bluuebunny/crossref_metadata_2025 huggingface dataset:
Immobilized-enzyme membrane sandwich reactor used in automated micro-scale assay of plasma uric acid.
Authors: Kamoun P , Douay O
Published: January 2020

We describe the automated microassay of plasma uric acid by use of an immobilized uricase-membrane sandwich reactor. Hydrogen peroxide, formed when uric acid is oxidized, oxidatively couples two molecules of p-hydroxyphenylacetic acid in the presence of peroxidase to produce a highly fluorescent compound. The specificity of the uricase reaction is coupled with a significantly lower cost of analysis.
Effect of diluent temperature on creatine kinase values found for lyophilized controls and reference sera.
Authors: Feld R D , Brown L F , Neri B P , Witte D L
Published: January 2020

We report the effect of temperature of diluent on creatine kinase activity in several lyophilized controls. Creatine kinase activity was significantly greater when the lyophilized control was reconstituted with diluent at 4 degrees C as compared to 25 degrees C. This is an additional source of variation in creatine kinase controls.
Should blood samples for assay of plasma renin activity be chilled?
Authors: Emanuel R L , Williams G H
Published: January 2020

Collecting blood on ice for renin determination reportedly may produce falsely high results. To assess the probability of this occurring under actual collection conditions, we measured renin activity in duplicate aliquots of plasma from blood samples from 25 hypertensive patients, both supine and upright, and in 10 supine normotensive controls. One aliquot of the blood was collected on ice and processed at 4 degrees C, the other at room temperature. The two aliquots showed no significant differences in renin activity. If anything, values for samples collected at room temperature were higher. Repeat determination on the same specimens stored at--20 degrees C for nine and 12 months revealed no significant changes in results for any samples, although the amount of angiotensin I found in the sample before incubation at 37 degrees C significantly increased. We conclude that it makes little difference at what temperature one collects blood for renin determination, but because of the wide fluctuations in "room" temperature we recommend that blood samples be collected on ice.
Creatine kinase isoenzyme of high relative molecular mass in serum of a cancer patient.
Authors: Yuu H , Takagi Y , Senju O , Hosoya J , Gomi K , Ishii T
Published: January 2020

We describe an atypical serum creatine kinase isoenzyme in the serum of a woman with cancer of the left breast. This isoenzyme migrated toward the cathode, closely following the MM isoenzyme on agarose gel electrophoresis. Its relative molecular mass was estimated to be about 325,000, fourfold that of normal creatine kinase. It is more heat-stable and is inhibited more by urea than the normal MM isoenzyme. Isoenzyme monomer B activity was observed to be 20 U/liter in the serum, as measured with use of an antibody against the M monomer. On anion-exchange column analysis, creatine kinase activity was observed only in the MM fraction, in spite of the fact that B activity was observed in the patient's serum. Results of the immunological investigation make it unlikely that the atypical isoenzyme is linked to immunoglobulin or beta-lipoprotein. It may have been present as the result of modification of normal creatine kinase by the therapeutic radiation the patient was receiving.
Effect of membrane potential on dialysis of calcium in a continuous-flow system.
Authors: Staley M J , Farrance I
Published: January 2020

Electical potentials across the membrane in a modified AutoAnalyzer dialyzer (Technicon) have been determined under various conditions and related to the dialysis of 45Ca. The results suggest that anomalies in the diffusion of calcium from protein and nonprotein containing streams could be due to factors other than "Donnan equilibrium effects."
Assay of an antitumor protein, neocarzinostatin, and its antibody by fluorescence polarization.
Authors: Maeda H
Published: January 2020

I evaluated use of the fluorescence polarization technique to measure neocarzinostatin, a proteinaceous antitumor antibiotic, and its antibody, in serum. The antigen (neocarzinostatin), labeled with fluorescein isothiocyanate, was allowed to interact with its antibody in a cuvet, in the instrument, yielding an increase in the fluorescence polarization value. Antibody content was determined in the presence of a definite amount of the labeled antigen, fluorescence polarization values increasing in parallel with each addition of antibody. Antigen content was determined with a known amount of antibody, which reacted at first with an unknown amount of antigen in samples, followed by addition of a definite amount of the labeled antigen (competition). I used the method to determine a pharmacokinetic parameter, the apparent volume of distribution for neocarzinostatin in rabbits, using drug-injected rabbit sera. I evaluated precision, accuracy, and reproducibility, using various samples or possible interfering substances such as bilirubin and hemoglobin, and also compared results for antigen with those by single radial immunodiffusion assay. The present assay is fast (less than 2 min), sensitive (less than 10 nmol/liter can be detected), and simple (there is no separation step before readout of the results).
Analytical precautions in measurement of blood cyanide.
Authors: Rodkey F L , Robertson R F
Published: January 2020

Solutions of KCN in 0.1 mol/liter NaOH, stored for six months at 4 degrees C, were unchanged as determined by silver titration and color development with pyridine-pyrazalone reagent. At room temperature the cyanide concentration of such solutions decreased by 0.079% per day (half life = 880 days) and the extent of color development changed in direct proportion to the change in silver titer. Rubber stoppers adsorb (or dissolve) HCN when in contact with this gas. The cyanide tends to come out of the stopper long after the source of the gas is removed. Rubber stoppers and expired air contaminated by HCN must be carefully avoided during cyanide analysis. Expired air contains HCN, generally in larger amounts for persons who smoke.
Microchromatographic methods for hemoglobin A2 quantitation compared.
Authors: Brosious E M , Wright J M , Baine R M , Schmidt R M
Published: January 2020

