bluuebunny/crossref_metadata_2025 huggingface dataset:
Quantification of cotinine in plasma and saliva by liquid chromatography.
Authors: Machacek D A , Jiang N S
Published: January 2020

Measurement of cotinine, a nicotine metabolite, has been studied as a method for monitoring smoking behavior and determining smoking status. We describe a specific, sensitive method for quantifying it in plasma and saliva by reversed-phase paired-ion liquid chromatography and detection by absorbance at 257 nm. The cotinine is extracted with methylene chloride, and 2-phenylimidazole is the internal standard. Cotinine peak heights are linearly related to the amount on the column from 0 to 500 ng. The mean (+/- SD) concentration of cotinine in plasma of 31 passively exposed nonsmokers was 2.1 +/- 1.6 micrograms/L (range, 0-7.9 micrograms/L). The regression of saliva cotinine concentration (y) on plasma cotinine concentration (x) at 0, 24, and 48 h in 10 smokers who refrained from smoking for 48 h was y (micrograms/L) = 1.155x (micrograms/L) + 0.245 (r = 0.986). The efficiency of cotinine as a biological marker was determined at 0, 24, and 48 h of smoking abstinence. Within-run CVs were 3.5% (n = 5) and day-to-day CVs 4.4% (n = 6) at 150 micrograms/L.
Isolation and quantification of apolipoproteins C-I, C-II, and C-III0-2 in very-low-density lipoproteins by "high-performance" anion-exchange chromatography.
Authors: Weisweiler P , Friedl C , Schwandt P
Published: January 2020

Using "high-performance" anion-exchange chromatography, we isolated and quantified human C apolipoproteins (apo C) from very-low-density lipoproteins (VLDL) in serum. As revealed by isoelectric focusing and double immunodiffusion against monospecific antisera, apo C-I, apo C-II, apo C-III0, apo C-III1, and apo C-III2 were purified to homogeneity. Assay reproducibility (coefficients of variation) ranged between 0.9% and 4.7%. The relative percentages of apo C subspecies in VLDL from normal and hypertriglyceridemic serum samples agreed with data obtained by alternative methods. This precise technique is suitable for detecting and analyzing abnormalities of C apolipoproteins in VLDL.
Stability of total cholesterol, high-density-lipoprotein cholesterol, and triglycerides in frozen sera.
Authors: Stokes Y M , Salmond C E , Carpenter L M , Welby T J
Published: January 2020

When human serum must be stored for extended periods before analysis, conditions must be such that samples give results comparable with those for "fresh" (unstored) samples. In a balanced experiment with statistically pre-determined sample size, we assessed, at three-weekly intervals for 18 weeks, the stability of total cholesterol, high-density-lipoprotein cholesterol, and triglycerides in human serum stored at -15 degrees C. No significant changes were observed.
The porphyrias: recent advances.
Authors: Hindmarsh J T
Published: January 2020

Recent research has elucidated several of the hitherto poorly understood steps in heme synthesis. This review describes this metabolic pathway and pinpoints the enzymatic blockages in the various porphyrias. Recent advances in the understanding of the etiology of porphyria cutanea tarda are discussed, as are the abnormalities of porphyrin metabolism seen in chronic renal failure and in lead poisoning. An outline is given of the clinical and biochemical abnormalities seen in the porphyrias. Included is an algorithm to aid in the differential diagnosis of these diseases, and a brief review of the new analytical techniques available for the identification and quantification of porphyrins and their precursors in body fluids.
Biochemical markers in the assessment of protein-calorie malnutrition in premature neonates.
Authors: Nadeau L , Forest J C , Masson M , Morrissette I , Larivière F , Caron M
Published: January 2020

We studied 135 premature newborns of 26 to 36 weeks gestation, divided into three groups: the control group, 66 premature infants with uncomplicated course; 51 premature neonates with appropriate birth weight for gestational age (AGA), who suffered from clinical problems that delayed oral feeding; and 18 premature infants with small birth weight for gestational age (SGA). When neonates of the same postnatal age were compared, prealbumin concentrations were the lowest in the SGA group at the third and fourth postnatal week. Although the AGA group had the most infants with serious illnesses and the lowest protein-calorie intakes, their prealbumin concentrations did not differ significantly from those of the control group. But when the infants of each group were subdivided on the basis of intakes and weight gain regardless of postnatal age, those with greater intakes showed significantly higher prealbumin values; however, in all groups, the infants with higher intakes were also significantly older. Total proteins and albumin showed similar changes in all groups. Prealbumin concentrations showed great interindividual variability in infants of the same postnatal age. We conclude that prealbumin, albumin, and serum total proteins are not sufficiently sensitive biochemical markers to assess alterations of the nutritional status of premature infants.
Simultaneous liquid-chromatographic determination of five antiarrhythmic drugs and their major active metabolites in serum.
Authors: Proelss H F , Townsend T B
Published: January 2020

We report an isocratic "high-performance" liquid chromatographic method for the simultaneous measurement of tocainide, lidocaine, procainamide, quinidine, disopyramide, and their major active metabolites in serum. The drugs are extracted from 200 microL of serum at pH 9.5 with 1.5 mL of 1,2-dichloromethane, concentrated by evaporation, and separated on a CN-bonded-phase column at 40 degrees C (flow rate 2 mL/min) with a pH 7.1 mobile phase of acetonitrile/methanol/phosphate buffer (60/7/33, by vol); the phosphate buffer contains 10 mmol of KH2PO4 and 0.5 mmol of triethylamine per liter. The antiarrhythmic drugs elute in the K' (capacity factor) range of 1.43 (lidocaine) to 5.7 (disopyramide). Results vary linearly with drug concentration to at least 30 mg/L; the detection limit is 0.1-0.2 mg/L. Within-run precision (CV) ranges from 0.9% to 5.0% and day-to-day precision from 2.1% to 6.2%, depending on the specific drug and its concentration in serum. Extraction efficiencies vary from 76% to 85% and analytical recoveries from 98.5% to 103%. At toxic serum concentrations, several basic drugs may interfere with the assay of some antiarrhythmics, but only hydroxyzine, verapamil, and certain local anesthetics interfere at therapeutic concentrations.
Evaluation of a monoclonal immunoenzymometric assay for alpha-fetoprotein.
Authors: Chan D W , Kelsten M , Rock R , Bruzek D
Published: January 2020

