, cocaine, opiates, and phencyclidine. These laboratories confirmed all positive drug results with a different analytical method. Ten urine samples were sent to each laboratory, which resulted in 1486 trials. There were no false-positive results. The overall accuracy rate was 97%. Our study demonstrates that urine drug testing can be accurate when performed by qualified staff, using up-to-date screening and confirmation methods, appropriate quality-assurance measures, and a chain of custody.
Bedside diagnosis of alcohol intoxication with a pocket-size breath-alcohol device: sampling from unconscious subjects and specificity for ethanol.
Authors: Falkensson M , Jones W , Sörbo B
Published: January 2020

We describe a novel mouth-cup device for sampling breath from unconscious subjects and analysis with a hand-held breath-alcohol instrument, the "Alcolmeter SD-2." This equipment was evaluated in healthy volunteers after they drank a moderate dose of alcohol. Three kinds of breath were analyzed: (a) end-expired air from a conventional mouth-tube, (b) breath sampled from the mouth-cup, and (c) air from a nasal tube supplied with the breath analyzer. The ethanol concentration in breath from the mouth-cup was slightly less than in end-expired air but significantly greater than in nasal air. Results with mouth-tube and mouth-cup correlated highly with blood-ethanol concentration as determined by gas chromatography; nasal-tube air correlated less well. The Alcolmeter responded not only to ethanol but also to methanol, 1-propanol, and 2-propanol, whereas ethylene glycol gave no response. The time-response curve for methanol was different, and this might permit differential diagnosis of methanol poisoning.
Development of a confirmatory enzyme-linked immunosorbent assay for HIV-1 antibodies.
Authors: Chiang C S , Grove T , Cooper M , Cuan J , Kowalski A , Parcells K , Tsunokawa M , Rosenberg M , Arcuri E , Franklin S
Published: January 2020

We subcloned six discrete protein-coding regions representing the gag (Kp24 and Kp55), env (Kp41, Kp120N, and Kp120CC), and pol (Kp66/31) gene products of the human immunodeficiency virus type 1 (HIV-1) and expressed them in Escherichia coli as fusion proteins with the first 56 residues of galactokinase. An enzyme-linked immunosorbent assay for confirming the presence of HIV-1 antibodies was developed by coating the six purified antigens on individual wells of a microtiter plate (the HIVAGEN assay). This assay yielded no false-negative results and fewer indeterminate results than the Western blot assay for 143 specimens from patients with acquired immunodeficiency syndrome (AIDS) or AIDS-related complex (ARC). Analysis of 1016 specimens from seronegative donors by the HIVAGEN assay yielded no false-positive results, and the rate of indeterminate results was substantially lower than for the Western blot assay. The HIVAGEN assay is well suited for routine confirmation of the presence of HIV-1 antibodies because it is objective, quantitative, rapid, precise, and readily automatable.
Development and validation of a two-site immunoradiometric assay for human atrial natriuretic factor in unextracted plasma.
Authors: Lewis H M , Ratcliffe W A , Stott R A , Wilkins M R , Baylis P H
Published: January 2020

This two-site immunoradiometric assay (IRMA) for human atrial natriuretic factor (ANF)99-126 in plasma utilizes a mouse monoclonal antibody raised against rat (r) ANF103-125 with specificity directed towards the ring structure of ANF, and a rabbit antiserum to human (h) ANF99-126. The monoclonal antibody is radioiodinated, and the IgG fraction of the antiserum is coated onto wells of a microtiter plate. Plasma or standard hANF99-126 (150 microL) is incubated with the radioligand in coated wells for 24 h. The detection limit is 0.9 pg per well, corresponding to 2 pmol/L, with a working range (CV less than 10%) from 4.5 to 540 pmol/L. Intra- and interassay precision are 7% and 9%, respectively, and the assay is unaffected by plasma matrix. In humans, the IRMA is specific for hANF99-126, the major circulating form of ANF, and does not cross-react with metabolites having deletions at the carboxy terminus. Plasma IRMA values in normal seated subjects were 6.6 +/- 1.5 (SEM) pmol/L and results correlated with those of an extraction RIA (r = 0.81, P less than 0.001).
Glycated hemoglobin in fractionated erythrocytes.
Authors: Nakashima K , Nishizaki O , Andoh Y , Takei H , Itai A , Yoshida Y
Published: January 2020

