bluuebunny/crossref_metadata_2025 huggingface dataset:
Aminoglycoside antibiotic measurement by bioluminescence, with use of plasmid-coded enzymes.
Authors: Daigneault R , Larouche A , Thibault G
Published: January 2020

We describe a bioluminescent assay for gentamicin in serum that is applicable to the measurement of other aminoglycosides as well. The assay is based on the measurement of residual ATP with the luciferase reaction after incubation of the antibiotic with a plasmid-coded enzyme. Two aminoglycoside-inactivating enzymes were used: an adenylytransferase and an acetyltransferase coupled to S-acetyl coenzymeA synthetase. We investigated the latter system further because of the good stability of the acetyltransferase, its recovery in high yield from bacteria, and its more favorable ATP/gentamicin mass ratio. Serum ATPases were inactivated at 60 degrees C for 20 min. The operating range of the assay was 0-15 mg of gentamicin per liter. The precision (CV) was 10.1% at a concentration of 2 mg/L and 1.1% at 10 mg/L. The method correlated well with a radio-enzymatic assay for mock unknown sera (r = 0.981). The results were available within 2 h.
A new screening system for nonsteroidal anti-inflammatory drugs based upon inhibition of chemiluminescence produced from human cells (granulocytes).
Authors: Van Dyke K , Van Dyke C , Udeinya J , Brister C , Wilson M
Published: January 2020

A new screening system for nonsteroidal anti-inflammatory drugs that involves use of human phagocytic cells has been developed in which chemiluminescence measurement is used. Luminol-dependent chemiluminescence is measured after the addition of opsonized (coated with antibodies and complement) zymosan particles to human granulocytic leukocytes in the presence or absence of drugs. Of all the compounds tested, indomethacin was the most potent in blocking chemiluminescence, with measurable inhibitory activity at 5 mumol/L. The order of inhibitory potency at 0.1 mmol/L and in the presence of Ca2+ and Mg2+ was indomethacin greater than sodium salicylate greater than fenoprofen Ca greater than tolmetin greater than naproxen greater than ibuprofen. It is likely that the active compound itself must be added to the system because aspirin did not inhibit chemiluminescence, whereas its metabolite, sodium salicylate, was markedly inhibitory. Dexamethasone and methylprednisolone also did not inhibit chemiluminescence. The drugs that inhibit chemiluminescence are also known inhibitors of prostaglandin synthase (cyclooxygenase portion).
Maximizing the efficiency of an admissions test profile in a 680-bed hospital.
Authors: Cole G W
Published: January 2020

I describe the development and improved use of an efficient planned and scheduled approach to laboratory testing in a large tertiary-care hospital, which anticipates virtually all the usual needs of patient and clinician and which has been demonstrated to provide better service at lower cost.
Immunoradiometric serum ferritin concentration compared with stainable bone-marrow iron as indices to iron stores.
Authors: Forman D T , Vye M V
Published: January 2020

Immunoradiometric analysis has made it possible to measure ferritin reliably in serum. The determination is a sensitive means of assessing body iron because concentrations of ferritin closely parallel the body's reticuloendothelial iron stores. The information provided is similar to that obtained from bone-marrow aspirates or biopsies stained for iron with Prussian Blue. Comparative studies indicate good correlation between iron content in the marrow and serum ferritin, except possibly in patients with leukemia and metastatic tumors to the bone marrow. The clinical value of the assay in relation to other indicators of iron status (i.e., hemoglobin, erythrocyte indices, serum iron and iron-binding capacity, and erythrocyte protoporphyrin) is in its specificity and sensitivity for assaying physiological iron stores.
Computer-assisted analysis of two-dimensional electrophoreses of human lymphoid cells.
Authors: Lester E P , Lemkin P , Lipkin L , Cooper H L
Published: January 2020

Two-dimensional electrophoresis in polyacrylamide gel provides a highly reproducible display of biosynthetically pulse-labeled cellular polypeptides, whose relative rates of synthesis can be judged from the relative spot densities they produce in fluorographic images of the electrophoreses. When experimental and control cell populations synthesize protein at differing rates, either the sample load per gel or the duration of exposure of gel to film may be adjusted to produce comparable experimental and control images with no alteration in the rank order of relative spot density. However, variation in the duration of pulse-labeling may significantly alter relative spot density. Film density saturation and nonlinear response to radiation may also alter relative spot density for polypeptides with very large or very small amounts of radioactivity. Stimulation of human peripheral blood lymphocytes with phytohemagglutinin alters the relative synthesis rates of certain polypeptides visualized on the gels. These can be quantitated by using an automated series of computer algorithms to analyze the images. Data from this analysis confirm statistically significant alterations in the relative densities produced by certain polypeptides. Various human lymphoblastoid cell lines may also be distinguished from each other by this technique.
Diagnosis of viral infections by multicomponent mass spectrometric analysis.
Authors: Abbott R , Anbar M , Faden H , McReynolds J , Rieth W , Scanlon M , Verkh L , Wolff B
Published: January 2020

Metabolic profiles of urine extracts of humans with viral infections, as well as of media of virus-infected human tissue cultures, have been analyzed by non-fragmenting mass spectrometry and compared with corresponding controls. The spectra were then subjected to several alternative computerized statistical procedures to detect diagnostic biochemical profiles. Controlled longitudinal studies on fully informed, consenting volunteers who received sandfly fever virus demonstrate the onset of a characteristic metabolic pattern that precedes the onset of symptoms and subsides when the patients overcome the infection. Longitudinal studies of human tissue cultures infected with poliomyelitis virus demonstrate characteristic metabolic patterns within a few hours after infection. Non-fragmenting mass spectrometry may thus provide the clinical laboratory with a sensitive, reliable test for viral infections significantly faster than attainable by current techniques.
Continuous-flow zone electrophoresis: experimental demonstration and discussion of potential clinical and research applications.
Authors: Fosslien E , Anderson D N , Nastir A
Published: January 2020