A monoclonal immunoenzymometric assay for alpha-fetoprotein was evaluated for detection and monitoring of hepatocellular carcinoma (HCC). We studied 1343 serum specimens from 759 patients with various neoplastic and non-neoplastic diseases. The interassay CV for this assay (M-AFP) ranged from 3.5 to 5.5%, with a minimum detectable concentration of 2.2 micrograms/L, as compared with 10 micrograms/L for a polyclonal (P-AFP) RIA for AFP. The calibration curve (0-300 micrograms/L) was linear, and serum dilutions paralleled it. The reference interval (0-9 micrograms/L) was established from data on 111 healthy subjects. Regression analysis of the AFP concentration (0-300 micrograms/L) of HCC patients obtained with the M-AFP assay (y) and the P-AFP RIA (x) yielded the equation y = (1.125)x - 0.52 (r = 0.9395, n = 165), with a considerable number of discrepant results for AFP less than 100 micrograms/L. By M-AFP immunoassay, AFP was above-normal (greater than 9 micrograms/L) in most HCC patients (80%), and to a lesser extent in other liver tumors (48%). AFP was within the normal reference interval for most patients with germ-cell tumors or benign liver disease and for other disease groups. For maximum diagnostic efficiency (90%) for HCC the decision level was increased to 100 micrograms of AFP per liter. Changes in serum AFP were correlated with changes in tumor volume in most HCC patients.
Assays for thyrotropin-receptor binding and thyroid-stimulating antibodies in sera from patients with Graves' disease.
Authors: Massart C , Hody B , Mouchel L , Edan G , Nicol M
Published: January 2020

We compared the activities of thyroid-stimulating antibodies (TSAb) measured in cultures of human thyrocytes and the values for thyrotropin-receptor antibodies (TRAb) as measured with a commercial kit based on use of radiolabeled receptors. Sera were obtained from patients with Graves' disease before, during, and after therapy with carbimazole (1-methyl-2-thio-3-carbethoxyimidazole). We found a significant correlation between the measurements of these two antibodies in patients: before treatment (r = 0.74, p less than 0.01, n = 44), after three months of treatment (r = 0.76, p less than 0.01, n = 21), and during relapse after the drug was discontinued (r = 0.64, p less than 0.01, n = 19). In all three situations, our TSAb technique was more sensitive than the TRAb method. We conclude that, even though the TRAb technique is simpler and quicker, this commercial kit is too insensitive to replace measurement of TSAb in fresh human thyrocyte cultures for management of drug therapy of patients with Graves' disease.
Effects of therapeutic hypothermia on activity of some enzymes in cerebrospinal fluid of patients with anoxic-ischemic brain injury.
Authors: Vaagenes P
Published: January 2020

I assessed the effect of therapeutic hypothermia on the activity in cerebrospinal fluid of creatine kinase (EC 2.7.3.2) and its brain isoenzyme (CK-BB), lactate dehydrogenase (EC 1.1.1.27), and aspartate aminotransferase (EC 2.6.1.1.) as markers of cerebral damage in patients with transient anoxic-ischemic brain injury. Moderate hypothermia (30-32 degrees C) lasting more than 24 h resulted in disproportionately greater activity of creatine kinase during the post-insult period than in patients not treated with hypothermia but having similar insults and outcome (p less than .01 for survivors, and p less than .005 for nonsurvivors). No differences were observed for the thermostable enzymes lactate dehydrogenase and aspartate aminotransferase, which demonstrates that the effect of hypothermia must be taken into account when thermolabile enzymes are used as sole markers of brain damage in such patients.
Use of the caffeine reagent in direct spectrophotometry of bilirubin.
Authors: Vink K L , Schuurman W , van Gansewinkel R
Published: January 2020

The molar absorptivity of unconjugated bilirubin in caffeine reagent is independent of the protein matrix. This finding, together with the simplicity of a dilution step for direct spectrophotometry, will improve the calibration methods of bilirubin and make them more nearly accurate. This is encouraging for the development of a new method for bilirubin determination in neonates; moreover, the caffeine reagent has a clearing influence on the turbidity of human sera. These findings should also be important for standardization, especially because the method of Jendrassik and Gróf is also protein-independent. Therefore, the introduction of one reliable, inexpensive, "universal" standard of bilirubin in bovine serum albumin will be of importance for both methods.
The Stratus immunofluorometric assay system evaluated for quantifying human choriogonadotropin in serum.
Authors: Rogers L C , Kahn S E , Oeser T H , Bermes E W
Published: January 2020