Erythrocytes were fractionated, by centrifugation, on the basis of cell density, which is proportional to cell age. Glycated hemoglobin (Hb A1c) in the fractionated cells was measured and compared with the Hb A1c in whole blood and with the fructosamine concentrations in plasma of normal and diabetic subjects. In normal subjects, Hb A1c increased according to the age of the erythrocytes, but hyperbolically rather than linearly, indicating the slow conversion of Hb A to Hb A1c with saturability. The greatest correlation of Hb A1c with plasma fructosamine was in younger middle-aged cells. A subgroup of subjects with impaired glucose tolerance had high Hb A1c in older cells. Hb A1c of the young cells reflects recent glycemic status, for monitoring the initial stage of the treatment of diabetics, whereas Hb A1c of the older middle-aged cells more specifically reflects the longer term glycemic status, for screening and for the long-term monitoring index for treatment of diabetes.
Cocaine and pregnancy: clinical and toxicological implications for the neonate.
Authors: Chasnoff I J , Lewis D E , Griffith D R , Willey S
Published: January 2020

Recent studies show that the rate of cocaine use by pregnant women in the United States is much higher than realized hitherto, and an increasing number of infants are being born to cocaine-using mothers. In an ongoing research project to study the effects of cocaine on pregnancy outcome, we studied 70 infants born to cocaine-using women. These infants were matched to a drug-free comparison group selected from the population of the same hospital: children of pregnant women of a similar racial and socioeconomic distribution, but with no history or evidence of licit or illicit drug use during pregnancy. Cocaine-exposed infants had lower birth weight, shorter gestation, and a smaller head circumference than control infants. Cocaine-exposed infants also had neurobehavioral abnormalities at initial evaluation and a higher rate of perinatal complications. Toxicological evaluation of urines of neonates born to cocaine-using women showed that benzoylecgonine, a primary metabolite of cocaine, persisted in the urines for as long as 120 h after delivery. We discuss the role of the immature fetal and neonatal system in the clinical and toxicological outcome of the infant, and emphasize that further long-term studies of this will be needed.
HPLC measurement of chlorophenoxy herbicides, bromoxynil, and ioxynil, in biological specimens to aid diagnosis of acute poisoning.
Authors: Flanagan R J , Ruprah M
Published: January 2020

A simple high-performance liquid-chromatographic assay for eight chlorophenoxy (2,4-D and related compounds) and two benzonitrile (bromoxynil and ioxynil) herbicides has been developed to aid in the diagnosis of acute poisoning. Sample (whole blood, plasma/serum, urine, or tissue homogenate) or standard (100 microL) is vortex-mixed (ca. 5 s) with 20 microL of internal standard solution [1.00 g/L 2,4,5-TP in 0.02 mol/L Tris buffer, pH 9.6:methanol (1 + 1)]. Dilute (0.2 mL/L) hydrochloric acid in methanol, 200 microL, is added and the mixture is again vortex-mixed (30 s). After centrifugation (9950 X g, 2 min) a 10-20 microL portion of the supernate is analyzed on a 250 X 5 mm (i.d.) Spherisorb S5 Phenyl column, with aqueous potassium dihydrogen orthophosphate (50 mmol/L, pH 3.5) and acetonitrile (3 to 1 by vol) at a flow-rate of 1.8 mL/min as eluent. The method is capable of resolving the chlorophenoxy/benzonitrile mixtures (2,4-D/MCPP, 2,4-D/DCPP, 2,4-D/ioxynil, 2,4-D/MCPP/DCPP, 2,4-D/2,4,5-T, and MCPP/ioxynil) encountered in the U.K. The limit of detection (at 240 nm) is 20 mg/L (10 mg/L for bromoxynil and ioxynil). Intra-assay and interassay CVs were less than 5% and less than 8%, respectively, for all analytes. Plasma:whole blood distribution ratios ranged from ca. 1.7 for 2,4-DB to ca. 2.0 for 2,4-D, emphasizing that results of whole-blood measurements must be multiplied by a factor of ca. 2 for comparison with plasma/serum data.
Standardization of apolipoprotein B and A-I measurements.
Authors: Albers J J , Marcovina S M
Published: January 2020

