bluuebunny/crossref_metadata_2025 huggingface dataset:
Evaluation of pteroylglutamic acid and n-5-methyltetrahydrofolic acid reference standards in a new "No-Boil" radioassay for serum folate.
Authors: Theobald R A , Batchelder M , Sturgeon M F
Published: January 2020

Serum folate, pteroylglutamic acid standards, and N-5-methyltetrahydrofolate standards extract differently during sample preparation for radiometric assay. B/B0 values for each material were determined before and after an alkaline extraction procedure. Serum folate most closely resembled N-5-methyltetrahydrofolate standards in that it exhibited a slightly greater ability to displace iodinated folate derivative in competition for beta-lactoglobulin binder. Pteroylglutamic acid standards displaced folate derivative less well, so that serum folate values were lower than when N-5-methyltetrahydrofolate was used as the standard material.
A strategy for radioimmunoassay of plasma progesterone with use of a homologous-site 125I-labeled radioligand.
Authors: Corrie J E , Hunter W M , Macpherson J S
Published: January 2020

In this sensitive, specific 125I radioimmunoassay for plasma progesterone, an identical glucuronide bridge is used in the immunogen and radioligand. This apparently provides a system in which the bridge is poorly recognized and thereby avoids the excessively high affinity of the tracer for the antiserum and the consequent poor sensitivity that commonly is found in other homologous-bridge situations. The glucuronide bridge is potentially useful for attachment of other labeling probes.
Adsorbent treatment and immunoaffinity chromatography compared for removing estradiol-17 beta from plasma.
Authors: McBride J H , Davies R J
Published: January 2020

Removal of estradiol-17 beta from plasma by immunoaffinity chromatography on anti-estradiol antibodies coupled to Sepharose was investigated and compared with the use of charcoal adsorbent. Both methods completely removed added [3H]estradiol from pooled male plasma. In contrast to the adsorbent treatment, which caused marked changes in the biochemical composition of the plasma, immunoaffinity chromatography caused only minor perturbations. We discuss the advantages of using plasma, stripped of endogenous estradiol or other steroids by immunoaffinity chromatography, in construction of radioimmunoassay calibration curves.
Method variation in lactate dehydrogenase isoenzyme determination.
Authors: Papadopoulos N M
Published: January 2020

Differences between methods for determining lactate dehydrogenase isoenzymes are illustrated, which can account for discrepant results. They should be taken into consideration in the interpretation of test results for the diagnosis of myocardial infarction.
New Perspectives in Coagulation Testing
Authors: Fareed Jawed , Messmore Harry L , Bermes Edward W
Published: January 2020

Vol. 26

p 1386, Table 5, column 3, entry two should read "71 ± 26," not "17 ± 26."

p 1933, under the heading Notes, change first sentence to read "...equal to the numerical value of the molar..."

Vol. 27

p 202, The last equation for sT2 should read: 5W2 + SD2 [n(k - 1)]/(kn - 1)

p 204, col. 2, fourth full paragraph "25 mg/L" should read "25 g/L"

Because of a postal delay, one paper was printed without the authors' corrections, which are:

p 287, para. 1, fifth line "Theoretical...," insert the word "and" after "alternates."

Table 1, column 4: last entry should read "+7.57(0.974)"; column 5: first entry should read "±6.5." Amend title of Table 2 to read "Quantitative Performance of Second- and Fourth-Derivative Assays for Paraquat Dichloride Extracted from Horse Serum," and change "hight" to "higher" in footnote a.

p 288, under "Procedure," in two places the words "setting the instrument on" should be changed to "using as reference."

p 289, under "Aqueous...," in first paragraph, fourth line, delete ",dissolved" and insert "solution." In the next paragraph delete the words "is used...satellite" and insert instead "measure (to the long-wavelength satellite) is used." In the fourth line of the first paragraph in column 2, change "is" to "in."

p 290, line 10 under "Discussion": correct spelling is "Åkerblom."

p 297, In the equation, E is activity of the enzyme in serum at time t, less the basal activity.

p 317, In first sentence of "Discussion," the binding constants should be 1010 to 1011 L/mol.

p 343: The following Table was inadvertently omitted from the Letter

See table in PDF file

p 422, Under "Reagents," dilute the 4 mL of D-glucarate solution with 16 (not 46) mL of 10 mmol/L HCl.

p 425, Table 4, last column should read: 5.07 (1.72) and 5.06 (1.55).

p 508, See Letter.
Characterization and intermethod relationships of materials containing purified human pancreatic and salivary amylase.
Authors: Sampson E J , Duncan P H , Fast D M , Whitner V S , McKneally S S , Baird M A , MacNeil M L , Bayse D D
Published: January 2020

We describe the preparation and characterization of materials containing human pancreatic and salivary alpha-amylase (EC 3.2.1.1) and examine their relationship to endogenous amylase in human serum. Amylase was purified from human pancreas and saliva by solvent- and salt-fractionation and column chromatography to specific activities of 63 and 279 kU/g, respectively. Four liquid pools, differing only in activity, were prepared from each source of amylase, each in a matrix containing, per liter: 30 g of human albumin, 50 mmol of sodium chloride, 1 mmol of calcium chloride, and 50 mmol of Tris hydrochloride buffer, pH 7.4. Characterization of the pools showed that the amylase activity in the materials was stable for at least six months at 25 degrees C; among-vial variability of amylase activity was less than or equal to 0.5% (2 CV); and the pools were free from eight possible contaminating enzymes. Plots of salivary vs pancreatic amylase activity measure in our materials with eight commercially available methods showed least-squares slopes ranging from 0.51 to 1.0. The intermethod "commutability" of the materials (i.e., how closely they mimic endogenous serum amylase) was examined in relationship to approximately 100 human sera.
Laser nephelometry of orosomucoid in serum of newborns: reference intervals and relation to bacterial infections.
Authors: Bienvenu J , Sann L , Bienvenu F , Lahet C , Divry P , Cotte J , Bethenod M
Published: January 2020

