bluuebunny/crossref_metadata_2025 huggingface dataset:
Automated fluorescent analysis procedure for enzymatic mutation detection
Authors: Del Tito Benjamin J , Poff Herbert E , Novotny Mark A , Cartledge Donna M , Walker Ralph I , Earl Christopher D , Bailey Anne L
Published: January 2020

The Enzymatic Mutation Detection™ (EMD) assay detects mutations or polymorphisms in DNA. The assay procedure takes <1 h and is followed by electrophoretic detection. We report an automated procedure, using fluorescently labeled probe and quantitative analysis on the ABI Prism™ 377 DNA Sequencer, that improves on earlier methods (1,2) by eliminating the need for sample purification, shortening the hybridization time, and increasing the signal-to-noise ratio. The EMD assay uses the bacteriophage resolvase T4 endonuclease VII, which cleaves the heteroduplex molecules at the mismatch site, forming two shorter fragments that are resolved by gel electrophoresis. Unlike existing mutation techniques, the EMD method uses a single protocol to identify point mutations, deletions, and insertions for all DNA fragments. Test DNA samples are assayed directly from PCR reactions, and fragments up to 4 kb in size have been assayed successfully. A independent analysis on the p53 tumor suppressor gene from clinical samples has shown 100% sensitivity and 94% specificity. Because the fluorescent EMD assay has been optimized for high signal-to-noise ratios, mutations can be identified in mixed samples containing up to a 20-fold excess of normal DNA.
Reference standardization and triglyceride interference of a new homogeneous HDL-cholesterol assay compared with a former chemical precipitation assay
Authors: Cobbaert Christa , Zwang Louwerens , Ceriotti Ferruccio , Modenese Annalisa , Cremer Peter , Herrmann Wolfgang , Hoss Gerhard , Jarausch Jochen , Türk Regina , März Winfried , Nauck Matthias
Published: January 2020

A homogeneous HDL-c assay (HDL-H), which uses polyethylene glycol-modified enzymes and sulfated α-cyclodextrin, was assessed for precision, accuracy, and cholesterol and triglyceride interference. In addition, its analytical performance was compared with that of a phosphotungstic acid (PTA)/MgCl2 precipitation method (HDL-P). Within-run CVs were ≤1.87%; total CVs were ≤3.08%. Accuracy was evaluated in fresh normotriglyceridemic sera using the Designated Comparison Method (HDL-H = 1.037 Designated Comparison Method + 4 mg/L; n = 63) and in moderately hypertriglyceridemic sera by using the Reference Method (HDL-H = 1.068 Reference Method − 17 mg/L; n = 41). Mean biases were 4.5% and 2.2%, respectively. In hypertriglyceridemic sera (n = 85), HDL-H concentrations were increasingly positively biased with increasing triglyceride concentrations. The method comparison between HDL-H and HDL-P yielded the following equation: HDL-H = 1.037 HDL-P + 15 mg/L; n = 478. We conclude that HDL-H amply meets the 1998 NCEP recommendations for total error; its precision is superior compared with that of HDL-P, and its average bias remains below ±5% as long as triglyceride concentrations are ≤10 g/L and in case of moderate hypercholesterolemia.
Analytical approaches of European Union laboratories to drugs of abuse analysis
Authors: Badia Roser , de la Torre Rafael , Corcione Sergio , Segura Jordi
Published: January 2020

We report a survey on urine drug testing within a total of 269 laboratories of the European Union. Clinical laboratories predominated over forensic laboratories (59.5% vs 28.5%). Screening without identification/quantification was the common approach used by clinical laboratories, whereas screening with identification/quantification was the approach used by almost all forensic laboratories. Screening was primarily performed by immunoassay in both types of laboratories. Gas chromatography coupled to mass spectrometry was the main analytical method used for specific identification/quantification of drugs, but other methods (including immunoassays) were also used. Cutoff values applied varied by laboratory type, country, and method used. A high percentage of laboratories did not use or report cutoff values. Overall, countries of the European Union vary significantly in regards to drugs tested, analytical approach, and screening and identification cutoff values. It is recommended to clearly state the analytical method and the cutoff values used when reporting results for drugs of abuse testing.
Fully automated assay for total homocysteine, cysteine, cysteinylglycine, glutathione, cysteamine, and 2-mercaptopropionylglycine in plasma and urine
Authors: Pastore Anna , Massoud Renato , Motti Corradino , Russo Anna Lo , Fucci Giorgio , Cortese Claudio , Federici Giorgio
Published: January 2020