On 20 consecutive work days during four weeks, one technologist performed 24 microchromatographic determinations of hemoglobin A2 (Hb A2) by each of four methods: the Efremov procedure requiring Tris/HCl buffer, the original Huisman technique with use of glycine developer, and two commercial test kits in which a modified glycine developer is used. The bloood samples tested were obtained from 12 adults with no hematological abnormality and from 12 beta-thalassemia carriers previously diagnosed by familial and hematologic studies. Results by the first method and the two commercial kits (one from Helena Laboratories and one from Isolab, Inc.) did not differ significantly in precision for either the normal or beta-thalassemia trait samples. For both sample types, the second method yielded larger coefficients of variation than those obtained with the other methods. Moreover, the second method was the only one with which values overlapped for normal samples and samples with above-normal Hb A2 concentrations.
Use of a programmable calculator in processing and interpreting serum cholinesterase phenotypes.
Authors: Brown P , McQueen M J , Patterson M C
Published: January 2020

Cholinesterase phenotyping is done at 25 degrees C with use of benzoylcholine as substrate and dibucaine, fluoride, chloride, and succinyldicholine as inhibitors. We wrote a diagnostic program in modified BASIC language for the processing and interpretation of these cholinesterase phenotypes. We used the HP 9830A calculator. The diagnostic aspect of the program uses 646 words, a further 86 words being used to run this program. The traditional and the programmed reporting procedures were duplicated for 296 consecutive patients, with no case of disagreement between the two reporting methods as to the appropriate phenotype. The programmed system requested nine repeat analyses more than did the traditional method. Seven of these were due to the tight limits set in the program for the fluoride inhibitor numbers. These tight limits were set to ensure that there was as limited an overlap as possible between the phenotypes E1uE1u and E1uE1f. The remaining two repeats were due to the restrictions placed on the succinyldicholine inhibitor numbers that would be acceptable before a patient was designated as having the relatively rare phenotype, E1aE1f.
Immobilized-enzyme nylon-tube reactors for routine determination of urea and citrulline in serum.
Authors: Sundaram P V , Igloi M P , Wassermann R , Hinsch W , Knoke K J
Published: January 2020

A continuous-flow clinical analyzer for the routine estimation of urea is described that makes use of an immobilized-enzyme nylon-tube reactor as part of a flow-through system (a Technicon AutoAnalyzer I). Results of blood-urea analyses by use of the immobilized urease are compared with determinations made with the diacetyl monoxime method and the urease solution method. Clinical trials carried out routinely with the immobilized enzyme nylon tube reactor give reliable and reproducible results with high precision and low cost. The reactors are stable to intermittent or continued use for at least four months or for 2000 tests. A method is described in which differential colorimetry is used for determining citrulline in blood and which makes use of the immobilized urease, albeit indirectly.
Creatine kinase in serum: 4. Differences in substrate affinity among the isoenzymes.
Authors: Szasz G , Gruber W
Published: January 2020

The goal of this work was to find out whether it is possible to measure all three creatine kinase isoenzymes under the same reaction conditions in spite of their different kinetic properties. We found the tightest substrate binding for purified human BB, followed by the MB And MM isoenzyme preparations for both creatine phosphate and ADP. An increase in substrate concentration usually resulted in an inhibition. Nevertheless, it was possible with a method optimized for the MM isoenzyme also to measure the BB and MB isoenzymes at a rate of inhibition of only 6 and 3%, respectively. Marked differences in the apparent Km values between purified and native MM isoenzyme in human serum may indicate that the enzyme declined in substrate affinity during the isolation procedure. The use of enzyme preparations for standardization purposes, therefore, is only suitable if their kinetic properties are close to those of the enzyme in serum. Difficulties in the calculation of the apparent Km values are discussed and the graphical procedures of Lineweaver and Burk and of Eisenthal and Cornish-Bowden compared.
Gas-chromatographic method for plasma acetate analysis in acetate-intolerance studies.
Authors: Nielsen L G , Ash K O , Thor E
Published: January 2020

We describe a modified gas-chromatographic method for acetate in serum or plasma, intended for use in the investigation of acetate intolerance in hemodialysis patients. The assay may be adapted for use with a single-column gas chromatograph equipped with a flame ionization detector. The analysis, made isothermally, requires only 0.5 ml of plasma or serum. Only one deproteinizing step is required to prepare the sample for analysis. Additionally, we present preliminary findings of an ongoing acetate-intolerance study.
Preparation of ligand-free human serum for radioimmunoassay by adsorption on activated charcoal.
Authors: Carter P
Published: January 2020

I describe a convenient method for stripping hormones and other ligands from human serum. Formation of a serum/charcoal colloid is avoided by incubating serum on a limited surface area with activated charcoal, in a lateral oscillating shaker. Particle-free serum of negligible turbidity is obtained without ultracentrifugation by ejecting the centrifugate through a cellulose ester membrane. By this technique, more than 80% of thyrotropin, triiodothyronine, thyroxine, cortisol, insulin, digoxin, folate, estriol, or an ether derivative of testosterone is removed from serum. About 70% of follitropin and somatotropin are removed, as well as half the choriogonadotropin. Serum protein remains intact throughout the adsorption process.
Determination of 5-hydroxytryptophan in plasma by high-performance liquid chromatography and fluorometric detection after phthaldialdehyde reaction.
Authors: Enbaek F , Magnussen I
Published: January 2020