We evaluated the Stratus (American Dade, Miami, FL), an automated immunofluorometric assay system, for the quantification of human choriogonadotropin (hCG) in serum or plasma. The assay is based on the "sandwich" (two-site) immunoassay methodology: use of two monoclonal antibodies, one specific for the alpha subunit and the other for the beta subunit, results in an assay that is specific for the intact hCG molecule. Results for the first sample are obtained in 7 min; subsequent additional values are produced at 1-min intervals. Inter-run precision (CV), estimated from replicate determinations of sera, was 4.5% at an hCG concentration of 38 int. units/L, 4.9% at 114, and 6.1% at 194. Intrarun CV was less than 2% at all three concentrations. Correlations of results for 127 specimens analyzed in duplicate with the Stratus (y) and by a radioimmunoassay (x) for beta hCG (Gamma Dab M [cf931125I] beta-hCG, Travenol-Genentech Diagnostics, Cambridge, MA) yielded the following regression equation: y = 0.969x - 6.0 (r = 0.995). The Stratus immunofluorometric system provides a rapid and convenient assay of hCG in serum or plasma.
A case of immunoglobulin G conjugated with lactate dehydrogenase, producing both loss of enzyme activity and an abnormal isoenzyme pattern.
Authors: Sudo K , Maekawa M , Watanabe H , Matsumoto K , Mori Y , Sasaki T , Kanno T
Published: January 2020

We present a patient with an inhibitor of lactate dehydrogenase (LD; EC 1.1.1.27) in serum, who had pericarditis and myocardial hypertrophy. The patient's LD activity in serum was low and the LD isoenzyme pattern was abnormal. The relative molecular mass of this abnormal LD-IgG complex was approximately 490 000. The patient's IgG reacted with all five LD isoenzymes.
Measurement of ionized calcium in serum with ion-selective electrodes: a mature technology that can meet the daily service needs.
Authors: Bowers G N , Brassard C , Sena S F
Published: January 2020

This article reviews key advances in ion-selective electrode technology that have made potentiometric measurements of ionized calcium (Ca2+) reliable and precise. Our use of two second-generation Ca2+ analyzers (Radiometer ICA1 and NOVA 8) made possible uninterrupted service as volume increased to 31 640 patient tests in 1985. The lower results on the NOVA 8 were adjusted upwards to match those of the ICA1 to give identical results. Both analyzers were evaluated under working conditions of high volumes and multiple operators to establish downtime, electrode life, and costs. We have classified all Ca2+ analyzers into first-, intermediate-, and second-generation instruments, the better to understand their differences. Results for large numbers of patients' sera were shown to be systematically different when any two analyzers were compared. These differences are the consequence of each manufacturer's unique choices of the following: (a) the matrix of the calcium calibration solutions, (b) the type and configuration of the reference electrode, and (c) the salt-bridge solution. Elimination of each analyzer's biases will require agreement on a reference system that defines the accuracy of Ca2+ measurements on serum, plasma, or whole blood. The sound analytical performance of today's second-generation Ca2+ analyzers has allowed us to exploit the inherent superiority of Ca2+ over total calcium (CaT) measurements in the daily care of patients. We report on the preference of Ca2+ over CaT by physicians at our hospital since the introduction of second-generation Ca2+ analyzers. Therefore, we state unequivocally from our very satisfactory experience over the past five years that Ca2+ is a clinical laboratory test whose time has come!
Detection of anti-laminin antibodies in sera by latex agglutination.
Authors: Bernard A M , Foidart J M , Mahieu P , Viau C , Lauwerys R R
Published: January 2020

We describe a latex particle agglutination assay for detecting circulating antibodies against laminin, a noncollagenous glycoprotein of basement membranes. Polystyrene latex particles on which laminin has been adsorbed are incubated with serum for about 25 min at 42-45 degrees C. The agglutination is then measured by counting residual unagglutinated particles. Polyethylene glycol 6000 enhances the agglutination. The assay is fully automated, yielding results in about 45 min, for 50 samples per hour. Addition of purified laminin abolishes the agglutination of laminin-coated particles in practically all positive sera. The anti-laminin antibody titers obtained by this latex immunoassay and by radioimmunoassay correlated well in 161 sera from patients with suspected or established renal diseases. The agglutination assay more frequently gave positive results for cases of glomerulonephritis with linear deposits (20/22 cases) than for glomerulonephritis with granular deposits (7/68) or glomerulonephritis with no glomerular deposits (2/13). The finding of low anti-laminin antibody titers in sera from about 15% (34/230) of the healthy subjects suggests that these autoantibodies are pathogenic only in certain circumstances.
Two new monoclonal antibody-based enzyme-linked assays of apolipoprotein B.
Authors: Young S G , Smith R S , Hogle D M , Curtiss L K , Witztum J L
Published: January 2020

We describe two new monoclonal antibody-based, solid-phase immunoenzymometric assays for the quantification of apolipoprotein (apo) B in plasma: a competitive assay and a direct assay. For both, we utilize 96-well microtiter plates and native low-density lipoprotein (LDL) for preparing the standard curve. A single monoclonal antibody, MB24, is used in the competitive assay. The direct assay involves use of two monoclonal antibodies, MB24 and MB47. These two antibodies bind to distinct, unrelated apo B epitopes expressed by all LDL particles, and both antibodies detect apo B in very low-density and intermediate-density lipoproteins as well as LDL. With the two-step competitive assay, which involves use of LDL-coated microtiter plates, the intra- and interassay reproducibility in plasma apo B measurements averaged 6% and 12%, respectively. In the one-step direct assay, which takes less than 2 h for completion, plasma samples and peroxidase-labeled MB24 are incubated simultaneously on MB47-coated microtiter wells. The amount of labeled MB24 bound to lipoproteins trapped by MB47 is proportional to apo B concentration. With the direct assay, intra- and interassay CVs averaged 7% and 12%, respectively. These assays are simple, reproducible, involve convenient incubation intervals, and do not require radioisotopes; thus they can be widely applied in clinical laboratories.
Incidence and specificity of interference in two-site immunoassays.
Authors: Boscato L M , Stuart M C
Published: January 2020

