bluuebunny/crossref_metadata_2025 huggingface dataset:
Defects in diagnostic kits for determination of urate in serum
Authors: Whitehead T P , Bevan E A , Miano L , Leonardi A
Published: January 2020

Certain diagnostic kits that measure serum urate by the Barham and Trinder principle of enzymic liberation of oxygen and its combination with chromogens can give results for urate in fresh serum that are approximately 20% lower than results from serum stored at ambient temperature for 72 h. In fresh serum, antioxidants compete with chromogen for liberated peroxyl-oxygen. We postulate that during storage the interfering antioxidant substances are destroyed. In some diagnostic kits, L-ascorbate oxidase is added to the reaction, eliminating some but not all of this effect. We discuss defects of several commercially available kits for determination of serum urate and recommend comparing results of these kits with results from the phosphotungstic acid method as a precaution against falsely low results.
Quantification of virus-specific antibodies in cerebrospinal fluid and serum: sensitive and specific detection of antibody synthesis in brain
Authors: Reiber Hansotto , Lange Peter
Published: January 2020

Specific antibody synthesis in brain could be detected with maximal sensitivity by combining an advanced enzyme immunoassay with a sophisticated evaluation method that involves calculating the ratio between the cerebrospinal fluid (CSF)/serum quotients for specific antibodies (Qspec) and total IgG (QIgG). This Antibody Index (AI = Qspec/QIgG) discriminates between a blood-derived and a pathological, brain-derived specific antibody fraction in CSF and takes into account individual changes in blood/CSF barrier function. For local synthesis of polyclonal IgG in the central nervous system (QIgG greater than QLim), we propose the correction AI = Qspec/QLim (QLim represents that IgG fraction in CSF originating only from blood, calculated from the individual albumin quotient of a single patient). The normal reference range for the AI was between 0.7 and 1.3 (n = 250 control patients for each antibody species). Values of AI greater than or equal to 1.5 indicated a local specific antibody synthesis in the central nervous system. Sensitivity and precision were greatest if we analyzed the virus-specific antibodies in CSF and serum simultaneously with an enzyme immunoassay in continuous concentrations (arbitrary units) instead of titer steps. We have applied the method successfully to antibodies to measles, rubella, herpes simplex, varicella-zoster, human immunodeficiency virus (HIV), and cytomegalovirus, and to anti-Toxoplasma or -Borrelia antibodies. Clinical relevance is demonstrated for an acute zoster virus infection (monospecific response), chronic diseases such as HIV encephalitis with acute opportunistic Toxoplasma infection, and multiple sclerosis (secondary polyspecific response).
Determination of serum cholesterol by isotope dilution mass spectrometry with a benchtop capillary gas chromatograph/mass spectrometer: comparison with the National Reference System's Definitive and Reference Methods
Authors: Eckfeldt John H , Lewis Linda A , Belcher John D , Singh Jasbir , Frantz Ivan D
Published: January 2020

We developed an isotope dilution mass spectrometric cholesterol method with [25,26,27-13C]cholesterol as internal standard and a benchtop gas chromatograph/mass spectrometer (GC/MS) that is much easier and less time consuming than previously described Reference and Definitive Methods for cholesterol. The internal standard, cholesterol standards, and unknown specimen are delivered volumetrically with an automated dilutor and the saponifying reagent. After saponification, extraction, and derivatization, specimens are injected into a benchtop quadrupole MS with an autosampler. Unknown cholesterol concentrations are calculated automatically by comparing the peak area ratio of the m/z = 368, 371 ion pair with the ratios for the cholesterol standards (0 to 12.93 mmol/L). We found within-run and day-to-day (overall) imprecision of 0.44% and 0.95%, respectively, when specimens were assayed singly. In several lyophilized and frozen Standard Reference Material (SRM) pools, cholesterol results with our GC/MS method averaged 0.4% less than the National Institute for Standards and Technology definitive GC/MS result performed about three years earlier. Our GC/MS results averaged 1.3% and 2.0% less than results by the National Reference System (NRS) Abell-Levy-Brodie-Kendall (ALBK) results from clinical specimens and the SRM pools, respectively. These results are consistent with the previously reported bias between the NRS Reference and Definitive Methods and the 0.1% per year decrease in cholesterol concentrations in SRM pools as determined by GC/MS analysis. These results further emphasize the small but consistent bias between cholesterol results by isotope dilution mass spectrometry and the ALBK Reference Method, the latter being the basis for the National Cholesterol Education Program guidelines and population reference values.
Effect of weight loss on serum lipoprotein(a) concentrations in an obese population
Authors: Corsetti James P , Sterry Judith A , Sparks Janet D , Sparks Charles E , Weintraub Michael
Published: January 2020

To assess the distribution of serum concentrations of the atherogenic lipoprotein(a) [Lp(a)] in an obese population and the possible effects of weight reduction, we determined Lp(a) in 52 white, moderately obese subjects (16 men and 36 women). The subjects were participating in a weight-reduction program of diet, exercise, and behavior modification plus combination anorectic drug therapy (fenfluramine and phentermine). This placebo-controlled, double-blind study lasted 104 weeks. We also determined concentrations of fasting insulin, triglycerides, total cholesterol, high-density lipoprotein cholesterol, apolipoprotein (apo) A-I, low-density lipoprotein cholesterol, and apo B. Results showed the following: a highly skewed, nongaussian distribution of serum Lp(a) values in the obese population, virtually identical to that reported for healthy adults; no clinically significant change in Lp(a) concentrations with weight loss; and further evidence of normalization of insulin, lipid, and lipoprotein concentrations with weight loss.
Massive overdoses with sustained-release lithium carbonate preparations: pharmacokinetic model based on two case studies
Authors: Friedberg Richard C , Spyker Daniel A , Herold David A
Published: January 2020