We report continuous-flow sample application and continuous electrophoretic separation on a moving cellulose acetate tape. This very simple automated electrophoretic system requires minimum electronic control circuitry. Samples and calibrators are pumped by a peristaltic pump from sample containers onto a continuously moving cellulose acetate tape. Applied samples are interspaced with rinse solution (buffer) and small air segments for sample integrity. Electrophoretic separation, staining, and destaining are all continuous functions performed on the continuously moving tape. The system appears suitable for separation of proteins. Continuous-flow application could be combined with discrete sample application onto the same tape for continuous counter-immunoelectrophoresis; moreover, counter-electrophoresis of two continuously applied proteins, with one applied in a gradient, could potentially produce a system for automated gradient counter-immunoelectrophoresis. It should also be possible to design a simple system for automated immunofixation electrophoresis.
Combined liquid chromatograph/mass spectrometer for involatile biological samples.
Authors: Blakley C R , Carmody J C , Vestal M L
Published: January 2020

A new liquid chromatograph/mass spectrometer has been developed in our laboratory for application to analysis of biological molecules of extremely low volatility. Oxyhydrogen flames rapidly vaporize the total liquid-chromatographic effluent, and molecular and particle beam techniques are used to efficiently transfer the sample to the ionization source of the mass spectrometer. This new instrument is comparable in cost and complexity to a combined gas chromatograph/mass spectrometer, but extends the capabilities of combined chromatography/mass spectrometry to a broad range of compounds not previously accessible. We are currently testing biologically significant samples with this instrument, using reversed-phase liquid-chromatographic separation and both positive and negative ion chemical-ionization mass spectrometry. Results have been obtained from mixtures of nucleic acid components—bases, nucleosides, and nucleotides—and from amino acids, peptides, saccharides, fatty acids, vitamins, and antibiotics. In all cases investigated to date, ions indicative of molecular mass are obtained in at least one of the operating modes available. Detection limits are typically in the 1-10 ng range for full mass scans (about 80-600 amu); sub-nanogram quantities are usually detectable with single-ion monitoring.
Specific detection of primary amines in the effluent of a gas-chromatographic column by on-line measurement of fluorescence.
Authors: Chow F , Karmen A
Published: January 2020

Compounds with primary amino groups react with o-phthalaldehyde in solution to yield highly fluorescent products. This reaction is now in wide use for detecting amino acids and amines in liquid-chromatography effluents. We report here a method for using o-phthalaldehyde to detect primary amines in the effluent of a gas-liquid-chromatographic column. The effluent gas, nominally 20 mL/min, is delivered to a scrubber consisting of a small-bore, simulated capillary chromatographic column that is simultaneously supplied with 1 mL/min of the reagent solution. The liquid effluent of the scrubber, separated from the gas, is drawn through the flow cell of a fluorometer. Short-chain amines and ammonia were quantitatively scrubbed. The response of the fluorometer was directly proportional to the number of nanomoles injected into the column. Less than a nanomole of amine was detectable. Comparison of results with those from a hydrogen flame-ionization detector showed minimal additional peak broadening or compromise of resolution. These results demonstrate the feasibility of using highly specific, as well as sensitive, liquid-chromatographic detection methods for gas-liquid chromatography.
Liquid chromatography with amperometric reaction detection involving electrogenerated reagents: applications with in-situ generated bromine.
Authors: King W P , Kissinger P T
Published: January 2020

We describe the use of electrogenerated reactants for continuous on-line reaction detection with thin-layer hydrodynamic amperometry. The reagent is introduced into the liquid-chromatographic column effluent at a constant rate by using controlled-current electrochemistry. After the effluent passes through a short reaction coil, the reagent concentration is monitored at the detector. Reaction of eluted compounds with bromine is signalled by changes in the current detected. The direct electrochemical control of the reagent concentration allows changes to be made, even during the course of obtaining a chromatogram. Depending on the specific reagent or reaction, the reagent is supplied either by addition of a second stream or by direct generation in the mobile phase. The latter configuration provides sufficient baseline stability to permit detection of the uptake of as little as 10 pmol of reagent bromine. The technique has been used to detect nanograms of underivatized fatty acids, prostaglandins, and phenols after separation by reversed-phase liquid chromatography.
Electrophoretic, kinetic, and immunoinhibition properties of gamma-glutamyltransferase from various tissues compared.
Authors: Shaw L M , Petersen-Archer L , London J W , Marsh E
Published: January 2020