Using a modified "two-site" immunoradiometric assay, termed an "interference assay," we have demonstrated the occurrence of non-analyte antibody-binding substances in approximately 40% of serum samples. These substances multivalently bind antibodies from any of several species at a site other than the antigen-binding site. Using a two-site immunoradiometric assay for human choriogonadotropin, we have investigated their effect on analyte quantification. In this system these antibody-binding substances mimic the presence of analyte; when analyte is actually present, they can also cause over- or underestimates. Addition of excess nonspecific immunoglobulin from an appropriate species eliminates this interference. However, the amount of nonspecific immunoglobulin added to an assay system may not always be enough to block interference in all samples.
Effect of phospholipase C on high-molecular-mass alkaline phosphatase in serum.
Authors: Sykes E , Kiechle F L , Epstein E
Published: January 2020

Electrophoresis of some serum samples on polyacrylamide gel, followed by staining for alkaline phosphatase (EC 3.1.3.1), produces a band of activity at the gel origin. This high-Mr band consists of liver membrane fragments containing alkaline phosphatase and other enzymes. Alkaline phosphatase is closely associated with phosphatidylinositol in liver plasma membranes, and we have found that phospholipase C (EC 3.1.4.3) from Bacillus cereus, known to possess some phosphatidylinositol specificity, was able to release liver alkaline phosphatase from the high-Mr band. Two preparations of phospholipase C from Clostridium perfringens, however, which has no phosphatidylinositol specificity, had no effect on the alkaline phosphatase activity in the high-Mr band.
A new method for reporting the sources of abnormal activities of lactate dehydrogenase in serum.
Authors: Politser P E , Powell S H , Fink J
Published: January 2020

We have developed and tested a new method to increase the diagnostic usefulness of measurements of lactate dehydrogenase (LDH; EC 1.1.1.27) isoenzymes. The method estimates the separate contributions from enzymatically distinct organ clusters (e.g., heart/kidney/erythrocyte, liver/muscle, lung) to the total activity of LDH in serum. To test this method, we monitored serum LDH isoenzymes over the entire hospital course of 73 patients admitted to the intensive-care unit with chest pain, myocardial infarction, or serious hemodynamic disturbances. The organ-specific estimates provided useful information beyond measurements of the original isoenzymes. The sensitivity and specificity of this new method in detecting acute myocardial infarction, as well as concomitant disorders involving the liver or lung, are significantly greater than those of other diagnostic indices or pathologists' judgments. Serial plots of the organ-specific estimates may provide additional insight into evolving pathophysiological processes.
Assessment of immunochemical methods for determining low concentrations of albumin in urine.
Authors: Watts G F , Bennett J E , Rowe D J , Morris R W , Gatling W , Shaw K M , Polak A
Published: January 2020

Four immunochemical methods (radioimmunoassay, RIA; radial immunodiffusion, RID; immunoturbidimetry, IT; enzyme-linked immunosorbent assay, ELISA) for measuring urinary albumin at low concentrations were assessed for their assay characteristics and practicability. Precision and accuracy were comparable between the methods when studied individually. We made a method comparison, with RIA as reference, using urine samples from diabetic patients with albumin concentrations ranging from 1 to 120 mg/L. There was no significant systematic difference between RID and RIA, but IT and ELISA gave consistently lower values than RIA, the mean differences being 1.8 (p less than 0.01) and 9.7 mg/L (p less than 0.001), respectively. Random error, compared with that for RIA, was in increasing order: RID (residual SD = 3.8 mg/L); IT (4.3 mg/L); ELISA (7.3 mg/L). The difference between the methods increased with the albumin concentration. Operational cost was highest with IT, lowest with RIA. Capital cost was highest with RIA and lowest with RID, which required most technical skill. ELISA had intermediate overall costs.
Alkaline phosphatase isoenzymes resolved by electrophoresis on lectin-containing agarose gel.
Authors: Schreiber W E , Whitta L
Published: January 2020

With this electrophoretic method the liver, biliary, and bone isoenzymes of alkaline phosphatase are clearly separated on agarose gels. Wheat-germ lectin, incorporated in the gel matrix, binds the bone isoenzyme selectively, forming a precipitate near the origin. Neither liver nor biliary isoenzyme is affected. Activity staining with an indigogenic dye substrate reveals the liver isoenzyme migrating nearest the anode, followed by the biliary and bone isoenzymes. Results are generally similar to those of electrophoresis on cellulose acetate. However, the lectin-agarose gels better resolve the liver and bone isoenzymes, and heat treatment of samples is not required before electrophoresis.
CEDIA, a new homogeneous immunoassay system.
Authors: Henderson D R , Friedman S B , Harris J D , Manning W B , Zoccoli M A
Published: January 2020

Genetic engineering of beta-galactosidase (EC 3.2.1.23) has led to the development of a new homogeneous assay system, CEDIA. The Z gene of the lac operon of Escherichia coli encodes a large enzymatically inactive polypeptide that spontaneously aggregates and folds to form active beta-galactosidase. Using recombinant DNA techniques, we have been able to engineer beta-galactosidase protein into a large polypeptide (an enzyme acceptor, EA) and a small polypeptide (an enzyme donor, ED). The EAs and EDs are both enzymatically inactive, but spontaneously associate to form enzymatically active tetramers. In the assay, hapten or analyte is attached to an ED, and an analyte-specific antibody is used to inhibit the spontaneous assembly of active enzyme. Analyte in a patient's serum competes with the analyte in the analyte-ED conjugate for antibody, modulating the amount of beta-galactosidase formed. The signal generated by enzyme substrates is directly proportional to the analyte concentrations in the patient's serum. We describe quick (5-15 min) colorimetric tests for digoxin, requiring no serum pretreatments or predilutions and suitable for use with centrifugal and random-access analyzers.
Four immunoassay methods and standards compared for measuring fibronectin.
Authors: Brubaker D B , Blick K E , Romine M
Published: January 2020

