bluuebunny/crossref_metadata_2025 huggingface dataset:
In vitro cholesterol esterification in human serum.
Authors: Yao J K , Dyck P J
Published: January 2020

We report a simple, convenient, and reproducible method, involving the use of radiolabeled cholesterol dispersed in Tween 20 as a tracer and endogenous lipoproteins as a substrate, for measuring the rate of serum cholesterol esterification in vitro. The reaction of lecithin acyltransferase (EC 2.3.1.43) was enhanced by the presence of Tween 20, which probably accelerates the exchange between radiolabeled cholesterol and endogenous lipoprotein cholesterol. In sera from 65 normal subjects, the in vitro cholesterol esterification rate was significantly correlated (r=0.47,P=0.001) with age. The mean rate of esterification of 31 subjects 30 years old or younger was significantly lower than that of 34 subjects 31 to 64 years of age. We found no significant difference in the rate of esterification between men and women. The rate of cholesterol esterification (nmol/ml per h) was significantly correlated with the concentration of endogenous free cholesterol in serum, but the fractional rate (the percentage of radiolabeled cholesterol esterified per hour) was inversely proportional to the endogenous free cholesterol. The fatty acid composition of the cholesteryl esters formed by the acyltransferase reaction may provide an index in recognizing some specific disorder.
Evaluation of serum triiodothyronine and adjusted triiodothyronine (free triiodothyronine index) in pregnancy.
Authors: Parslow M E , Oddie T H , Fisher D A
Published: January 2020

We measured serum thyroxine (free and total), triiodothyronine (free and total), thyroxine-binding globulin, and triiodothyronine uptake by talc in 97 normal men and 50 pregnant women. Mean serum thyroxine and triiodothyronine concentrations were higher in the pregnant subjects (104 vs. 78 mug/liter and 1.69 vs. 1.30 mug/liter) because of a higher mean thyroxine-binding globulin concentration (70 vs. 38 mg/liter). Mean triiodothyronine uptake by talc was lower in the pregnant subjects (0.82 vs. 1.03). Mean free thyroxine concentrations were similar in the two groups, but mean free triiodothyronine concentrations were 10% lower in the pregnant subjects. Triiodothyronine uptake by talc and the diayzable thyroxine and triiodothyronine fractions were highly correlated (r = 0.85 and r = 0.82, P less than 0.001). Calculated free thyroxine index and free triiodothyronine index values (hyroxine and triiodothyronine indirectly adjusted, using triiodothyronine talc uptake to compensate for differences in thyroxine-binding globulin concentration), were statistically similar (84 vs. 82 and 1.38 vs. 1.34) in pregnant and male subjects. The results indicate that the total triiodothyronine concentration can be normalized on the basis of the triiodothyronine uptake by talc to correct for variations in thyroxine-binding globulin concentration.
Lipoxygenic micromethod for specific determination of lipase activity in serum and duodenal fluid.
Authors: Proelss H F , Wright B W
Published: January 2020

We propose a rapid enzymatic micromethod for the specific determination of lipase (EC 3.1.1.3) activity in serum and duodenal fluid. Free linoleic acid produced during 10-min incubation of 10 mul of sample with 1 ml of substrate (trillinolein emulsion) at 30 degrees C is converted by lipoxygenase (EC 1.99.2.1), in a coupled reaction, to its hydroperoxide, which is measured photometrically after solubilizing the reaction mixture in ethanol. Lipase activity is calculated from the rate of hydroperoxide formation, with linoleic acid as primary standard. The velocity of the reaction is greatest at pH 8.8, 35-37 degrees C, and a deoxycholate concentration of 3.6 mmol/liter. The energy of activation is 6.7 kcal/mol. The differing "apparent" Km values obtained for lipase in undiluted serum (4 X 10(-5) mol/liter) and in albumin-based diluents (1 X 10(-5) mol/liter) indicate the presence of a competitive inhibitor in the serum matrix. We detected no lipase activity in urine. Results by the proposed method correlate well with those by a copper soap extraction method (r = 0.95), but values are significantly higher for pancreatitis patients' sera (slope 1.6). The linear dynamic range extends to 1000 U/liter. Hemolysis, lipemia, and hyperbilirubinemia do not interfere. The normal range is 40-60 U/liter. Lipase activity of pancreatitis patients generally exceed 1000 U/liter during the acute phase and 250 U/liter for as long as 10 days after it.
Hydrolysis of steroid glucuronides with beta-glucuronidase preparations from bovine liver, Helix pomatia, and E. coli.
Authors: Graef V , Furuya E , Nishikaze O
Published: January 2020

We determined the enzymic activity of beta-glucuronidase preparations from bovine liver, Helix pomatia, and Escherischia coli with steroid glucuronides and nonsteroid glucuronides as substrates. We also studied the effect of Na2SO4 on the enzymic hydrolysis of several substrates with the three preparations of beta-glucuronidase. Na2SO4 increases the rate of hydrolysis of all substrates with beta-glucuronidase from bovine liver. Hydrolysis of a steroid glucoronide with beta-glucuronidase from Helix pomatia and E. coli is inhibited by Na2SO4. None of the three enzyme preparations gives complete hydrolysis of urinary steroid conjugates, because urine contains inhibitors, which can be removed by absorption chromatography of the urine on a column of neutral polystyrene resin Amberlite XAD-2. But when Amberlite XAD-2 is not used, hydrolysis of urinary glucuronides of androsterone, etiocholanolone, pregnanediol, estriol, and 17-hydroxycorticosteroids proves that, given an incubation time of 24 h, the beta-glucuronidase preparation from bovine liver, in the presence of Na2SO4, is suited for determining all of the above steroids except esriol; the preparation from Helix pomatia is good for determining estriol and 17-hydroxycorticosteroids; the preparation from E. coli is good for determining androsterone, 17-hydroxycorticosteroids, and especially estriol, the glucuronide, of which is maximally hydrolyzed in 2 h.
Faster estimation of reserve bilirubin binding capacity of serum from the neonate by thin-layer chromatography on sephadex.
Authors: Trivin F , Odièvre M , Lemonnier A
Published: January 2020