We describe a 6-min HPLC method to measure the total concentrations of the most important thiols in plasma and urine–cysteine, homocysteine, cysteinylglycine, and glutathione–as well as the concentrations in plasma and urine, respectively, of cysteamine and 2-mercaptopropionylglycine, two compounds used to treat disorders of cysteine metabolism. Precolumn derivatization with bromobimane and reversed-phase HPLC were performed automatically by a sample processor. Throughput was up to 100 samples in 24 h. The within-run CV ranged from 0.9% to 3.4% and the between-run CV ranged from 1.5% to 6.1%. Analytical recovery was 97–107%, with little difference between plasma and urine samples. The detection limit was ∼50 nmol/L for all the analytes studied. Thiol concentrations were determined in the plasma of 206 healthy donors and in the urine of 318 healthy donors distributed for age and sex. Mean values of plasma cysteine and homocysteine were significantly lower in infants (ages, <1 y) compared with other age groups (P <0.005). In adults, mean plasma homocysteine values were higher in males than in females (9.2 vs 6.7 μmol/L, P <0.0001) and in the 6- to 10-year-old group (P <0.05). Mean values for glutathione and cysteinylglycine were not sex- and age-dependent. In urine, both cysteine and homocysteine showed a wide range of variation.
A stochastic approximation method for assigning values to calibrators
Authors: Schlain Brian
Published: January 2020

A new procedure is provided for transferring analyte concentration values from a reference material to production calibrators. This method is robust to calibration curve-fitting errors and can be accomplished using only one instrument and one set of reagents. An easily implemented stochastic approximation algorithm iteratively finds the appropriate analyte level of a standard prepared from a reference material that will yield the same average signal response as the new production calibrator. Alternatively, a production bulk calibrator material can be iteratively adjusted to give the same average signal response as some prespecified, fixed reference standard. In either case, the outputted value assignment of the production calibrator is the analyte concentration of the reference standard in the final iteration of the algorithm. Sample sizes are statistically determined as functions of known within-run signal response precisions and user-specified accuracy tolerances.
Optimized serum pancreolauryl test for differentiating patients with and without chronic pancreatitis
Authors: Domínguez-Muñoz J Enrique , Malfertheiner Peter
Published: January 2020

The serum pancreolauryl test has limited sensitivity for detecting mild pancreatic insufficiency. The aim of this study was to optimize the serum pancreolauryl test so as to increase the probability of positive results in patients with chronic pancreatitis. The study had three parts. First, the sampling time was optimized by analyzing retrospectively the frequency of fluorescein peaks at different times from 0 to 240 min in 560 consecutive patients. Second, the calculation of serum fluorescein concentrations by means of a standard calibration factor was prospectively compared in 271 consecutive patients with a modification involving a specimen-specific calibration factor for each patient. Third, the clinical utility of the intravenous injection of secretin before ingestion of the test meal was prospectively evaluated in a further 32 patients. As a result, the optimized serum pancreolauryl test developed differs from the former version of the test in utilizing intravenous administration of secretin before the test meal, calculation of serum fluorescein based on specimen-specific calibration factors, and blood samples taken only at 0 (basal), 120, 150, 180, and 240 min. This optimized pancreolauryl test was abnormal more frequently in patients with chronic pancreatitis than was the formerly used test, especially for cases of mild and moderate disease.
Misleading serum free thyroxine results during low molecular weight heparin treatment
Authors: Stevenson Heather P , Pooler G , Archbold R , Johnston Paul , Young Ian S , Sheridan Brian
Published: January 2020