gamma-Glutamyltransferase (EC 2.3.2.2) from human liver, kidney, pancreas, and duodenum migrated in acrylamide gels (65 g/L) as a single band with the following decreasing order of electrophoretic mobility: liver > pancreas > kidney > duodenum. The initial velocity kinetic constants of pancreatic and duodenal gamma-glutamyltransferase and of the enzyme in human serum were determined and compared with those we previously established for the enzyme from human kidney, liver, and serum. The greatest differences were in the glycylglycine competitive-inhibition constants: kidney gamma-glutamyltransferase was the most strongly inhibited and pancreatic enzyme the second most strongly inhibited by high concentrations of glycylglycine, with the liver and duodenal isoenzymes only slightly inhibited and the enzyme in serum not inhibited over the concentration range (0-150 mmol/L) of glycylglycine used. Differences between the other kinetic constants of these isoenzymes were much smaller. Human liver gamma-glutamyltransferase was obtained in a highly purified form by a six-step procedure that included papain digestion of the original homogenate. Rabbit antisera raised against this preparation inhibited liver, kidney, and pancreatic gamma-glutamyltransferase activity equally well (78, 76, and 78% inhibition, respectively), but inhibited the hog-kidney enzyme only slightly (1%). We conclude that the polypeptide portions of the isoenzyme molecules are structurally similar but that the carbohydrate moieties differ significantly in structure and topography.
Rapid radioimmunoassay for prostate-specific acid phosphatase in human serum.
Authors: Vihko P , Kostama A , Jänne O , Sajanti E , Vihko R
Published: January 2020

We describe a rapid radioimmunoassay for human prostatic acid phosphatase (EC 3.1.3.2) in serum, with use of monospecific antisera raised in rabbits against the primary highly purified acid phosphatase (pl 4.9) from human prostates, and with a second antibody-polyethylene glycol porecipitation. This radioimmunoassay is sensitive and can be performed within 5 h. Concentrations of the immunoreactive acid phosphatase in sera of healthy men (n = 394) ranged from 0.3 to 3.6 microgram/L (mean 1.94, SD 0.66 microgram/L). Concentrations of the enzyme in sera of men with benign prostatic hyperplasia (n = 56) or with carcinoma of nonprostatic origin (n = 24) were identical with those of the reference group. Serum concentrations of immunoreactive prostatic acid phosphatase of patients with occult, non-metastatic, and metastatic prostatic carcinoma varied from 1.7 to 9.3 (n = 9), 4.2 to 59.4 (n = 12), and 20 to 198 (n = 10) microgram/L, respectively. The amount of immunoassayable prostatic acid phosphatase was unchanged for at least five days in serum stored at 4 degrees C.
Gas chromatographic-mass spectrometric identification of isosaccharino-1,4-lactone in human serum and urine.
Authors: Niwa T , Maeda K , Ohki T , Saito A , Sakakibara J , Kobayashi K
Published: January 2020

We have identified alpha-isosaccharino-1,4-lactone and beta-isosaccharino-1,4-lactone in the ethyl acetate extract of an ultrafiltrate of blood and of normal human urine, using high-resolution gas chromatography-mass spectrometry. The ultrafiltrate of blood was obtained from three patients on hemodialysis, one with psoriasis vulgaris, one with uremia, and one with amyotrophic lateral sclerosis. The concentration of beta-isosaccharino-1,4-lactone in the ultrafiltrate was two- to threefold that of alpha-isosaccharino-1,4-lactone. Mass fragmentography showed that isosaccharino-1,4-lactone is normally present in human serum in a concentration of 1.7 (SD 1.5) mg/L. In physiological fluid, isosaccharino-1,4-lactone apparently is present in its hydrated form, isosaccharinate.
Modified aca method for determination of iron chelated by deferoxamine and other chelators.
Authors: Steinmetz W L , Glick M R , Oei T O
Published: January 2020

Iron in serum and urine specimens containing deferoxamine (a chelator used in treating acute iron intoxication) can be measured by the DuPont aca discrete-analyzer method for iron if 0.5 mL of a 100 mL/L solution of thioglycolic acid (mercaptoacetic acid) is added to the IRN-1 and IRN-2 test packs before their use. This reagent releases the unreactive chelated iron, allowing reliable colorimetry. Because about 50% of hemoglobin iron is measured by the modified method, hemolysed samples should not be used (or results should be corrected for hemoglobin iron). The modification allows emergency determination to total serum iron and assessment of urinary iron during chelation therapy, as well as measurement of plasma iron in samples treated with sodium citrate or oxalate plus fluoride (the unmodified aca iron method gives falsely low iron values in the presence of these chelators). About 60% of iron present as the dextran complex (Imferon) is measured by the modified aca method, as compared with <5% by the unmodified aca technique.
Advantage of Florisil over charcoal separation in a mechanized testosterone radioimmunoassay.
Authors: Harman S M , Tsitouras P D , Kowatch M A , Kowarski A A
Published: January 2020

We have facilitated radiommunoassay of testosterone by mechanizing every pipetting step and separating bound from free hormone by shaking assay tubes with activated magnesium silicate (Florisil) in a multi-tube Vortex mixer. Compared with dextran-charcoal separation, Florisil separation has the advantage that testosterone is only negligibly adsorbed except during active shaking, so that the interval for adding adsorbent does not produce discrepancies in bound hormone estimates between first and last assay tubes in a series. Our method gave CVs of 5% (within assay) and 6% (between assay) for samples containing 4.0 mug of testosterone per liter, considerably better than those obtained with charcoal (respectively 12 and 15%). The assay also had low blank values and demonstrated excellent accuracy.
Improved ultrafiltration method for determining unbound cortisol in plasma.
Authors: Jerkunica I , Sophianopoulos J , Sgoutas D
Published: January 2020