Knowledge of the reserve bilirubin-binding capacity is useful in management of hyperbilirubinemia in the newborn. We describe thin-layer gel chromatography that permits measurement of this variable, with a 250-mul sample of serum, within 50 min. Single samples from four different infants can be analyzed on the same plate. The capacity of 40 sera from jaundiced and of 19 sera from nonjaundiced infants was measured. Results by thin-layer Sephadex gel chromatography and column Sephadex chromatography correlated well. Dithiothreitol, added to the standard bilirubin solution, protected bilirubin from oxidation for at least six days, which obviated the time consuming preparation of standard solution before such determination.
Evaluation of the Du Pont aca alpha-amylase procedure.
Authors: James G P , Passey R B , Fuller J B , Giles M L
Published: January 2020

The procedure used with the Du Pont aca for alpha-amylase (1,4,-alpha-D-glucan glucanohydrolase, EC 3.2.1.1) was evaluated in our laboratory and compared with the Roche Diagnostics "Amylochrome" and Perkin-Elmer Coleman 91 amylase assays. The within-run coefficients of variation (CV) for samples of fresh normal sera were: aca 5.8% and 4.3% on two different lots of reagent, Amylochrome 7.4%, and Coleman 91 3.3%. In sera with abnormally high amylase activity, the respective CV's were: aca, 1.2% and 0.8%; Amylochrome, 2.0%; and Coleman 91, 2.8%. Day-to-day precision studies on fresh and lyophilized normal and abnormal sera gave CV's in the following ranges: aca, 1.8% to 6.7%; Amylochrome, 3.0% to 5.2%; and Coleman 91, 4.5% to 5.9%. Results by the aca procedure were linearly related to activity to about 10-fold the upper limit of normal amylase activity. For serum, correlations were: r = 0.977 for aca vs. Coleman 91 and r = 0.974 for aca vs. Amylochrome. For urine they were: r = 0.978 for aca vs. Coleman 91 and r = 0.975 for aca vs. Amylochrome. Mean recovery from 53 supplemented samples was 98%. Icterus, hemolysis, and lipemia did not interfere with method correlation of aca vs. Coleman 91 or Amylochrome.
Measurement of Procainamide and N-Acetylprocainamide in Serum by High-Performance Liquid Chromatography
Authors: Shukur Lillian R , Powers James L , Marques Roberto A , Winter Michael E , Sadèe W
Published: January 2020

We measured procainamide (I) and its metabolite, N¬acetylprocainamide (II), in human serum samples by sol¬vent extraction, high-performance liquid chromatography on a reverse phase column, and detection at 280 nm, with use of external standards. The method requires 0.2 ml of serum and is sensitive to 0.3 mg of I and 0.6 mg of II per liter of serum, with intra-assay standard deviations of 0.22 and 0.24 mg/liter, respectively, at 5 mg/liter (n = 10) and inter-assay standard deviations of 0.63 and 0.81 mg/liter, respectively, at 7.5 mg/liter (CV 8.4 and 10.5 % , respec¬tively, n = 20). Concentrations measured by high-perfor¬mance liquid chromatography and by an established flu¬orescence technique correlated well (r = 0.98 for I and 0.97 for II). No interfering substances were found in 20 randomly selected sera from patients receiving a large number of other drugs. Of the pure drug substances tested, only sulfathiazole interfered with the assay of II. The method is therefore suitable for routinely monitoring these compounds in serum in a clinical laboratory. The high concentrations of the metabolite in a significant number of patients demonstrate the need to consider it as well as the parent drug as guides in optimizing dosage regiments for I.
Determination of Nonsuppressible Insulin-Like Activity in Human Serum by a Sensitive Protein-Binding Assay
Authors: Zapf Juergen , Kaufmann Ueli , Eigenmann Eugen J , Froesch E Rudolf
Published: January 2020

We describe a sensitive protein-binding assay for non-suppressible insulin-like activity in human serum. It can detect as little as 0.2 microunits (corresponding to 0.5 ng) of the activity in 0.4 ml of the assay mixture. It is measured in a low-molecular-weight fraction (termed "biological material") obtained by chromatography of serum on Se¬phadex G-50 in 1 mol/liter acetic acid. This fraction has been shown earlier to contain nearly all this biologically active material that is present in serum. A partially purified carrier protein from human serum is used as the binding protein; different concentrations of a partially purified preparation of material with the activity serve as standards, which compete with 1251-labeled tracer for binding. Bio¬logical material dilutes more or less in parallel with the standard over a 10-fold concentration range. In the chro¬matographed serum fractions, displacing activity appears between 50 and 80% bed volume, with the peak at 60%, and coincides with the distribution and the peak of radio¬activity obtained by chromatography of tracer. A good correlation (γ = 0.88) is observed between the values determined for this activity in the rat fat-pad assay and the protein-binding assay, although the latter yields about twofold higher results (160 ± 37 milliunits/liter vs. 345 ± 65 milliunits/liter, mean values for 18 normal sera). Values determined in the protein-binding assay are decreased in hypopituitary patients (183 ± 27 milliunits/liter) and in¬creased in acromegalics (486 ± 88 milliunits/liter), in accord with the results of the bioassay (68 ± 21 milli-units/liter for hypopituitary patients, 293 ± 53 for acro¬megalics).
Determination of Serum Bilirubin Concentration during Phototherapy of Newborns and In Vitro: Results Compared by the Direct Spectrometric Method and the Diazo Method
Authors: Ebbesen Finn
Published: January 2020