Apolipoprotein B, the major protein of low-density lipoprotein, and apolipoprotein A-I, the major protein of high-density lipoprotein, can serve as important predictors of atherosclerotic cardiovascular disease. However, these apolipoprotein measurements have not realized their full potential because of inadequate standardization. Purified apolipoprotein A-I of known absolute mass, in lyophilized form, can serve as primary standard for apolipoprotein A-I, and freshly isolated low-density lipoprotein of narrow density range can serve as primary standard for apolipoprotein B. These primary standards can be used to assign target values to secondary reference material and calibrators by reference laboratories using standardized, validated immunoassay procedures. Lyophilized serum can serve as secondary reference material for apolipoprotein A-I. Freshly frozen serum pools should be used as reference material for apolipoprotein B until more practicable materials that do not exhibit matrix interactions are developed. Implementation of proper standardization procedures can lead to significant improvements in apolipoprotein measurements.
Lipoprotein(a) quantified by an enzyme-linked immunosorbent assay with monoclonal antibodies.
Authors: Labeur C , Michiels G , Bury J , Usher D C , Rosseneu M
Published: January 2020

This new, sensitive, specific "sandwich"-type enzyme-linked immunosorbent assay (ELISA) for quantifying lipoprotein(a) [Lp(a)] in human serum and in ultracentrifugal lipoprotein fractions is based on use of a monoclonal antibody raised against apolipoprotein(a) as coating protein and a polyclonal antibody, raised against either apo B or against Lp(a) and conjugated with peroxidase, for detection of bound Lp(a). Mean intra- and interassay CVs for assay of 16 samples were 3.0% and 5.6%, respectively. Sample pretreatment with urea did not enhance Lp(a) immunoreactivity, and treatment with nonionic detergents decreased binding to the monoclonal antibody. Results correlated well (r = 0.99, n = 38) with those by radial immunodiffusion (RID). The ELISA assay, however, detects amounts corresponding to Lp(a) contents of 10 to 1000 mg/L in plasma samples diluted 1000-fold, compared with 100-500 mg/L for RID. For 92 normolipidemic subjects, the mean Lp(a) concentration was 120 (SD 130) mg/L. In patients undergoing coronary angiography, Lp(a) concentrations increased with the severity of the disease but were not correlated with either HDL cholesterol, triglycerides, apo A-I, or apo B, and only weakly with plasma cholesterol and apo A-II. These two correlations were even weaker in normal subjects, and only the correlation with total cholesterol was valid. Lp(a), measured at birth and at seven days and six months, steadily increased with age. This assay is well suited for measuring Lp(a) in plasma and in lipoprotein fractions and also for screening programs evaluating this significant genetic risk factor for the development of atherosclerosis.
Improved radioimmunoassay of atrial natriuretic peptide in plasma.
Authors: Poiesi C , Rodella A , Mantero G , Cannella G , Ferrari R , Albertini A
Published: January 2020

We describe a radioimmunoassay (RIA) for measurement of atrial natriuretic peptide (ANP), based on one-step incubation and a simplified extraction procedure. The extraction was performed on a "Supelclean LC 18" column, with 2-mL plasma samples. Use of a diiodinated tracer improved the sensitivity of the RIA method. The minimal detectable value was 5 ng/L. Simplification of the extraction procedure and simultaneous incubation of the reagents provide a method more suitable for routine standard assay of ANP than those currently available. Intra- and interassay CVs were 6% (n = 12) and 11% (n = 10), respectively. The mean concentration of ANP in plasma of 32 healthy volunteers was 33 (SEM 4) ng/L. The ANP values for plasma after one-step incubation correlated well with those determined by a commercial RIA kit: r = 0.971, slope = 1.099, intercept = 1.949 ng/L (n = 25).
Re-evaluation of the diagnostic utility of serum total creatine kinase and creatine kinase-2 in myocardial infarction.
Authors: Leung F Y , Galbraith L V , Jablonsky G , Henderson A R
Published: January 2020