Clinically significant delayed absorption after lithium overdose has been reported previously without adequate explanation. We have studied two patients after they took massive intentional lithium overdoses. The first patient presented shortly after ingesting 74 g of lithium carbonate. Pharmacokinetic analysis with a multicompartmental model of 29 serum lithium concentrations during 300 h (including hemodialysis) established absorption and elimination kinetics. Lithium absorption was both slow (peak concentration 33 h after the initial overdose) and delayed (a second peak occurred at 148 h, 30 h after initiation of oral tube feedings). The delayed absorption of a large fraction of lithium implicated a gastrointestinal drug reservoir. Study of the pharmacokinetics in a second patient, who ingested 98 g of lithium carbonate, provided additional evidence of an endogenous reservoir. This patient's medical management was guided by experience gained from the initial case. Appropriate management for a predicted endogenous drug reservoir may have shortened intensive care and hospitalization. In treating overdoses of sustained-release drug preparations, clinically significant delayed absorption triggered by enteral fluids must be considered as a contributor to delayed absorption.
Modified enzyme-based colorimetric assay of urinary and plasma oxalate with improved sensitivity and no ascorbate interference: reference values and sample handling procedures
Authors: Wilson David M , Liedtke Robert R
Published: January 2020

The measurement of oxalate in urine and plasma continues to be difficult, particularly in the presence of ascorbate. We have modified and validated a colorimetric assay involving the use of oxalate oxidase (EC 1.2.3.4). Modification of an HPLC spectrophotometric detector improved sensitivity (to as much as 1000-fold that of conventional spectrophotometers) and allowed measurement of oxalate concentrations less than 1 mumol/L. This provided more than enough sensitivity for measurement of normal concentrations of plasma oxalate. We established reference values for oxalate concentrations in urine and plasma, studied sample handling, and established conditions to avoid ascorbate interference in urine and plasma measurements. Mean analytical recovery of [14C]oxalate from plasma to the filtrate was 86 (SD 10)%; recovery of unlabeled oxalate from filtrate was 87 (SD 9)%. Urinary oxalate excretion rates in apparently healthy controls were 0.11-0.46 mmol/24 h. Plasma concentrations in control subjects were 2.5 (SD 0.7) mumol/L, similar to concentrations determined by recent gas chromatographic and isotope dilution methods. Frozen and acidified urine samples showed no interference from ascorbate when excess ascorbate was avoided. Ingestion of 2 g of ascorbate daily did not increase urinary oxalate in healthy control subjects, but during storage ascorbate was converted to oxalate in all conditions tested.
Isocratic reversed-phase HPLC method to measure pyrimethamine extracted from plasma of infants treated for toxoplasmosis
Authors: Zytkovicz T H , Salter J , Hennigan L , Timperi R , Maguire J , Hoff R
Published: January 2020

An isocratic HPLC method for measuring pyrimethamine extracted from infant plasma is reported. The method is an improvement over previously published methods by requiring lower volumes of plasma (100 microL) and having increased sensitivity to pyrimethamine at 210 nm. The procedure, which entails a basic organic extraction and subsequent HPLC chromatography of the reconstituted extract, can detect 1.4 ng and quantify 4.0 ng of pyrimethamine per 40-microL injection, with two analyses per 100-microL sample. Analytical recovery of pyrimethamine added to plasma at 10, 50, and 125 ng/100 microL averaged 80%, 92%, and 101%, respectively (n = 20). Within- and between-day CVs were less than 7%. Studies of various plasma samples from adults and infants (n = 15) revealed no interference from other plasma peaks with the analyte of interest.
Serum profiles of steroid hormones in patients with Cushing's syndrome determined by a new HPLC/RIA method
Authors: Ueshiba H , Segawa M , Hayashi T , Miyachi Y , Irie M
Published: January 2020

We developed a method for simultaneously measuring steroid hormones in very small volumes of serum, using a combination of high-performance liquid chromatography (HPLC) and radioimmunoassay (RIA). By this method, aldosterone, cortisol, 11-deoxycortisol, estrone, estradiol, androstenedione, dehydroepiandrosterone, deoxycorticosterone, 17-hydroxyprogesterone, testosterone, pregnenolone, and progesterone could be determined in a single 100-microL aliquot of serum from normal adults and patients with Cushing's syndrome. The steroid profile associated with Cushing's syndrome caused by adrenal adenoma was quite distinct from that associated with the syndrome caused by adrenal hyperplasia. Serum concentrations of androstenedione, dehydroepiandrosterone, estrone, estradiol, 17-hydroxyprogesterone, pregnenolone, and testosterone were significantly higher in patients with adrenal hyperplasia than in those with an adenoma. We compared the results of this HPLC/RIA method with those of 125I RIAs. The use of a HPLC/RIA system to obtain an accurate and sensitive profile of a range of serum steroids, as described here, obviates the need for large volumes of blood.
Predictive, error-compensating kinetic method for enzymatic quantification of creatinine in serum
Authors: Bacon B L , Pardue H L
Published: January 2020