During direct illumination of a serum bilirubin solution the bilirubin concentration decreased markedly, both as de-termined by the direct spectrometric method and (even more so) by the diazo method. In contrast, I found the same values for serum bilirubin concentrations as determined by these two methods for serum from untreated, "single light," and "double light" treated full-term infants with neonatal hyperbilirubinemia without blood type immuni¬zation. The same was true for untreated and "single light" treated premature infants with this disease. Furthermore, no difference was found in the above-mentioned rela¬tionship between "single light" treated infants with rhesus hemolytic disease, "double light" treated infants with the same disease, and untreated infants with neonatal hyp¬erbilirubinemia without immunization, all born at term. This is important, because the direct spectrometric method is simpler and requires less serum than does the diazo method
The Gallium Melting-Point Standard: Its Application and Evaluation for Temperature Measurements in the Clinical Laboratory
Authors: Bowers George N , Inman Stanford R
Published: January 2020

I discuss the traceability of calibration of electronic ther-mometers to thermometric constants of nature or to the National Bureau of Standards, from a manufacturer's basic standards through the manufacturing process to the user's laboratory. Useful electrical temperature sensors, their advantages, and means for resolving their disadvantages are described. I summarize our development of a cell for realizing the melting phase equilibrium of pure gallium (at 29.770 °C) as a thermometer calibration fixed point, and enumerate its advantages in the routine calibration veri-fication of electrical thermometers in the clinical chemistry laboratory.
Photometric Determination of Total Protein in Lipemic Sera
Authors: Chromy Vratislav , Fischer J
Published: January 2020

We compared various procedures and biuret reagents suggested for determination of protein in lipemic sera. A modified procedure, consisting of the precipitation of serum protein with acetone and dissolution of the pre¬cipitate in a biuret reagent, has been developed. The sample (0.02 ml) is mixed with 0.1 ml of distilled water and 2 ml of acetone. The mixture is shaken, centrifuged, and the precipitate dissolved in 1.5 ml of ethylenediaminete¬traacetate-chelated biuret reagent. This method is suitable for both clear and lipemic sera. Values obtained with the procedure proposed are in good agreement with values by the Kjeldahl method.
Stability of commonly used thiols and of human creatine kinase isoenzymes during storage at various temperature in various media.
Authors: Nealon D A , Henderson A R
Published: January 2020

We assessed the stability--at 20, 4 --20, and --80 degrees C--of 2-mercaptoethanol, dithioerythritol, and dithiothreitol, and of semi-purified creatine kinase isoenzymes of human origin in the presence and absence of the three compounds. The isoenzymes plus the sulfhydryl compounds were also assessed an aqueous buffer, in heat-inactivated pooled sera, or in fresh pooled sera with low endogenous total creatine kinase. We also examined the influence of thawing conditions from--20 or--80 degrees C, of fast freezing, and of exposure to fluorescent light. At 20 and 4 degrees C some--SH groups are oxidized. At--20 and--80 degrees C this loss is diminished. It is a function of both temperature and diluent. For stability, creative kinase isoenzymes stored at any temperature require the presence of a suitable sulfhydryl compound. Periodic addition of fresh sulfhydryl solutions stabilized creatine kinase isoenzymes suspended in the two protein-based diluents by 10-30%; for those in an aqueous buffer such additions decreased the activity to stored creatine kinase isoenzymes. Fast freezing does not increase recovery of creatine kinase isoenzyme activity over slow freezing at--80 or--20 degrees C. Thawing should be done as infrequently as possible and should be done rapidly, at 37 degrees C, for the residual enzyme activity to be maximal.
Intra-individual variation of some serum constituents and its relevance to population-based reference ranges.
Authors: Pickup J F , Harris E K , Kearns M , Brown S S
Published: January 2020

The advent of high-capacity multi-channel analyzers allows estimation of long-term variability in serum constituents of large numbers of subjects. By frozen storage of specimens with subsequent analysis in a single machine run, long-term analytical variation may be eliminated, thus sharpening the estimates of intra-individual variation. In the present study we used the Vickers M-300 analyzer to obtain the data for such estimates from 37 male volunteers, each bled once a week for 22 weeks. Secimens were analyzed in random order to eliminate any biasing effect of analytical drift during the 4-h machine run. Ten serum constituents were measured. Storage-induced linear trends were small or negligible during the period of specimen collection. Using the ratio of average within-subject variance to the variance among subjects as a guide, serum alkaline phosphatase was found to show the greatest individuality, sodium and potassium the least. Other constitutents showed varying degrees of individuality, but for all these analytes, the usual population-based reference ranges were found to be either insensitive or irrelevant to the study of concentration changes over time within most healthy subjects. Our results generally confirmed those of smaller but comparable earlier studies.
Myoglobin concentrations and muscle-enzyme activities in serum after myocardial infarction and cardiac arrhythmia.
Authors: Rosano T G , Sanders L A , Johnson E S , Kenny M A , Clayson K J , Strandjord P E
Published: January 2020

