bluuebunny/crossref_metadata_2025 huggingface dataset:
Stabilization of ascorbic acid in human plasma, and its liquid-chromatographic measurement.
Authors: Margolis S A , Davis T P
Published: January 2020

We describe two independent HPLC procedures for the rapid, accurate analysis for ascorbic acid in human plasma. No sample extraction or phase separation is required. We also describe a procedure for preparing a human plasma reference material for use in clinical laboratory analysis for ascorbic acid. The ascorbic acid in plasma can be determined in 15 min, with as little as 50 microL of sample. Analytical recoveries are near 100% with direct injection of deproteinized plasma. Extensive stability data under several conditions, with dithiothreitol as a preservative (antioxidant), indicate that ascorbic acid remains stable in stored plasma for as long as 57 weeks. CVs for round-robin analysis of 11 normal human blood samples by two independent methods were between 0.1% and 5.3%. These clinical samples appear to be stable for at least 50 days under the described conditions of stabilization and sample treatment. Finally, because ascorbic acid prepared by the described procedures is stable at room temperature for at least 18 h, these methods can be readily adapted to clinical laboratory automation at room temperature.
Nosocomial dysnatremia.
Authors: Neithercut W D , Spooner R J
Published: January 2020

Patients who exhibited both hyponatremia and hypernatremia during a single admission were identified by a review of their biochemistry data. The mortality of patients who developed either hypernatremia or hyponatremia, followed by the opposite abnormality within less than or equal to 10 days, was 42%. This was greater than the 8.2% mortality for an age-, sex-, and admission-matched normonatremic control group (P less than 0.01, chi 2). Eight of the 10 patients who died had had a change in the sodium concentration in serum greater than 20 mmol/L. The interval (days) between the maximum and minimum sodium concentrations in the survivors and those who died was not statistically different. We found no specific clinical features that could be used to predict the development of this biochemical abnormality.
Analytical problems encountered in determining aluminum status from hair in controls and hemodialyzed patients.
Authors: Chappuis P , Duhaux L , Paolaggi F , de Vernejoul M C , Rousselet F
Published: January 2020

The problems involved in evaluating aluminum concentrations in hair are reviewed, especially those concerning removal of contaminating metals, a critical factor. In the few published studies of Al concentrations in hair, acetone was usually used for its removal. Here, its use in the washing sequence was found to give less precise results and higher Al values than the use of isopropanol. With isopropanol, the whole analysis can be done in a single tube. We compared results with those in the literature. We found that the Al concentration in the hair of control subjects was not related to sex or hair color and that there was a highly significant (P less than 0.001) difference between values for control subjects and hemodialyzed patients: 126 (SD 58) nmol/g, n = 49, vs 226 (SD 104) nmol/g, n = 39, respectively.
Amidolytic kinetic assay of protein C by selective spectrophotometry in a centrifugal analyzer.
Authors: Kobayashi I , Amemiya N , Endo T , Motegi J , Kurihara A , Hamaoka S , Tamura K , Kume S
Published: January 2020

This rapid, simple amidolytic assay of protein C activity in whole plasma involves activation by protein C activator from the venom of Agkistrodon contortrix contortrix (Protac) and use of a Cobas Fara spectrophotometer programmed for kinetic assay. Plasma is incubated with activator venom in the presence or absence of antibody to human protein C in the instrument, chromogenic substrate (S-2366) is added, and the absorbance is measured at 405 nm. The difference between the absorbance of the sample plasma with and without antibody to human protein C correlated well with protein C antigen as assayed by enzyme-linked immunosorbent assay (ELISA) and the Laurell rocket technique in normal subjects, patients being treated with warfarin, and patients with liver cirrhosis or disseminated intravascular coagulation. Our mean value for protein C in normal subjects is 115.9 (SD 16.7)% for amidolytic activity, 103.0 (SD 17.4)% for ELISA, and 97.2 (SD 18.1)% for the rocket technique. The high value for normal subjects presumably includes some nonspecific amidolytic activity activated by the activator venom, as indicated by measurable activity in immuno-depleted protein C-deficient plasma. Within-run and between-run CVs were less than 5% at low, normal, and high concentrations of protein C amidolytic activity.
The stability of uric acid in ammonium hydroxide.
Authors: Ellerbe P , Cohen A , Welch M J , White E
Published: January 2020

We examined the stability of uric acid in dilute aqueous ammonium hydroxide solution by mass spectrometry. Uric acid decomposes in ammonium hydroxide even as dilute as 15 mmol/L when the mole ratio of ammonium hydroxide to uric acid is 50:1. There are at least four products of the decomposition, two of which have been identified as allantoin and urea. The slope of the decomposition curve indicates that uric acid is destroyed at an initial rate of 2-3% per hour. In ammonium hydroxide at a concentration of 1 mmol/L and a mole ratio of ammonium hydroxide to uric acid of less than or equal to 3.4, uric acid is not detectably decomposed. Evidently, any method for determination of uric acid that involves treating the analyte with ammonium hydroxide before analysis may destroy it. Therefore, a published method described as being "definitive" for uric acid (J Clin Chem Clin Biochem 1985; 23:129-35) could produce incorrect results because it involves storing the uric acid in 15 mmol/L ammonium hydroxide at a mole ratio of ammonium hydroxide to uric acid of greater than 120:1.
Assay of dipeptidyl peptidase IV in serum by fluorometry of 4-methoxy-2-naphthylamine.
Authors: Scharpé S , De Meester I , Vanhoof G , Hendriks D , van Sande M , Van Camp K , Yaron A
Published: January 2020