Several companies have developed commercial kits to measure plasma fibronectin rapidly and inexpensively with readily available laboratory equipment. In two of these kits (Cooper Biomedical and Boehringer-Mannheim) an immunoturbidimetric method is used. In a third kit (Biomedical Technologies, Inc.) an enzyme immunoassay method is used. To evaluate these commercial kits for fibronectin assay, we selected nephelometry as a comparison method for ranking the kits with regard to precision and accuracy. We also compared antibody and fibronectin cross reactivity. The antibodies from various manufacturers appear similar, but the fibronectin standards from different sources showed significant variation. Rate nephelometry and the Boehringer-Mannheim kit had the best within-run precision (CVs of 0.38% and 5.5% respectively). Between-run precision for nephelometry was excellent (CV = 1.9%) and somewhat high for the Boehringer-Mannheim kit (CV = 15.4%). This study demonstrates a need for further standardization of antigen (fibronectin) and antibody in commercial kits and the development of suitable stable quality-control material.
pK1' and bicarbonate concentration in plasma.
Authors: Flear C T , Roberts S W , Hayes S , Stoddart J C , Covington A K
Published: January 2020

Values for pK1' were determined from pH measured at 37 degrees C with three blood-gas analyzers and from calculated pco2 values in 443 freshly separated plasmas, tonometered at 37 degrees C. Plasma was taken from healthy volunteers, seriously ill patients, and hyponatremic patients. pK1' values varied by considerably more than 0.06 in healthy volunteers as well as in very ill patients, and bicarbonate concentrations ([HCO3]p) calculated by blood-gas analyzers based on the pK1' value of 6.1 could be in error by some +/- 60%. pK1' was similarly determined for tonometered (37 degrees C) replicate dilutions of plasma samples. By adding weighed amounts of dry NaCl and NaHCO3 to the diluted samples we increased the Na+ concentration to approximately 150 mmol/L and bicarbonate concentrations to values ranging from approximately 2.5 to approximately 52.5 mmol/L. pK1' values decreased when [HCO3]p was increased in dilutions of plasma kept at constant ionic strength. At any given [HCO3]p, pK1' values were higher at high than at low values of pco2.
Technique for gas-chromatographic measurement of volatile alkanes from single-breath samples.
Authors: Zarling E J , Clapper M
Published: January 2020

For detection of small quantities of alkanes that are present in expired breath, these gases have hitherto been concentrated, either by passing large volumes of breath through a liquid-nitrogen-cooled precolumn or by use of a closed collection system. Here, we describe a technique for analyzing small volumes of gas from single-breath samples from humans, in which no precolumn is required. Results are linearly related to sample concentrations of ethane, propane, butane, and pentane in the range 0 to 13 nmol per liter of air (r = 0.999). Within-run coefficients of variation were less than 15%. Breath samples could be stored for as long as 10 h without loss of the alkanes. We also report alkane concentrations in samples of alveolar gas and total breath collected from normal subjects. This technique appears to be well suited for measuring alkane concentrations in single-breath samples.
Turbidimetric determination of transthyretin (prealbumin) with a centrifugal analyzer.
Authors: Hamlin C R , Pankowsky D A
Published: January 2020

We describe a simple, rapid turbidimetric method for measuring transthyretin in human serum by use of a centrifugal analyzer (Cobas-Bio) and commercially available antiserum. The immunoturbidimetric reaction is monitored at 340 nm for 180 s, and the changes in absorbance after the antiserum is added are used to prepare a stable standard curve. The range of measurement is 10 to 520 mg/L. The CV for between-day precision was less than 5.0% for normal and low controls. Bilirubin up to 310 mumol/L and hemoglobin up to 225 mumol/L did not interfere, but triglycerides greater than 2.3 mmol/L falsely depress the results. The normal reference interval for adults, derived from data on subjects 20 to 65 years old (both sexes), is 193 to 376 mg/L. Concentrations are lower in children (67-302 mg/L) and in adults 65 or more years old (113-292 mg/L).
Rate nephelometry and radial immunodiffusion compared for measuring serum prealbumin.
Authors: Goldsmith B M , Munson S
Published: January 2020

We compared a rate-nephelometric method and a radial immunodiffusion (RID) assay for measurement of prealbumin (transthyretin) in 55 samples of serum from healthy children. The mean prealbumin concentration as measured by the Beckman Auto ICS nephelometer was 188 mg/L (range 128-350); the mean by RID was 221 mg/L (range 125-419). This difference was statistically significant by Student's t-test (p less than 0.05), but the correlation coefficient (r) was 0.95. To determine a reference interval for prealbumin in children by the Auto ICS method, we assayed samples from 93 healthy children between the ages of one day and 18 years (55 boys, 38 girls). The mean was 191 mg/L, the reference interval (mean +/- 2 SD) 109-273 mg/L. There was no significant difference in prealbumin concentrations between girls and boys (Student's t-test, p greater than 0.05). Evidently the Beckman Auto ICS method measures prealbumin in serum rapidly and accurately.
Clinical application of a sensitive non-isotopic immunometric assay of thyrotropin.
Authors: Sheppard M C , Black E G
Published: January 2020