We describe a modified ultrafiltration method for measuring unbound cortisol in diluted or undiluted plasma or serum. After equilibration at 37 degrees C with purified [3H]cortisol, plasma or serum with or without buffer was placed in the ultrafiltration cell and two successive 0.2-mL fractions of protein-free ultrafiltrate were obtained. Under our conditions, free ligand concentration was independent of flow rate. The second fraction (the first is discarded) was used for determining the proportion of unbound cortisol. The assay is rapid (less than 2 h), practical (no more than 1.5 mL of plasma or serum is necessary), and reproducible (CV: 4.5% within assay and 5.2% in different assays). Samples from normal men and women (blood taken at 0800 and 1600 hours), from pregnant women, and from patients with Cushing's disease and adrenal insufficiency gave results that agreed with those obtained by equilibrium dialysis.
Assay for trimethoprim in serum and urine by means of ion-pair chromatography.
Authors: Watson I D , Shenkin A , McIntosh S J , Cohen H N
Published: January 2020

We describe a rapid, precise, and reliable procedure for assay of trimethoprim in serum and urine by ion-pair chromatography. Trimethoprim concentrations in urine are determined by an externally standardized, direct-injection procedure; assay in serum involves a simple preliminary extraction and internal standardization. The assays are suitable for pharmacokinetic studies and have been applied to determination of trimethoprim concentrations in serum and urine during therapy with Co-trimoxazole, a sulfonamide/trimethoprim preparation.
Variability in unconjugated and total estriol in serum during normal third trimester pregnancy.
Authors: Penney L L , Klenke W J
Published: January 2020

We determined unconjugated and total estriol concentrations in serum during the third trimester in 34 normal gestations. Data were obtained weekly for 13 women for as long as nine weeks; 21 others were studied daily for up to 15 days. No correlation between birth weight and either estriol fraction was demonstrated. Between-patient variability was less for unconjugated estriol and was similar for weekly and daily data. Within-patient variability was also less for unconjugated estriol and, as expected, was less for daily than for weekly data. The CV for daily samples, within patient, averaged 13.0% for unconjugated estriol and 20.3% for total estriol. Our data support the daily determination of unconjugated estriol in serum, evaluated for percent changes from single or mean preceding values, as the preferred method of monitoring estriol during pregnancy.
Free amino acid analysis of untimed and 24-h urine samples compared.
Authors: Tsai M Y , Marshall J G , Josephson M W
Published: January 2020

We measured 11 amino acids in untimed urine samples, to determine whether such samples are suited for use in diagnosis of aminoacidurias. Results for untimed samples varied by as much as 25% more than for 24-h collections when amino acid excretions were expressed in terms of urinary creatinine. Values decreased with increasing age for either type of specimen. Urinary amino acid excretions were also determined with untimed or 24-h samples from patients with cystinuria. Lowe’s syndrome, nonketotic hyperglycinemia, or phenylketonuria. In all cases studied, the amino acids diagnostic of the diseases significantly exceeded the reference interval obtained for 260 control subjects in six age categories. We conclude that untimed urine samples can be used for diagnosis of these inborn errors of amino acid metabolism, but further studies are needed to evaluate their usefulness for other metabolic disorders.
Concurrent measurements of carcinoembryonic antigen, glucosephosphate isomerase, gamma-glutamyltransferase, and lactate dehydrogenase in malignant, normal adult, and fetal colon tissues.
Authors: Munjal D D
Published: January 2020

Carcinoembryonic antigen and activities of glucosephosphate isomerase (EC 5.3.1.9), γ-glutamyltransferase (EC 2.3.2.2), and lactate dehydrogenase (EC 1.1.1.27) were measured in aqueous extracts of fetal, normal adult, and malignant human colon tissues. Fetal colon, as well as primary and metastatic colon tumor tissue, showed higher activities of these analytes than did normal adult human colon. Liver metastases of colon cancer gave the highest values, normal adult human colon the lowest. Statistically, these differences were more striking in the case of carcinoembryonic antigen and glucosephosphate isomerase than for γ-glutamyltransferase or lactate dehydrogenase. In contrast to the other markers, γ-glutamyltransferase activity was lower in fetal organs than in normal adult colon and colon tumors. These results are consistent with earlier observations that activities of these markers are significantly increased in the blood of patients with metastatic colon cancer.
Macro creatine kinase: a study on CK-linked immunoglobulin.
Authors: Yuu H , Ishizawa S , Takagi Y , Gomi K , Senju O , Ishii T
Published: January 2020

We describe three cases in which creatine kinase (CK, EC 2.7.3.2) was linked to immunoglobulin in serum. In this study, its prevalence was 0.8%. Enzyme-immunofixation electrophoresis revealed that the heavy chain of CK-linked immunoglobulins was of class alpha in all cases. The light-chain type was identified as lambda in two cases and as both lambda and kappa in one case. The complexes were dissociated at pH 3.4 and re-formed with CK isoenzymes MM and MB at pH 7.4. The complex fraction obtained by gel filtration was not inhibited by anti-CK-M antibodies. Treatment of the serum with urea after heating shows residual CK-MM activity; in contrast, normal CK activity disappeared entirely after this treatment. The present study suggests that CK-linked immunoglobulins may be one of the circulating immune complexes and must be distinguished from other macro-CK in the strict sense. The results obtained show that the presence of the complexes results in false-positive CK-B activity in the immuno-inhibition test, and they may provide interesting insights into the mode of binding of the CK-linked immunoglobulins.
Measurement of ultrafiltrable calcium in serum with use of the "Worthington Ultrafree Anticonvulsant Drug Filter".
Authors: Eckfeldt J H , Koehler D F
Published: January 2020