A new fluorometric assay for determining dipeptidyl peptidase IV (DPP IV; EC 3.4.14.5) was developed. The synthetic substrate glycyl-L-proline-4-methoxy-2-naphthylamide (20 mmol/L), Tris buffer (50 mmol/L, pH 8.3), and serum (20 microL) are mixed and incubated. The reaction is stopped with citrate (100 mmol/L, pH 4.0) and the released 4-methoxy-2-naphthylamine is measured fluorometrically. The mean value of DPP IV activity in serum for 64 healthy subjects was 58 (SD 16) mumol of 4-methoxy-2-naphthylamine released per liter of serum per minute. The proposed procedure is sensitive, rapid, and accurate and can easily be automated.
Evaluation of four commercially available assays for free thyroxin.
Authors: Ooi D S , Mahadevan M S , Greenway D C , Gertler S Z
Published: January 2020

We assessed two two-step and two analog assays for measuring free thyroxin (FT4) in serum: Clinical Assays' "GammaCoat Free/Total T4" (CA), Vitek's "KinetiCount Phase II Free T4" (VTK), Diagnostic Products Corporation's "Coat-a-Count Free T4" (DPC) kit (June 1987 version), and Amersham's "Amerlex-M Free T4" (AMX). The VTK assay is automated except for the initial pipetting step. Interassay results correlated well except for samples with abnormal serum albumin concentrations. FT4 values for hypoalbuminemic samples showed a highly significant (P less than 0.0001) correlation with serum albumin concentration in the DPC and AMX assays. The relationships are described by the equations y = 0.382albumin (g/L) + 0.81 pmol/L and y = 0.450albumin (g/L) - 3.20 pmol/L, respectively. When we used an equation derived from the Law of Mass Action to adjust FT4 values to values expected at an ideal albumin concentration, the observed correlation of albumin and FT4 was abolished completely in the DPC assay, and partly so in the AMX assay. The precision of CA was comparable with that of the analog assays; the CV for the VTK assay was approximately twice that for the other three assays.
Liquid-chromatographic determination of fleroxacin in serum and urine.
Authors: Awni W M , Maloney J A , Heim-Duthoy K L
Published: January 2020

This highly sensitive, accurate, and reproducible HPLC method for determining fleroxacin in human serum and urine makes use of a common C18 column, a fluorescence detector, and an internal standard. Serum samples require a simple extraction procedure; urine must be diluted. The method, which we have used extensively for pharmacokinetic assessment of fleroxacin in patients, measures concentrations as low as 5 micrograms/L.
Time-resolved pulsed fluorescence immunometric assays of carcinoembryonic antigen.
Authors: Dakubu S , Hale R , Lu A , Quick J , Solas D , Weinberg J
Published: January 2020

A time-resolved pulsed fluorescence immunometric assay (TR-PFIA) for carcinoembryonic antigen is described in which either Eu(III) or Tb(III) chelate is used as label. Described in detail is the assay involving the well-documented format of microtiter matrix and Eu(III) fluorescence enhanced with a beta-diketone and quantified in a commercial time-resolving fluorometer. We have also used the same basic assay, but one with a Tb(III) chelate as label, and we read the fluorescence signal directly off a surface without the application of enhancement solution. The Tb(III) fluorescence is then brought into solution by using an analog of dipicolinic acid in an enhancement solution. The latter approach demonstrates the scope of the methodology, which invokes the extra complexity of enhancement only when increased sensitivity might be required. The power and versatility of the enhancement methodology are demonstrated.
A reflectance photometer with a square photodiode array detector for use on multilayer dry-film slides.
Authors: Neeley W E
Published: January 2020

This semiautomated prototype reflectance photometer measures reflected light from multilayer dry-film slides. The instrument makes use of a square photodiode array detector, a Hewlett-Packard desktop computer, and a modified mechanical transport mechanism from an Ektachem DT60 analyzer. When 2 microL of serum is placed on a dry-film slide, a colored spot is formed. The slide is automatically transported to an incubation area and then to the photometer area. There the spot is illuminated with dual tungsten lamps, and the reflected light passes through an interference filter, where it is focused on a square photodiode array containing 10,000 individual detectors. The analog signal from each detector is digitized and transmitted to a computer for calculation of the percentage of reflectance. I used a series of algorithms to locate the spot, estimate spot area, correct for minor variations in sample volume, and compute the average reflectance from a central spot area. To evaluate the instrument's performance, I ran parallel glucose determinations in the Beckman Astra; results correlated well. The small sample size along with no dead sample volume makes the system useful for small sample volumes.
Increased urinary excretion of digoxin-like immunoreactive substance by insulin-dependent diabetic patients: a linkage with hypertension?
Authors: Giampietro O , Clerico A , Gregori G , Bertoli S , Del Chicca M G , Miccoli R , Lucchetti A , Cruschelli L , Navalesi R
Published: January 2020