Sera from patients with myocardial infarction and cardiac arrhythmias were analyzed for myoglobin concentration and the activities of total creatine kinase, creatine kinase isoenzyme-2, and lactate dehydrogenase isoenzyme-1 at the time of hospital admission and during the first few days of hospitalization. The nine patients with a final diagnosis of myocardial infarction had abnormally high values for total creatine kinase, creatine kinase-2, lactate dehydrogenase-1, and myoglobin. Myoglobin concentrations were highest on admission in six patients and on the day after admission in the other three patients. Creatine kinase-2 manifested maximum activity on the day after admission for all patients with myocardial infarction. Lactate dehydrogenase-1 did not reach maximal values until the second or third day after admission. The six patients with arrhythmias did not show any significant increases in creatine kinase-2 or lactate dehydrogenase-1. Myoglobin and total creatine kinase, however, were increased in the four patients who had received cardioversion. The specificity and diagnostic usefulness of these serum measurements are discussed.
Comparison of several methods for semiquantitative determination of urinary protein.
Authors: Gyure W L
Published: January 2020

Two types of urine protein dipsticks and the sulfosalicylic acid method were compared for their accuracy and specificity, with use of urine samples supplemented with various proteins. Dipsticks yield accurate results when the protein under consideration is restricted to albumin; the sulfosalicylic acid method accurately determines many kinds of proteins in addition to albumin. Detergents affect each of the methods, but changes in salt concentration only affect results by dipstick procedures. Dipsticks, which are based on the protein-error principle for indicators, are subject to some of the conditions that apply to the bromcresol green method for serum albumin determination.
A stopped-flow clinical analyzer in which immobilized-enzyme reaction loops are used.
Authors: Joseph M D , Kasprzak D J , Crouch S R
Published: January 2020

A stopped-flow clinical analyzer is described that makes use of a reaction loop containing immobilized enzyme(s) for the determination of the analyte/substrate. The analyzer has been evaluated by determining glucose with immobilized glucose oxidase. The stopped-flow mixing system was constructed at a current cost of less than $500. The analyzer separates the enzymatic reaction from a followup, spectrophotometric indicator reaction. This separation allows the enzymatic reaction to be used in either a fixed-time, kinetic mode or in an equilibrium mode. Likewise, the indicator reaction can be used in either mode. Results for glucose in blood serum indicate that good precision and accuracy can be obtained.
Micromethod for measuring hexachlorophene in whole blood by gas-liquid chromatography.
Authors: Dodson W E , Tyrala E E , Hillman R E
Published: January 2020

We describe a micromethod for measuring hexachlorophene by use of gas-liquid chromatography with a 63Ni electron capture detector. The procedure requires 100 micronl of blood for extractions of hexachlorophene, and dichlorophene is added as an internal standard. CV is 3.4% over the concentration range of 500 to 1300 microng of hexachlorophene per liter of whole blood. This procedure permits repeated measurements of hexachlorophene in newborns who are being washed with soap containing hexachlorophene.
Evaluation of the dual-precipitation method by comparison with the ultracentrifugation method for measurement of lipoproteins in serum.
Authors: Demacker P N , Vos-Janssen H E , Jansen A P , van 't Laar A
Published: January 2020

We evaluated the dual-precipitation method for quantitative measurement of lipoproteins as described by Wilson and Spiger [J. Lab. Clin. Med. 82, 473 (1973)] for normo- and hyperlipemic sera, by comparison with the results obtained with ultracentrifugation. If serum with an above-normal triglyceride concentration is analyzed, the very-low-density lipoprotein cholesterol value obtained with the precipitation method is usually too low. For measurement of high-density lipoprotein cholesterol the ultracentrifugation and precipitation procedures give comparable results, but the latter method is preferred because sinking pre-beta-lipoproteins present in the high-density lipoprotein fraction isolated by means of the ultracentrifuge may result in falsely high values for cholesterol in that fraction. Therefore, at least for the determination of very-low-density lipoprotein cholesterol in hyperlipemic serum, the use of an ultracentrifuge remains necessary. Because few laboratories have an ultracentrifuge at their disposal, it seemed important to look at the stability of sera in view of the forwarding of samples. Also, a way of increasing the efficiency of the ultracentrifuge was studied. Sera can be stored for a week at 4 degrees C or for 54 h at room temperature without noticeable effect on lipoprotein values. Moreover, reliable values can be obtained with an ultracentrifugation time of 8 h (0.8 X 10(8) g-min).
Radioimmunoassay for free and conjugated urinary metanephrine.
Authors: Lam R W , Artal R , Fisher D A
Published: January 2020

We describe a convenient and specific radioimmunoassay for urinary metanephrine, in which we used an antiserum generated in rabbits by injecting with synephrine conjugated to bovine serum albumin as described by Grota and Brown [Endocrinology 98, 615 (1976)]. The antiserum is specific for metanephrine and epinephrine. Epinephrine cross reaction is minimized to 1% by adding 2 ng of epinephrine to each assay tube. Incubation time is 12 h. The sensitivity of the assay is 40 pg. Within-and between-assay coefficients of variation are 7.0 and 9.7%, respectively. We assayed first-voided morning urine and 24-h urine specimens from 15 normal subjects before and after acid hydrolysis, to assess total and free metanephrine concentrations. Mean (+/-SD) total and free urinary metanephrine excretion were 17 +/- 11 and 3.5 +/- 1.0 micrograms/24 h in these subjects; corresponding values for the morning specimens were 13.4 +/- 8.3 and 3.5 +/- 1.5 micrograms/liter, respectively.
Optimal conditions for assaying human lactate dehydrogenase by the lactate-to-pyruvate reaction: Arrhenium relationships for lactate dehydrogenase isoenzymes 1 and 5.
Authors: Buhl S N , Jackson K Y , Lubinski R , Vanderlinde R E
Published: January 2020