The diagnostic utility of total creatine kinase activity (I), creatine kinase-2 isoenzyme activity (II), and II as a percentage of I, was examined by receiver-operating characteristic curve and likelihood ratio (LR) analyses in 310 persons admitted to the Coronary Care Unit (151 proven cases of myocardial infarction and 159 non-myocardial infarction controls), from whom blood was sampled at 6-h intervals for 48 h after the onset of chest pain. I was ineffective either as a "rule-in" or as a "rule-out" test within the first 6 h of the onset of chest pain; thereafter, it was an effective test. II was the most effective test during the entire 48-h period. III was more effective than I in the first 24-h period, but was less effective than I during the next 24-h period. The decision threshold for high test sensitivities varies with time over the entire 48-h period, but remains constant for high test specificities. It is essential to tabulate the LR(+) and LR(-) values for both test sensitivity and specificity at constant values to determine the utility of each test at each time interval for respectively ruling out or ruling in a diagnosis of myocardial infarction.
Chemiluminescent assay of alkaline phosphatase applied in an ultrasensitive enzyme immunoassay of thyrotropin.
Authors: Bronstein I , Voyta J C , Thorpe G H , Kricka L J , Armstrong G
Published: January 2020

We compared a chemiluminescent assay and a colorimetric endpoint assay for measuring an alkaline phosphatase (EC 3.1.3.1) label in an enzyme immunoassay of thyrotropin (TSH). The substrate in the chemiluminescent assay is a derivative of adamantyl 1,2-dioxetane phosphate. On dephosphorylation, catalyzed by alkaline phosphatase, the 1,2-dioxetane decomposes further and emits a glow of light (lambda max 470 nm). We modified the Hybritech Tandem-E TSH High Sensitivity assay for chemiluminescent detection of bound alkaline phosphatase label by using this substrate (with 20-, 40-, and 60-min incubations). Detection limits (mean +2 SD of zero standard) were 6.0, 5.2, and 4.5 micro-int. units/L for these incubation periods, respectively, vs 20 micro-int. units/L for the conventional colorimetric version of the assay. Comparison of results for 44 clinical specimens assayed by the chemiluminescent (20-min incubation, y) and colorimetric (60-min endpoint, x) TSH immunoassays gave statistical values of: slope = 1.17, intercept = -0.22, and r = 0.98. Hemoglobin, but not bilirubin, lipids, or protein, interfered; but these interferents were removed by the washing steps in the enzyme immunoassay.
Elimination of caffeine interference in HPLC determination of urinary nicotine and cotinine.
Authors: Thuan N T , Migueres M L , Roche D , Roussel G , Mahuzier G , Chretien J , Ekindjian O G
Published: January 2020

We report an analytical reversed-phase liquid-chromatographic procedure for quantifying nicotine and cotinine in urine, taking into account the presence of interfering caffeine frequently encountered in such specimens. These analytes are extracted from the alkalinized urine with chloroform. After evaporation of the chloroform, the residue is dissolved in methanol and injected into a chromatographic C18 column. Extraction recoveries averaged 80% to 97%. Chromatographic conditions were investigated to obviate caffeine interference. The proposed eluent mobile phase is a polar mixture of water, acetonitrile, methanol, and a pH 4 acetoacetate buffer (65/2/29/4 by vol) adjusted to pH 4.30 +/- 0.02 with triethylamine. High resolution and linearity were obtained for each analyte up to a concentration of 200 mg/L. The minimum detectable amount of each compound was 20 ng per injection, corresponding to 10 micrograms per liter of urine. Correlation with results of gas-liquid chromatography was excellent (r = 0.99). This simple, rapid procedure allows routine screening of tobacco exposure with acceptable precision: within- and between-run coefficients of variation were less than 2% and less than 5%, respectively.
Urinary organic acid excretion by babies born before 33 weeks of gestation.
Authors: Walker V , Mills G A
Published: January 2020