Excretion of digoxin-like immunoreactivity (DLIS) was measured by RIA in timed overnight urine collections from 91 normotensive nondiabetic subjects and 104 normotensive insulin-dependent diabetic (IDDM) patients. The mean +/- SD DLIS excretion rate for the diabetic patients significantly exceeded that for the controls (73 +/- 41 vs 63 +/- 36 pg/min, P = 0.024). In both groups, the mean DLIS excretion rates for men were significantly higher (P = 0.0014, P = 0.006) than for women. In the controls, the DLIS excretion rate significantly correlated with the urinary excretion rate of creatinine (P less than 0.01), Na+ (P less than 0.05), and K+(P less than 0.05), and with the subjects' body weight (P less than 0.01), body mass index (P less than 0.05), and systolic blood pressure (P less than 0.05). In the diabetics, the DLIS excretion rate was significantly correlated with body weight (P less than 0.05) and with urinary excretion rates for albumin (P less than 0.01), creatinine (P less than 0.01), Na+ (P less than 0.05), and K+(P less than 0.05). Our data indicate that: (a) increased amounts of a cardiac glycoside-like substance (or a group of substances) are excreted in the urine of IDDM patients; (b) the urinary excretion of DLIS seems to depend on glomerular filtration rate and physiocochemical properties of glomerular membrane, as well as on subjects' body mass; and (c) because cardiac glycoside-like substances may increase peripheral vascular resistance, increased urinary excretion of DLIS by IDDM patients may indicate a tendency to develop hypertension.
Creatine kinase:aspartate aminotransferase activity ratio as an indicator of the source of an increased creatine kinase activity.
Authors: Dufour D R
Published: January 2020

Although measurements of creatine kinase isoenzyme 2 (CK-MB) are often used to diagnose acute myocardial infarction, their sensitivity and specificity are less than 100%. Because skeletal muscle contains more CK and less aspartate aminotransferase (AST) than cardiac muscle, the CK/AST ratio might provide a useful adjunct in evaluating the source of a supranormal value for CK. I established the following decision levels in a retrospective study of 342 patients: ratios less than 14 (if total CK was 300-1200 U/L), less than 20 (CK 1201-2000 U/L), or less than 25 (CK greater than 2000 U/L) suggested myocardial infarction, with a sensitivity of 95% and a specificity of 65%. In a validation study with 277 additional patients, liver disease and alcohol abuse caused erroneous results, leading to exclusion of 22% of these patients. In the remaining cases, sensitivity was 94%, specificity 90%. The CK/AST ratios changed little with time, suggesting that a single value would be adequate for evaluating patients with increased CK.
Concomitant purification of prostatic carcinoma tumor markers from human seminal fluid under nondenaturing conditions.
Authors: Rusciano D , Berardi A , Ceccarini C , Terrana B
Published: January 2020

We report a protocol for concomitant purification to homogeneity of both prostatic acid phosphatase [orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2] and prostate-specific antigen, from human seminal fluid. The method requires only two chromatographic steps: passage through an Affigel-Blue column and gel filtration HPLC. This is a fast, efficient procedure for purification of these two important tumor markers, which are specific for prostatic cancer.
Improved liquid chromatographic/immunoassay of digoxin in serum.
Authors: Stone J A , Soldin S J
Published: January 2020

This HPLC/immunoassay procedure measures digoxin in serum with no interference from digoxin metabolites or digoxin-like factors. We used solid-phase (C18 and Diol) extraction, a C18 column with a tetrahydrofuran/water mobile phase, and final quantification by fluorescence polarization immunoassay. Deslanoside and gitoxigenin were used as the internal standard and the retention-time marker, respectively. The average CV for 300-microL samples at digoxin concentrations between 0.9 and 3.9 nmol/L was 9.3%. Minimum column lifetime with daily use was three months. We also compared results, for 49 samples from patients taking digoxin, obtained with the Abbott "TDx FPIA digoxin I" and the present procedure. Discrepancies between the two methods were substantial for 20% of the samples.
Assay of pancreatic amylase with use of monoclonal antibodies evaluated.
Authors: Zaninotto M , Bertorelle R , Secchiero S , Plebani M , Burlina A
Published: January 2020

To evaluate a new method for measuring pancreatic amylase in serum, in which the salivary isoenzyme is inhibited with a specific monoclonal antibody, we determined the activity of pancreatic and salivary amylase in sera from 103 healthy subjects and from 114 hospitalized patients having a wide range of total amylase activities. CVs for the proposed method ranged from 0.8% to 5.1% (within day) and from 2.3% to 6.6% (day to day). Results correlated well with those obtained by the wheat-germ inhibition method (r = 0.998) and by electrophoresis on cellulose acetate. Analytical-recovery studies confirmed the good specificity of the monoclonal antibody for salivary amylase (97%) and its low cross-reactivity (0.6%) toward pancreatic amylase. The assay procedure presents a wide range of linearity (141-1817 U/L) and can easily be adapted to an automated kinetic system. We found the proposed method suitable for routine determinations of pancreatic amylase.
Autoantibodies to thyroxin and triiodothyronine in the immunoglobulin G fraction of serum.
Authors: Li Calzi L , Benvenga S , Battiato S , Santini F , Trimarchi F
Published: January 2020

Thyroid hormone antibodies (THAbs)--i.e., antibodies to thyroxin (T4) and triiodothyronine (T3)--are detected rarely in human serum, where they are searched for, possibly because of a quantitatively minimal interaction between thyroid hormones (the haptens) and serum IgGs (the antibodies). The weak binding could result from these facts: (a) there are already six physiological carrier proteins for thyroid hormones; (b) THAbs usually account for a very small fraction of the total serum IgGs; (c) THAbs may have--as reported in the literature--a relatively low affinity. To ascertain whether THAbs could pass undetected in serum, we measured antibodies to T3 and T4 in both the serum and the corresponding IgG fraction of six normal persons and 45 patients with various thyroid diseases (Graves' disease, idiopathic myxedema, Hashimoto's thyroiditis, subacute thyroiditis, tumors), using radioimmunoprecipitation. The prevalence of antibodies to T4 was 0/51 in both the sera and the IgG fractions; the prevalence of antibodies to T3 was 1/51 in both materials. Because all of the sera that tested THAb negative were confirmed to be so in the THAb assay of the IgG fraction, we conclude that the prevalence of serum THAbs is not underestimated and that autoimmunization against thyroid hormones is really a rare phenomenon.
Differential precipitation of isolated human plasma lipoproteins with heparin and manganese chloride.
Authors: Ruiz-Albusac J M , Velázquez E , Montes A
Published: January 2020