Measured free thyroxine concentrations in serum increase markedly after intravenous heparin administration, but the effect of heparin administered subcutaneously has not been adequately documented. We found in vitro increases of up to 63% in measured FT4 after a single dose of subcutaneous heparin (enoxaparin, 2000 units) in nine healthy volunteers, and the magnitude of these increases was correlated with initial serum triglyceride concentrations (r = 0.93, P <0.005) and in vitro free fatty acid release (r = 0.88, P <0.005). In 10 cardiac inpatients receiving repeated doses of enoxaparin (2000 units twice daily), measured FT4 increased by up to 171% in specimens taken 2–6 h after injection. When specimens were obtained 10 h after injection, the effect appeared to be minimized, with in vitro increases of <40%, but such increases may still be sufficient to cause interpretative errors. If FT4 estimation is absolutely necessary in patients receiving enoxaparin, specimens should be taken ≥10 h postdose and analyzed within 24 h.
Standards of laboratory practice: analgesic drug monitoring
Authors: White Suzanne , Wong Steven H Y
Published: January 2020

Analgesics are the most commonly consumed over-the-counter preparations in the United States. They are used in the treatment of various pain syndromes and other medical conditions. Although analgesics are generally perceived to be safe agents, serious toxicity may occur in the setting of acute overdose, chronic abuse, or overuse. The indications for therapeutic drug monitoring in patients using these medications appropriately is as yet not well defined. The emphasis of this discussion, therefore, is on recommendations for monitoring in situations where toxicity is suspected. Preanalytical, analytical, and practice issues including drug interactions, frequency of monitoring, pertinent ancillary tests, reporting, and special patient groups at risk for toxicity are reviewed. Recent information from a major manufacturer of evacuated tubes arguing against the use of gel tubes for blood collection for drug monitoring is included. Colorimetric/enzymatic/immunoassays for the routine/stat monitoring of acetaminophen and salicylate and diflunisal cross-reactivity with most of the currently used salicylate assays are presented. Achiral and chiral chromatographic assays and newly introduced columns such as restricted access media and/or automated chromatographic systems are reviewed for the analysis of ibuprofen, naproxen, and the recently introduced tramadol. Finally, concepts regarding future directions including drug chirality and chiral analysis are presented.
A homogeneous fluorescence assay for PCR amplicons: its application to real-time, single-tube genotyping
Authors: Whitcombe David , Brownie Jannine , Gillard Helen L , McKechnie Doug , Theaker Jane , Newton Clive R , Little Stephen
Published: January 2020

We have developed a method whereby a single TaqMan™ probe can be used for many PCR reactions. We demonstrate its application as an integrated system for the direct measurement of allele-specific amplicon generation coupled to the suppression of primer-dimer accumulation in PCR. The system uses a 5′-exonuclease assay of amplicon annealed fluorogenic probes that operates in conjunction with the Amplification Refractory Mutation System, whereby relative changes in reporter fluorescent emission are monitored in real-time using an analytical thermal cycler. We have called this system Three-STAR, and it is universal in that it can either use a single probe for the detection of any one target DNA sequence or a single pair of probes for genotyping any bi-allelic polymorphism. Three-STAR is, therefore, particularly useful for the single-tube genotype analysis of a variety of human DNA polymorphisms and mutations.
Evaluation of a homogeneous assay for high-density lipoprotein cholesterol: limitations in patients with cardiovascular, renal, and hepatic disorders
Authors: Simó Josep M a , Castellano Isabel , Ferré Natàlia , Joven Jorge , Camps Jordi
Published: January 2020

We evaluated the performance of a homogeneous assay for the automated measurement of high-density lipoprotein cholesterol (HDL-C) and compared it with a conventional precipitation technique in the following groups of people: control subjects (group A), clinically-healthy elderly (group B), myocardial infarction patients (group C), nephrotic syndrome patients (group D), and liver cirrhosis patients (group E). The performance of the technique was acceptable with respect to precision, accuracy, linearity, and detection limit. Triglycerides up to 40 mmol/L and bilirubin up to 150 μmol/L did not cause interferences. Hemoglobin decreased HDL-C measurements. Samples were stable at −20 °C for up to four months. Bland–Altman plots showed a good agreement between both techniques in the control group but with a progressive divergence in the patient groups B to E. Results indicate limitations of the technique in certain clinical conditions and, coincidentally, the need for reliable calibration materials.
Analytical and clinical performance of a homogeneous enzymatic LDL-cholesterol assay compared with the ultracentrifugation-dextran sulfate-Mg2+ method
Authors: Rifai Nader , Iannotti Elizabeth , DeAngelis Kristen , Law Terence
Published: January 2020