Using analytical procedures that are widely used by laboratories investigating metabolic disorders, we investigated urinary organic acid excretion by premature neonates who were receiving the usual clinical care. Our purpose was to provide a basis for the diagnosis of inherited organic acid defects. We analyzed 127 random (untimed) urine samples collected weekly from 22 infants of 25-32 weeks of gestation (median, 28 weeks). A wide variety of organic acids was excreted. After oximation, they were extracted with ethyl acetate and diethyl ether, derivatized to trimethylsilyl forms, and analyzed by gas-liquid chromatography on a nonpolar fused silica capillary column, with mass spectrometry for identification. Profiles for individual babies varied markedly on different occasions, reflecting their metabolic status and bacterial activity in the gut. There was no significant ketonuria. Three metabolites identified for the first time in urine from normal neonates were 2,3-butanediol, 3-hydroxy-2-butanone (acetoin), and 4-hydroxy-3-methoxyphenyllactic acid. Significantly increased excretion of 4-hydroxyphenyllactic acid and other phenolic acids occurred during parenteral feeding.
Dimethylmethylene blue-based spectrophotometry of glycosaminoglycans in untreated urine: a rapid screening procedure for mucopolysaccharidoses.
Authors: de Jong J G , Wevers R A , Laarakkers C , Poorthuis B J
Published: January 2020

Glycosaminoglycans (GAGs) are measured in urine to screen for mucopolysaccharidoses. Other assay procedures are only qualitative (spot tests), can give false-negative results (spot tests, turbidity tests), or are relatively laborious (uronic acid-carbazole test). The present spectrophotometric procedure, based on the color reaction with dimethylmethylene blue (DMB), can be performed directly on untimed urine samples without prior precipitation. Reference values were age dependent. We tested urines of 27 patients with various mucopolysaccharidoses and compared results by three other procedures (cetylpyridinium chloride turbidity tests at pH 4.8 and at pH 7.0, and the uronic acid-carbazole test). In the DMB assay, GAGs were increased in 26 of the 27 patients. The exception was a Morquio A patient, whose activity of the defective enzyme was higher than in classical Morquio patients. Uronic acid, measured in precipitated GAG by the carbazole test, was increased in 23 of the 25 patients so tested. In the turbidity test at pH 7.0, values were increased in 24 of the 27 patients. In contrast, with the citrate-buffered (pH 4.8) turbidity measurement, GAG content was increased in only 19 of the 27 patients. This rapid and easy DMB method is a reliable screening procedure for mucopolysaccharidoses and compares well with procedures used hitherto.
Depressed activities of purine enzymes in lymphocytes of patients infected with human immunodeficiency virus.
Authors: Renouf J A , Wood A , Frazer I H , Thong Y H , Chalmers A H
Published: January 2020

Enzyme activities were studied in peripheral blood lymphocytes from patients infected with, or at risk for, infection with human immunodeficiency virus (HIV). No significant differences were observed in the HIV-infected and HIV-seronegative high-risk patients with regard to enzyme activities of hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8) and purine nucleoside phosphorylase (EC 2.4.2.1) in peripheral blood. Adenosine deaminase (EC 3.5.4.4) was significantly (P less than 0.02) depressed in asymptomatic HIV-seropositive patients and HIV-seronegative patients at high risk of HIV infection as compared with a healthy HIV-seronegative population. Adenosine kinase (AK, EC 2.7.1.20) was significantly increased in the asymptomatic seropositive (P less than 0.02) and also in the HIV-seronegative high-risk groups (P = 0.01) compared with the normal controls. AK activity was significantly lower in subjects with AIDS than in the asymptomatic (P less than 0.002) and high-risk groups (P less than 0.01). Taken together, these results indicate that adenosine deaminase and AK activities are influenced by the health of the patient, and that measurement of AK activity may prove useful in monitoring the clinical progress of patients with HIV infection.
Fluorometric determination of magnesium in serum with 2-hydroxy-1-naphthaldehyde salicyloylhydrazone.
Authors: Ioannou P C , Konstantianos D G
Published: January 2020