We describe the sensitive and precise determination 5-hydroxytryptophan in plasma from patients treated with this compound. The method is based on solvent extraction of plasma, high-performance liquid chromatography and continuous fluorometric detection after reaction with phthaldialdehyde in strong acid. The combined time for chromatography and reaction is 25 min. The sensitivity of the method is adequate to quantitate 0.05 mumol of 5-hydroxytryptophan per liter, in 750 microliter of plasma. No interference by other hydroxyindole compounds was found.
Measurement of chloride in sweat by use of a selective electrode and strip-chart recorder.
Authors: Warwick W J , Hansen L G
Published: January 2020

We describe a method for direct potentiometric measurement of sweat chloride by use of a chloride-selective electrode, with strip-chart recording of the measurement to facilitate interpretation of the measurement. Comparison of this method and the Gibson and Cooke method [Pediatrics 23, 545 (1959)] gave a correlation coefficient of 0.86. Recording of the same test at various pressures showed that different pressures had little effect if they were kept constant.
Radioimmunoassay of creatine kinase isoenzymes in human serum: isoenzyme MM.
Authors: Van Steirteghem A C , Zweig M H , Schechter A N
Published: January 2020

Measurement of the mass concentration of serum enzymes by radioimmunoassay provides direct quantitation of specific isoenzymes and may be less subject to some of the limitations of traditional assay procedures for enzymes. We describe the development of a sensitive and specific radioimmunoassay for the muscle isoenzyme of creatine kinase, CM-MM, in human serum. CK-MM, purified from human skeletal muscle, was used to raise high-titer antisera and for iodination by the Chloramine T method. The radioimmunoassay required 50 microliter of sample, utilized a double-antibody separation method, and was completed in 24 h. Cross reactivity with CK-BB was virtually zero, 3--17% with CK-MB. The mass concentration of CK-MM in the serum of healthy subjects ranged from 36 to 1668 microgram/liter and correlated closely with total CK enzymatic activity. Serum concentrations of CK-MM from casually selected patients correlated less well with total CK enzymatic activity, suggesting the existence of other CK isoenzymes or the presence of inactive forms.
Archival storage of clinical laboratory data.
Authors: Soffer S , Entine S , Wolfe T , Eggert A , Friedman R
Published: January 2020

This paper describes a data-base system for the maintenance of cumulative clinical data. A unique aspect of this data base is its ability dynamically to alter its two-level format to create an alternative three-level hierarchical structure in order to optimize retrieval efficiency. The relative advantages of these two types of data-base structures are discussed and examples of their use in clinical resrarch are presented.
Adenylate kinase inhibition by adenosine 5'-monophosphate and fluoride in the determination of creatine kinase activity.
Authors: Meiattini F , Giannini G , Tarli P
Published: January 2020

The current methods for the determination of creatine kinase (EC 2.7.3.2) activity are derived from Oliver's method, in which AMP is used to decrease interference by adenylate kinase (EC 2.7.4.3). Recently, Szasz et al. and Rosano et al. described methods in which diadenosine pentaphosphate and fluoride, respectively, are used to reduce this interference. However, diadenosine pentaphosphate does not sufficiently inhibit such activity of hepatic origin, while fluoride alone can only inhibit it at concentrations at which the fluoride tends to precipitate as MgF2. Finally, Szasz et al., the Committee on Enzymes of the Scandinavian Society for Clinical Chemistry and Clinical Physiology, and the German Society for Clinical Chemistry have proposed methods in which both AMP and diadenosine pentaphosphate are used to inhibit adenylate kinase. We have found that by using low concentrations of AMP and fluoride together, we can greatly diminish this interference without significant loss of creatine kinase activity and with no precipitation of MgF2.
Improved method for fractionating gamma-glutamyltransferase by electrophoresis on cellulose acetate.
Authors: Burlina A
Published: January 2020

I describe a method of fractionating gamma-glutamyltransferase on cellulose acetate. The tracing is obtained in parallel with that of serum protein, and the gamma-glutamyltransferase bands are characterized by correspondence with the major protein fractions. The overall pattern of the isoenzyme activity in normal sera is one of activity in the alpha1- and alpha2-globulin regions. In hepatic diseases four bands are usually present, but some more specific observations are possible, e.g., the presence of an intense beta-globulin band in occlusive icterus, intra- or extrahepatic, and a marked alpha2-globulin band in alcoholism. The potentialities of this technique as a diagnostic and prognostic aid together with its simplicity prompt me to recommend its use in the clinical laboratory.
Use of Values for Calcium and Protein Serum, and of a Derived Index Obtained from a Probability Population Sample
Authors: Kelly Anthea , Munan Louis , PetitClerc Claude , Ho Kok Ping , Billon Bernard
Published: January 2020

Volume 22

p 1726: In the footnote to Table 3, the last half of the sentence should read " for SI units, the index becomes (80 calcium - 120)/protein."