Here we describe an error-compensating kinetic-based method for the enzymatic quantification of creatinine in serum. The method, which has a large linear range and very low dependency on experimental variables that influence enzyme activity, is based on the use of creatinine amidohydrolase in a four-step coupled reaction sequence to generate a product that is monitored photometrically. We collected data for absorbance vs time during two to four half-lives of each reaction and fit a first-order model to the data to compute the total absorbance change that would be measured if the reaction were monitored to completion. Computed values of absorbance change agreed well with measured values and varied linearly with creatinine concentration in the sample throughout the range examined: 44 to 1326 mumol/L. A twofold change in enzyme activity in the final reaction mixture (3.3 to 6.6 kU/L) produced changes of only 12% and 4% for creatinine concentrations of 44 and 353 mumol/L, respectively. Results (y) for 39 serum samples that contained creatinine concentrations between 20 and 1800 mumol/L agreed well with liquid-chromatographic results (x), yielding linear least-squares statistics of y = (1.03 +/- 0.01) x + (5 +/- 5) mumol/L (r = 0.995, Sy.x = 37 mumol/L). We conclude that the predictive kinetic approach is a robust method for the quantification of creatinine in serum.
Measurement of the ratio of lecithin to sphingomyelin in amniotic fluid by fast atom bombardment mass spectrometry
Authors: Shimizu A , Ashida Y , Fujiwara F
Published: January 2020

We describe a fast atom bombardment mass spectrometric method to determine the ratio of lecithin to sphingomyelin in amniotic fluid. Between m/z 400 and 1000, amniotic fluid extracts showed clear peaks for only sphingomyelin and lecithin. The molecular species of fatty acid in the phospholipids were analyzed quantitatively and quickly in a small volume of the extracts. The dipalmitoyllecithin/palmitoylsphingomelin ratio, determined by the proposed method, showed a reasonable correlation to the lecithin/sphingomyelin ratio measured by thin-layer chromatography. This method allows rapid and sensitive measurement of these phospholipids.
Computerized approach to monitoring aminoglycosides
Authors: Leal T , Parez J J , Vanbinst R , Wallemacq P E
Published: January 2020

Aminoglycosides are still used extensively in the treatment of nosocomial infections with Gram-negative bacteria. However, the treatment is associated with several adverse effects. Aminoglycosides monitoring is therefore essential to prevent toxic accumulations and to reach therapeutic concentrations. A computer program, PHARMONITOR, has been developed to optimize aminoglycosides monitoring, responding to the demands of most clinical daily situations. This program, based on a one-compartment open pharmacokinetics model, is developed for IBM PC-compatible computers, using D-Base III+. It can calculate t1/2, Vd, Cldrug, Cpmax, and the theoretical optimal dose and interval and also evaluates the creatinine clearance. The program has been conceived to allow maximal speed, flexibility, and reliability by the use of (e.g.) a linear least-squares analysis, the possible reference to previous protocols, the extensive use of keywords to classify and recall patients according to their pathologies, the development of messages recommending maximal dose or minimal dosing interval, and increasing the safety of the analysis. We consider the program a valuable tool for adjusting aminoglycoside dosage in individuals.
Serum lactate dehydrogenase isoenzyme 4/5 ratio discriminates between hepatocarcinoma and secondary liver neoplasia
Authors: Castaldo G , Oriani G , Cimino L , Topa M , Budillon G , Salvatore F , Sacchetti L
Published: January 2020

Total lactate dehydrogenase (LD; EC 1.1.1.27) and its five isoenzymes were determined in sera from (a) 98 cases of cirrhosis at various stages classified according to Child and Turcotte; (b) 37 cases of hepatocarcinoma (HC) at different stages of the Okuda classification; (c) 17 patients with secondary liver neoplasia (SLN), mainly from an abdominal primary site; and (d) 19 cases of abdominal neoplasia without liver metastasis, in an attempt to contribute to the differential diagnosis between these conditions. LD-4 was enhanced in SLN and LD-5 in HC, thus indicating the LD-4/LD-5 ratio as a potential index with which to differentiate between HC and SLN patients. At a cutoff value of 1.05, 91% of these patients were correctly classified (82% for SLN and 95% for HC). Consequently, this biochemical index appears to be an efficient and rapid indicator to distinguish HC from SLN. On the other hand, the LD isoenzymes are unable to discriminate between HC and cirrhosis or between abdominal neoplasia with and without liver metastases.
Effects of storage time and temperature on urinary enzymes
Authors: Matteucci E , Gregori G , Pellegrini L , Navalesi R , Giampietro O
Published: January 2020

We measured three renal tubular brush-border enzymes (lactate dehydrogenase, LDH, EC 1.1.1.27; gamma-glutamyltransferase, GGT, EC 2.3.2.2; and alkaline phosphatase, AP, EC 3.1.3.1) in morning urine samples from 48 healthy subjects to check whether different storage times and temperatures could modify enzyme concentrations. Short-term (24 h) storage time at room temperature or 4 degree C does not affect urinary enzyme activity. A few days of freezing, at -20 or -70 degrees C, dramatically lowers LDH and AP values; GGT is partially preserved only at -70 degrees C, if the sample has been previously centrifuged. Urinary enzymes investigated in this study are extremely labile at low temperatures.
Time Course of pH Regulation in Free Thyroxin Determinations in Serum
Authors: Wilcox R Bruce , Nelson Jerald C
Published: January 2020

Vol 37:

p. 1299: Left column: The HEPES buffer concentration on lines 12 and 24 should be 48 mmol/L. Middle column: References 2 and 7 cite J Clin Endocrinol Metab. Right column: The "many studies" in line 7, second paragraph, are references 5-7 in the response. The third sentence after this should read: Dialysis against this buffer resulted in a mean FT4 concentration of 18.7 pmol/L in healthy subjects, compared with 14.8 pmol/L in our recent study, and 15.8 pmol/L reported by Ekins and Ellis (3); a different HEPES buffer and a greater dilution were used in the latter two studies.
Benign inherited hyperphosphatasemia of intestinal origin: report of two cases and a brief review of the literature
Authors: Panteghini M
Published: January 2020