This simple, rapid, sensitive fluorometric method for determining magnesium is based on formation of a fluorescent complex of magnesium with 2-hydroxy-1-naphthaldehyde salicyloyl-hydrazone in alkaline ethanolic medium (lambda ex = 420 nm, lambda em = 460 nm). The detection limit of the method is 0.05 microgram/L (2 nmol/L) and the relative standard deviations (CVs) are 0.8%, 1.1%, and 2.5% at magnesium concentrations of 50, 5.0, and 0.50 microgram/L, respectively. The standard curve is linear from 0 to 250 micrograms/L (0-10 mumol/L). We investigated the chemistry of the reaction, and have applied the method to determine magnesium in 100-fold-diluted, otherwise untreated serum samples. Within-run CVs for the method were 1.7%, 1.1%, and 1.3% at mean magnesium concentrations of 15.7, 26.4, and 36.2 mg/L, respectively. Day-to-day CVs were 2.7%, and 3.6% at mean magnesium concentrations of 10.1 and 25.5 mg/L, respectively. Samples from 96 hospitalized patients in intensive-care units were analyzed by the proposed method (y) and by an automated colorimetric method involving Magon sulfonate reagent (x). Linear regression analysis of the results yielded: y = 1.01x - 0.04 (r = 0.982, Sxy = 0.90).
Evaluation of the biotinylated (Blugene) vs 32P-labeled cDNA probes of beta-glucocerebrosidase: relative sensitivities in genomic and other systems.
Authors: Strasberg P
Published: January 2020

The sensitivity, rapidity, and ease of use of biotinylated (Blugene, Bethesda Research Laboratories) and 32P cDNA probes have been compared, the probe being the cDNA for beta-glucocerebrosidase (EC 3.1.2.45). With the Blugene kit I could detect 2 pg of biotinylated DNA on dot blots. However, under conditions of hybridization, the lower limit of detection for unlabeled cDNA (transblotted onto nitrocellulose) by its labeled counterpart was 5000-fold smaller (10 pg vs 50 ng) for the isotopically labeled probe. 32P- and Blugene-probes hybridized detectably with 0.5 and 10 micrograms, respectively, of transblotted EcoR 1-digested genomic DNA, making the radioactive method 20 times as sensitive. However, color development was complete within 30 min to 3 h, whereas radioautoradiography required 12 h to one week. Blugene was also safer, easy to use, and effective under appropriate conditions. The 32P method is expensive, hazardous, time-consuming, and technically difficult. This nonisotopic procedure represents a desirable improvement in biotechnology.
Tumor-derived activated cells: preliminary laboratory and clinical results.
Authors: Oldham R K , Maleckar J R , Friddell C S , Lewko W M , West W H , Yannelli J R
Published: January 2020

It is well known that T lymphocytes can mediate significant anti-tumor responses. A limiting factor has always been the ability to expand T cells, whether from the peripheral blood, spleen, or tumor. The recent availability of recombinant interleukin-2 (r-IL2) has demonstrated the feasibility of expanding T cells and the clinical efficacy of these cells as anti-tumor effectors in murine models. Concomitantly, researchers discovered that lymphokine-activated killer cells--peripheral blood cells functionally distinct from T cells--could be cultured, expanded, and re-infused in patients, with significant clinical effects. For many years, the infiltrating lymphocytes have been recognized in tumor biopsies and known to be cytolytically active. Major limiting factors were the ability to culture large numbers of these infiltrating cells and the limited understanding of the tumor antigens involved for T-cell stimulation. Restimulation by antigen (tumor cells) appears to provide the ongoing antigen stimulation needed to maintain selective killing of tumor cells. By defining various factors in the medium that support and enhance T-cell growth and activation, the components are becoming available to develop a broad attack on advanced cancer by using this laboratory-based technology of stimulation and expansion of tumor-derived activated cells.
Radioimmunoassay of testosterone not bound to sex-steroid-binding protein in plasma.
Authors: Déchaud H , Lejeune H , Garoscio-Cholet M , Mallein R , Pugeat M
Published: January 2020