We describe a method for ultrafiltrable calcium involving the use of Worthington Ultrafree Anticonvulsant Drug Filters. When measured at 37 °C, with mineral oil covering the sample to prevent loss of CO2, values for ultrafiltrable calcium correlated reasonably well (r = 0.91) with those for ionized calcium as measured with an ion-selective electrode. All patients’ samples with significantly high or low values for ionized calcium were identified by the ultrafiltration method, including one specimen for which the McLean-Hastings protein correction could not explain the discrepancy between ionized and total calcium. The method requires about 2 mL of serum, yields about 100 microL of protein-free ultrafiltrate, and with it any laboratory with a semi-micro calcium method can measure ultrafiltrable calcium.
Multivariate analysis of an enzymic profile for the differential diagnosis of viral hepatitis.
Authors: Plomteux G
Published: January 2020

Differential diagnosis of acute viral hepatitis, persistent chronic hepatitis, aggressive chronic hepatitis, and post-necrotic cirrhosis can reasonably be achieved on the basis of three well-known liver-function tests: aspartate aminotransferase, alanine aminotransferase, and glutamate dehydrogenase. With use of principal-component analysis, these four liver diseases can be characterized by two criteria: a “cytolytic” criterion, correlated particularly with a membrane-permeability test—namely, alanine aminotransferase activity—and a “mitochondrial damage” criterion, which is associated with above-normal ornithine carbamyltransferase and glutamate dehydrogenase activities.
Apparent mitochondrial creatine kinase in the serum of a patient with metastatic cancer to the liver.
Authors: Heinz J W , O'Donnell N J , Lott J A
Published: January 2020

Creatine kinase (CK, EC 2.7.3.2) activity in the serum of a patient with metastatic carcinoma migrated as two distinct bands cathodal to the origin and to CK-3 on agarose gel electrophoresis. The more cathodal isoenzyme (CKm-2) is of high molecular mass, is precipitated by ammonium sulfate at 30% of saturation, and is not retarded by Sephadex G-100. Treatment with urea at a concentration of 6 mol/L caused CKm-2 to elute with proteins of lower molecular mass on a G-100 column and shifted the electrophoretic migration to a position just cathodal to the origin (CKm-1). Antibody to CK-1 and CK-2 did not affect the activity of either CKm-1 or CKm-2. Similarities between these cathodal bands of CK activity and mitochondrial CK suggest the mitochondrial origin of these isoenzymes. These cathodal CK isoenzymes reacted unpredictably with different commercial reagent systems for determination of CK activity in serum or in agarose gel.
Total creatinine kinase and isoenzyme MB activity in serum and skeletal muscle of a patient with dermatomyositis.
Authors: Tsung S H , Huang T Y , Han D , Loh W P
Published: January 2020

Total creatine activity was greatly increased, and creatine kinase MB isoenzyme was measurable in the serum of a patient with dermatomyositis. In contrast, total CK activity in an extract of skeletal muscle from this patient was markedly decreased as compared with normal muscle extracts, but contained about the normal proportion of the MB isoenzymes, 1.5%. We believe skeletal muscle to be the source of the MB isoenzyme in the serum, because the patient’s electrocardiographic pattern was normal and she had no signs or symptoms of myocardial infarction.
Liquid-chromatographic determination of norepinephrine and epinephrine in human plasma.
Authors: Yui Y , Fujita T , Yamamoto T , Itokawa Y , Kawai C
Published: January 2020

We describe a “high-performance” liquid-chromatographic method for measuring picogram amounts of norepinephrine and epinephrine simultaneously in human plasma. Alumina-treated samples are injected onto a strong cation-exchange column (Zipax SCX) for the separation of catecholamines, with a sodium phosphate buffer/acetonitrile mixture as mobile phase. The separated catecholamines then enter a continuous-flow system and the reagents for the trihydroxyindole reaction are added sequentially; the fluorescent products are then measured with a spectrofluorophotometer. Analytical recoveries from plasma averaged 66% for norepinephrine, 68% for epinephrine. Amount and response (peak height) were linearly related up to 1000 pg. For catecholamines in human plasma, within-run and day-to-day CV's were 0.2% and 1.0%, respectively, for norepinephrine, and 0.3% and 0.9%, respectively, for epinephrine. Average values for norepinephrine and epinephrine in apparently normal human plasma were 185 +/- 29 ng/L and 32 +/- 8 ng/L, respectively (mean +/- SEM, n = 10). This relatively rapid and highly sensitive system is suitable for routine use.
Amylolysis of a chromogenic substrate, Cibachron Blue F3GA-amylose: kinetics and mechanism.
Authors: Klein B , Foreman J A
Published: January 2020

We compared the modes of action of human pancreatic, human salivary, and porcine pancreatic amylases on Cibachron Blue F3GA-amylose. Both human enzymes showed similar catalytic activity with almost equal Vmax but dissimilar apparent Km's. The ratios of soluble dyed oligosaccharides to reducing substances were identical. Porcine pancreatic amylase exhibited less than half the Vmax of the human enzymes and a smaller apparent Km. Reducing substances were formed faster than were the soluble dyed products. These differences in amylolytic action can be explained by differences in the degree of the “multiple attack" mechanism. Introduction of dye substituents into the amylose molecule did not alter the substrate characteristics of amylose toward human serum amylase.
Enzymic inhibition assay for methotrexate with a discrete analyzer, the ABA-100.
Authors: Brown L F , Johnson G F , Witte D L , Feld R D
Published: January 2020