LDL-cholesterol (LDL-C) concentration is currently determined in most clinical laboratories by the Friedewald calculation. This approach has several limitations and may not meet the current total error requirement in LDL-C measurement of ≤ 12%. We evaluated the analytical and clinical performance of the direct N-geneous LDL-C assay (Equal Diagnostics). The N-geneous method correlated highly with the modified beta-quantification assay (r = 0.95; y = 0.91x + 70.6 mg/L; n = 199), showed no significant effect of increased triglyceride or other common interferants, and performed adequately in serum samples from nonfasting individuals. This assay demonstrated a mean total error of 6.75% over a wide range of LDL-C concentrations. In addition, at the medical decision cutoff points, this LDL-C assay showed positive predictive values of 78–95% and negative predictive values of 84–99%. We conclude that the N-geneous LDL-C meets the currently established analytical performance goals and appears to have a role in the diagnosis and management of hypercholesterolemic patients.
Determination of ciprofloxacin in plasma and urine by HPLC with ultraviolet detection
Authors: Kamberi Marika , Tsutsumi Kimiko , Kotegawa Tsutomu , Nakamura Koichi , Nakano Shigeyuki
Published: January 2020

A simple, sensitive isocratic method for the detection and quantification of ciprofloxacin in plasma and urine has been developed. The assay consisted of reversed-phase HPLC with ultraviolet detection. Plasma proteins were removed by a fast and efficient procedure. For the urine samples, the only required sample preparation was dilution. Separation was achieved on a C18 reversed-phase column. The quantification limit was 0.01 mg/L in plasma and 0.5 mg/L in urine. This method was sufficiently sensitive for pharmacokinetic studies.
Non-separation assay for glycohemoglobin
Authors: Blincko Stuart , Edwards Raymond
Published: January 2020

The determination of glycohemoglobin [HbA1c, HbA1, or total glycohemoglobin (GHb)] has become an established procedure in the management of diabetes mellitus. Here, we describe the development of a simple, fluorescence, non-separation assay for the percentage of GHb (%GHb). The fluorescence of an eosin-boronic acid derivative when it was mixed with hemolysates of unwashed erythrocytes was quenched in proportion to the percentage of glycohemoglobin. Measurement of the fluorescence intensity gave an estimate of GHb in the sample, and measurement of light absorbance gave an estimate of total hemoglobin. A combination of the two measurements gave the assay response. Comparison with HPLC (Menarini-Arkray HA-8140 fully automated analyzer) for the percentage of HbA1 (%HbA1) gave %GHb(NETRIA) = 1.1(SD ±0.03)%HbA1 +0.6(SD ±0.3), Sy‖x = 0.821, r = 0.972, n = 80; comparison for HbA1c gave %GHb(NETRIA) = 1.3(SD ±0.04)%HbA1c + 1.8(SD ±0.3), Sy‖x = 0.813, r = 0.973, n = 80. Precision, estimated as the percentage of the CV of the %GHb assay results, was <2% (intraassay, range 5–22% GHb) and <4.2% (interassay, range 4–16% GHb). Dilution of a high-percentage GHb sample lysate showed that the assay was linear, and addition of glucose (60 mmol/L), bilirubin (250 μmol/L), and triglycerides (14 mmol/L) to low, medium, and high %GHb samples showed no clinical interference in assay results.
Rapid sequencing of the p53 gene with a new automated DNA sequencer
Authors: Bharaj Bhupinder S , Angelopoulou Katerina , Diamandis Eleftherios P
Published: January 2020