Optimal reaction conditions to sassay human lactate dehydrogenase (lactate-to-pyruvate) were established for isoenzymes 1 and 5 at 25, 30, and 37 degrees C in diethanolamine and 2-amino-2-methyl-1,3-propanediol. Different substrate concentrations are required at each temperature. The conditions permit measurement of lactate dehydrogenase 1 and 5 with the lowest substrate concentrations that allow for the highest equal sustainable efficiency in measuring both isoenzymes. About 95% of each isoenzyme activity is measured if the assay is performed within the first minute after the reaction is initiated even for activities as high as triple the upper limit of normal. The Arrhenius relationship is different for each isoenzyme, but results obtained for each at one temperature can be compared with results at another temperature by use of simple conversion equations. Assays at 25 and 30 degrees C are more economical and less variable than assays at 37 degrees C.
Improved assessment of plasma lipoprotein patterns. III. Direct measurement of lipoproteins after gel-electrophoresis.
Authors: Neubeck W , Wieland H , Habenicht A , Müller P , Baggio G , Seidel D
Published: January 2020

Based on a previously described technique [Clin. Chem. 19, 737 (1973)] of precipitating plasma lipoproteins with polyanions after their electrophoretic separation in gels, a new method is presented for measuring normal plasma lipoproteins densitometrically. The method is fast and easy; the CV for beta-, pre-beta-, and alpha-lipoproteins was less than 5% in one series. Results are linearly related to concentration up to 10 g of total lipoprotein per liter. No unusual equipment is required. Standardization is done with the aid of a commercially available filter. Total plasma cholesterol and cholesterol calculated from quantified lipoprotein fractions were highly (r = 0.963) correlated.
Simultaneous determination of iron, zinc, selenium, rubidium, and cesium in serum and packed blood cells by neutron activation analysis.
Authors: Versieck J , Hoste J , Barbier F , Michels H , De Rudder J
Published: January 2020

We determined the iron, zinc, selenium, rubidium, and cesium concentrations in serum and packed blood cells by instrumental neutron activation analysis without chemical separations. Lyophilized samples were irradiated for 12 days at a flux of 10(13) neutrons-cm-2-s-1, mineralized by wet digestion, and measured two times with a high-resolution Ge(Li) detector--for 6 h about a month after the irradiation and for 15 h two or three months after the irradiation, The following values were obtained: 163 +/- 0.43 mg/liter (serum iron), 1025 +/- 136 mg/kg wet wt (packed cells iron), 1¿ +/- 0.20 mg/liter (serum zinc), 11.15 +/- 1.83 mg/kg wet wt (packed cells zinc), 0.13 +/- 0.02 mg/liter (serum selenium), 0.16 +/- 0.03 mg/kg wet wt (packed cells selenium), 0.17 +/- 0.04 mg/liter (serum rubidium), 4.28 +/- 0.98 mg/kg wet wt (packed cells rubidium), 0.74 +/- 0.20 microgram/liter (serum cesium), and 4.82 +/- 2.10 microgram/kg wet wt (packed cells cesium).
Measurement of thiamylal and thiopental in plasma by electron capture and flame photometric gas-liquid chromatography.
Authors: Smith R H , MacDonald J A , Thompson D S , Flacke W E
Published: January 2020

We describe an isothermal gas-liquid chromatographic procedure developed for measuring thiamylal and thiopental in plasma. The unchanged drug is extracted into ethyl acetate from acidified plasma, together with an internal standard. The solvent is removed, the residue methylated, aliquots, diluted with benzene, are injected into a 183-cm gas-liquid chromatographic column containing 3% OV-17. Sensitivity of detection is in the nanogram to picogram range.
Elimination of glucose interference and improved precision in a continuous-flow analysis for inulin.
Authors: Torelli J A , Middleton B , Stein R M
Published: January 2020

A modified fermentation procedure based on one described by Homer Smith in his textbook was used to eliminate glucose interference in the automated detrmination of inulin. The manifold of Fjeldbo and Stamey [J. Lab. Clin. Med. 72, 353 (1968)] was modified to enable us to insert the fermentation procedure without sacrificing precision in continuous-flow analysis for inulin. Analytical recoveries of inulin from plasma and urine with the modified manifold were 100.1 +/- 0.8% (SD) and 99.9 +/- 1.6%. Corresponding recoveries of inulin from specimens of plasma and urine containing 5 g of glucose per liter and treated with yeast were 99.6 +/- 1.5% and 100.0 +/- 1.6%.
Therapeutic monitoring of tricyclic antidepressants in plasma by gas chromatography.
Authors: Dorrity F , Linnoila M , Habig R L
Published: January 2020