Orosomucoid was evaluated by laser-nephelometry in 1790 sera collected from 1170 newborns. Within-run precision (CV) was 2.1 to 4.2%, between-run 2.9 to 5.2%. Results correlated well with radial immunodiffusion (r = 0.989). Results can be obtained within 1 h. Orosomucoid concentrations in serum at birth range from 130 to 200 mg/L and are influenced by gestational age during the first two days of postnatal life. Thereafter, the values increase very rapidly in the first week of life, concentrations being the same as in adults by about 10 months. In 66 of 78 cases of severe bacterial infections, orosomucoid concentrations were above normal. Evidently, serum orosomucoid constitutes an useful index in diagnosis and monitoring of bacterial infections in the neonatal period.
Interference of levodopa and its metabolites with colorimetry of uric acid.
Authors: Bierer D W , Quebbemann A J
Published: January 2020

Reportedly, levodopa (L-DOPA) administration produces spuriously high values for plasma uric acid as measured by the commonly used phosphotungstic acid-hydroxylamine colorimetric method. We confirm this interference, not only by L-DOPA but also by three of its major metabolites: dopamine, 3,4-dihydroxyphenylacetic acid, and 3-methoxy-4-hydroxyphenylacetic acid. However, at therapeutic concentrations in plasma (less than 5 mg/L), the maximum spurious uric acid concentration due to L-DOPA is less than 2 mg/L. Also, at reported peak plasma concentrations of L-DOPA plus three of its major metabolites, the maximum spurious uric acid concentration due to all four compounds combined is less than 8.5 mg/L. Therefore, the hyperuricemia observed with this method in some patients who are chronically receiving L-DOPA cannot be attributed only to interference by L-DOPA and its metabolites in the colorimetric determination of uric acid. Evidently L-DOPA may increase laboratory values for plasma uric acid concentrations, both by pharmacological and chemical mechanisms.
Clinical evaluation of a radiometric assay specific for creatine kinase isoenzyme MB.
Authors: Kwong T C , Rothbard R L , Biddle T L
Published: January 2020

A new radiometric assay specific for creatine kinase isoenzyme MB was evaluated with respect to its precision and agreement with a conventional electrophoretic CK-MB assay for the diagnosis of myocardial infarction. The reference interval we find for serum CK-MB in healthy subjects is 0--30 micrograms/L. The coefficients of variation at 197 and 40 micrograms of CK-MB per liter, were 5.2 and 11.5%, respectively. In a clinical study of 52 consecutive patients admitted into a Coronary Care Unit with a diagnosis of suspected myocardial infarction, there was overall agreement in CK-MB results by the two assays for 51 of 52 patients. A more sensitive and quantitative assay that is specific for CK-MB can be helpful in cases where diagnosis of myocardial infarction is equivocal.
Quantitative determination of human plasma apolipoprotein A-I by laser immunonephelometry.
Authors: Rosseneu M , Vercaemst R , Vinaimont N , Van Tornout P , Henderson L O , Herbert P N
Published: January 2020

A technical procedure is described for quantitation of human apolipoprotein A-I (apo A-I) in normal plasma or serum by immunonephelometry. Dilution of the plasma samples with 6 mol/L guanidine chloride ensures maximum exposure of the antigenic sites of the apoprotein and enables optimum quantitation of the apo A-I without requiring extraction with organic solvents. Similar data are obtained by this assay and with radioimmunoassay for normal subjects (1.2--1.5 g/L), and the results obtained on 31 patients are correlated with a coefficient of 0.92. The apo A-I values are correlated with values for plasma high-density lipoprotein cholesterol (r = 0.64). The interassay CV for immunonephelometry is about 7% and the standard curve is linear between 0.1 and 1.0 microgram of apo A-I per sample, corresponding to a 150-fold dilution of serum or plasma. The assay is applicable to plasma samples containing as much as 4 g of triglycerides per liter. At higher concentrations plasma delipidation is required.
High-molecular-mass alkaline phosphatase in serum and bile: physical properties and relationship with other high-molecular-mass enzymes.
Authors: Crofton P M , Smith A F
Published: January 2020

High-molecular-mass alkaline phosphatase has similar properties in serum and bile. It is of sufficiently large size to be excluded by Sepharose 6B and carries a high negative charge at alkaline pH, largely accounted for by the presence of sialic acid residues. It is slightly more heat labile than the lower-molecular-mass liver isoenzyme. Other enzymes originating from the hepatocyte plasma membrane are present as similar high-molecular-mass forms in normal bile, and in both bile and serum from patients with liver disease. The activities of these relatively large enzymic forms in serum were correlated with one another and we show they are converted similarly to lower-molecular-mass forms by solubilization with detergents. However, they carry slightly different net charges, thereby ruling out the possibility that they all consisted of a single homogeneous multi-enzyme complex. These findings are discussed in the light of two current hypotheses that the high-molecular-mass enzymes represent (a) membrane fragments released into the circulation in liver disease or (b) low-molecular-mass enzymes that aggregate, after release, with the lipid and protein in the circulation.
Column affinity chromatography for bound/free separation in ligand assays. I. Radioimmunoassay of choriomammotropin (human placental lactogen).
Authors: Cornale P , Bonazzi M , Multinu C , Romelli P , Vancheri L , Pennisi F
Published: January 2020