Two families with benign hyperphosphatasemia of intestinal origin were studied and compared with six other cases reported in the literature. No evidence of clinical abnormalities or explanations for the unusual enzyme concentrations were found. Agarose gel electrophoresis of alkaline phosphatase (ALP, EC 3.1.3.1) isoenzymes in serum demonstrated markedly increased intestinal isoforms (the "soluble" and the "hydrophobic" forms), which accounted for approximately 60% of total ALP activity. The description of these families demonstrated patterns suggesting autosomal-dominant inheritance, even if the precise genetic background of the abnormality affecting the enzyme production or the control mechanisms for its entry into the circulation could not be determined. Exact recognition of this benign biochemical abnormality should help to avoid unnecessary investigation.
Detection of two abnormal hemoglobins, Hb Manitoba and Hb G-Coushatta, during analysis of glycohemoglobin (A1c) by high-performance liquid chromatography
Authors: Wong S C , Tesanovic M , Poon M C
Published: January 2020

This report concerns the detection of two abnormal hemoglobins (Hb), Hb Manitoba and Hb G Coushatta, during analysis for glycohemoglobin (Hb A1c). Blood samples from two diabetic patients, analyzed for Hb A1c by HPLC, were found to contain additional Hb peaks. The presence of an abnormal Hb was confirmed in both instances by hemoglobinopathy studies. Structural studies determined the two Hb variants to be Hb Manitoba (alpha 2 102 Ser----Arg beta 2) and Hb G-Coushatta (alpha 2 beta 2 22 Glu----Ala). The significance of the presence of an abnormal Hb in Hb A1c analysis is discussed.
Chemiluminescent and bioluminescent techniques
Authors: Kricka L J
Published: January 2020

Light-emitting chemical reactions (chemiluminescence, CL) and biological reactions (bioluminescence, BL) have a diverse range of analytical applications but relatively few have been adopted by routine clinical laboratories. Advantages of CL and BL assays include sensitivity (attomole and sub-attomole detection limits), speed (signal generated in a few seconds and in some cases stable for several hours), nonhazardous reagents, and simple procedures. The most promising clinical applications are in immunoassay, protein blotting, and DNA probe assays. Chemiluminescent molecules exploited as labels include luminol, isoluminol, acridinium esters, thioesters and sulfonamides, and phenanthridinium esters. Separation and nonseparation assays have been devised, based on isoluminol and acridinium ester labels. The combination of the amplification properties of an enzyme and a CL or BL detection reaction provides a highly sensitive analytical system. Since 1983, CL and BL methods have been developed for many enzyme labels, e.g., alkaline phosphatase, glucose-6-phosphate dehydrogenase, horseradish peroxidase, Renilla luciferase, and xanthine oxidase. Currently, the most successful enzyme assays are the enhanced CL method for a peroxidase label involving a mixture of luminol, hydrogen peroxide, and an enhancer (e.g., p-iodophenol) and the direct CL method for alkaline phosphatase, with an adamantyl 1,2-dioxetane phenyl phosphate as substrate. Both systems are very sensitive (the detection limit for alkaline phosphatase when using the dioxetane reagent is 0.001 amol) and produce long-lived light emission (greater than 30 min), which is ideal for membrane applications in which light emission is detected with photographic film or a charge-coupled device camera.
Detection of genetic variants of alpha 1-antitrypsin with site-specific monoclonal antibodies
Authors: Zegers N D , Claassen E , Gerritse K , Deen C , Boersma W J
Published: January 2020

The serum protein alpha 1-antitrypsin (alpha 1-AT) serves as the major inhibitor of neutrophil elastase. The most common allele of the alpha 1-AT gene is designated as PiM. The Z mutation is a single-base substitution of the normal M allele, causing a Glu----Lys change at position 342 in the molecule. The ZZ phenotype is associated with a severe deficiency of alpha 1-AT, serum concentrations of the protein being 10% of normal. Individuals with an alpha 1-AT deficiency are at an increased risk of developing emphysema. To generate antibodies that specifically detect the 342 position in the context of the flanking sequences, we synthesized several peptides that included the 342 position for both the M and the Z variant. Immunization with variant-specific peptide-carrier conjugates elicited alpha 1-AT variant-specific responses, as determined in a direct enzyme-linked immunoassay. Monoclonal antibodies (MAbs) were selected with different specificity for the 342 region: MAbs F43 recognize only the alpha 1-AT sequence with 342Glu, i.e., all variant proteins that are non-Z, either from hetero- or homozygous individuals; MAbs F50 recognize only the sequence with 342Lys, i.e., all Z-variant proteins in ZZ or heterozygous individuals; MAbs F46 recognize alpha 1-AT with either 342Lys or 342Glu, all variant proteins with sequences as in the peptides used. Z homo- and heterozygotes were detected with our MAbs in a rapid and simple immunoblot assay. Other variants (M, S, and F) can also be assigned on the basis of the electrophoretic pattern. This sensitive detection method is very easy, rapid, and straightforward and provides a powerful tool for diagnosis of the alpha 1-AT deficiencies, allowing early treatment (augmentation of alpha 1-AT) and proper advice on lifestyle practices.
Reaction tray and noncontact transfer method for heterogeneous chemiluminescence immunoassays
Authors: Khalil O S , Hanna C F , Huff D , Zurek T F , Murphy B , Pepe C , Genger K
Published: January 2020

We describe a reaction tray for a heterogeneous chemiluminescence (CL) immunoassay having the following features: separate sample incubation and signal detection wells; a design that allows for noncontact transfer of the reaction mixture from incubation wells to detection wells; surface features to mate with a detector and create a light-tight seal for CL detection; and self-contained means for liquid removal. The reaction mixture is transferred by injecting a wash solution from a group of nozzles into the incubation well. Quantitative transfer of microparticles (transfer efficiencies greater than 95% and CV less than 5%) is achieved by injecting two 300-microL pulses of transfer solution at a rate of 2.1 m/s. The performance of the tray and method of transfer is tested by determining the precision of CL signal for a sample containing a concentration of anti-hepatitis B core antigen (anti-HBc) or hepatitis B surface antigen (HBsAg) close to the cutoff value for the assay.
High-dose methadone and the need for drug measurements in plasma
Authors: Wolff K , Hay A , Raistrick D
Published: January 2020