We describe a comprehensive gas chromatographic analysis for therapeutic concentrations of amitriptyline, nortriptyline, imipramine, desipramine, doxepin, and desmethyldoxepin in plasma, with use of a nitrogen detector. All these drugs are extracted and chromatographed under identical conditions. Each tertiary amine tricyclic is well resolved from its secondary amine metabolite on a mixed-phase column and the concentrations of both are determined simultaneously, without derivatization. The lower limit of sensitivity is 10 microgram/liter of plasma (2-ml sample). Analytical recoveries of the tertiary and secondary amines are 100 and 80%, respectively. Between-run CV's for all of the drugs ranged between 5 and 7%.
Positive interference with the Jaffé reaction by cephalosporin antibiotics.
Authors: Swain R R , Briggs S L
Published: January 2020

Cephaloglycin, cephalothin, cephaloridine, cefoxitin, and cephacetrile are cephalosporin antibiotics that react with alkaline picrate solution to give a creatinine-like response. The mechanism of this reaction is not known, and several structurally similar compounds do not react under these same conditions. Because large quantities of a cephalosporin antibiotic may be administered to a patient with limited ability to excrete the drug (as occurs with compromised renal function), the magnitude of this interference can be appreciable. It does not appear possible to avoid the cephalosporin-caused interference by the use of "kinetic" creatinine methods.
Heparin pretreatment suppresses norepinephrine concentrations in dogs in endotoxic shock.
Authors: Devereux D F , Michas C A , Rice S
Published: January 2020

Mongrel dogs were treated intravenously with either 1000 units of beef-lung heparin per kilogram of body weight or with isotonic saline, before intravenous administration of E. coli endotoxin. We found significant differences in circulating norepinephrine concentrations between a heparin-pretreatment group (1.89 +/- 0.39 microgram/liter) and the control group (9.83 +/- 4.64 microgram/liter), but none with respect to epinephrine. Systolic blood pressures at 360 min were also significantly (P less than 0.05) different, 148 +/- 6 mmHg as compared with 118 +/- 13.4 mmHg. Evidently heparin pretreatment can decrease circulating norepinephrine concentrations in the endotoxic state and changes in circulating catecholamine concentrations can affect physiological variables.
Column cation-exchange separation of melanin-related metabolites in urine from cases of melanoma.
Authors: Banda P W , Sherry A E , Blois M S
Published: January 2020

We describe recent developments in the use of a stable free radical, diphenylpicrylhydrazyl, as a colorimetric detector of reducing metabolites in melanoma urines. A procedure for buffering aqueous/solvent reagent solutions is presented, and examples of the baseline stability achieved for reference chromatograms and urine samples are provided. Chromatograms of phaeomelanin precursors and of an extract of a highly pigmented hamster melanoma are also presented. Identities are tentatively assigned for some of the chromatographic peaks that have previously been correlated with disease, including isomers of cysteinyldopa, and observations of new pigment- and tumor-related metabolites in the chromatograms are noted.
Enzyme immunoassays with the miniature centrifugal fast analyzer.
Authors: Lasky F D , Ahuja K K , Karmen A
Published: January 2020

We studied the EMIT (Enzyme Multiplied Immunoassay Technique, Syva) procedures for the assay of phenytoin and phenobarbital in serum, adapting them to the miniature Centrifugal Fast Analyzer. For different concentrations of drug, each rate of reaction decreased continuously with time, tending to converge on a single common value. The rate was most affected by the concentration of drug almost immediately after the reagents were mixed, less so thereafter. The antibody evidently is present in sufficient excess to bind all the enzyme-labeled drug ordinarily present, but the antibody-bound enzyme was only 75% inhibited; this helps explain the appreciable residual activity when no drug is present. The reaction course was the same whether the serum and enzyme-labeled drug were added to the antibody sequentially or simultaneously, which suggests that antibody is bound to drug appreciably faster than to enzyme-labeled drug. The reaction rates 15 to 30 s after mixing were used as the measure of the drug concentrations. These results were confirmed by noting the rates at successive 15-s intervals. The analyzer yielded a run-to-run CV of 10% for phenobarbital at 30 mg/liter, and 9% for phenytoin at 15 mg/liter, as compared to the 15% quoted by Syva.
Microchemical analytical techniques of potential clinical interest.
Authors: Glick D
Published: January 2020

Microchemical techniques with the capability of single-cell analysis will become increasingly important in clinical laboratory practice. One of the current moves underway in this direction is determination of enzyme activities of cells by microscale spectrophotometry and spectrophotofluorometry for prenatal diagnosis of inherited enzyme deficiencies. Three less-common microanalytical techniques are considered here that are of potential interest in laboratory medicine. The first is dilatometry, which provides a means for enzyme or substrate assay involving measurement of the change in volume or density accompanying chemical reaction in solution. In this connection, the measurement of specific gravity itself to obtain certain chemical information is also of interest. The second of these techniques is the use of spectrophotometry to measure oxygen uptake for functional assays of cells. The third is the use of luminometry in a general system of analysis for determination of many important biochemical substances and activities. Each of these techniques can be used for microscale analysis without sacrifice of precision or accuracy; each is relatively simple, instrumentally ant technically, and could be automated.
Enzyme kinetics in single cells: concept and model.
Authors: Markovic N , Markovic O , Young D S
Published: January 2020

We describe a technique whereby it is possible to measure enzyme activity in a singel cell. The model chosen involved the measurement of myeloperoxidase activity in a polymorphonuclear neutrophil leukocyte. Details of the apparatus are described. The experiments necessary to optimize the reaction conditions are summarized. The nature of the phases of the reaction are described. The technique appears to have further application in measurement of the activities of other enzymes in leukocytes or other cells.
Nucleotide pyrophosphatase and phosphodiesterase. I. Organ distribution and activities in body fluids.
Authors: Haugen H F , Skrede S
Published: January 2020