Volume 23

p 1779: In column two of "corrections," the word "malate" should be substituted for "maleate."

p 2127: In column two, last line, change "chinic" to "chenodeoxycholic." On the next page, change the last part of the sub-legend to Figure 7 to read "(5) ursodeoxycholic acid, (6) cholic acid."

p 2205: col. two, line 32. For "mg/g body weight" read "mg/kg body weight."

p 2288: In the last sentence of the abstract, change ".... 0.3 to 7.8 mole..." to "... 0.3 to 7.8 mmol...."

p 2289: Table 1, footnote a should read: "Millimoles of 4NP injected/270 x 10-3 moles of 4NPP. This is calculated as if the 4NP were present in 270 nmol of 4NPP...."

p 2290: In Table 2, in column A "7.76d" should read "7.76c"

p 2356: The reader may incorrectly infer that the "Gamma-Coat Kit" (CA 535, 536; 555, 556) is intended for the analysis of dried blood samples on paper in screening for congenital hypothyroidism in infants. Actually, that kit is intended for serum thyroxine assay. A modified kit dedicated to dried blood-sample screening for hypothyroidism is currently under development and will be introduced shortly as CA-538, 558.

Further, in Tables and text, the "3.5-mm" disks referred to should read "3.18 mm" (⅛''). This is of significance, because the error in diameter produces a 20% error in the apparent blood volume or thyroxine content per disk in the Tables.

Inadvertent omissions from the list of invited reviewers (p 2360) and our annual index are, respectively:

Jocelyn M. Hicks and Royden Rand

and

"Direct Spectrophotometric Determination of α-Amylase Activity in Saliva, with p-Nitrophenyl α-Maltoside as Substrate," by Baiba K. Gillard, Henry C. Markman, and Stephen A. Feig. (p 2279)
A radioimmunoassay for nortriptyline (and other tricyclic antidepressants) in plasma.
Authors: Maguire K P , Burrows G D , Norman T R , Scoggins B A
Published: January 2020

The radioimmunoassay for nortriptyline described here can detect as little 1 microgram/liter of plasma. Within-day precision and day-to-day precision (CV) were +/- 6 and +/- 11%, respectively, over the concentration range 100-200 microgram/liter. The major metabolite hydroxy-nortriptyline, does not cross react with the antiserum. Results so obtained correlate closely with results by a double-isotope derivative dilution technique. The major advantages of this technique over currently available methods are its sensitivity, convenience (many samples can be processed in one day), simplicity, and cost. Further, prior extraction of plasma samples is not required. Cross-reactivity studies have been carried out with all other available tricyclic antidepressants. The antiserum has the ability to bind these drugs, thus radioimmunoassay for all the tricyclic antidepressant drugs can be set up because concurrent use of more than one of these drugs is rare.
Measurement of sodium in albumin solutions with ion-selective electrodes.
Authors: Mohan M S , Bates R G , Hiller J M , Brand M J
Published: January 2020

We have investigated the variations in the potentials of sodium-selective glass-membrane electrodes in various concentrations of albumin. These protein solutions serve as a simple model for measurements of electrolytes in whole blood, plasma, or serum with such an electrode. The results indicate that albumin-sodium binding and variations in liquid-junction potentials are of negligible importance. Mostly, the effect of albumin on the sodium glass-electrode potential is attributable to variations in the activity of sodium, determined by the water content of the solutions. We have calculated apparent fractional protein volumes in albumin solutions from measurements of sodium glass-electrode potential and find they compare well with the classical McLean-Hastings formulation. Our results indicate the need to correct experimentally determined potentiometric electrolyte concentrations for protein volumes.
Enzymatic determination of blood ethanol, with amperometric measurement of rate of oxygen depletion.
Authors: Cheng F S , Christian G D
Published: January 2020

A rapid electrochemical measurement of blood ethanol is proposed. Alcohol is oxidized by NAD+ in the presence of alcohol dehydrogenase; and the NADH produced is aerobically oxidized by horseradish peroxidase. The rate of depletion of buffer-carried oxygen, which is directly proportional to the alcohol concentration in the sample, is amperometrically monitored with a membrane oxygen-sensing electrode. Only a 5-microliter sample of whole blood is required, with no deproteinization, incubation, extraction, or dilution. Results, obtained in less than 1 min, correlate well with those obtained by gas-chromatographic and spectrophotometric methods.
Detection and elimination of contaminations interfering with the determination of zinc in plasma.
Authors: Reimold E W , Besch D J
Published: January 2020

We undertook detailed examination of the practices and procedures involved in the analysis of serum for zinc by atomic absorption spectrophotometry, to identify possible sources of zinc contamination. Some degree of contamination was found in almost all steps of the procedure. These findings are presented and alternative materials and procedures are described that significantly decrease or eliminate contamination from extraneous zinc. The plasma of 76 normal adults was analyzed by the revised procedure, and a lower mean value was obtained than has been previously reported.
Determination of cadmium in urine by graphite-furnace atomic absorption spectroscopy.
Authors: Vesterberg O , Wrangskogh K
Published: January 2020

A simple procedure for graphite-furnace atomic absorption analysis of cadmium in urine, based on the use of a temperature-controlled heating unit, is described. The temperature of the carbon rod is measured by resistance and controlled by electronic feedback. We studied the influence of various heating times and temperatures of ashing and atomization, as well as suitable diluents. Urine samples were diluted with an equal volume of 0.3 mol/liter HNO3, and 1 to 5 microliter of the mixture was analyzed, with background correction. Signals were evaluated electronically, giving values for integrated peaks as well as for peak heights. The detection limit was 1.2 nmol/liter. The method appears to be accurate and reproducible. The CV averaged 8% for concentrations in the range 20 to 100 nmol/liter. Correlations with a chelation-extraction method was 0.994.
Two immunofluorescent methods compared with a radial immunodiffusion method for measurement of serum immunoglobulins.
Authors: Blanchard G C , Gardner R
Published: January 2020