We adapted an inhibition assay for methotrexate, involving dihydrofolate reductase from bovine liver, for use with a discrete analyzer (the ABA-100). The analyzer was used both for dilution and a 5-min pre-incubation of the sample with NADPH--enzyme reagent, and for the assay itself. The standard curve was linear between 10 and 120 microgram/L. Without pre-incubation the standard curve was nonlinear. The presence of albumin in the NADPH--enzyme reagent enhanced both enzyme activity and stability. Within-run precision (CV) was 2.0% (n = 24), run-to-run precision 7.1% (n = 49). Results obtained on patients' samples (29 sera, 15 urines, 18 cerebrospinal fluids) by the present method and a radioimmunoassay method did not differ statistically (p greater than 0.05) when the paired data were analyzed by use of the sign test and Wilcoxon's ranked sign test.
Radioimmunoassay of thyroxine in 10 microliters of serum, with use of aggregated antithyroxine antibodies.
Authors: Collins S , Brooks M , Bermes E W
Published: January 2020

We describe a sensitive radioimmunoassay for rapidly determining the concentration of thyroxine in 10 microL of human serum. Aggregated antithyroxine antibodies are used to separate bound and free hormone. This speeds the assay and economizes on reagents without loss of sensitivity, specificity, or precision. Results for normal subjects and patients with thyroid disease agree well with those obtained by other, well-established techniques.
Alkaline phosphatase of high and low molecular mass in human serum and bile: a comparative study of kinetic properties.
Authors: Crofton P M , Smith A F
Published: January 2020

We studied the kinetic properties of high- and low-molecular-mass forms of alkaline phosphatase purified from serum and bile, to clarify their interrelationships. They were found to share virtually identical kinetic properties, and to obey the same general kinetics as the liver-derived isoenzyme from serum and the low-molecular-mass isoenzyme from bile with regard to optimum conditions of assay, activation by magnesium ions, inhibition by L-homoarginine, inhibition by nickel and zinc ions, and inactivation by urea. Most of the characteristics such as Km (at low magnesium ion concentrations), Ki for L-homoarginine, and half-life for urea inactivation, were closely similar for low- and high-molecular-mass alkaline phosphatase. We conclude that these forms of alkaline phosphatase in plasma and bile are closely related. We discuss the possible nature of this relationship.
Amniotic fluid alkaline phosphatase, gamma-glutamyltransferase, and 5’-nucleotidase activity from 13 to 40 weeks’ gestation, and alkaline phosphatase as an index of fetal lung maturity.
Authors: Brocklehurst D , Wilde C E
Published: January 2020

Reference ranges for amniotic fluid alkaline phosphatase, gamma-glutamyltransferase, and 5-nucleotidase are described from 13 to 40 weeks’ gestation. Gamma-glutamyltransferase and 5-nucleotidase activities peak early in the second trimester and then decrease to low values. Alkaline phosphatase shows a similar pattern of activity from 13 to 29 weeks’ gestation, but thereafter activity increases to term; this late increase is mainly related to the heat-labile particulate form of alkaline phosphatase. Total and heat-labile alkaline phosphatase alone or expressed as a ratio with gamma-glutamyltransferase can be used with or as an alternative to lecithin/sphingomyelin ratios in the investigation of fetal lung maturity. A total alkaline phosphatase activity of 0.36 mukat/L and an alkaline phosphatase/gamma-glutamyltransferase ratio greater than 2 indicate pulmonary maturity.
Graphical determination of specific activity, binding constants, and antibody-site concentrations for radioimmunoassays, with application to thymopoietin.
Authors: Verhoff F H , Lisi P J , Fischer C D , Teipel J W , Goldstein G , Schiffman M B
Published: January 2020

A graphical procedure for determining the specific activity of radiolabeled ligands has been developed for use with radioimmunoassays. Although with this procedure we utilize the same experimental information required for displacement analysis, we are also able to determine both the specific activity and the binding constants of the labeled and unlabeled materials without assuming that these constants are equal; the concentration of antibody-binding sites can also be calculated. Thus, this graphical technique permits calculation of additional information without additional experimentation. We applied this procedure to the labeled materials used in a thymopoietin assay, testing two different preparations of radiolabeled material, and saw negligible differences between the two. The specific activity determined from the displacement analysis correlated well with that calculated by the graphical procedure.
Interference by the 4-hydroxylated metabolite of propranolol with determination of metanephrines by the Pisano method.
Authors: Chou D , Tsuru M , Holtzman J L , Eckfeldt J H
Published: January 2020

Measurements of urinary metanephrines by the Pisano procedure (Clin. Chim. Acta 5: 406, 1960) are unreliable in patients who are taking propranolol owing to the presence of 4-hydroxypropranolol in the urine. Three properties of this propranolol metabolite lead to interference: (a) it is absorbed and eluted from ion-exchange resins under the conditions Pisano used for metanephrine isolation, (b) at high pH it absorbs at 350 to 360 nm, and (c) it is oxidized by periodate to a substance with negligible absorption in this region. Because 350 to 360 nm is the wavelength used to quantitate vanillin, the product formed from periodate oxidation of metanephrines, and because the unoxidized eluate is used as a specimen blank, the presence of 4-hydroxypropranolol spuriously decreases the measured urinary metanephrines; this has special significance because patients being tested for increased metanephrines are also likely to be receiving propranolol for hypertension.
Liquid-chromatographic detection of thiazide diuretics in urine.
Authors: Tisdall P A , Moyer T P , Anhalt J P
Published: January 2020