We estimated nucleotide pyrophosphatase and phosphodiesterase I activities in human and rat organs and in body fluids from man and dog. The highest organ activities were found in epididymis, kidney, liver, and intestine. In body fluids, the activity was highest in seminal plasma, followed by intestinal lymph, serum, heart lymph, cerebrospinal fluid, milk, and urine. The ratio nucleotide pyrophosphatase/phosphodiesterase I and the urea resistance of phosphodiesterase I differed among human organs, body fluids, and blood cells. Different isoenzymes probably exist. The activities in serum share several properties with those in several organs--e.g. pH-optimum 9.6-9.8, dependency on Zn2+, and the effects of inhibitors. Phosphodiesterase I in erythrocytes, which has not been described previously, differs from enzyme from other sources by lower pH optimum (8.5), dependency on Mg2+, inhibition by Zn2+, and stimulation by dithiothreitol.
Lactate dehydrogenase inhibitors in NADH preparations.
Authors: Margolis S A , Howell B F , Schaffer R
Published: January 2020

The presence of a new lactate dehydrogenase inhibitor on the trailing edge of the NADH peak from chromatography on diethylaminoethyl-celluose [Loshon et al., Clin. Chem., this issue] was verified. It was resolved from the NADH by high-performance liquid chromatography on muBondapak C18. When the new inhibitor was present in a reaction mixture to the extent that, of the initial 260-nm absorbance, about 5% was contributed by the inhibitor, the rate of NADH oxidation by lactate dehydrogenase decreased by 65%. The inhibitor absorbs at 260 and 340 nm, and is different from the Strandjord-Clayson inhibitor [J. Lab. Clin. Med. 67, 144 (1966)] by both types of chromatography. Because this new inhibitor has ultraviolet properties similar to those of NADH and chromatographs with the NADH on DEAE-cellulose, the high-performance liquid chromatographic method must be used to ensure its absence in preparations of NADH used for clinical assay.
Simultaneous radioassay of serum vitamin B12 and folic acid.
Authors: Gutcho S , Mansbach L
Published: January 2020

The radioassays of vitamin B12 and folic acid can be carried out in a single tube to give the simultaneous assay of both vitamins in 100 microliter of serum. Release of bound vitamins from their endogenous binders and the destruction of these binders are effected by a heating step at pH 9.3. The subsequent binding reactions with hog intrinsic factor and milk binder protein proceed advantageously and simultaneously in the same tube at pH 9.3. A single set of dual reagents replaces two sets of reagents that would normally be used for separate radioassays. Complete separation of bound radioactivities, [57Co]cyanocobalamin and 125I-labeled folate derivative, is obtained in a dual-channel gamma counter with no requirement for any correction for spill-over of counting data. Analytical results are comparable to those found for previously developed individual radioassays. The simultaneous assay has decreased technical time of analysis for these interrelated vitamins by about 50%.
Evaluation with the centrifugal fast analyzer of a chemical activation procedure for creatine kinase MB isoenzyme.
Authors: Bostick W D , Mrochek J E
Published: January 2020

The differential activation method for the determination of the "myocardial" isoenzyme of creatine kinase (MB) is based on the computed difference in activity of serum aliquots activated by the combination of dithiothreitol and glutathione (skeletal plus myocardial creatine kinase) and by glutathione alone (skeletal creatine kinase). The unique ability of the Centrifugal Fast Analyzer to perform analyses in parallel is used to precisely measure (CV = 0.1-1.5%) the activity of the two chemically activated aliquots. Statistical considerations concerning the reliability of this difference estimation are discussed with respect to both the precision of the rate measurements and to the relative amount of isoenzyme MB present. Another potential source of error in the analysis of the two differently activated aliquots, namely variation in lag phases, is circumvented by use of a linear-search FOCAL software package. This program searches the data for a linear segment of maximum slope, automatically rejecting those data that appear in lag or depletion regions of the curve representing the progress of the reaction. Correspondence of the results obtained with those of comparison techniques (chromatography and electrophoresis) are discussed.
Determination of morphine by electron capture gas-liquid chromatography.
Authors: Nicolau G , Van Lear G , Kaul B , Davidow B
Published: January 2020

We describe a micromethod for determining as little as 0.1 microgram of morphine per liter of human urine. The procedure is about 20-fold more sensitive than are currently used gas-liquid chromatographic methods. It is particularly suitable for use as a confirmatory method for large-scale radioimmunoassay screening tests. The procedure involves extraction of morphine from urine and analysis of its heptafluorobutyryl derivative by gas-liquid chromatography with electron capture detection. Morphine can be determined by this procedure over a range of 0.1-200 microgram/liter of urine, 2 pg of the pure derivative being the lowest amount detectable. Urine samples from patients receiving methadone were analyzed by thin-layer chromatography, radioimmunoassay, and our procedure. The results by our procedure agreed well with those obtained by radioimmunoassay.
Comparison of the Hoesch and the Watson-Schwartz tests for urinary porphobilinogen.
Authors: Pierach C A , Cardinal R , Bossenmaier I , Watson C J
Published: January 2020