We studied the precipitation of isolated lipoproteins with heparin and MnCl2. Lipoproteins were isolated from human plasma by preparative ultracentrifugation and their free cholesterol was labeled. Each lipoprotein fraction was then precipitated at various pHs, with or without bovine serum albumin (60 g/L) present. Under no set of conditions was one class of lipoproteins completely separated from the other two. Specifically, under standard conditions for precipitation of serum lipoproteins (pH 7.4 and protein 60 g/L), 12% of the very-low-density lipoprotein (VLDL) and 8% of the low-density lipoprotein (LDL) remained in the supernatant liquid, and 30% of the high-density lipoprotein (HDL) was precipitated. These results indicate that, under these conditions, so-called HDL cholesterol may be a mixture of VLDL, LDL, and HDL, although the sum of the amount of these three fractions remaining in the supernate is fortuitously very close to the value for HDL cholesterol isolated by ultracentrifugation.
Specific 3H radioimmunoassay with a monoclonal antibody for monitoring cyclosporine in blood.
Authors: Ball P E , Munzer H , Keller H P , Abisch E , Rosenthaler J
Published: January 2020

A specific radioimmunoassay involving a mouse monoclonal antibody to cyclosporine has been developed for monitoring the parent drug in blood. Pretreatment with methanol removes cyclosporine from the erythrocytes. The limit of detection is about 12 micrograms/L, sample volume is 50 microL of blood, and within- and between-assay CVs are less than 7%. Assay results correlated well with those obtained by "high-performance" liquid chromatography (HPLC) for liver (n = 42), for heart (n = 64), for bone-marrow (n = 36), and for kidney (n = 140). For blood specimens obtained from patients treated with cyclosporine postoperatively for as long as 65 months, the mean RIA/HPLC ratio in all with transplant indications was close to 1. Therefore, the specific radioimmunoassay apparently can be used instead of HPLC to measure the parent drug in blood.
The chemistry of aluminum and silicon in relation to Alzheimer's disease.
Authors: Birchall J D , Chappell J S
Published: January 2020

Aluminosilicates have been identified at the core of senile plaques in Alzheimer's disease, and aluminum has been found within neurons bearing neurofibrillary tangles. Here we show that aluminum species interact with silicic acid, Si(OH)4--a normal component of plasma--to form aluminosilicate species solubilized by citrate. A switch in the binding of aluminum from silicate to phosphate at pH less than 6.6 calls attention to the strong binding of cationic aluminum species to proximate phosphate groups, as in the inositol phosphates, and to the potential effect on the activity of the phosphoinositide-derived intracellular messenger system. The chemistry may throw light on the debated relationship between aluminum and Alzheimer's disease.
A homogeneous, liposome-based signal amplification for assays involving enzymes.
Authors: Pinnaduwage P , Huang L
Published: January 2020

Exploiting the fact that phosphatidylethanolamine (PE) liposome can be stabilized by a membrane lipid or protein, and that destruction of this stabilizer leads to rapid lysis of the liposome, we have designed a liposome-based signal enhancement mechanism for assays that involve enzyme as the final read-out step. Stable liposomes with entrapped glucose-6-phosphate dehydrogenase (G6PDH) were prepared with unsaturated PE stabilized with 5 mol percent of ganglioside GM1. Addition of beta-galactosidase caused rapid (3-5 min) lysis of liposomes, revealing the latent G6PDH activity, owing to the enzymatic degalactosylation of GM1. We have used Microgenic's CEDIA assay for digoxin as an example. The magnitude of signal was 25 mA/min per microgram of digoxin per liter for the unamplified assay and 1000 mA/min per microgram of digoxin per liter for the liposome-enhanced assay--i.e., a 40-fold amplification. This simple, rapid, and homogeneous signal-amplification mechanism is likely to be useful in many enzyme-dependent assays, such as ELISA, CEDIA, gene-probe assays, and immunoliposome assays.
Quantitative X-ray diffraction analysis of urinary calculi by use of the internal-standard method and reference intensity ratios.
Authors: Wandt M A , Rodgers A L
Published: January 2020

The internal-standard method and the powder diffractometer have been applied here to the quantitative determination of urinary stone constituents by x-ray diffraction (XRD). Reference intensity ratios determined for six stone substances were used in the reduction of intensity data. Constituent concentrations calculated for 21 stones were compared with values obtained from an element-sensitive technique. We conclude that XRD analysis alone cannot be regarded as a routine technique for the quantitative characterization of uroliths, but that semiquantitative XRD analysis supplemented by accurate quantitative elemental data is more suitable for the precise determination of true stone composition.
Effects of reaction variables on nephelometric and turbidimetric responses for the immunochemical reaction of immunoglobulin G.
Authors: Skoug J W , Pardue H L
Published: January 2020