A method is described for separating antibody-bound from free fractions in ligand assays by column affinity chromatography, and its application to radioimmunoassay of choriomammotropin. In the method, 70 x 10 mm (i.d.) polypropylene columns containing about 150 mg of immunosorbent (goat anti-rabbit gamma-globulins covalently linked to Sepharose CL-4B) are used. Standards or unknowns, tracer and antiserum, pipetted into bottom-capped columns, are kept separated from the immunosorbent bed by a porous polyethylene disc and allowed to react for 15 min at room temperature. The reaction mixture is then allowed to pass through the columns by removing the bottom caps. Free antigen is eluted by washing the column, and discarded; antibody-bound fractions remain bound to the immunosorbent. The radioactivity in the columns is counted. The major advantages of the present technique, arising from the liquid-phase reaction combined with the solid-phase separation by column affinity chromatography, are the very low nonspecific binding (less than 1%), good sensitivity (0.02 mg/L), good precision (CV 3.4%), and simple and fast (30-min) assay. For 50 clinical samples so assayed (gamma) and compared with a polyethylene glycol precipitation technique (x), the regression equation was: y - 0.14 + 0.98x (r = 0.994). The assay method was clinical validated by 3493 determinations.
Specific spectrophotometric assay for the M isoenzymes of pyruvate kinase in plasma samples containing mixtures of the muscle (M) and liver (L) isoenzymes.
Authors: Edwards R J , Watts D C
Published: January 2020

Plasma pyruvate kinase (EC 2.7.1.40) activity contains two major isoenzymes. The "L" type originates from liver (erythrocytes contain a similar isoenzyme), the "M" type predominantly from skeletal muscle. The diagnostic use of pyruvate kinase as an index of muscle leakage requires an assay of only the M type. An M-type-selective assay for mixtures of the two isoenzymes was devised, based on the differences that the L form has a higher Km for phosphoenolpyruvate and is inhibited by about 95% by a high concentration of ADP. All relevant conditions of the assay have been examined and the reaction system has been optimized at 30 degrees C. The assay yields the expected Km values for M type pyruvate kinase. An apparent increase in activity in hemolysed plasma samples was found to be derived from leukocytes.
A screening method for establishing laxative abuse.
Authors: de Wolff F A , de Haas E J , Verweij M
Published: January 2020

Abuse of laxatives, most of them belonging to the group of colonic stimulants or cathartics, can cause various disorders. Extensive diagnostic work can be avoided by early toxicological screening of the suspected patients with respect to laxatives. Because no screening method of this kind was available, we developed a procedure with which all phenolic and anthraquinone laxatives--except sodium picosulfate--can be detected in urine. This method is based on high-performance thin-layer chromatography in two systems after pretreatment of a 20-mL urine sample with beta-glucuronidase and subsequent column extraction. The procedure is very sensitive: at least 32 h after a single dose of bisacodyl, danthron, phenolphthalein, or sennoside, the drug can be detected in the urine. Bisoxatin and oxyphenisatin are still detectable in the urine 18 h after intake. The method is also highly specific; none of 73 other drugs interfered in either of the two chromatographic systems. This procedure can be helpful for the early diagnosis of laxative abuse.
Effect of centrifugation on fluorescence polarization of amniotic fluid.
Authors: Simon N V , Elser R C , Levisky J S , Polk D T
Published: January 2020

Fluorescence polarization was measured on amniotic fluids of different gestational ages before and after centrifugation at relative centrifugal forces of 34 to 4955 x g. Centrifugation significantly increased the fluorescence polarization values over those of uncentrifuged amniotic fluids. The greatest increase was observed in the most "mature" fluids centrifuged at 34 to 230 x g. Almost without exception, use of centrifugal forces greater than 1239 x g did not further increase fluorescence polarization values.
Response-surface-optimized, zinc-enhanced assay for serum alkaline phosphatase.
Authors: Thompson J C , Hodges C T , Dobler G L , Williamson J A
Published: January 2020

An optimized assay for alkaline phosphatase (EC 3.1.3.1) is reported. A discrete analyzer, the DuPont Automatic Clinical Analyzer (aca), was used for this study. The assay is based on results of response-surface experimental co-optimization techniques, and response is enhanced over the present aca assay. A key feature is the incorporation of zinc ions, both to fully optimize the assay and to reduce the sensitivity of measured activity to zinc-binding impurities in the buffer, 2-amino-2-methyl-1-propanol. In addition, a simple technique is described for measuring relative concentrations of zinc-binding impurities in this buffer. These features should be considered in the design of any assay for alkaline phosphatase that is based on p-nitrophenyl phosphate as substrate and 2-amino-2-methyl-1-propanol as buffer.
Spectrophotometric assay for urinary N-acetyl-beta-D-glucosaminidase activity.
Authors: Horak E , Hopfer S M , Sunderman F W
Published: January 2020