A comparison of the Hoesch and the Watson-Schwartz tests shows that the latter, although slightly more complicated, generally yields more concise results and is superior in sensitivity and specificity for porphobilinogen. The recommendation of the Hoesch test for use as a "bedside screening" method seems unrealistic. Before the diagnosis of an "inducible" porphyria is made, a positive Hoesch test requires that indoles, indoleacetic acid, methyldopa, end-stage alcoholic malnutrition, and phenazopyridine HCl be excluded, to avoid misinterpretation.
Quantitative method for estimating myoglobin in urine.
Authors: Markowitz H , Wobig G H
Published: January 2020

A simple, rapid, and quantitative immunochemical technique is described for quantitation of urinary myoglobin. The test is specific for myoglobin in the presence of hemoglobin or tissue constituents, can be completed within 1 h or less, and can detect 1 to 2 mg/liter. Normal urinary myoglobin concentrations are less than this.
Sensitive, optimized assay for serum AMP deaminase.
Authors: Pedersen R C , Berry A J
Published: January 2020

A sensitive, optimized assay for serum AMP deaminase (EC 3.5.4.6) is presented, which is based on a colorimetric indophenol determination of ammonia liberated during the enzyme reaction. An average enzyme activity in serum of 2.7 U/liter (n = 36, range 0-6.2) for healthy adults was established. Both the enzyme and colorimetric reactions were optimized to give an assay sensitivity of 0.2 U/liter of serum, a 98-99% analytical recovery in the normal serum enzyme concentration range, and a CV of 7.9% in-run and 10.6% between-run. This optimization included studies on specific buffer, pH, ionic strength, and potential activators. The Km for the enzyme in serum was determinated to be 1.4 X 10(-3) mol/liter, which agrees well with the value reported for human skeletal muscle enzyme. Above-normal serum AMP deaminase activity may be present in various myopathies and could be useful in detecting carriers of the X-linked dystrophies.
Urinary organic acids quantitated by age groups in a healthy pediatric population.
Authors: Thompson J A , Miles B S , Fennessey P V
Published: January 2020

Urine samples from 92 individuals of a healthy pediatric population ranging in age from less than 24 h to 12 years were analyzed for organic acid content (amino acids excepted). A few samples from adults were included for purposes of comparison. Acids were extracted by using an anion-exchange procedure, converted to trimethylsilyloxime derivatives, and analyzed by gas chromatography with the help of a small computer system. Identifications were confirmed by using a combined gas chromatograph/mass spectrometer/computer system. The data has been tabulated according to five pediatric age groups. Despite large individual variation within age groups, significant differences in amounts of several acids excreted (based on urinary creatinine concentration) have been determined between age groups. Such differences might be due to one or a combination of several factors, including changes with age in creatinine excretion, in physiologic maturity, and in diet. Such data may help to provide a basis for diagnosing metabolic abnormalities in pediatric patients.
Comparison of enzyme immunoassay and gas chromatography for determination of carbamazepine and ethosuximide in human serum.
Authors: Sun L , Szafir I
Published: January 2020

Patients' sera were analyzed for carbamazepine and ethosuximide by enzyme immunoassay (x) and gas chromatography (y), and the results were compared. The correlation coefficients were: for carbamazepine, x vs. y 0.94 (n = 93); for ethosuximide, x vs. y 0.99 (n = 30). These results suggest that the two methods could be used interchangeably. Ten serum samples supplemented with carbamazepine (2.5 to 12.5 mg/liter) and ethosuximide (20.0 to 130.0 mg/liter) were analyzed by both methods. The correlation coefficients were: x vs. y 0.99 (n = 10) for carbamazepine and x vs. y 0.99 (n =10) for ethosuximide.
Improved direct determination of serum cholesterol in low-density lipoproteins with use of polycations.
Authors: Heuck C C , Middelhoff G , Schlierf G
Published: January 2020

The previously described procedure of direct determination of LDL-cholesterol after selective extraction of VLDL and HDL with polycations and a polycation-exchange resin has been improved by using dodecoylated poly(ethyleneimine) instead of poly(ethyleneimine). The acyl derivative has a higher affinity for lipoproteins. Disc-electrophoretic patterns of lipoproteins separated by this procedure correspond to patterns of apolipoproteins in VLDL and HDL, a finding that corroborates the conclusion that the mechanism of the selective binding of certain lipoproteins to lipophilic water-soluble polycations is based on hydrophobic interaction between the polymer and lipids located at the surface of lipoprotein particles. The procedure offers a simple possibility for direct LDL-cholesterol and LDL-triglyceride determination and for the isolation of VLDL and HDL. The mechanism of the reaction has a biochemical correlate in the action of phospholipase A2 on phospholipids.
Serum alkaline phosphatase: normal values by sex and age.
Authors: Eastman J R , Bixler D
Published: January 2020

We report here the normal range of serum alkaline phophatase activity as measured by the method proposed by Hausamen et al. [Clin. Chim. Acta 15, 241 (1967)] with a much larger sample size than used in previous investigations. In the statistical analysis the sample population is subdivided by sex and age, two variables which are known to influence enzyme activity. No statistically significant influence of blood type on enzyme activity was observed. The normal range of enzyme activity is reported in percentiles.
Influence of methotrexate on results by three methods for determining protein in cerebrospinal fluid.
Authors: Zweig M H
Published: January 2020

Because high intravenous and intrathecal doses of methotrexate are used in treatment for various neoplasms, this drug can reach high concentrations in cerebrospinal fluid. At those encountered clinically, it produced significant positive interference with values for cerebrospinal fluid protein as determined with the du Pont aca. A small interference was found with the Folin-Lowry method, and virtually no interference with an automated immunochemical procedure.