We describe a liquid-chromatographic procedure for detection in urine of all thiazide drugs currently used clinically. Urine is treated initially with NaBH4 to convert any chlorothiazide present to hydrochlorothiazide. The urine is acidified with NaH2PO4 (1.0 mol/L, pH 5), and thiazides are extracted with ethyl acetate. Interfering substances are then removed in two washes with 0.1 mol/L Na2HPO4 at pH 8. The ethyl acetate is evaporated and the residue redissolved in mobile phase. Thiazides are assayed by reversed-phase chromatography, with detection by ultraviolet absorption. Analytical recovery of thiazides ranged from 53 to 93%. Urines from 48 patients were so studied, and the results were compared with results by the currently used spectrophotometric method. The two methods agreed for 56% of samples. Evaluation of the discrepancies by review of patients’ histories clearly showed liquid chromatography to have correctly identified seven of eight positive urines that the spectrophotometric method failed to detect. Ultraviolet scanning incorrectly identified as positive two samples, whereas liquid chromatography did not falsely identify any urines as positive. Our method was more sensitive and more specific than the spectrophotometric method.
Liquid-chromatographic method for simultaneous determination of phenytoin and 5-(4-hydroxyphenyl)-5-phenylhydantoin in plasma and urine.
Authors: Sawchuk R J , Cartier L L
Published: January 2020

We describe a liquid-chromatographic assay for phenytoin and its para-hydroxylated metabolite, 5-(4-hydroxyphenyl)-5-phenylhydantoin, in plasma and urine. Unlike previously reported methods, this procedure is sensitive enough to allow quantitation of the unconjugated metabolite in plasma at concentrations observed clinically (50-500 microgram/L). Simultaneous measurements of the drug and its metabolite in plasma and urine may be helpful in explaining changes in the apparent clearance of phenytoin, assumed to occur through its metabolite. The method may also be used, without modification, for analysis for phenobarbital and carbamazepine in plasma. The accuracy of our method for these three drugs was assessed for 25 months in quality-assurance program for determining drug concentrations. Regression slopes for measured vs reported values for phenytoin, phenobarbital, and carbamazepine were 1.02, 0.99, and 0.99, respectively.
Radioimmunoassay of thyrotropin and thyroxine in dried blood by use of Staphylococcus aureus containing "protein A.".
Authors: Mitchell M L , McKenna J J , McIver J
Published: January 2020

We report the use of Staphylococcus aureus rich in protein A as an immunoadsorbent in the radioimmunoassay of thyrotropin and thyroxine in blood collected from newborn infants and dried on filter paper. The procedures for thyrotropin and thyroxine, which differ mainly with respect to concentration and volume of the Staphylococcus aureus suspension, are reliable, rapid, and technically simple. The ease of this method, coupled with its dependability, offers certain advantages over some of the commonly used techniques of separation, and therefore it provides a unique approach to measurement of these analytes in laboratory screening programs for neonatal hypothyroidism.
Simultaneous measurement of lecithin, sphingomyelin, phosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine, and phosphatidylserine in amniotic fluid.
Authors: Painter P C
Published: January 2020

A one-dimensional thin-layer chromatographic method is described by which the ratios and proportions of lecithin, sphingomyelin, phosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine, and phosphatidylserine in amniotic fluid may be simultaneously determined. Commercial chromatographic plates, 10 X 7.5 cm, pretreated with cupric chloride, are used. The solvent system is chloroform/methanol/ammonium hydroxide (30/17/1.5 by vol). The plates are sprayed with a cupric acetate (350 g/L)/phosphoric acid (80 mL/L) reagent and charred on a hot plate to make the phospholipids visible. The percentage of each phospholipid present is determined by direct reflectance or transmission densitometry. The correlation coefficients for the lecithin/sphingomyelin, phosphatidylinositol/sphingomyelin, and phosphatidylglycerol/sphingomyelin ratios of the two scanning methods are 0.971, 0.980, and 0.955, respectively, for between-day analyses.
Effect of serum pH on storage stability and reaction lag phase of human creatine kinase isoenzymes.
Authors: Nealon D A , Pettit S M , Henderson A R
Published: January 2020

We report the effect of serum pH on the storage stability of the human creatine kinase isoenzymes and on the creatine kinase assay lag phase (Szasz et al., Clin. Chem. 22: 650, 1976). We also investigated the effect of including mercaptoethanol, N-acetyl-L-cysteine, monothioglycerol, ethylenediaminetetraacetate, or ethylene glycol bis(betaaminoethyl ether)-N,N,N',N'-tetraacetate at 20, 4, and --20 degrees C. Storage stability of the isoenzymes is profoundly affected by pH. For patients' samples and semi-purified human creatine kinase isoenzymes added to heat-inactivated sera, increasing diluent pH above 7.0 decreases creatine kinase stability. The thiol agents or chelators generally give little or no protection above pH 7.5; at pH 8.5 they contribute significantly to isoenzyme instability. Storage at 4 degrees C provides greater stability than storage at 20 degrees C, particularly in the case of creatine kinase isoenzyme BB. The lag phase was minimum at a serum pH of 6.5, in the presence of 10 mmol of monothioglycerol per liter. Increasing serum pH to 8.5 prolongs the reaction lag phase by about 1 min over the minimum. We recommend that, before they are stored at 4 degrees C, the pH of patients' samples be adjusted to 6.5 and oxidation of SH-groups be minimized by adding monothioglycerol to the sample.
Gas-chromatographic determination of urinary oxalate.
Authors: Park K Y , Gregory J
Published: January 2020