An improved assay for N-acetyl-beta-D-glucosaminidase activity in urine is described that involves (a) gel filtration to separate the enzyme from inhibitors in urine, (b) enzymic hydrolysis of p-nitrophenyl-N-acetyl-beta-D-glucosaminide at pH 4.4, and (c) spectrophotometry of the liberated p-nitrophenylate. Measurements of activity of the enzyme in 58 urine specimens correlated closely (r = 0.9998) with results by an established procedure. The within-run coefficient of variation (CV) was 3.7%; the between-run CV averaged 6.8%. Reference values for the activity were established by assays of urine specimens from 135 healthy persons, age two weeks to 52 years. Efficacy of the assay for detection of nephrotoxicity was demonstrated in rats after experimental induction of reversible renal insufficiency by intraperitoneal injection of nickel chloride. Clinical application of the assay in approximately 1000 patients corroborated its utility for detection and monitoring of renal disorders.
Reversed-phase C18 cartridge for extraction of estrogens from urine and plasma.
Authors: Heikkinen R , Fotsis T , Adlercreutz H
Published: January 2020

We describe a simple method for extraction of estrogens that is efficient over the whole polarity range of estrogen metabolites in urine and plasma. A small reversed-phase C18 cartridge (Sep-Pak C18, Waters Associates, Inc.) is used for this purpose. After application of the biological fluid, followed by an aqueous wash, estrogens are eluted in 3 mL of methanol. For urine the pH should be kept at 3 before the methanol elution. When estrogens are extracted from plasma, the steroid-protein interaction is counteracted by the presence of triethylammonium sulfate and D-norgestrel at pH 5. The recoveries of added 3H-labeled estrogens are essentially quantitative. The capacity of the column is high and estrogens can be extracted from large volumes of pregnancy urine without loss in the washing water. Because of the high flow rate (2 mL/min) and the small volume of methanol in which the estrogens are eluted, the whole procedure is convenient and rapid.
Precision of "high-performance" liquid-chromatographic assays with sample pretreatment. Error analysis for the Technicon "FAST-LC" system.
Authors: van der Wal S , Snyder L R
Published: January 2020

A comprehensive theory has been presented elsewhere (Anal. Chem. 53: 877-884, 1981) for the various contributions to assay imprecision in procedures that are based on sample extraction followed by "high-performance" liquid chromatography. Experimental data (1800 assays, 8400 results) for the Technicon FAST-LC system are used with this theory in an effort to understand and control the precision of clinical-laboratory procedures, both automated and manual. This study provides specific conclusions and recommendations on matters such as: standardization procedures and protocols, physical properties requires in internal standards, the relative importance of different sources of imprecision and means for improving precision, the relative importance of temperature control in pretreatment and liquid-chromatographic analysis, and the precision obtainable with small sample volumes or with samples containing very low concentrations of analyte (e.g., in assays for free drugs). Automation evidently can lead to twofold greater assay precision, other factors being equal, for liquid-chromatographic procedures that include sample pretreatment. Similarly, internal standardization, properly applied, can decrease assay imprecision by twofold.
Plasma glucose concentrations of whole blood, as determined with a multilayer-film analytical element.
Authors: Ohkubo A , Kamei S , Yamanaka M , Arai F , Kitajima M , Kondo A
Published: January 2020

A new type of multilayer film analytical element--consisting of a spreading layer, a blocking layer, an enzymic reagent layer, and a transparent layer--has been developed for blood glucose determination. After a spot of whole blood is dropped on the film, the plasma glucose concentration is determined in 6 min without further manipulations. Precise measurement of sample volume spotted on the film is not necessary for good results, nor is knowledge of the hematocrit in the range 10-46%. The minimum concentration of glucose detectable by this method is 100 mg/L. The coefficients of variation, within-run and between-run, were all less than 2.5% for 800-4000 mg/L glucose concentrations. Macromolecules and hydrophobic substances in blood did not interfere. This method seems suitable for emergency use, especially.
Immunological studies of ribonuclease C from human urine.
Authors: Cranston J W , Hoover H C , Crisp E R
Published: January 2020

Antiserum to human urinary RNase C [ribonuclease (pancreatic), EC 3.1.27.5], developed in rabbits, was used to characterize this enzyme through studies of inhibition of RNase C-catalyzed poly(C) hydrolysis and of competition in a RIA. By either assay, the antiserum failed to cross react with human urinary RNase U (EC 3.1.27.-) or bovine pancreatic RNase A (EC 3.1.27.5). RNase C is immunologically identical to the poly(C)-active RNase in various human sera, including samples obtained from normal individuals, patients with pancreatic carcinoma, pancreatitis, or other malignant and nonmalignant diseases. This conclusion is based on the finding of superimposable antibody dose-inhibition curves for poly(C) hydrolysis and parallel competition RIA curves for RNase C and the various sera. There was a positive correlation (r = 0.73; p less than 0.001) between concentrations of RNase C as determined by poly(C) hydrolysis and competition RIA in serum samples from 102 patients. Therefore, the latter technique provides al alternative method for measuring RNase C in sera.
Measurement of alkaline phosphatase activity: characterization and identification of an inactivator in 2-amino-2-methyl-1-propanol.
Authors: Rej R , Bretaudiere J P , Jenny R W , Jackson K Y
Published: January 2020

An inactivator of alkaline phosphatase (EC 3.1.3.1) in 2-amino-2-methyl-1-propanol is demonstrated and characterized. This time-dependent inactivation results from chelation of enzyme-bound Zn2+; it is reversed by addition of Zn2+ and, to a lesser extent, other divalent metal ions. Cu2+ is an effective spectral indicator and can be used to determine the presence and quantity of inactivator. Data obtained from enzyme inactivation, Cu2+ absorbance spectra, "high-performance" liquid chromatography, thin-layer chromatography, Fourier-transform infrared spectroscopy, and mass spectroscopy indicate that the inactivator is 5-amino-3-aza-2,2,5-trimethylhexanol. This compound, even in trace amounts (less than 0.05% on a molar basis), shown to inactivate alkaline phosphatase.
Hyperlabile diabetes accompanied by insulin resistance.
Authors: Renie A , Hamilton R G , Adkinson N F , Rendell M S
Published: January 2020