We report a case of high-dose methadone prescribed to a heroin addict for pain control. The patient was prescribed methadone during convalescence from surgery and subsequently for maintenance treatment. Dosing was started at 360 mg of methadone per day and reduced over 12 days to an 80 mg/day maintenance dose. Although the patient was drowsy on the initial dose, his recovery was uneventful. However, there were complaints of pain and withdrawal discomfort when the plasma concentration decreased to less than 1 mg/L. Measurements of methadone in plasma were helpful for monitoring the recovery of this patient after surgery and are likely to prove useful in similar cases.
Association between Magnesium, Calcium, Phosphorus, Copper, and Zinc in Umbilical Cord Plasma and Erythrocytes, and the Gestational Age and Growth Variables of Full-Term Newborns
Authors: Speich Michelle , Bousquet Bernard , Auget Jean-Louis , Gelot Simone , Laborde Olivier
Published: January 2020

We determined reference values in umbilical cord plasma and erythrocytes for magnesium, total calcium, phosphorus, copper, and zinc, and then calculated correlations and stepwise-regression equations in 66 white full-term newborn infants (35 boys, 31 girls). Only infants meeting certain optimal criteria and benefiting from excellent maternal conditions and uncomplicated pregnancies were included. There were no significant sex-related differences at birth among the variables studied. Gestational age was positively correlated with erythrocyte zinc (P less than 0.001), and plasma calcium was positively correlated with erythrocyte copper (P less than 0.001). Plasma copper proved to be the most significant variable in the stepwise-regression equation for birth height as the dependent variable. The most significant regressors accounting for birth weight were erythrocyte zinc followed by plasma zinc.
Screening for Defined Cystic Fibrosis Mutations by Solid-Phase Minisequencing
Authors: Jalanko Anu , Kere Juha , Savilahti Erkki , Schwartz Marianne , Syvänen Ann-Christine , Ranki Marjut , Söderlund Hans
Published: January 2020

We have developed a rapid method for the quantitative detection of point mutations and deletions. In this minisequencing method, enzymatically amplified DNA, 5'-biotinylated in one strand, is bound to a solid phase and denatured. A detection primer, constructed to end immediately before the mutation, is annealed to the immobilized single-stranded template and elongated with a single, labeled deoxynucleoside residue. We have applied the solid-phase minisequencing method to the detection of the major mutation, delta F508, causing cystic fibrosis (CF). In the presence of the allele with the delta F508 mutation, [3H]dTTP is incorporated; with the nonmutated allele, [3H]dCTP is incorporated. Thus, samples from heterozygous individuals allow the incorporation of both labels. The method was evaluated by analyzing 59 coded DNA specimens collected from 20 Finnish CF patients and their parents. The ratio of [3H]C to [3H]T gave unambiguously the allele combination. The solid-phase minisequencing method was also applicable to the analysis of three CF mutations simultaneously, i.e., delta F508, G542X, and G551D. We conclude that the microtiter-plate-based minisequencing test is an accurate method for the screening of defined sequence alterations in the CF gene.
Concentrations of Human Choriogonadotropin, Its β-Subunit, and the Core Fragment of the β-Subunit in Serum and Urine of Men and Nonpregnant Women
Authors: Alfthan H , Haglund C , Dabek J , Stenman U H
Published: January 2020

Sensitive, specific time-resolved immunofluorometric assays were used to measure the concentrations of human choriogonadotropin (hCG), free beta-subunit (beta-hCG), and the core fragment of beta-hCG (c beta-hCG) in serum and urine of men and nonpregnant women without evidence of cancer. Concentrations of hCG and beta-hCG were measurable in 59-70% of serum samples and in 50-59% of urine samples. c beta-hCG was mostly undetectable in serum but measurable in 81% of urine samples. Concentrations were higher in women than in men, and hCG concentrations increased with age. Therefore, reference ranges based on the 97.5 percentile were calculated separately for women and men and for those < 50 and > 50 years. However, concentrations of hCG correlated much more strongly with those of follicle-stimulating hormone than with age. hCG concentrations in serum were similar to those reported before, but beta-hCG concentrations were below the detection limit of earlier assays, and the upper reference limit was one-fifth to one-tenth the cutoff concentrations used earlier. In urine, hCG and c beta-hCG were the major forms of hCG, and their concentrations were similar to those of hCG in serum.
Direct Determination of Cadmium and Lead in Whole Blood by Potentiometric Stripping Analysis
Authors: Ostapczuk P
Published: January 2020

The commercially available equipment for potentiometric stripping analysis (PSA) was tested for routine lead and cadmium determination in whole-blood samples. In contrast to anodic stripping voltammetry, PSA is not subject to background interferences from organic electroactive constituents in the sample or to the presence of dissolved oxygen (i.e., oxygen removal is not necessary). To determine lead and cadmium by PSA, it is sufficient to dilute the blood sample with an appropriate supporting electrolyte (0.5 mol/L HCl). The detection limit changes with deposition time and volume of blood sample used. For 1 mL of blood and a 1-min deposition time, the detection limit is 1 microgram/L for both elements. If the deposition time increases to 10 min, cadmium can be determined at its normal concentration in blood (the detection limit is improved to < 0.1 microgram/L). Procedures for routine determination of lead and cadmium in whole blood are presented.
Magnesium, Total Calcium, Phosphorus, Copper, and Zinc in Plasma and Erythrocytes of Venous Cord Blood from Infants of Diabetic Mothers: Comparison with a Reference Group by Logistic Discriminant Analysis
Authors: Speich M , Murat A , Auget J L , Bousquet B , Arnaud P
Published: January 2020