We found high correlations when the values for immunoglobulins G, A, and M, obtained from the analysis of a group of 50 or more human sera by a measured-time radial immunodiffusion method, were compared to values found by two new commercial immunofluorescent methods. The Fiax and the Immuno-Fluor systems make use of fluorescently labeled antibodies and solid-phase immunoadsorbents and offer simple, precise, and objective methods for immunoglobulin quantitation. The Fiax procedure is less time consuming than is the Immuno-Fluor and gives IgG and IgA concentrations that are in close agreement with those found by radial immunodiffusion and IgM values that are significantly lower than the radial immunodiffusion values. The IgG results determined by the Immuno-Fluor method compare well with the radial immunodiffusion results, uhile the IgA values are significantly higher and IgM values significantly lower than those obtained by radial immunodiffusion.
Posture and the composition of plasma.
Authors: Dixon M , Paterson C R
Published: January 2020

For a group of normal medical students, we examined the effect of posture on the concentration of a number of constituents in plasma. On standing, there is a significant increase in plasma total protein, albumin, calcium, alkaline phosphatase, bilirubin, and cholesterol--all proteins or substances bound to protein. Although it is possible to make an allowance for postural variations in plasma calcium, no correction is possible for changes in protein concentrations and care is needed whenever the precise values are important, as in the follow-up of patients with the nephrotic syndrome.
A preliminary evaluation of tissue polypeptide antigen in serum or urine (or both) of patients with cancer or benign neoplasms.
Authors: Menendez-Botet C J , Oettgen H F , Pinsky C M , Schwartz M K
Published: January 2020

We evaluated assays of Tissue Polypeptide Antigen in serum and urine, as an index to the presence of cancer. In the assay, serum, which is first absorbed with human albumin-labeled sheep erythrocytes, or untreated urine (diluted with an equal volume of TPA-free serum) is incubated with antibody specific to Tissue Polypeptide Antigen and then reacted with sheep erythrocytes labeled with Tissue Polypeptide Antigen. We found an increased concentration of Tissue Polypeptide Antigen in the serum of 378 of 513 (74%) and in the urine of 49 of 77 (64%) patients with cancer, as compared with 40/112 (36%) and 7/29 (24%), respectively, for individuals with benign neoplasms. Normal individuals were defined as those with less than 0.09 unit of the antigen per milliliter of specimen. Concentrations exceeding this were found in 2/67 (3%) sera and 6/56 (11%) urines from supposedly normal persons. Tissue Polypeptide Antigen was found in above-normal concentrations in patients with cancer, regardless of neoplasm type and extension, with a higher proportion of abnormal values in patients with distal metastases.
Enzyme-coupled measurement of ethanol in whole blood and plasma with a centrifugal analyzer.
Authors: Jung G , Férard G
Published: January 2020

We describe an automated enzymic method for ethanol determination with a centrifugal analyzer (the GEMSAEC) by measuring the rate of the reaction catalyzed by alcohol dehydrogenase and coupled to aldehyde dehydrogenase. The detection limits, reproducibility, and accuracy of the method have been evaluated. It can be applied to whole blood or plasma, with or without previous deproteinization. Our results, compared with those by an automated alcohol dehydrogenase method in the presence of semicarbazide, show an improved linearity, sensitivity, and rapidity of determination.
Biochemical changes in a porcine model of acute pancreatitis.
Authors: Marenberg S P , Lott J A , Pflug B K , Kibbey W E , Carey L C
Published: January 2020

Pancreatitis was induced in 11 miniature pigs by infusing a bile salt-trypsin solution into the pancreatic duct. Seven animals served as sham-operated controls. Serum ionized calcium, total calcium, albumin, total protein, inorganic phosphorus, urea nitrogen, magnesium, insulin, glucagon, and hematocrit were determined every six to 12 h over a period of one week in both test and control animals. We observed significant decreases in ionized and total calcium, modest decreases in albumin, and significant increases in the inorganic phosphorus, urea nitrogen, and hematocrit in the pancreatitic pigs. The latter two findings were consistent with early acute hypovolemia. Glucagon and insulin appeared to play no role in the hypocalcemia. Glucagon concentrations increased to the same degree in both test and control animals, probably as a result of the stress of being handled and operated on. The highest concentrations of inorganic phosphorus and the lowest concentrations of both ionized and total calcium were seen 18 h after the induction of pancreatitis in the test animals. These findings suggest that parathyrin (parathormone) was not being secreted in adequate amounts, or that the target organs were unresponsive to parathyrin.
Isolation of gamma-glutamyltransferase from human liver, and comparison with the enzyme from human kidney.
Authors: Shaw L M , London J W , Petersen L E
Published: January 2020