It is generally held that high insulin antibody concentrations, by "buffering" abrupt swings in free insulin concentrations after injections of exogenous insulin, tend to stabilize blood glucose variations in diabetic patients. However, we encountered a patient with extremely labile diabetes coexisting with insulin resistance. This patient's injections were switched to pure porcine insulin from his usual mixed bovine/porcine insulin, in an effort to decrease his insulin requirement. This treatment was successful, and, as his insulin dosage decreased, his diabetic lability diminished substantially. His diabetes was eventually considered stabilized on about 22 units of porcine insulin daily. The serial decrease in his insulin antibody concentrations, monitored by use of solid-phase radioimmunoassay, paralleled the disappearance of his diabetic lability as well as the decrease in his insulin requirement.
Glucose oxidase immunoenzyme methodology as a substitute for fluorescence microscopy in the clinical laboratory.
Authors: Rathlev T , Hocko J M , Franks G F , Suffin S C , O'Donnell C M , Porter D D
Published: January 2020

Enzymes as markers for antigens or antibodies in immunohistochemical procedures have several advantages over commonly used fluorochrome labels. These include use of a regular light microscope and the ability to get permanently stable slide preparations. Glucose oxidase (EC 1.1.2.3.4), being absent in mammalian tissue, provides no background staining, such as that seen with the commonly used horseradish peroxidase (EC 1.11.1.7) owing to peroxidase-like activity in tissues. A glucose oxidase histochemical method is detailed that is useful for detection of human antibodies; it can be easily used in clinical laboratories as a substitute for fluorescent techniques.
Relationships between immunogen structure and antisera specificity in the narcotic alkaloid series.
Authors: Findlay J W , Butz R F , Jones E C
Published: January 2020

We report the production and comparative specificities of antisera raised to different immunogens containing codeine, morphine, and oxycodone. Antisera raised to bovine serum albumin (BSA) conjugates of codeine-6-hemisuccinate, ethylmorphine-6-hemisuccinate, or oxycodone-6-carboxymethyloxime had greatest recognition of structural changes around the piperidine ring nitrogen atom and th 14-position. N-carboxypropylnormorphine-BSA, N-carboxypropylnorcodeine-BSA, and norcodeine-BSA (directly coupled) conjugates elicited antisera that recognized structural changes at the 3- and 6-positions best, but also clearly discerned changes in the 14-substituent. Attachment of codeine to BSA via the 8-position gave a conjugate that elicited antisera with specificity characteristics similar to those of the antiserum to N-carboxypropylnorcodeine-BSA. Thus clear relationships existed between immunogen structure and antiserum specificity. The utility of these antisera was illustrated by the application of antiserum to codeine-6-hemisuccinate-BSA and solvent extraction to a study of codeine disposition in the dog. The specific antisera of N-carboxypropylnormorphine-BSA and to norcodeine-BSA were applied directly to an examination of the distribution of codeine and metabolically produced morphine in the milk and plasma of a nursing mother.
On-line cleavage of urinary estriol conjugates with immobilized beta-glucuronidase before liquid-chromatographic analysis.
Authors: Bowers L D , Johnson P R
Published: January 2020

Urinary estriol measurement has been widely accepted as a useful indicator of fetoplacental status. Classically, glucuronide conjugates of estriol have been cleaved with soluble beta-glucuronidase (EC 3.2.1.31) before extraction and measurement. We have developed a system in which urine is injected directly into a "high-performance" liquid chromatograph and the conjugates are cleaved on-line in an immobilized beta-glucuronidase reactor. Estriol is quantitated with fluorescence detection after separation from other interfering species with a reversed-phase column. The stability of the immobilized enzyme under storage conditions and in the presence of the mobile phase are discussed. Only 150 mL/L methanol could be pumped through the reactor and onto the analytical column, but this allowed on-column preconcentration of the free estriol produced. Gradient elution bypassing the immobilized enzyme reactor eluted the compounds of interest without damaging the enzyme. Comparison with radioimmunoassay results yielded a slope of 0.97 (r = 0.996, n = 19).
Improved isocratic liquid-chromatographic simultaneous measurement of phenytoin, phenobarbital, primidone, carbamazepine, ethosuximide, and N-desmethylmethsuximide in serum.
Authors: Szabo G K , Browne T R
Published: January 2020

We describe an improved "high-pressure" liquid-chromatographic assay for simultaneous determination in serum of the five major antiepileptic drugs (ethosuximide, primidone, phenobarbital, phenytoin, and carbamazepine) and N-desmethylmethsuximide (the compound that must be quantitated for therapeutic drug monitoring of the antiepileptic drug methsuximide). Serum protein is precipitated with an acetone solution containing 5-ethyl-5-(p-methylphenyl)barbituric acid as the internal standard. The centrifuged supernate is injected onto the chromatographic column. Drugs and internal standard are eluted at 30 degrees C with mobile phase containing acetonitrile/methanol/phosphate buffer (17/28/55 by vol) at a flow rate of 0.7 mL/min, monitored at 195 nm. Analysis time is about 20 min. Quantitation is by measurement of peak areas. Analytical and absolute recoveries varied from 95 to 104%. Within-day coefficients of variation ranged from 1.6 to 5.4%, between-day CVs from 0.0 to 3.4% in subtherapeutic, therapeutic, and toxic samples. Resolution of therapeutic concentrations of all six drugs was complete. As yet, we have found no drug or drug metabolite that interferes.
Laboratory evaluation of the Boehringer Mannheim "Diagnostic M" automated discrete analyzer.
Authors: Robinson C A , Proelss H , Stabler T V
Published: January 2020