To measure the concentration of testosterone (T) that is not bound to sex-steroid-binding protein (SBP) in plasma, we quantified by radioimmunoassay the T in the supernates of plasma samples after precipitation with 50%-saturated ammonium sulfate. The concentrations of non-SBP-bound T. directly measured with this assay, correlated significantly (P less than 0.001) with those deduced from measurement of the percentage of non-SBP-bound T determined with [3H]T as tracer or from mathematical models according to the law of mass action. It also correlated significantly with the ratio of T to SBP and with the concentration of nonbound T. As determined with this assay, the mean concentration of non-SBP-bound T in normal men was higher in young (4.67, SD 2.68 nmol/L; n = 30) than in older (greater than 40 years) subjects (2.48, SD 1.61 nmol/L; n = 35; P less than 0.001) and lower than normal in hyperthyroid (1.61, SD 0.91 nmol/L; P less than 0.01) or infertile men (3.28, SD 1.70 nmol/L; P less than 0.01). In women, non-SBP-bound T was higher in hirsute patients (0.24, SD 0.11 nmol/L; P less than 0.01) and was lower during pregnancy (0.09, SD 0.05 nmol/L; P less than 0.05) than in normal women during the follicular phase (0.16, SD 0.07 nmol/L). We conclude that this direct measurement of non-SBP-bound T in plasma is suitable for routine use and represents a reliable index of androgenicity in human pathology, particularly when alterations of the binding capacity of SBP modify the concentrations of total T.
Unusually high concentrations of Zn alpha 2-glycoprotein and the lack of alpha 2HS-glycoprotein in human ejaculates.
Authors: Bürgi W , Simonen S , Baudner S , Schmid K
Published: January 2020

Ejaculates of 132 nonvasectomized and 129 vasectomized men were assayed for their Zn alpha 2- and alpha 2HS-glycoprotein contents by radial immunodiffusion. The respective mean concentrations of Zn alpha 2-glycoprotein were 319 and 309 mg/L, respectively, but these fluids were devoid of alpha 2HS-glycoprotein. In comparison, the concentration of these two proteins in serum of normal men were 62 and 617 mg/L for Zn alpha 2- and alpha 2HS-glycoprotein, respectively. Because albumin in the ejaculates is 100-fold less concentrated than in serum, the fivefold increase in the concentration of Zn alpha 2-glycoprotein of the ejaculates represents about a 500-fold enrichment of this protein in the ejaculates over the concentration expected by comparison with albumin.
Oligoclonal immunoglobulins in HIV infection.
Authors: Sinclair D , Galloway E , McKenzie S , Follett E A , Wallace L
Published: January 2020

We tested 150 patients infected with human immunodeficiency virus (HIV) for the presence of oligoclonal bands in serum, prompted by reports that these abnormal proteins may have prognostic significance. Sixty HIV-negative individuals from "at-risk" groups were tested along with 80 HIV-negative, healthy blood donors for the presence of these bands. All sera were tested by isoelectric focusing, because it is more sensitive for this purpose than more-conventional electrophoretic techniques. In the HIV-positive group, 61% of the sera had oligoclonal bands; in the HIV-negative "at-risk" group, 36% had bands. No bands were detectable in sera from the healthy blood-donor group. Some patients were also followed for differing periods throughout their infection, and changes in their oligoclonal banding patterns could not be correlated with disease progression. The fact that oligoclonal bands were found to be present without HIV infection in a substantial number of individuals from within the "at-risk" groups leads us to conclude that the presence of oligoclonal bands in HIV infection is of limited prognostic significance.
Urinary excretion of transferrin by non-insulin-dependent diabetics: a marker for early complications?
Authors: Cheung C K , Cockram C S , Yeung V T , Swaminathan R
Published: January 2020

We measured concentrations of transferrin (TRF, in micrograms), and creatinine (Cr, in millimoles) in samples of untimed urine from 53 healthy subjects and 157 non-insulin-dependent diabetic (NIDD) subjects. The urinary TRF/Cr ratio was significantly higher in the NIDD group (P less than 0.001). If NIDD subjects are grouped according to their Alb/Cr ratio into normal albuminuria (Group A, Alb/Cr less than 2.5 mg/mmol), microalbuminuria (Group B, Alb/Cr 2.5-26.8 mg/mmol), and macroalbuminuria (Group C, Alb/Cr greater than 26.8 mg/mmol), the TRF/Cr ratios in all three groups exceeded those for healthy controls. Moreover, this ratio was higher in Group B than in Group A and higher in Group C than in Group B. The value for TRF/Cr was clearly abnormal (i.e., exceeded the 95th percentile value found in healthy subjects) in 61%, 95%, and 100% of Group A, B, and C subjects, respectively. The TRF/Cr ratio was significantly higher in those NIDD subjects with clinical retinopathy, and it correlated with arterial pressure. Evidently, TRF/Cr may be increased early in NIDD subjects, and it may be a sensitive marker for detecting development of complications of diabetes.
Variations in protein binding of drugs in plasma and serum.
Authors: Ohshima T , Hasegawa T , Johno I , Kitazawa S
Published: January 2020