We describe a simple, rapid method for determining urinary oxalate: isolation by precipitation with calcium chloride and conversion to dimethyl oxalate, which then is measured by gas chromatography. To each sample, tracer amounts of 14C-labeled oxalic acid are added, to determine the analytical recovery of urinary oxalate. Analytical recovery of [14C]oxalic acid added to urine specimens ranged from 15 to 95% (mean, 80%), and corrected recovery, based on calculation isotope-dilution techniques, ranged from 98 to 100%. The urinary excretion of oxalic acid by 18 normal men, ages 23 to 43 years, ranged between 9 and 23 mg/24h, with a mean value of 16 mg; that by 68 patients with small-bowel bypass ranged from 60 to 210 mg/24 h, with a mean of 127 mg.
Use of the conjugated polyene fatty acid, parinaric acid, in assaying fatty acids in serum or plasma.
Authors: Berde C B , Kerner J A , Johnson J D
Published: January 2020

We describe a method for rapidly assaying fatty acid concentrations in plasma or serum. This method is particularly appropriate for monitoring increased concentrations of fatty acids that may be present during intravenous lipid infusion, especially in neonates. Binding cisparinaric acid (9, 11, 13, 15-cis-trans-trans-cis-octadecatetraenoic acid) to albumin shifts the absorption spectrum of the acid. If fatty acids are present in serum, they will compete with parinaric acid for binding, changing the extent of this absorption shift. The measurement requires a spectrophotometer, 30 microL of serum or plasma, and knowledge of the sample's albumin concentration.
Turbidimetry of inorganic sulfate, ester sulfate, and total sulfur in urine.
Authors: Lundquist P , Mårtensson J , Sörbo B , Ohman S
Published: January 2020

We describe simple and precise methods for the determination of inorganic sulfate, ester sulfate, and total sulfur in urine. The methods are based on turbidimetry of sulfate as barium sulfate in the presence of a small amount of preformed barium sulfate and with polyethylene glycol as a stabilizing agent. Inorganic sulfate is directly determined, whereas ester sulfate is measured after removal of inorganic sulfate, followed by acid hydrolysis. Total sulfur is determined after wet oxidation of the sample with nitric acid and perchloric acid in the presence of vanadate as a catalyst. We also report excretion values for healthy persons on a self-selected diet. Men excrete significantly higher amounts of inorganic sulfate and total sulfur than women, but this sex-related difference becomes insignificant if excretion values are expressed relative to creatinine.
Assay for erythrocyte uroporphyrinogen I synthase activity, with porphobilinogen as substrate.
Authors: Ford R E , Ou C N , Ellefson R D
Published: January 2020

We describe a convenient, economical procedure for measuring uroporphyrinogen I synthase (EC 4.3 1.8) activity in erythrocytes, the results of which can be used to diagnose acute intermittent porphyria, in either its latent or acute stage. By limiting the test reaction sequence to the conversion of porphobilinogen to porphyrins, we eliminated several disadvantages of alternative methods in which delta-aminolevulinate is used as substrate. The latter assay was inhibited by lead; our procedure was not. Our procedure also gave greater porphyrin synthesis with less substrate. Erythrocytes of healthy women presented a mean activity of 12.4 nmol of porphyrin formed per liter per second; erythrocytes of healthy men presented a mean activity of 11.0 nmol/L per second. The within-run coefficient of variation (CV) for our assay was 1.8%; the between-run CV was 3.1%.
beta 2-Microglobulin clearance as measured by radioimmunoassay.
Authors: Woo J , Floyd M , Longley M A , Cannon D C
Published: January 2020

We describe a radioimmunoassay for beta 2-microglobulin (beta 2 mu) in serum and urine. We incubated aliquots of diluted samples at room temperature for 1 h with 125I-labeled beta 2 mu and a rabbit antiserum monospecific for human beta 2 mu, and separated the phases by the double-antibody technique. The logit-log transformed dose-response curve was linear in the range 2 to 64 ng, equivalent to 0.5 to 16 mg/L of serum and 0.5 to 320 mg/L of urine. Assay sensitivity was 2.4 ng of beta 2 mu. Validation studies included tests of precision, accuracy, antibody specificity, and parallelism of the dose-response curves for standard and unknown. In a study of 25 normal individuals, serum and urine beta 2 mu ranged from 1.1 to 2.3 mg/L and 40 to 360 micrograms/24 h; the clearance of beta 2 mu was 8 to 130 microL/min. In 21 renal allograft recipients tested one to five weeks after transplantation, serum and urine beta 2 mu ranged from 3.9 to 15.6 mg/L and 7.2 to 611 mg/24 h; beta 2 mu clearance was 0.60 to 33.3 mL/min. Values for both serum and urine correlated well with severity of allograft rejection.
Immunonephelometric assay of human apolipoprotein AI.
Authors: Lopes-Virella M F , Virella G , Evans G , Malenkos S B , Colwell J A
Published: January 2020

We report an immunonephelometric assay for apolipoprotein AI, which involves use of a laser nephelometer and an antiserum prepared in our laboratory. Coefficients of variation were 2.5% within-run, 6.3% between-run. Several types of manipulation of the samples (heating, delipidation, dissociation with urea), reportedly necessary for accurate determinations by radioimmunoassay or gel diffusion immunoassay, did not substantially affect results obtained with our technique. The assay is sensitive, reproducible, and simple, and appears suitable for routine use.