We evaluated a new multiple-channel chemistry analyzer, the Boehringer Mannheim "Diagnostic M." This instrument can perform 25 tests at the rate of 120 1.3-mL serum samples per hour. The instrument may be run in either a profile mode or single-test mode. In the single-test mode only the necessary reagent is pumped. the instrument is computer controlled. We compared it with the Technicon SMAC, SMA 12/60, and SMA 6/60. It demonstrated excellent precision, linearity, lack of interference, ease of operation, and satisfactory comparison with values obtained by the Technicon methods.
Assays of serum lipase by the "BALB-DTNB method" mechanized for use with discrete and continuous-flow analyzers.
Authors: Furukawa I , Kurooka S , Arisue K , Kohda K , Hayashi C
Published: January 2020

We successfully adapted the dimercaprol (BAL) tributyrate-5,5'-dithiobis(2-nitrobenzoic acid) method (J. Biochem. 81: 361, 1977) for assay of lipase in human serum to a discrete analyzer (the TBA 880) (I) or a continuous-flow analyzer (AutoAnalyzer, Type II) (II). In both, BAL-tributyrate is used as substrate, in combination with serum esterase inhibitors and a chromogenic reagent for the SH group of the liberated BAL. Serum lipase activities of patients with pancreatic diseases, measured at 90 or 40 samples per hour by I or II, respectively, correlated well with those measured by the corresponding manual method or by Kaplan's radioassay (Anal. Biochem. 33: 213, 1970). The correlation coefficients were all greater than 0.95, and the coefficients of variation were less than 8%, showing the practical usefulness of these procedures.
Enzyme immunoassay of thyroxin with a centrifugal analyzer.
Authors: Izquierdo J M , Sotorrío P , Quirós A
Published: January 2020

We have applied a homogeneous enzyme immunoassay for determination of thyroxin in serum to the "Cobas Bio" centrifugal analyzer. To unbind thyroxin from its protein complex, serum is treated for 20 min with a solution of NaOH containing "Lipex," an agent for sequestering free fatty acids. The immunoenzymic reaction is then automatically performed by the analyzer at 37 degrees C. To 20 microL of sample mixture is added 125 microL of reagent (thyroxin antibodies and NAD+) and this mixture is incubated for 10 s. Then 25 microL of start reagent (enzyme-thyroxin conjugate and malate substrate) is added and the change in absorbance is monitored at 340 nm. The standard curve is linear up to at least 200 micrograms of thyroxin per liter. Within-assay precision (CV) varied from 1.1 to 2.9%, between-assay precision from 3.1 to 7.8%. Analytical recovery of thyroxin was complete. The deviation of control samples from target values ranged from -2.1% to 7.0%. Interference by hemoglobin or bilirubin is negligible. Results compare favorably with those by radioimmunoassay.
Comparative evaluation of two calcium ion-selective electrode systems, and their utility for monitoring steady-state changes in [Ca2+].
Authors: Drop L J , Tochka L N , Misiano D R
Published: January 2020

We compared ionized calcium concentrations ([Ca2+]) as measured with two ionized-calcium analyzers: the NOVA 2 and the Orion SS-20. Samples were obtained from 43 human volunteers, 213 patients, and five dogs (106 samples). In the [Ca2+] range of 0.85 to 1.8 mmol/L, [Ca2+] measurements in whole blood with the NOVA 2 consistently exceeded those measured with the Orion SS-20. However, in the normal range, this difference appeared to be smaller when we compared values for plasma or serum, and was absent over the entire range when we compared aqueous solutions. The normal human [Ca2+] in whole blood as measured with the NOVA 2 is 1.22 +/- 0.01 mmol/L (mean +/- SEM) and that with the Orion SS-20 is 1.12 +/- 0.01 mmol/L (p less than 0.0001 by paired t-test); the 95% confidence intervals were from 1.14 to 1.30 and from 1.02 to 1.22 mmol/L, respectively. Using dogs, we also tested the usefulness of the ionized-calcium electrode for monitoring [Ca2+] during infusion of either citrate or calcium chloride solutions, to produce steady-state alterations in [Ca2+] equilibrium. Frequent successive [Ca2+] measurements were essential to appropriately adjust the infusion rates of these solutions to achieve steady-state [Ca2+].
Hemoglobin A1 in hemolysates from healthy and insulin-dependent diabetic children, as determined with a temperature-controlled minicolumn assay.
Authors: Kaplan L A , Cline D , Gartside P , Burstein S , Sperling M , Stein E A
Published: January 2020