We isolated gamma-glutamyltransferase [(gamma-glutamyl)-peptide:amino acid gamma-glutamyltransferase, EC 2.3.2.2] from human liver and compared some of its properties with the same enzyme prepared from human kidney. The enzymes from these two sources are very similar with respect to initial velocity kinetic constants, pH optima of the transpeptidation and autotransfer reactions, heat stability, competitive inhibition by glutathione of the colorimetric assay in which gamma-glutamyl-4-nitroanilide is substrate, stability of catalytic activity to trypsinization, and relative rates of transfer of the gamma-glutamyl moiety from gamma-glutamyl-4-nitroanilide and L-[glycine-2-3/]glutathione to some amino acids and small peptides. The kidney enzyme is inhibited more by the gamma-glutamyl acceptor substrate, glycylglycine, as reflected in a sevenfold lower value for the inhibition constant KiA. Major differences were observed in the lectin-binding properties of liver gamma-glutamyltransferase compared to the kidney enzyme. Lectin-binding property differences are retained for the trypsinized form of the liver and kidney enzymes, although the degree of precipitation was less for certain lectins as compared to the untreated enzyme. Lectin-binding properties were reversed by carbohydrates specific for each lectin. We adapted the histochemical staining technique of Rutenberg et al. [J. Histochem. Cytochem. 17, 517 (1969)] to the detection of gamma-glutamyltransferase activity in acrylamide gels; diffusion artifacts are minimized and the color produced is stable for several days. Untreated and trypsinized forms of the liver enzyme both migrated faster in acrylamide gels (as single bands) than did the corresponding forms of the kidney enzyme.
Rapid determination of light halogenated hydrocarbons in urine.
Authors: Youssefi M , Faust S D , Zenchelsky S T
Published: January 2020

We describe a rapid method for determining light halogenated hydrocarbons (LHH) in urine by electron capture gas-liquid chromatography. The hydrocarbons are extracted from urine into pentane. A 2-microliter injection of the pentane extract provides a detection limit of less than 1 microgram/liter.
High-performance liquid-chromatographic separation and fluorescence measurement of biogenic amines in plasma, urine, and tissue.
Authors: Davis T P , Gehrke C W , Gehrke C W , Cunningham T D , Kuo K C , Gerhardt K O , Johnson H D , Williams C H
Published: January 2020

We describe a high-performance liquid-chromatographic method for measuring histamine, norepinephrine, octopamine, normetanephrine, dopamine, serotonin, and tyramine in plasma (2 ml), brain (0.2 g), or urine. These amines are modifed by pre-column derivatization with o-phthalaldehyde, which stabilizes the molecules, facilitates extraction, and improves detection of nanogram amounts. Before separation, samples were neutralized with KOH and immediately derivatized and extracted into ethyl acetate, in which derivatives were stable for longer than 24 h. Interfering amino acids were removed from ethyl acetate by partitioning twice with Na2HPO4 buffer (pH 10.0). Separation was complete in about 90 min on a "mu Bondapak/phenyl" column, with which a stepwise gradient of methanol/phosphate buffer (pH 5.1) was used. A variable-wavelength fluorometer was used (exciting wavelength, 340 nm; emission wavelength, 480 nm). Amount and response were linearly related from 1 to 200 pmol. Precision (CV) for retention times was 1%, for derivatization and injection 2.5%. Analytical recoveries of the seven amines from 2 ml of plasma fortified with 200 pmol averaged 65% (CV approximately 8%). Data on rat-brain tissue samples are compared with results by the trihydroxyindole method. Application of the method to urine from normal persons and a patient with a brain tumor is demonstrated.
Determination of drugs in plasma by high-performance thin-layer chromatography.
Authors: Fenimore D C , Davis C M , Meyer C J
Published: January 2020

High-performance thin-layer chromatography was used to determine chlorpromazine, amitriptyline, nortriptyline, imipramine, desipramine, phenobarbital, and phenytoin in plasma, to demonstrate the utility of this technique for routine analysis. We quantitated the separated components by use of ultraviolet reflectance spectrometry with detection limits as low as 1 microgram/liter. Regressions of psychoactive agents extracted from plasma were linear over the range of 0 to 300 microgram/liter. The anti-convulsant drugs, phenobarbital and phenytoin, were determined over a range of 0 to 50 mg/liter. Analyses were rapid, reproducible, and well-suited to large-scale programs. Separated components also can be identified in situ by ultraviolet spectrophotometry.
Improved enzymatic assay of chloramphenicol.
Authors: Smith A L , Smith D H
Published: January 2020

We partly purified R-factor-encoded chloramphenicol acetyltransferase (EC 2.3.1.28) from a highly choloramphenicol-resistant mutant derived from Escherichia coli W677/R5. The preparation permitted rapid quanitation of chloramphenicol by use of [14C]acetylcoenzyme A, removing the diacetylated product by selective adsorption onto micropore filters. Succinyl and glucuronyl 3-hydroxyl esters of chloramphenicol were not active as substrates for the preparation, nor were they active as inhibitors. The enzyme was free of chloramphenicol reductase activity, and utilizes other biologically active chloramphenicol analogs. Other antibiotics, at concentrations commonly found in human sera, and blood preservatives, at concentrations 10-fold that found in blood-collection tubes, did not interfere with the enzymic quantitation of chloramphenicol. We conclude that this enzyme preparation permits rapid clinical quantitation of chloramphenicol.
Micro-scale extractive alkylation procedure for the gas-chromatographic measurement of theophylline in serum and saliva, with use of a nitrogen detector.
Authors: Joern W A
Published: January 2020