Here we report a kinetic study of the immunoprecipitin reaction involving immunoglobulins G, A, and M. We used stopped-flow mixing adapted for simultaneous monitoring of nephelometric and turbidimetric signals for some studies and centrifugal mixing for others. The variables having the most significant effects on the kinetic responses in the regions of excess antibody and excess antigen are polyethylene glycol concentration, ionic strength, and the ratio of antigen to antibody. We document effects of these variables on maximum velocities and signal changes over a fixed-time interval for both monitoring modes and on the maximum signal change for nephelometry. We use response-surface methodology to help identify interactive effects among these variables (polyethylene glycol, NaCl, and antibody concentrations) and to select the best combination to use to quantify antigen in all regions of the immunoprecipitin curve. We also observe that turbidimetric responses are more reproducible and much simpler than are nephelometric responses. Implications of these results for quantification of the immunoglobulins are discussed.
Kinetic turbidimetric method for the immunochemical quantification of immunoglobulins, including samples with excess antigen.
Authors: Skoug J W , Pardue H L
Published: January 2020

Here we describe a kinetic approach for quantification of the immunoglobulins (IgG, IgA, IgM) in all regions of the immunoprecipitin curve. We use centrifugal mixing and report results for maximum-velocity, two-point, and multipoint curve-fitting methods as well as the use of rate coefficients obtained from the curve-fitting process to differentiate among regions of excess antibody, equivalence, and excess antigen. We show that it is possible to quantify each immunoglobulin over a concentration range from a large excess of antibody to moderate excesses of antigen with a single set of measurements made on a single dilution of each sample. Results for standard additions of the immunoglobulins to pooled sera have relative standard deviations (coefficients of variation) in the range of 1% to 3%, with analytical recoveries in the range of 95% to 106%. Correlations among determined and reported values in individual sera are quite good, with slopes ranging from 0.86 to 1.03 and no intercepts differing from zero by more than two standard deviation units. Concentrations quantified in 14 pathological sera by the proposed method correlated well with concentrations quantified by a fluorescence immunoassay method.
Is serum fructosamine assay specific for determination of glycated serum protein?
Authors: Schleicher E D , Mayer R , Wagner E M , Gerbitz K D
Published: January 2020

We compared the fructosamine activity in sera from healthy and diabetic subjects with the degree of protein glycation detected by a liquid-chromatographic method. The latter technique measures furosine as a specific product after hydrolysis of epsilon-amino-fructose-lysine. Our results indicate that the fructosamine assay measures the extent of glycation of purified human serum albumin correctly. On the other hand, we found no correlation between the two methods for sera from healthy subjects, although for diabetics' sera the values obtained with both methods were related. However, only about half of the reducing activity (fructosamine) was due to specific nonenzymatic glycation of proteins in healthy subjects and well-controlled diabetics. The remaining unspecific activity varied from serum to serum. It was not reducible with NaBH4 and was independent of the glycation of albumin, which normally accounts for about 80% of glycated serum proteins. The fructosamine assay is therefore of limited specificity for the exact measurement of glycated proteins in serum.
A rapid and accurate spectrofluorometric method for quantification and screening of urinary porphyrins.
Authors: Westerlund J , Pudek M , Schreiber W E
Published: January 2020

We describe a fluorescence method for screening and quantifying urinary porphyrins. New and effective approaches are used to oxidize prophyrinogens, correct the baseline, and ensure that uroporphyrin (uro) and coproporphyrin (copro) are equally detected, mole for mole. No preliminary purification is required. A 45-microL aliquot of urine is oxidized with 3 mmol/L iodine in 3 mol/L HCl to convert porphyrinogens to porphyrins, and then decolorized with 5 mL of 0.45 mmol/L sodium thiosulfate. An excitation scan is done from 350 nm to 440 nm, monitoring emission at 650 nm. Total porphyrin content is determined at the isosbestic point for uro and copro, and the mole fractions of uro and copro are estimated from the wavelength of the signal maximum. There is no interference from protein, glucose, bilirubin, or hemoglobin in high concentration. The limit of detection is less than 30 nmol/L and linearity is maintained up to 3200 nmol/L. Recoveries and precision are excellent. This is a rapid, sensitive screen for porphyrinuria as well as an accurate and precise quantitative method. We compared the method with existing methods and discuss some shortcomings common to many of them.
Laboratory evaluation of the Beckman Synchron CX3 clinical chemistry analyzer.
Authors: Peake M J , Pejakovic M , White G H
Published: January 2020

In this evaluation of the Beckman Synchron CX3, the multi-analyte clinical chemistry analyzer exhibited high precision, good linearity, and no carryover for each of the eight analytes measured. Results obtained correlated well with those produced by our routine instrumentation (Beckman Astra, Varian atomic absorption spectrophotometer). The instrument can process up to 75 samples per hour (600 tests per hour if all tests available are requested) and, after calibration, can provide urgent results for the complete panel of tests within 2 1/2 min. The performance characteristics of this instrument make it ideal as a routine or a "stat" analyzer for commonly requested tests in the clinical chemistry laboratory.
Getting involved in the legal system: choice or chance?
Authors: Labowitz D I
Published: January 2020

The legal system and the laboratory are becoming involved with each other on a daily basis today and the technologist is, willingly or unwillingly, being drawn into this unfamiliar arena. This article will explain some of the manner by which a part of that arena operates, specifically the trial courts. Whether civil or criminal, these courts are governed by rules and methods of operation for the persons in them. In the case of the laboratorian, the role is that of witness, and this article attempts to dispel any fears of that role and teaches how to best prepare for it.
A simple method for clinical assay of superoxide dismutase.
Authors: Sun Y , Oberley L W , Li Y
Published: January 2020