The free fractions of five drugs were determined in plasma treated with EDTA and in serum. The free fractions of phenytoin, sodium valproate, and phenobarbital in serum were significantly higher than in plasma (P less than 0.05). In contrast, the free fractions of carbamazepine and theophylline were significantly lower in serum than in plasma. Although we observed minor differences in the protein patterns obtained with two-dimensional electrophoresis, these did not have an important influence on protein binding. No significant differences were observed between plasma and serum in the physiological data, except for pH. Binding of these drugs by protein was shown to be pH-dependent. We conclude that serum may be better than plasma for determination of the free fraction, because the free fraction in plasma is affected by anticoagulants such as EDTA salts and heparin.
Measurement of cyclosporine in plasma from patients with various transplants: HPLC radioimmunoassay with a specific monoclonal antibody compared.
Authors: McBride J H , Rodgerson D O , Park S S , Reyes A F
Published: January 2020

This study compares cyclosporin A (CsA) concentrations in plasma from patients receiving various transplants, as measured by HPLC and RIA with a monoclonal antibody for CsA and an 125I-labeled ligand. The RIA was restandardized with in-house standards because it overestimated CsA by an average of 23%. The RIA was sensitive to 2 micrograms/L, the standard curve was linear from 20 to 500 micrograms of CsA per liter, analytical recovery was 98%, and CVs were less than 8% for intra- and interassay precision. RIA (y) vs HPLC (x) for 283 plasma samples from 145 patients gave a slope = 1.1256, r = 0.979. When the results were segregated according to transplant type, CsA in liver and heart recipients was overestimated by RIA as compared with HPLC: slope = 1.202, r = 0.973 and slope = 1.1477, r = 0.983, respectively. Adult and pediatric CsA values were acceptable when RIA and HPLC were compared: slope = 1.0755, r = 0.977 and slope = 1.0563, r = 0.980, respectively. For six samples (four heart, two liver recipients) where HPLC and RIA values demonstrated wide discrepancies, repeat HPLC and analysis of eluate fractions gave CsA concentrations nearer values by the initial HPLC assay. We conclude that this RIA cannot be substituted for HPLC in the case of heart and liver recipients. The need for each laboratory to standardize the RIA is obvious.
Neopterin as a predictive marker for disease progression in human immunodeficiency virus type 1 infection.
Authors: Fuchs D , Spira T J , Hausen A , Reibnegger G , Werner E R , Felmayer G W , Wachter H
Published: January 2020

We assessed the value of urinary neopterin concentrations for prognosis of disease progression in HIV-1-infected patients. Sixty-eight anti-HIV-1 seropositive homosexuals with lymphadenopathy syndrome were tested for urinary neopterin and T-cell subset counts in 1982-83, and the incidence rate at which they developed acquired immunodeficiency syndrome (AIDS) between then and May 1988 was evaluated. Overall, 21 of 68 (30.9%) cases progressed to AIDS, with a yearly progression rate of 4-9%. The predictive value of urinary neopterin concentrations was higher (P = 0.0042) than that of CD4+ T-cell counts (P = 0.015) or the CD4+/CD8+ T-cell ratio (P = 0.022). Counts of CD8+ T-cells failed to show predictive significance (P = 0.29). Similarly, multivariate-regression analysis indicated that neopterin concentrations and CD4+ T-cell numbers were significant copredictors. Produced by human macrophages activated by interferon gamma, neopterin is thus a marker of macrophage activation via T cells. We conclude that these data demonstrate a correlation between the amount of T-cell-macrophage activation, as measured by urinary neopterin concentrations, and the progression of the disease.