Concentrations of magnesium (Mg), total calcium (Ca), phosphorus (P), copper (Cu), and zinc (Zn) were investigated in plasma (Pl) and erythrocytes (Erc) of venous cord blood of 44 infants of diabetic mothers (IDMs). These same concentrations plus total glycohemoglobin and fructosamine were determined at delivery in a subset of 15 mothers of these infants. Mineral results for IDMs were compared with those for 66 apparently healthy newborns. The duration of gestation in the two groups was significantly different (P < 0.001). After adjustment for gestational age, the mean (+/- SD) differences between groups were significant for birth weight, head circumference, Erc-Mg (1.71 +/- 0.17 for IDMs vs 1.76 +/- 0.15 mmol/L for control subjects), Pl-Ca (1.96 +/- 0.32 vs 2.48 +/- 0.22 mmol/L), Pl-P (1.99 +/- 0.40 vs 1.57 +/- 0.25 mmol/L), and Erc-Cu (10.9 +/- 2.41 vs 12.9 +/- 3.00 mumol/L), but not for Erc-Zn (33.0 +/- 18.3 vs 40.4 +/- 13.6 mumol/L). The variable that best discriminated between the two infant groups after adjustment for gestational age was Pl-Ca. In the 15 mothers, Pl-Mg (0.67 +/- 0.07 mmol/L) and Pl-Ca (1.66 +/- 0.21 mmol/L) concentrations were low, Pl-Zn (9.81 +/- 3.40 mumol/L) was normal, and Pl-Cu (33.5 +/- 10.7 mumol/L) was above normal. Correlations between total glycohemoglobin and mineral values of the mothers or paired IDM mineral values were not significant. The concentration of Pl-Ca was positively correlated with Erc-Cu (P < 0.001) and Pl-Cu (P < 0.05) in the comparison group newborns but not in the IDMs.
Specific and Sensitive Measurement of FK506 and Its Metabolites in Blood and Urine of Liver-Graft Recipients
Authors: Christians U , Braun F , Schmidt M , Kosian N , Schiebel H M , Ernst L , Winkler M , Kruse C , Linck A , Sewing K F
Published: January 2020

A specific and sensitive assay for quantifying the immunosuppressant FK506 and its metabolites in blood and urine was developed. 32-O-Acetyl FK506 was synthesized and used as internal standard. FK506 and its metabolites were purified from the samples by solid-liquid extraction and were injected into a high-performance liquid chromatographic (HPLC) system linked to a mass spectrometer (MS) by particle-beam interface. The FK506 derivatives were separated from interfering material by use of a 100 x 4 mm C8 analytical column and water/acetonitrile or water/methanol gradient elution; they were detected by negative chemical ionization with methane as reagent gas. The limit of detection was 25 pg in a standard solution, and the limit of quantification in blood was 250 pg (extracted from 1 mL of blood). The CV was 11.3% at 5 ng, and no interferences with other drugs were found.
Automated Determination of Fluvoxamine in Plasma by Column-Switching High-Performance Liquid Chromatography
Authors: Härtter S , Wetzel H , Hiemke C
Published: January 2020

A column-switching system with high-performance liquid-chromatographic separation and ultraviolet detection is described for automated determination of fluvoxamine in human plasma or serum. Samples were injected and the drug was retained in a clean-up column [20 x 4.6 mm (i.d.)] filled with C8 reversed-phase material (10-micron particles). After unwanted material was washed out, the drug was eluted and separated with an analytical chromatography column, 4.6 x 250 mm (i.d.), filled with Nucleosil 100 CN (5-micron particles) with an acetonitrile:methanol:0.01 mol/L phosphate buffer eluent (188:578:235 by vol) at a flow rate of 1.5 mL/min for < 20 min and detected by spectrometry at 214 nm. With oxaprotiline as internal standard, fluvoxamine could be easily quantified, and it was well separated from endogenous plasma constituents and various psychoactive drugs. The detection limit was 10 micrograms/L (31.6 nmol/L), the analytical recoveries were 97-100%, and the relationship between drug concentration and detector response was linear from 0 to 1000 micrograms/L (3160 nmol/L). The automated method is suitable for therapeutic monitoring of fluvoxamine in the treatment of psychiatric patients.
Standardization with Synthetic 22-KDa Monomer Human Growth Hormone Reduces Discrepancies Between Two Monoclonal Immunoradiometric Assay Kits
Authors: Banfi G , Marinelli M , Pontillo M , Bonini P
Published: January 2020

Discrepancies among different methods for assaying human growth hormone have been described in various studies. The two major sources of discordant results are the heterogeneity of the antibodies and the different standardization bases used by the assay manufacturers. We propose standardizing assays with 22-kDa biosynthetic monomer human growth hormone diluted with the diluents supplied by the kit manufacturers. In a study of two monoclonal immunoradiometric assays (Hybritech, specific for the 22-kDa monomer; Sorin, recognizing also a 20-kDa variant hormone), standardization with 22-kDa monomer human growth hormone reduced by 63% the differences in results for 44 serum samples from children. The use of 22-kDa human growth hormone as a common standard, highly pure and easily available in large quantities, could help limit the interpretative problems in growth diagnostics.
Improving Lactate Analysis with the YSI 2300 Gl: Hemolyzing Blood Samples Makes Results Comparable with Those for Deproteinized Whole Blood
Authors: Foxdal P , Bergqvist Y , Eckerbom S , Sandhagen B
Published: January 2020