This assay for superoxide dismutase (SOD, EC 1.15.1.1) activity involves inhibition of nitroblue tetrazolium reduction, with xanthine-xanthine oxidase used as a superoxide generator. By using a reaction terminator, we can determine 40 samples within 55 min. One unit of activity of pure bovine liver Cu,ZnSOD and chicken liver MnSOD was expressed by 30 ng and 500 ng of protein, respectively. The mean concentrations of Cu,ZnSOD as measured by this method in blood from normal adults were 242 (SEM 4) mg/L in erythrocytes, 548 (SEM 20) micrograms/L in serum, and 173 (SEM 11) micrograms/L in plasma. The Cu,ZnSOD concentrations in serum and plasma of patients with cancer of the large intestine tended to be less and greater than these values, respectively, but not statistically significantly so.
Assessment of proton nuclear magnetic resonance spectroscopy for detection of malignancy.
Authors: Wilding P , Senior M B , Inubushi T , Ludwick M L
Published: January 2020

Water-suppressed proton nuclear magnetic resonance spectra were generated (by using 360 and 500 MHz systems) from human plasma and serum samples taken from 35 apparently healthy individuals, 52 patients with overt malignancies, and 37 patients with hypertriglyceridemia (triglycerides greater than 200 mg/dL or 2.26 mmol/L). The line widths from the lipoprotein-lipid methylene and methyl resonances at approximately 1.3 and 0.9 ppm were averaged by the method of Fossel et al. (N Engl J Med 1986;315:1369-76), but, contrary to their findings, we were unable to distinguish normal individuals from those with malignant tumors (e.g., mean +/- SD line width at 360 MHz: normal group = 32.9 +/- 3.6 Hz, malignant group = 28.3 +/- 4.9 Hz). The average line-width measurements (y), however, varied with the triglyceride content (x, mg/dL) of the plasma or serum as follows (logarithmic transformation of the data determined at 360 MHz and regression analysis): y = 110 (x-0.27). Data from both nonmalignant and malignant specimens fit this equation, the coefficient of correlation being -0.91. These findings suggest that considerable caution should be used in interpreting water-suppressed proton NMR spectra for cancer detection.
Urinary enzymes and low-molecular-mass proteins as indicators of diabetic nephropathy.
Authors: Jung K , Pergande M , Schimke E , Ratzmann K P , Ilius A
Published: January 2020

We measured the excretion rates of six urinary enzymes that either originate from the proximal renal tubule, like alanine aminopeptidase (EC 3.4.11.2), alkaline phosphatase (EC 3.1.3.1), gamma-glutamyltransferase (EC 2.3.2.2), and N-acetyl-beta-D-glucosaminidase (EC 3.2.1.30), or that are typical low-molecular-mass proteins, like lysozyme (EC 3.2.1.17) and pancreatic ribonuclease (EC 3.1.27.5). These rates were compared with those of total protein and albumin in urine of 36 insulin-dependent diabetic men and 30 healthy men. Seventeen of the diabetics had "clinical proteinuria," defined as excretion of more than 7.5 g of protein per mole of urinary creatinine (group B). Group A comprised the 19 diabetics without proteinuria. Except for gamma-glutamyltransferase, the excretions of enzymes and proteins were significantly higher in diabetics than in controls and were greater in group B than in group A. N-Acetyl-beta-D-glucosaminidase was the analyte most often increased in group A (89%), followed by albumin and alkaline phosphatase (each 32%). All patients in group B showed increased excretion of N-acetyl-beta-D-glucosaminidase. We conclude from the comparative data that this enzyme may be useful as an early predictor of diabetic nephropathy.
Interference by endogenous glycerol in an enzymatic assay of phosphatidylglycerol in amniotic fluid.
Authors: Herold D A , Reed A E
Published: January 2020

We investigated the possibility of interference by endogenous glycerol with the enzymatic measurement of phosphatidylglycerol in amniotic fluid. Phosphatidylglycerol is an important indicator of fetal lung maturity. The concentrations of glycerol and phosphatidylglycerol in amniotic fluid were measured by using a coupled enzymatic assay with and without phospholipase D (EC 3.1.4.4). The precision of the assay was acceptable (within-run CV = 1.2%, between-run CV = 4.8%). Endogenous glycerol content was demonstrated to be approximately 10-20 times that of phosphatidylglycerol. This high proportion of endogenous glycerol in amniotic fluid would preclude the accurate enzymatic determination of amniotic fluid phosphatidylglycerol unless the glycerol is first removed. Nor can the actual phosphatidylglycerol concentration be determined by subtracting the endogenous glycerol concentration from the total glycerol, which includes that glycerol derived from phosphatidylglycerol. With a usual range of 9 +/- 7 mumol/L, the error for a given phosphatidylglycerol measurement of +/- 6.6 mumol/L (+/- 2 SD) clearly is too high for this assay to be clinically useful. There was no correlation between concentration of endogenous glycerol or apparent phosphatidylglycerol in amniotic fluid and the lecithin/sphingomyelin ratio of the sample.
Thyrotroph function assessed by sensitive measurement of thyrotropin with three immunoradiometric assay kits: analytical evaluation and comparison with the thyroliberin stimulation test.
Authors: Wians F H , Jacobson J M , Dev J , Heald J I , Ortiz G
Published: January 2020