We have evaluated an immunometric assay of thyrotropin (TSH) based on enhanced chemiluminescence signal; its detection limit is 0.06 milli-int. unit/L. Values in 101 clinically euthyroid subjects with normal thyroid hormone concentrations ranged from 0.39 to 6.83 milli-int. units/L. TSH in 15 hypothyroid patients ranged from 10.3 to greater than 200 milli-int. units/L, whereas in 31 hyperthyroid subjects with increased concentrations of free thyroxin and free triiodothyronine, TSH was undetectable serum of all but one subject. Of 32 clinically and biochemically euthyroid patients with goiter, two had undetectable serum TSH and six had values below the normal range. In 19 clinically euthyroid patients from an intensive-care unit, TSH was undetectable in two and below the normal range in another two. This immunometric chemiluminescence assay distinguishes thyrotoxic from euthyroid subjects, but caution is required in interpreting TSH values alone in subjects with goiter or nonthyroidal illness.
Liquid-chromatographic method for determination of homovanillic acid in plasma, with electrochemical detection.
Authors: Zumárraga M , Andia I , Bárcena B , Zamalloa M I , Dávilla R
Published: January 2020

We describe a sensitive, simple method for measuring homovanillic acid in human plasma. The method is based on liquid chromatography with electrochemical detection. Sensitivity was 125 pg of homovanillic acid per injection. Samples were deproteinized and extracted with organic solvent before chromatography. Quantification was by the standard-additions technique. Analytical recovery of added HVA was 44.8 (SD 6.2%). We confirmed specificity by using serial amperometric detectors.
Automated enzymatic measurement of lecithin, sphingomyelin, and phosphatidylglycerol in amniotic fluid.
Authors: Bradley C A , Salhany K E , Entman S S , Aleshire S L , Parl F F
Published: January 2020

We describe methods for automated enzymatic measurement of lecithin, sphingomyelin, and phosphatidylglycerol in amniotic fluid. Phospholipase C (EC 3.1.4.3) and sphingomyelin phosphodiesterase (EC 3.1.4.12) are reacted with lecithin and sphingomyelin, respectively, to liberate phosphocholine. Phosphocholine is then reacted with alkaline phosphatase, choline oxidase, peroxidase, and 4-aminoantipyrine to form a colored complex, for which the absorbance at 500 nm is measured with a centrifugal analyzer. Phosphatidylglycerol is hydrolyzed by phospholipase D (EC 3.1.4.4) to form glycerol, which is subsequently reacted with ATP and NAD+ in the presence of glycerol kinase and glycerol-3-phosphate dehydrogenase to yield NADH. The absorbance of the NADH formed is measured at 340 nm. These methods provide a simple, rapid, and accurate alternative to thin-layer chromatography for determination of phospholipids in amniotic fluid for assessment of fetal lung maturity.
Measurement of acetylcholinesterase in erythrocytes in the field.
Authors: Magnotti R A , Eberly J P , Quarm D E , McConnell R S
Published: January 2020

We describe here a field method we developed for colorimetry of erythrocytic acetylcholinesterase (EC 3.1.1.7) in capillary blood samples. Three stable, premixed assay reagents and de-ionized water (but no centrifuge or balance) are required. This method, adapted for a microplate format, is essentially that of Ellman et al. (Biochem Pharmacol 1961;7:88-95) as modified by George and Abernethy (Clin Chem 1983;29:365-8). Assays were quantified and corrected for hematocrit by using a battery-powered colorimeter with a silicon carbide (blue) light-emitting-diode source. Advantages over existing field methods include better portability, ruggedness, greater precision, and lower cost per sample.
Determination of delta-aminobutyric acid and other amino acids in cerebrospinal fluid of pediatric patients by reversed-phase liquid chromatography.
Authors: Goldsmith R F , Earl J W , Cunningham A M
Published: January 2020

The reversed-phase liquid-chromatographic system described here is capable of resolving the neurotransmitter amino acids aspartic acid, glutamic acid, and gamma-aminobutyric acid (GABA) plus 21 other amino acids in cerebrospinal fluid (CSF) in a single analysis. The amino acids, derivatized with o-phthalaldehyde, are separated in 65 min. Concentrations of glutamine less than or equal to 600 mumol/L can be measured at the same time as GABA greater than or equal to 10 nmol/L. Using this method, we have determined reference intervals for amino acids, including GABA, in CSF in a group of pediatric patients who underwent lumbar puncture before myelography, and who were subsequently shown to have normal myelograms. These intervals are generally lower than those previously reported for childhood, but we believe this results from a more rigid selection of the control group. In addition, artifactual increases in concentrations of free neurotransmitters, caused by breakdown of amino acid conjugates, are minimized by (a) immediate freezing of the CSF samples to prevent enzyme-mediated changes, (b) omission of a deproteinization step, and (c) precolumn derivatization to reduce on-column breakdown of amide and peptide forms.
Determining methemoglobin in blood by zero-crossing-point first-derivative spectrophotometry.
Authors: Taulier A , Levillain P , Lemonnier A
Published: January 2020

We determined methemoglobin in blood by zero-crossing-point first-derivative spectrophotometry. After lysis of erythrocytes, hemoglobin was converted into oxyhemoglobin and the first derivative spectrum was recorded between 405 and 425 nm. At the exact point where the first-derivative spectrum of oxyhemoglobin was zero ("zero-crossing point"), the first-derivative value of oxyhemoglobin and methemoglobin mixture was proportional to the methemoglobin concentration. The standard curve was linear for all proportions of methemoglobin. Within-assay precision (CV) was 3.4% for a 20% methemoglobin content. Correlation with results by the Evelyn and Malloy method was very good for high proportions of methemoglobin (greater than 10%), but the proposed technique was far better for low methemoglobin percentages because of its linearity, its high sensitivity, and its low detection limit.
Liquid-chromatographic determination of five orally active cephalosporins--cefixime, cefaclor, cefadroxil, cephalexin, and cephradine--in human serum.
Authors: McAteer J A , Hiltke M F , Silber B M , Faulkner R D
Published: January 2020