We report age-specific glycohemoglobin values for nondiabetic infants, children, and young adults. These values were determined with an ion-exchange "mini-column" in a specially designed and constructed water bath that maintains column temperature at 23 +/- 0.1 degrees C. Two in-house-prepared controls with glycohemoglobin content amounting to 6.36% and 11.87% of total hemoglobin, stored at -20 degrees C, were used to assess long-term analytical precision. Between-day precision (CV) was 1.4% and 1.65%, respectively. We found a significant correlation (r = 0.981, p less than 0.01) between the glycohemoglobin value and the physicians' independent assessment of clinical control in 129 insulin-dependent diabetics, ages 3-23 years. There were significant differences (p less than 0.05) between glycohemoglobin values between patients with well-controlled or poorly controlled diabetes and those with intermediate control. Reported studies of 20 patients over three to 12 weeks showed that changes in clinical control were paralleled by changes in glycohemoglobin values.
Evaluation of a new serum separator.
Authors: Seckinger D L , Antonio Vazquez D , Rosenthal P K , Heller Z H
Published: January 2020

We tested the Centri-Sep filter (DADE) for its effectiveness in separating serum from clotted blood samples used for chemical analysis. Although statistical analysis by paired t-test showed differences in results for some analytes with the use of this device as compared with use of no separator or of a serum-decanting device, we concluded from the small bias of the paired means that the new separator device did not interfere with the clinical usefulness of reported values for the analytes studied. The separator is not an effective barrier for long-term storage of serum on its clot; however, we could obtain about 10% more serum with the separator than with decanting devices.
Proteins of human urine. II. Identification by two-dimensional electrophoresis of a new candidate marker for prostatic cancer.
Authors: Edwards J J , Anderson N G , Tollaksen S L , von Eschenbach A C , Guevara J
Published: January 2020

A protein series common to the urine and prostatic tissue of 16 of 17 patients with prostatic adenocarcinoma has been identified by high-resolution two-dimensional gel electrophoresis. These proteins, designated PCA-1, have a relative molecular mass in sodium dodecyl sulfate of about 40000. Analyses of urines from eight age-matched controls, seven patients with other ty pes of urogenital malignancies, two patients with benign prostatic hyperplasia, and five patients with malignancies not associated with the urogenital system failed to show PCA-1 in the patterns. These preliminary findings suggest that this protein should be systematically investigated as a candidate marker for prostatic adenocarcinoma in man.
Colorimetry of serum acetaminophen (paracetamol) in uremia.
Authors: Bailey D N
Published: January 2020

A popular colorimetric analysis for serum acetaminophen, based on ring-nitration of the drug, is demonstrated to yield both "false positives" and erroneously high results for serum from uremic patients. The interference appears to be anionic at physiological pH, and correlates significantly with serum creatine concentration and with the magnitude of the "anion gap." A modification of the analysis involving extraction of the acetaminophen with ether eliminates the interference. As little as 10 mg of drug per liter can be accurately measured in serum by the proposed procedure. Analytical recovery was 97% at a concentration of 1 g/L. Coefficients of variation for the analysis at respective concentrations of 100 and 500 mg/L were: within-run, 2.7% and 2.0%; between-run, 3.1% and 4.2%. I encountered no serious interferences from other drugs. The proposed method, rapid and reliable, is recommended for routine use in the clinical laboratory.
Liquid-chromatographic determination of the total thiamin content of blood.
Authors: Kimura M , Fujita T , Itokawa Y
Published: January 2020

A liquid-chromatographic method for determining the total thiamin content of blood is presented. Blood is deproteinized and incubated with Aspergillus oryzae carboxyl proteinase (EC 3.4.23.6; Takadiastase) to convert thiamin phosphate esters to free thiamin. An aliquot of the sample is applied to the column (Shodex OH-Pak M-414) of a high-performance liquid chromatograph. A 100 mg/L solution of potassium ferricyanide in 150 g/L sodium hydroxide is added to the column effluent with a proportioning pump, to convert thiamin into a fluorophore. The intensity of the fluorophore is measured with a spectrofluorophotometer and recorded graphically. The total thiamin in each blood sample appears as a single peak, and no co-eluting substance was detected. This method is simple, highly reproducible, and rapid, and its sensitivity is sufficient for determination of the thiamin content of 0.1-mL blood samples.
Stability of creatine kinase-3 in lyophilized materials.
Authors: Whitner V S , Morin L G , McKneally S S , Sampson E J
Published: January 2020

We examined the stability of creatine kinase (EC 2.7.3.2) isoenzyme-3 (CK-3) in lyophilized bovine albumin matrices in the presence and absence of various sulfhydryl compounds and ADP. We initially purified CK-3 from human myocardium and skeletal muscle by the batch-chromatographic technique and by gradient elution column chromatography to specific activities of 293 and 93 kU/g, respectively. To assess stability, we subjected the lyophilized materials to storage studies at 4, 25, 37, 42, 56, and 65 degrees C and compared first-order rate constants for the decay of creatine kinase activity at 42 degrees C. Our most stable matrix contained, per liter, 2 mmol of ADP and 10 mmol of N-acetylcysteine, and had an extrapolated first-order half-life (Arrhenius plot) at -20 degrees C of approximately 60000 years.
Morphine glucuronide hydrolysis: superiority of beta-glucuronidase from Patella vulgata.
Authors: Combie J , Blake J W , Nugent T E , Tobin T
Published: January 2020

beta-Glucuronidase from Patella vulgata, Helix aspersa, Helix pomatia, and bovine liver were evaluated for usefulness in routine hydrolysis of drug-glucuronic acid conjugates from equine urine samples. Factors affecting the reaction rate (enzyme concentration, ligand concentration, temperature, and pH) were optimized. A 3-h incubation at 65 degrees C with 5000 U of beta-glucuronidase from P. vulgata per milliliter of urine resulted in complete hydrolysis of all morphine glucuronide in the urine samples. Not only was the enzyme preparation from P. vulgata the most cost-effective beta-glucuronidase source studied, but also its thermal stability is such that it can be used at a temperature high enough to substantially shorten the incubation interval. Preliminary work on other drugs that form glucuronide conjugates indicates that this same procedure is similarly superior for use in their hydrolysis.
Interlaboratory surveys of the quantitation of thyroxin and thyrotropin (thyroid-stimulating hormone) in dried blood spot specimens.
Authors: Hearn T L , Hannon W H
Published: January 2020