We evaluated the analytical and clinical performance of three "sensitive" immunoradiometric assay (IRMA) kits (Tandem-R TSH HS, Hybritech, Inc.; EchoClonal TSH, Bio-Rad; Coat-A-Count TSH IRMA, Diagnostic Products Corp.) for measurement of thyrotropin (TSH) and compared their performance against a "regular" IRMA (ARIA-HT, Becton-Dickinson) to determine whether these assays might eliminate the need to perform the thyroliberin (TRF) stimulation test. We concluded that the Tandem and EchoClonal kits may obviate the need to perform TRF stimulation tests in some patients. Using only the basal TSH concentration to predict the TSH response to TRF, we found all three sensitive TSH assays to be useful for detecting abnormal thyrotroph function. Dose-response, linearity, analytical recovery, and specificity were acceptable for all kits, but intra-assay precision at very low TSH concentrations and analytical sensitivity differed considerably among the kits. Using the EchoClonal assay, we established a normal reference interval for TSH of 0.4-4.6 milli-int. units/L.
Semi-automated direct colorimetric measurement of creatine kinase isoenzyme MB activity after extraction from serum by use of a CK-MB-specific monoclonal antibody.
Authors: Landt Y , Vaidya H C , Porter S E , Whalen K , McClellan A , Amyx C , Parvin C A , Kessler G , Nahm M H , Dietzler D N
Published: January 2020

This semi-automated colorimetric assay for the MB isoenzyme of creatine kinase (EC 2.7.3.2) is based on a monoclonal antibody ("Conan-MB") specific for this isoenzyme and is a modification of a previously published method (Vaidya et al., Clin Chem 1986;32:657-63). A 0.64-cm bead coated with 2 to 3 micrograms of antibody is incubated with 100 microL of serum and 10 microL of 0.2 mol/L beta-mercaptoethanol for 1 h at room temperature, to extract CK-MB. The beads are washed with de-ionized water and incubated with CK substrate for 45 min at 37 degrees C. A solution containing trans-1,2-diaminocyclohexane-N,N,N', N'-tetraacetic acid, p-iodonitrotetrazolium violet, and diaphorase is added and the resulting colored product is measured at 492 nm. The standard curve is linear to 200 U of CK-MB per liter, and analytical recovery is 97-113%. Total assay CV for low (9.7 U/L) and high (50.7 U/L) quality-control materials was 14.1% (n = 1878) and 11.6% (n = 1842), respectively. CK-MB activity correlated well (r = 0.978, n = 226) with CK-MB measured by a two-site mass immunoassay, and 99.4% of samples with CK-MB greater than or equal to 12 U/L (n = 347) were verified by electrophoresis on agarose.
Drug testing in the military--technical and legal problems.
Authors: Irving J
Published: January 2020

The Armed Forces drug-testing program has been in operation since 1971. Since that time, its goals, capabilities, and methods of operation have undergone extensive evolution and change. As these laboratories developed, they helped to establish many of the currently accepted operating standards for drug screening. For laboratories considering entry into the business of drug screening, an examination of the evolution and current operation of the military laboratories will be of benefit. It will help to define the currently accepted standards and NIDA guidelines and assist them to avoid the types of problems that were encountered by the military laboratories.
Development and performance of a highly sensitive carboxyl-terminal-specific radioimmunoassay of calcitonin gene-related peptide.
Authors: Zaidi M , Girgis S I , MacIntyre I
Published: January 2020

The calcitonin genes encode a small family of peptides: the circulating hormone calcitonin; its flanking peptide, katacalcin; and a third novel peptide, calcitonin gene-related peptide (CGRP). CGRP is a potent vasodilator and a major circulating product from the calcitonin genes; it may be a physiologically important regulator of blood flow in humans. High concentrations of circulating CGRP are found in medullary thyroid carcinoma. We report the development and validation of a highly sensitive (detection limit 500 amol per tube) radioimmunoassay of CGRP involving a high-affinity antibody directed against the carboxyl terminus of the molecule and a highly pure tracer. The assay is precise, robust, and reproducible, and is therefore a potentially useful analytical method for studying the normal and abnormal physiology of this peptide.
Measurement of ferritin in serum by an indirect competitive enzyme-linked immunosorbant assay.
Authors: Polson R J , Kenna J G , Shears I P , Bomford A , Williams R
Published: January 2020

This indirect competitive enzyme-linked immunosorbant assay (ELISA) for ferritin, unlike other currently available ELISAS, does not require use of an anti-ferritin antibody-enzyme conjugate. Designed for use on microtiter plates, the method has a precision and sensitivity similar to those of other immunoassays. The detection limit is 20 pg of ferritin per test (corresponding to 2.0 micrograms/L in serum samples). Comparison of results obtained on serum from 57 patients by this method with those from a conventional radioimmunoassay gave a correlation coefficient of 0.92.
Genetic analysis of systemic lupus erythematosus: 1. Detection of disease-associated variant proteins by two-dimensional gel electrophoresis.
Authors: Morito F , Ohta A , Kaneoka H , Nagayoshi T , Hilda M , Yamaguchi M
Published: January 2020

Various genetic studies indicate that development of systemic lupus erythematosus (SLE) is regulated by the mode of multifactorial inheritance, i.e., by the overall effect of polygenes and environmental factors. To elucidate some variant genes involved in the polygenic system responsible for onset of SLE, we resolved and measured the protein components of lymphocytes and sera from inactive-SLE patients, their relatives, and normal controls, using two-dimensional gel electrophoresis. Intercomparison of polypeptide patterns between patients and controls revealed three major variations, two detected in lymphocytes and one in sera. These variations were present in 66-82% of the patients, in 20-36% of the control group, and in 41-64% of the relatives. In addition, nearly half of SLE patients, but only one of 19 normal controls, possessed all three SLE-associated traits, suggesting that these variant proteins may reflect in part the genetic factors contributing to development of SLE.
Direct determination of selenium in serum by graphite-furnace atomic absorption spectrometry with deuterium background correction and a reduced palladium modifier: age-specific reference ranges.
Authors: Jacobson B E , Lockitch G
Published: January 2020