We report an isocratic "high-performance" liquid-chromatographic (HPLC) procedure for measurement of five orally administered cephalosporins (cefixime, cefaclor, cefadroxil, cephalexin, and cephradine) in 0.1 mL of human serum. Serum protein is precipitated with acetonitrile, the sample is centrifuged, and the supernate is evaporated under nitrogen. The residue is reconstituted in 0.1 mL of mobile phase, and 50 to 80 microL of this is injected onto a reversed-phase Altex Ultrasphere Octyl (C8) column. The five cephalosporins are resolved by elution with a pH 2.6 mobile phase of methanol/monobasic phosphate buffer (20/80) by vol), flow rate 2 mL/min. The column effluent is monitored at 240 nm. Cefixime serves as the internal standard for the analysis of the four other compounds, cephalexin as the internal standard for cefixime. We used two standard curves for all compounds: a low-range curve for concentrations commonly observed clinically and a higher-range curve for higher concentrations. The former were linear from 1.0 to 10 mg/L for cefaclor, cefadroxil, cephalexin, and cephradine and from 0.1 to 1 mg/L for cefixime. The high-concentration curves were linear from 1 to 10 mg/L for cefixime and from 10 to 100 mg/L for the other compounds. The detection limits were 0.1 mg/L for cefixime, 1 mg/L for the other cephalosporins. Mean within-run and day-to-day CVs were always less than 15% for all compounds studied.
A latex agglutination assay for D dimer: evaluation and application to the diagnosis of thrombotic disease.
Authors: Hillyard C J , Blake A S , Wilson K , Rylatt D B , Miles S , Bunch R , Elms M J , Barnes A , Bundesen P G
Published: January 2020

Although latex agglutination assays have been used for some years to diagnose thrombotic disorders, only recently has it been possible to measure specifically the products of fibrin breakdown in the presence of fibrinogen degradation products, by using monoclonal antibodies. We have evaluated a preparation of latex particles coupled to the monoclonal antibody DD-3B6/22, which is specific for cross-linked fibrin degradation products (XDP) and allows accurate discrimination between normal and pathological conditions. Of samples from 515 apparently healthy volunteers, 97.7% failed to agglutinate the latex; the normal reference interval for XDP determined by enzyme immunoassay was less than 78-320 micrograms/L. The use of different anticoagulants with or without the addition of a protease inhibitor had no significant effects on the results of the latex assay. The latex preparation provides a useful, rapid diagnostic tool for assaying small numbers of samples or as an emergency test.
Identification and analysis of nine metabolites of cyclosporine in whole blood by liquid chromatography. 1: Purification of analytical standards and optimization of the assay.
Authors: Lensmeyer G L , Wiebe D A , Carlson I H
Published: January 2020

We describe an extraction and an isocratic "high-performance" liquid-chromatographic (HPLC) separation of cyclosporine (CsA) and nine metabolites (M1, M8, M17, M18, M21, M25, M26, M203-218, and MUNDF1) from whole blood. Metabolites (for standards) were purified from human bile with liquid-liquid and solid-phase extractions, chromatographed on a cyanopropyl (CN) semipreparative HPLC column, and further purified on octyl, CN, and silica columns. The identity of each metabolite was verified with authentic standards on three chemically different HPLC columns and on the basis of cross-reactivity data from radioimmunoassay. For the routine analytical method, 1 mL of whole blood is diluted, hemolyzed, and applied to a Bond Elut CN (500 mg) cartridge to extract CsA, metabolites, and cyclosporin C, the internal standard. Interferences are removed by using four wash solutions and an additional cartridge of octyldecyl sorbent introduced prior to elution. Analytes are separated on a Zorbax CN analytical column maintained at 58 degrees C, with detection at 214 nm. Analytical recovery, as tested with three lots of CN sorbent, ranged from 47% to 95% for the 10 cyclosporines. Between-run CVs are less than 10% at 200 micrograms/L (concentration of each compound) and the standard curves are linear to 1500 micrograms/L. We also report a study of the separation mechanisms.
Criteria for essential fatty acid deficiency in plasma as assessed by capillary column gas-liquid chromatography.
Authors: Siguel E N , Chee K M , Gong J X , Schaefer E J
Published: January 2020

To develop criteria for deficiency of essential fatty acids (EFA), we used capillary-column gas-liquid chromatography to determine fatty acids (percentage of total fatty acids) in plasma obtained in the fasting state from 56 reference subjects and from 10 patients with intestinal fat malabsorption and suspected EFA deficiency. Fatty acid evaluations (percentage of total fatty acids) that allowed for a clear distinction (P less than 0.01) between reference subjects and patients, based on values two standard deviations below or above the reference mean, included values for linoleic acid (18:2w6) below 27%, and values for palmitic acid (16:0), palmitoleic acid (16:1w7), oleic acid (18:1w9), vaccenic acid (18:1w7), and Mead acid (20:3w9) exceeding 21%, 2.6%, 23.3%, 2.1%, and 0.21%, respectively. Ratios of total EFA to total non-EFA of less than 0.60 and of Mead acid to arachidonic acid of greater than 0.025 also served to identify patients, and were not found in reference subjects. Significant inverse correlations between percentages of plasma EFA and plasma mono-unsaturated fatty acids were noted. Our reference-interval data can be used to assess normality of plasma EFA status.
Liquid chromatography and fluorescence polarization immunoassay methods compared for measuring flecainide acetate in serum.
Authors: Straka R J , Hoon T J , Lalonde R L , Pieper J A , Bottorff M B
Published: January 2020