In this 10-min gas-chromatographic assay for theophylline, a 25-microliter portion of serum or saliva sample, 100 microliter of extraction reagent, and 1.0 ml of solvent were mixed, the solvent separated and evaporated, and an aliquot of the reconstituted extract injected into the gas chromatograph. At a concentration of 11 mg/liter, within-run precision (CV) was 3.6% and run-to-run 2.9%. At 22 mg/liter, within-run precision was 2.8%. Concentration and instrument response are linearly related between 2 and 40 mg/liter. The limit of detection was 0.4 mg/liter. Results correlated well (r = 0.98) with those by another micro-scale gas-chromatographic procedure. Analytical recovery was estimated to exceed 90%, and no interferences from other xanthines or related drugs were observed.
Spectrodensitometry of nanogram quantities of cholesterol.
Authors: Touchstone J C , Dobbins M F , Hirsch C Z , Baldino A R , Kritchevsky D
Published: January 2020

In studies of hydrolysis and synthesis of cholesterol esters by aortic enzymes, conventional methods (aside from isotope methodology) are too insensitive to measure reaction products when few aortas are used. High-performance thin-layer chromatography coupled with spectrodensitometry of plates charred after spraying with a cupric acetate-phosphoric acid reagent permitted quantitation of 10-ng amounts of cholesterol, cholesterol oleate oleate, and oleic acid. Linear calibration curves were obtained after spectrodensitometry of chromatograms containing 10-200 ng of lipid. The results were verified by radioassay.
A fluorogenic substrate for angiotensin-converting enzyme in plasma.
Authors: Russo F S , Persson A V , Wilson I B
Published: January 2020

A simple, sensitive, and reproducible assay for angiotensin-converting enzyme is described. It is based on the hydrolysis of the minimally fluorescent substrate p-nitrobenzyloxycarbonylglycyl-L-tryptophylglycine to the products p-nitrobenzyloxycarbonylglycine and the highly fluorescent L-tryptophylglycine. The L-tryptophylglycine was analyzed by fluorometry (lambda excitation = 285 nm; lambda emission = 350 nm). The mean value for human plasma (serum) is 16.5 nmol of substrate hydrolyzed per minute per milliter of plasma under the described assay conditions.
Analysis for salicylic acid in serum by high-performance liquid chromatography.
Authors: Blair D , Rumack B H , Peterson R G
Published: January 2020

We report a specific and sensitive quantitative assay for salicylate in serum or plasma. As little as 3 microliter of sample can be analyzed by an external standard procedure and larger volumes by diluting with the internal standard (o-methoxybenzoic acid). No extraction or derivatization is necessary and salicylic acid and the internal standard elute in 6 and 3.75 min, respectively. Peak-height ratios are linearly related to concentrations between 10 and 500 mg/liter. By comparison with an existing fluorometric method, our procedure is faster, more specific, and more sensitive.
Gas-liquid chromatographic determination of bupivacaine and lidocaine in plasma.
Authors: Zylber-Katz E , Granit L , Levy M
Published: January 2020

A modified gas-liquid chromatographic method for determining plasma concentrations of bupivacaine and lidocaine is described, with cyclizine as an internal standard. The extraction procedure requires no solvent evaporation, thus overcoming the problem of drug volatility. Concentrations as low as 0.1 mg/liter can be determined. The plasma sample is made alkaline and extracted into n-hexane, re-extracted into a small volume of an aqueous acid phase, and finally extracted into 50 microliter of methylene chloride after alkalinization. The final extract is assayed by gas chromatography on a 5% OV-17 column. The extraction scheme of the present method eliminates interferences by endogenous plasma constituents.
Spurious brain creatine kinase in serum from patients with renal disease.
Authors: Coolen R B , Herbstman R , Hermann P
Published: January 2020

Creatine kinase isoenzyme I(BB) is generally not detectable in normal serum, and its occurrence in serum has been documented in only a few disease states. In particular, increased activity of this isoenzyme has been reported in association with chronic renal failure, hemodialysis, and renal transplantation. The present study demonstrates that the apparent creatine kinase observed in the serum of such renal patients is an artifact, observed as a result of measuring creatine kinase isoenzymes by fluorescence. Our observations resemble those of McKenzie et al. [Clin. Chim. Acta 70, 333(1976)] concerning an artifact in the fluorometric determination of lactate dehydrogenase isoenzymes in the sera of patients with end-stage renal failure. The artifact binds to albumin, is not a protein, and occurs in some normal sera at very low concentrations. This artifact can be mistakenly identified as isoenzyme I in renal-disease patients if CK isoenzymes are determined fluorometrically.
Emergency determination of acetaminophen.
Authors: Meola J M
Published: January 2020

I describe a senstive colorimetric micromethod in which a pH 11.0 carbonate buffer is used to induce acetaminophen to react with Folin-Ciocalteau reagent at room temperature. These reagents and a simple solvent extraction favor formation of a stable indophenol dye with acetaminophen, giving this procedure a good degree of specificity. Results correlate well with those by liquid-chromatographic procedures, and day-to-day precision is less than 8%.
Detergent-containing glucose oxidase reagent for use with the Beckman glucose analyzer.
Authors: Bajema L L , Lee W , Zebelman A M , Kenny M A
Published: January 2020