We describe the development of a direct method for determination of selenium in serum by graphite-furnace atomic absorption spectrophotometry with deuterium background correction. We include palladium modifier to stabilize selenium in the presence of a strong reducing agent. Spectral interferences from iron are not evident in this system. Because an analysis requires only 20 microL of serum or plasma, the method is applicable to neonatal populations. At a selenium concentration of 0.75 mumol/L (approximately the mean found in our pre-term infants), the within-run CV is 5.3%. For a concentration of 1.83 mumol/L, which approximates our normal adult mean, within-run and between-run CVs are 2.7% and 3.4%. Accuracy is demonstrated by analytical recoveries ranging from 96% to 102%. We present reference values for pre-term and term infants, and age-specific ranges for infants to adults.
Pre-analytical variation in alanine aminotransferase.
Authors: Ruby S G , Reiber N E , Lonser R E
Published: January 2020

To determine the effect of pre-analytical variation of alanine aminotransferase in blood specimens with normal activity concentrations of this enzyme, we stored serum and whole blood samples at 4 and 22 degrees C and assayed aliquots of each specimen at intervals up to 72 h. Analysis of variance revealed no statistically significant increase or decrease in activity of the enzyme for up to 72 h in either specimen type, whether stored at room temperature or refrigerated.
Amylase in urine as measured by a single-step chromolytic method.
Authors: Bertholf R L , Winn-Deen E S , Bruns D E
Published: January 2020

We studied a new single-step direct chromolytic method (Behring D.A.T.) for measuring urinary amylase (1,4-alpha-D-glucan glucanohydrolase, EC 3.2.1.1) activity, comparing results with those by a similar, but two-step, procedure that requires an auxiliary (coupling) enzyme. The two methods gave virtually identical relative responses to purified human pancreatic and salivary amylases. Assay of four quality-control materials to evaluate the total (day-to-day) precision of the new method yielded CVs of 4 to 7%, similar to those of the comparison method for each of the four quality-control samples. Amylase activity was measured by both methods in 110 random (i.e., untimed) urine specimens. Linear regression analysis provided a slope and y-intercept of 0.947 and 4 U/L (x = comparison method, y = direct method), respectively, and a standard error of the estimate of 25 U/L for specimens in which the amylase activities ranged from 11 to 1465 U/L (mean = 358 U/L) by the comparison method. The mean rate of amylase excretion in 2-h timed urine specimens from 95 healthy volunteers, as measured by the new method, was 7.18 (SD = 3.18) U/h, and the nonparametric (95% confidence interval) reference interval was 1.6 to 15.2 U/h. We consider the new method a promising alternative to other kinetic assays that require the use of auxiliary enzymes.
Comparison of Roche RIA, Roche EIA, Hybritech EIA, and Abbott EIA methods for measuring carcinoembryonic antigen.
Authors: Nisselbaum J S , Smith C A , Schwartz D , Schwartz M K
Published: January 2020

Using 600 clinical specimens, we compared the concordance of four methods for carcinoembryonic antigen: the Roche RIA (I); the Roche EIA (II); Hybritech EIA (III); and Abbott EIA (IV). EDTA-treated plasma was used for Methods I and II and serum for Methods III and IV. However, no significant difference was found between results for serum and plasma in Method II. The normal reference interval (in micrograms/L) was I (222 specimens), 1.94 +/- 1.54; II (57 specimens), 0.8 +/- 0.5; III (100 specimens), 2.94 +/- 2.47; and IV (614 specimens), less than 5.0. The precision of all four methods was acceptable. Concordance among all of the methods exceeded 90%.
Are increases in thyroxin-binding globulin in patients with acute hepatitis ascribable to synthesis by regenerating hepatocytes?
Authors: Shigemasa C , Tanaka T , Mitani Y , Ueta Y , Taniguchi S , Urabe K , Adachi T , Yoshida A , Abe K , Mashiba H
Published: January 2020

We describe a case of liver cirrhosis lacking the expected increase in serum thyroxin (T4)-binding globulin (TBG) despite abrupt, severe increases in aspartate and alanine aminotransferases (ASAT and ALAT) in serum. Sequential change in serum T4, triiodothyronine (T3), and TBG concentrations were also measured retrospectively in serum of 10 hospitalized patients with acute viral hepatitis. Although their mean T4 and TBG concentrations significantly exceeded those in 40 normal subjects (P less than 0.002 and P less than 0.001, respectively), these values were within the normal reference intervals in five patients. ASAT and ALAT concentrations were not significantly different in patients with increased TBG and patients with normal TBG, whereas mean concentrations of serum albumin and cholinesterase and mean prothrombin times (in percent) in the former group were significantly higher than those in the latter group (P less than 0.05, P less than 0.05, and P less than 0.001, respectively). For 60 samples with increased ASAT and ALAT, TBG and albumin or cholinesterase correlated significantly (r = 0.49, P less than 0.001 and r = 0.50, P less than 0.001, respectively), but not TBG and ASAT or ALAT. Collectively, these results suggest that the increase in serum TBG in acute hepatitis may reflect its synthesis in regenerating hepatocytes rather than a simple leakage from damaged hepatocytes.