Screening of newborns for hypothyroidism is mandated by law in most states. The screening usually consists of measuring thyroxin in dried blood spot specimens followed by measurement of thyrotropin if the results for thyroxin are suggestive of hypothyroidism. The first nationwide interlaboratory surveys designed to assess the proficiency of screening laboratories in the identification of euthyroid and hypothyroid specimens are reported here. Each survey consisted of three specimens mailed quarterly from late 1979 through 1980. A total of 88 laboratories participated in at least one of the four surveys. More than 17 different methods or diagnostic kits were used by participants in the surveys. Coefficients of variation ranged from 20% to 41% for thyroxin data and from 23% to 63% for thyrotropin. Despite the large analytical interlaboratory variation, most participants assigned clinical classifications consistent with the survey design. The incidence of mis-classifications was low.
Serum triglycerides determined colorimetrically with an enzyme that produces hydrogen peroxide.
Authors: Fossati P , Prencipe L
Published: January 2020

In this direct colorimetric procedure, serum triglycerides are hydrolyzed by lipase, and the released glycerol is assayed in a reaction catalyzed by glycerol kinase and L-alpha-glycerol-phosphate oxidase in a system that generates hydrogen peroxide. The hydrogen peroxide is monitored in the presence of horseradish peroxidase with 3,5-dichloro-2-hydroxybenzenesulfonic acid/4-aminophenazone as the chromogenic system. The high absorbance of this chromogen system at 510 nm affords useful results with a sample/reagent volume ratio as low as 1:150, and a blank sample measurement is not needed. A single, stable working reagent is used; the reaction is complete in 15 min at room temperature. The standard curve is linear for triglyceride concentrations as great as 13.6 mmol/L. Average analytical recovery of triglycerides in human sera is 100.1%, and within-run and between-run precision studies showed CVs of less than or equal to 1.6 and less than or equal to 3.0%, respectively. The method is suitable for automation.
Multipoint kinetic methods evaluated for quantitation of theophylline with prosthetic group label immunoassay.
Authors: Lin J D , Pardue H L
Published: January 2020

We describe the development and evaluation of a multipoint kinetic approach to the measurement and data-processing steps in immunoassays. Nonlinear regression methods are used to fit data obtained during the kinetic phase of the immunochemical reaction to a model that represents the kinetic behavior of the total system. This approach is evaluated for quantitation of theophylline with a new reaction system in which FAD is the label in a homogeneous reaction system. Competition for antibody binding sites between drug and labeled drug is monitored by the activity of glucose oxidase (EC 1.1.3.4) generated by the reaction of unbound label with apoglucose oxidase. The process is fit well by a parallel first-order/zero-order model and regression methods are used to predict steady-state velocities from non-steady-state portions of the response. One fitting method yields results in about 3 min after mixing, as compared with longer than 8 min for single-point fixed-time methods. Steady-state velocities vary linearly with theophylline concentration.
Improved DEAE-Sephadex column chromatography in measuring amylase in serous ovarian neoplasms, and results for 13 cases.
Authors: Zakowski J J , Bruns D E
Published: January 2020

We describe a column-chromatographic method for measuring amylase activity in cyst fluids of serous ovarian tumors. Using this technique, we confirm and extend the previous report of an "acidic" amylase in serous ovarian tumor cyst fluids. This form of the enzyme was eluted from DEAE-Sephadex mini-columns with a high-salt buffer. It accounted for 45 to 100% of the amylase in 13 cyst fluids. This "high-salt" amylase was also present in the tumor tissues. The acidic nature of the amylase does not appear to be due to sialic acid residues, because the chromatographic behavior of the amylase is unaffected by treatment with neuraminidase. We conclude that the "acidic" amylase reported previously in two serous ovarian tumors is a constant feature of these tumors and that it is distinct from the sialylated tumor amylase described by others.
Lactate dehydrogenase activity as measured by the SMAC is related to flow-cell energy.
Authors: Robertson E A , Wright E , Chesler R A , Elin R J
Published: January 2020

Determination of lactate dehydrogenase (LDH) activity in the SMAC (Technicon) is based on the change in NADH absorbance between two flow cells. We noted that results for patients' specimens and controls changed when the fiber optic terminations for the two LDH channel flow cells were adjusted or "peaked" at the colorimeter chopper assembly. The energy (intensity) of light reaching the flow cells was varied by adjusting the fiber optic terminations, and the absorbance readings for a series of solutions containing NADH and patients' specimens were recorded. For both flow cells, when the fiber optic terminations were adjusted to increase the zero absorbance light intensity from 20 lines to 60 lines, a significant (p less than 0.0001) proportional change was seen in the absorbance readings. Evidently the difference in absorbance between the two flow cells is related not only to the NADH concentrations but also to the difference in the light intensity at the two flow cells. Consequently, changes in the adjustment of the fiber optic terminations produce systematic changes in results for LDH in patients' sera. These systematic changes in LDH results may be minimized by maintaining equivalent settings of the fiber optic terminations for the two flow cells and by using the calibration material with an absorbance most similar to that of patients' specimens.