To obviate the well-documented problem of hematocrit dependency of the Yellow Springs Instruments (YSI) whole-blood lactate analyzer, we modified the dilution buffer by including a lysing reagent. This makes the results comparable with those of methods performed with deproteinized whole-blood samples. No centrifugation step is needed, thus preserving the convenience of the YSI instrument for stat and field use. The modification works equally well on plasma samples. Lactate concentrations measured in nonhemolyzed whole blood are not comparable with results for hemolyzed whole blood or protein-precipitated whole blood. We therefore recommend to all users of YSI equipment to lyse the erythrocytes before lactate determinations.
Cathepsin D Concentration in Breast Cancer Cytosols: Correlation with Disease-Free Interval and Overall Survival
Authors: Duffy M J , Reilly D , Brouillet J P , McDermott E W , Faul C , O'Higgins N , Fennelly J J , Maudelonde T , Rochefort H
Published: January 2020

Cathepsin D (CD) is an aspartyl protease implicated in cancer metastasis. In this study of 331 patients, we show that patients with primary breast carcinomas containing high concentrations of CD have a significantly shorter disease-free interval (chi-square = 4.28, P < 0.05) and overall survival (chi-square = 7.7, P < 0.01) than patients with low concentrations. CD as a prognostic marker for overall survival was equally valuable for women younger (chi-square = 4.39, P < 0.05) and older (chi-square = 3.97, P < 0.05) than 50 years. CD was also a significant prognostic marker for overall survival within the estradiol receptor (ER)-positive subgroup of patients (chi-square = 5.79, P < 0.025), but not in the ER-negative subgroup. Patients with tumors containing high concentrations of CD and low concentrations of ER had shorter disease-free intervals (chi-square = 15.1, P < 0.001) and lower overall survival (chi-square = 20.9, P < 0.001) than patients with high concentrations of ER but low concentrations of CD.
Concentrations and Molecular Forms of C-Type Natriuretic Peptide in Brain and Cerebrospinal Fluid
Authors: Togashi K , Kameya T , Kurosawa T , Hasegawa N , Kawakami M
Published: January 2020

To develop a radioimmunoassay (RIA) specific for human C-type natriuretic peptide (hCNP), we used a highly specific antiserum raised in rabbits. Quantitative inhibition tests with various natriuretic peptides demonstrated that the 50% inhibitory dose of hCNP was 15 fmol, whereas those of other natriuretic peptides were 10(5)-fold higher, indicating a specificity satisfactory for determining concentrations of hCNP in tissues. Using this antiserum, we detected immunoreactive hCNP (ir-hCNP) in various regions of human brain and spinal cord, as well as in cerebrospinal fluid (CSF). The ir-hCNP concentrations in human neural tissues were approximately 10-fold higher than those of immunoreactive human atrial natriuretic peptide (ir-hANP). The mean (+/- SD) concentration of ir-hCNP (72.0 +/- 17.8 ng/L) in CSF also was 10-fold higher than that of ir-hANP (5.2 +/- 2.1 ng/L). Using gel-permeation chromatography, we identified two molecular forms of ir-hCNP in brain and CSF: a 2-kDa form corresponding to mature hCNP, which is composed of 22 amino acid residues (hCNP-22), and a 5- to 6-kDa form corresponding to an N-terminally extended molecule (hCNP-53). The latter form was predominant in brain; the former was the main constituent of hCNP in CSF. These results support the hypothesis that hCNP is a major natriuretic peptide, is synthesized in human brain, and functions in human central nervous tissues.
Apolipoprotein(a) is Present in Urine and Its Excretion is Decreased in Patients with Renal Failure
Authors: Oida K , Takai H , Maeda H , Takahashi S , Shimada A , Suzuki J , Tamai T , Nakai T , Miyabo S
Published: January 2020

To investigate the relation between renal function and concentrations of lipoprotein(a) [Lp(a)] in serum, we measured Lp(a) in samples of serum and urine from patients with diabetes mellitus and in samples sent to a laboratory center for measurements of creatinine clearance. Serum Lp(a) concentrations were significantly increased in subjects with obvious renal dysfunction (serum creatinine > or = 176.8 mumol/L) compared with normal control subjects. Urinary Lp(a) excretion was decreased in subjects with obvious renal dysfunction compared with subjects without obvious renal dysfunction (serum creatinine < or = 88.4 mumol/L) and was negatively and positively correlated with serum creatinine and creatinine clearance, respectively. More than 80% of urinary Lp(a) was recovered in the d > 1.21 kg/L fraction. At least six bands for apolipoprotein(a) [apo(a)] fragments, which were smaller than native apo(a) in serum, were observed in urine by immunoblotting, and some of these were also detected in serum. Degraded apo(a) fragments are probably present in urine, and their excretion decreases in parallel with decreases in the glomerular filtration rate.
Rapid Nonradioactive Tracer Method for Detecting Carriers of the Major Ashkenazi Jewish Tay-Sachs Disease Mutations
Authors: Strasberg P M , Clarke J T
Published: January 2020