We analyzed 99 patients' serum samples for concentrations of a new antiarrhythmic agent, flecainide acetate, by fluorescence polarization immunoassay (FPIA) and "high-performance" liquid chromatography (HPLC). Within-day and between-day coefficients of variation at concentrations in the low and high ends of the therapeutic range were less than 7% for HPLC and less than 9% for FPIA. There was no statistical difference in the mean (+/- SD) concentrations of the clinical serum samples measured by the two methods (607 +/- 334 micrograms/L by HPLC, 602 +/- 344 micrograms/L by FPIA), but results by each differed by a mean of 0.13%. FPIA and HPLC measurements correlated significantly (r = 0.98, P less than 0.05), and were linearly related (slope = 0.970, intercept = 13 micrograms/L) as assessed by orthogonal regression. Both assay methods produced similar concentration measurements and were sufficiently accurate and precise to be used in therapeutic drug monitoring.
Time-resolved immunofluorometric assay of alpha-fetoprotein in serum and amniotic fluid, with a novel detection system.
Authors: Chan M A , Bellem A C , Diamandis E P
Published: January 2020

We describe a new "sandwich"-type nonisotopic immunoassay of alpha-fetoprotein (AFP) in serum and amniotic fluid. In the assay, AFP binds to a monoclonal antibody immobilized in a microtiter well and to a polyclonal soluble biotinylated antibody. A fluorescent product is created on the solid-phase after adding streptavidin labeled with a new Eu3+ chelate, 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA), and excess Eu3+. The fluorescent complex, monoclonal antibody-AFP-polyclonal antibody-biotin-streptavidin-BCPDA-Eu3+, is quantified by nitrogen laser excitation at 337.1 nm, the emission at 615 nm being monitored in an especially designed gated fluorometer working in a time-resolved mode. This assay of AFP has a broad dynamic range (up to 1 mg/L), and is precise and accurate. The detection limit is approximately 0.1 microgram/L. Results agree well with those obtained by established techniques.
Simultaneous determination of neopterin and creatinine in serum with solid-phase extraction and on-line elution liquid chromatography.
Authors: Werner E R , Fuchs D , Hausen A , Reibnegger G , Wachter H
Published: January 2020

This is a method for the simultaneous determination of neopterin, a product of interferon-gamma-activated macrophages, and creatinine in serum. Acidified, but not deproteinized serum is applied to a 4-propylbenzene sulfonic acid-modified silica sorbent cartridge, which quantitatively retains the analytes but not the serum proteins. The retained analytes are then eluted from the cartridge directly onto the liquid-chromatography column. Elution from the cartridge is facilitated by a pulse of 0.4 mol/L, pH 6.8 potassium phosphate buffer. On isocratic elution from an octadecylsilica column with potassium phosphate buffer (15 mmol/L, pH 6.0), neopterin is detected by its native fluorescence, creatinine by ultraviolet absorption. Detection limits are 0.5 nmol/L for neopterin, 1 mumol/L for creatinine at a sample volume of 100 microL. The standard curve is linear over the range of concentrations encountered in sera. For both neopterin and creatinine in serum, concentrations so measured agree well with results by established methods.
New assay of apolipoproteins A-I and B by rate nephelometry evaluated.
Authors: Maciejko J J , Levinson S S , Markyvech L , Smith M P , Blevins R D
Published: January 2020

We evaluated new, commercially available reagents for assaying apolipoproteins (apo) A-I and B by rate nephelometry (INA). Our initial linearity studies for apoA-I indicated that use of the commercial diluent resulted in incomplete immunoreactivity. Subsequent revision of the calibration line by the manufacturer compensated for this and improved the linearity for the apoA-I assay. We observed good linearity for the apoB assay. The within-run CVs were less than 4.0% and the between-run CVs were less than 5.5% for both assays. Results were 109% for apoA-I and 101% for apoB as compared with those measured for IUIS-WHO reference materials from the Centers for Disease Control. Recovery averaged 103% for apoA-I and 105% for apoB, for duplicate assays of three concentrations of purified apoA-I and low-density lipoprotein (LDL). Assaying sera from 45 patients, we demonstrated a good correlation between INA and radial immunodiffusion for both apoA-I (r = 0.92) and apoB (r = 0.95). Correlations between apoA-I and high-density lipoprotein cholesterol, and between apoB and LDL cholesterol compared favorably with previous reports. We conclude that these assays are accurate, precise, and easily automated for clinical application.
Evaluation of the Hitachi 704 automatic analyzer.
Authors: Lentjes E G , Harff G A , Backer E T
Published: January 2020

We evaluated the analytical performance of the Hitachi 704 automatic analyzer. The spectrophotometer showed a linearity of response at 340 nm up to 2.8 A. Photometric imprecision measured bichromatically at 340 and 376 nm was 0.49% at 0.16 A, 0.14% at 0.46 A, and 0.17% at 0.76 A. Imprecision of the sample probe was 0.4% for 5, 10, and 20 microL, and the volume delivered deviated -2.4%, -4.4%, and -4.2% from these preset volumes, respectively. Imprecision of the reagent probe over the range 50 to 500 microL ranged from 0.14% to 0.29%; volume delivered deviated from +1.7% to +4.4%). At equilibrium, the temperature in the cuvets was 29.8 (SD 0.05) degree C as measured by cresol red spectrophotometry. No sample carryover was detected. Reagent carryover was detected when a bilirubin assay was preceded by a total protein assay and when lactate dehydrogenase was measured after alanine aminotransferase. Imprecision for nine tests at three concentrations ranged from 1.1% to 4.4%. Comparison of methods with the SMAC II as reference method showed good results. Precision was better than reported for the Hitachi 705 automatic analyzer.