We describe a modified glucose oxidase reagent for use in the polarographic determination of glucose with the Beckman "Glucose Analyzer." The reagent contains 1 mL/L of a surfactant (Brij-35 250 g/L solution) as the wetting agent instead of glycerol. Precipitation of components associated with the formulation recommended by Fischl et al. [Clin. Chem. 21, 760 (1975)] does not occur with this reagent. It can be used immediately after preparation. When compared to analytical performance of the commercially prepared reagent, the precision was unchanged by the modified reagent, but the upper limit of accurate response was diminished (7.5 g/L vs. 6.7 g/L). The modified reagent is less expensive than are commercially prepared reagents.
Discrete modular system for automation of radioimmunoassay of serum choriomammotropin.
Authors: Hammond G L , Leinonen P , Vihko R
Published: January 2020

We describe a discrete automated radioimmunoassay system for determining choriomammotropin (placental lactogen) in human serum. With the present system it can be measured in as many as 37 unknown sera (50 muL) and three quality-control sera, in duplicate, within 1.5 h. The time required for sample preparation, incubation (15 min), and separation of free and bound radioactivities (a 150 mL/L polyethylene glycol solution is superior to a twofold volume of absolute ethanol) is less than 45 min. The remaining time required is for counting and data processing. Intra-assay precision is 4.6% (CV). The modular approach endows the instrumentation with much flexibility, and consequently is suitable for automation of a wide range of assay protocols.
Removal of contaminating substances before gas-liquid-chromatographic determination of aldosterone in urine.
Authors: Graef V , Furuya E , Nishikaze O
Published: January 2020

This paper deals with removal of contaminants before gas-chromatographic determination of aldosterone in urine. Urine is incubated with beta-glucuronidase, which hydrolyzes all beta-glucuronides except aldosterone-18-glucuronide. The contaminants (the aglycones released and other methylene chloride-soluble substances) are extracted with methylene chloride. Solvolysis of the aqueous phase liberates aldosterone from aldosterone-18-glucuronide, which then is extracted with methylene chloride and oxidized by use of periodic acid. The resulting lactone can be easily separated by one-dimensional thin-layer chromatography and determined by gas-liquid chromatography.
Improved determination of serum theophylline by gas chromatography with use of a nitrogen-phosphorus detector.
Authors: Vinet B , Zizian L
Published: January 2020

We describe a method for the 7-min determination of theophylline in less than 100 muL of serum. The procedure requires no centrifugation or solvent evaporation after extraction. Butylation is done on the gas-chromatographic column by injecting the serum extract followed by a butylating mixture which contains 1-iodobutane as the alkylating agent. The method is precise, accurate, and free of interference. Results correlate well with those by ultraviolet spectrophotometry.
Quality of sweat test performance in the diagnosis of cystic fibrosis.
Authors: Shwachman H , Mohmoodian A
Published: January 2020

The sweat test, correctly performed, appears to be reliable, but results were found to be unreliable about half the time in 84 small community hospitals assessed. The reasons are inexperienced and untrained technicians, the infrequency of test performance, the use of unstandardized equipment, and the lack of appropriate standards. It behooves all clinical laboratory directors to re-examine and standardize their procedures and use the recommendations of the Cystic Fibrosis Foundation. Physicians should be made aware of the current situation and send their patients only to laboratories that perform the test reliably.
Homogeneous enzyme immunoassay of serum digoxin with use of a bichromatic analyzer.
Authors: Eriksen P B , Andersen O
Published: January 2020

We applied the Enzyme Multiplied Immunoassay Technique (EMIT; Syva Corp., Palo Alto, CA) for determination of serum digoxin to the ABA-100 bichromatic analyzer. Assay conditions were almost exactly as prescribed for the manual procedure, but the ABA-100 offers high automation, smaller reagent volumes, and shorter reaction time. Precision studies gave CV's of less than 10%. Sixty patients' samples, analyzed for digoxin by radioimmunoassay and this enzyme immunoassay, gave a correlation (r) of 0.941. Results obtained with the ABA-100 were apparently slightly higher. One kit provides reagents for 250 assays, as compared to 70 assays with the manual procedure. In an emergency situation a result will be available about 60 min after the patient's sample is received; one operator can analyze about 120 samples in 8 h.
A liquid-chromatographic analysis for ritalinic acid [alpha-phenyl-alpha-(2-piperidyl) acetic acid] in serum.
Authors: Soldin S J , Hill B M , Cahn Y P , Swanson J M , Hill J G
Published: January 2020

We describe a "high-performance" liquid-chromatographic assay for measuring ritalinic acid in 300 muL of serum. The procedure includes adsorption of ritalinic acid and internal standard (alpha,alpha-dimethyl-beta-emthylsuccinimide) from serum onto charcoal, from which both compounds are eluted with methanol. The methanol extract is evaporated, the residue dissolved in a 93/7 (by volume) mixture of potassium phosphate buffer (20 mmol/L, pH 3.8) and acetonitrile, and an aliquot of this solution is chromatographed. We used a mu-Bondapak C-18 column with the above-mentioned buffer system as mobile phase, a flow rate of 2.0 mL/min, and a column temperature of 40 degrees C. Ritalinic acid and internal standard are detected by their absorbance at 192 nm and quantitated by measuring peak heights. The procedure allows for the reliable analysis for ritalinic acid in serum at concentrations greater than 50 microgram/L. Analytical recoveries were greater than 73% and the method affords good day-to-day precision (CV, less than 12%).