Tay-Sachs disease (TSD, GM2 gangliosidosis, Type I) is an autosomal recessive lysosomal storage disease caused by deficiency of beta-hexosaminidase A (Hex A) resulting from mutations in the gene (HEXA) encoding the alpha-subunit of the enzyme. Three mutations, in exons 7 and 11 and at the exon 12-intron 12 junction, account for > 90% of alleles identified in obligate Ashkenazi Jewish carriers. Mutation analysis requires amplification of available DNA by separate polymerase chain reactions (PCRs) and either restriction digestion and gel electrophoresis or 32P-labeled allele-specific oligonucleotide (ASO) probes. We developed a simple, nonradioisotopic method for rapidly identifying TSD carriers by a triplex PCR reaction followed by dot-blot analysis, using three wild-type and three mutant ASOs end-labeled with digoxigenin-dUTP (dig-ASO). Hybridization was demonstrated immunologically by reaction with an anti-digoxigenin-alkaline phosphatase conjugate followed by colorimetric demonstration of phosphatase activity. The results of analyses by the dig-ASO method of 65 carriers identified by serum enzyme activity and of 6 high-risk fetuses in prenatal testing were the same as those obtained by more conventional restriction analysis. Dig-ASO testing correctly reclassified 10 individuals who had tested inconclusively on analysis for leukocyte beta-hexosaminidase A activity; 3 were identified as carriers and 7 as noncarriers. The simplicity of the assay and the avoidance of the radioisotopes make this a potentially useful method for TSD carrier detection by mutation analysis in Ashkenazi Jews from populations in whom the identity and frequencies of the common TSD mutations are known.
Investigation of N-Methyl-D-Glucamine Buffer for Assay of Alkaline Phosphatase in Serum
Authors: Lewandrowski K , Lee-Lewandrowski E , Bowers G N , McComb R B
Published: January 2020

We evaluated N-methyl-D-glucamine (MEG) as a buffer for assay of alkaline phosphatase (ALP; EC 3.1.3.1) and compared the MEG-based assay with the current International Federation of Clinical Chemistry Reference Method for ALP (IFCC/RM/ALP), in which 2-amino-2-methyl-1-propanol (AMP) is the pH buffer. The ALP assay in MEG at 30 and 37 degrees C shows excellent correlation with the IFCC/RM/ALP at 30 degrees C, but yields proportionately higher ALP activities (8.2% at 30 degrees C and 57% at 37 degrees C). ALP is unstable in both MEG and AMP at 37 degrees C. Serum incubated in MEG undergoes a pH-dependent biphasic loss of ALP activity: an initial rapid 5% loss after 1 min of incubation and a 10% loss per hour thereafter. A similar pattern was seen for incubation with AMP. The use of a serum-initiated reaction (no preincubation of enzyme with buffer) eliminated the early loss in activity. The addition of the metal ion buffer N-hydroxyethylethylenediaminetriacetic acid, along with low concentrations of Zn and Mg, as used in the IFCC/RM/ALP, reduced the slow loss in activity over time, as did decreasing the reaction temperature to 30 degrees C, but had no effect on the early rapid decay in activity seen in the first minute. Moderate transphosphorylation (45%) and nonenzymatic hydrolysis (3.3 U/L) were observed with MEG under the conditions of the assay (37 degrees C). A comparison of different lots of MEG from two manufacturers showed no significant difference in ALP activities.
A New Case of IgE Myeloma
Authors: Alexander R L , Roodman S T , Petruska P J , Tsai C C , Janney C G
Published: January 2020

We report a case of IgE myeloma in a 78-year-old woman who presented with bone pain in the shoulder and hip and progressive weakness. Except for hypercalcemia, routine chemistry values were within normal limits. Hemoglobin was decreased and the leukocyte count slightly increased. Plasma cells were not observed in the peripheral blood. Serum protein electrophoresis showed a monoclonal protein in the beta-globulin fraction. Immunofixation confirmed the presence of an IgE kappa monoclonal protein. A bone marrow biopsy revealed an interstitial and nodular infiltration of abnormal plasma cells comprising 60% of nucleated cells present. Skeletal roentgenograms and bone scans of this patient showed osteolytic lesions and osteopenia of the thoracic and lumbar spine and osteolytic destruction of the right half of the sacrum. Flow-cytometric analysis of mononuclear cells isolated from peripheral blood showed that 15% of the lymphocytes bound IgE. Using cell-surface markers, we identified 45% of the IgE-positive cells as natural killer cells. Similar results have been found in other diseases marked by increased IgE. The clinical, radiological, and laboratory findings for this patient are compared with previously reported cases of IgE and other types of myeloma.
Use of Synthetic H Disaccharides as Acceptors for Detecting Activities of UDP-GalNAc:Fuc α1-->2Galβ-R α1-->3-N-Acetylgalactosaminyltransferase in Plasma Samples from Blood Group A Subgroups
Authors: Yazawa S , Takeya A , Hosomi O , Nakajima T , Shimoda N , Ohi H , Tachikawa T , Piscorz C F , Jain R K , Matta K L
Published: January 2020

Concentrations of blood group A-specified alpha(1-->3)-N-acetylgalactosaminyltransferase (A enzyme) were measured in human plasma of blood groups A1, A2, and A3 by using chemically synthesized H disaccharides and H type 1 and type 2 trisaccharides attached to hydrophobic aglycones as acceptors. When the trisaccharides were used as acceptors, enzyme activities were reduced in samples from A2 and A3. However, the H disaccharides were shown to be good acceptors even for enzymes from A2 and A3, and no significant difference in enzyme concentration was detected in any of the plasma tested.
Quantitative Determination of Methemoglobin by Measuring the Solvent-Water Proton-Nuclear Magnetic Resonance Relaxation Rate
Authors: Aime S , Dastrú W , Fasano M , Arnelli A , Castagnola M , Giardina B , Ascenzi P
Published: January 2020

We report a new method for the quantitative determination of human methemoglobin (metHb) based on the measurement of the solvent-water proton-nuclear magnetic resonance (NMR) relaxation rate R1 [normalized to 1 mmol/L hemoglobin (Hb) concentration]. MetHb (%) is estimated from the linear dependence of R1 on the metHb concentration, taking into account the simple relationship [MetHb] = [(R1 - R1HbO2)/(R1metHb - R1HbO2)].100, where R1HbO2 and R1metHb are values for the solvent-water relaxation rate of standard 1.0 mmol/L solutions of the oxygenated derivative of human hemoglobin (HbO2) and of metHb, respectively. The minimum metHb that may be determined from the analysis of the experimental data is 0.5 +/- 0.4%.