p53 is the most commonly mutated gene in human cancers. Approximately 90% of the p53 gene mutations are localized between domains encoding exons 5 to 8. Sequencing methods currently available are tedious and time-consuming and are not suitable for routine laboratory testing. In an effort to identify a simple and rapid sequencing method, we analyzed 16 preselected breast tumors and 18 preselected ovarian tumors, using a newly developed automated DNA sequencer. p53 gene mutations had been previously identified in these tumors, using a conventional automated sequencing procedure. Exons 5 to 8 were amplified by PCR, and the PCR products were subsequently subjected to cycle sequencing with the Sanger chain termination method, using Cy5.5-labeled primers. The sequencing mixture was then resolved on a newly developed automated DNA sequencer that can sequence ∼300 bases of DNA in 30 min. Of these 16 breast tumors, two had mutations in exon 5, four in exon 6, three in exon 7, and three in exon 8. Of the 18 ovarian tumors, two had mutations in exon 5, five in exon 6, two in exon 7, and three in exon 8. In all cases, we identified the same mutations by both the new and the conventional sequencing procedures. Most mutations affected an arginine codon. These data demonstrate that the new method has the capability to provide accurate sequencing information in a fraction of the time and labor in comparison with current automated sequencing techniques. When such procedures are used, DNA sequencing may become a routine tool for identifying clinically important mutations for diagnosis and prognosis of patients with genetic, malignant, infectious, and other diseases.
Highly specific radioimmunoassay for human insulin based on immune exclusion of all insulin precursors
Authors: Deberg Michelle , Houssa Paule , Frank Bruce H , Sodoyez-Goffaux Françoise , Sodoyez Jean-Claude
Published: January 2020

We describe a rapid and simple insulin RIA in which proinsulin and conversion intermediates do not interfere. Three monoclonal antibodies (S1, S2, and S53) were selected for their specificity (directed, respectively, against the B10 region, the junction between A chain and C-peptide, and the junction between B chain and C-peptide), their affinity constant (∼1010 L/mol), and their interactive properties in mixture. S2 and S53 were able to bind simultaneously to the same proinsulin molecule, whereas neither could bind simultaneously with S1. Preincubation of serum samples with an excess of S2 resulted in capture of proinsulin and conversion intermediates modified at the junction between B chain and C-peptide into immune complexes that no longer reacted with S1. Similarly, preincubation with S53 prevented proinsulin and conversion intermediates modified at the junction between A chain and C-peptide from reacting with S1. Preincubation with an excess of both S2 and S53 left insulin as the sole reactant with S1. Thus, separation of insulin precursors from insulin by mutually exclusive antibodies is feasible, and on the basis of this new principle, a highly specific RIA for insulin was designed. The detection limit was 11 pmol/L, and the inter- and intraassay coefficients of variation were 11% and 5%, respectively. The potential of the assay for use in clinical studies was verified by application to serum samples from control subjects and patients with diabetes or insulinoma.
Proton nuclear magnetic resonance spectral profiles of urine in type II diabetic patients
Authors: Messana Irene , Forni Franca , Ferrari Franco , Rossi Cristina , Giardina Bruno , Zuppi Cecilia
Published: January 2020

Serial urine samples of 33 type II diabetic patients and 20 control subjects were examined by 1H nuclear magnetic resonance (NMR). Metabolites including lactate, citrate, glycine, alanine, hippurate, trimethylamine-N-oxide, and dimethylamine were identified in all subjects although in higher concentrations in diabetic patients. Other analytes, such as creatine, acetate, betaine, and ketone bodies, were found more frequently and in greater concentrations in diabetics than in controls. In addition, although lactate, citrate, alanine, and hippurate concentrations increased with increasing glycosuria and glycohemoglobin, trimethylamine-N-oxide and dimethylamine were present at high concentrations even in diabetics with good metabolic control. 1H NMR spectroscopy permitted us to explore the relationships among the metabolites present in the urine samples and to obtain information about the disease status in type II diabetic patients.
Differences of bone alkaline phosphatase isoforms in metastatic bone disease and discrepant effects of clodronate on different skeletal sites indicated by the location of pain
Authors: Magnusson Per , Larsson Lasse , Englund Gunnar , Larsson Brita , Strang Peter , Selin-Sjögren Lena
Published: January 2020

We compared clodronate with placebo administration in 42 primarily or secondarily hormone-refractory prostate cancer patients with skeletal metastases and persisting pain. Serum total alkaline phosphatase (ALP), bone ALP isoforms, osteocalcin, cross-linked carboxy-terminal telopeptide of type I collagen, and prostate-specific antigen were analyzed before and after 1 month of treatment. Six ALP isoforms were quantified by HPLC: one bone/intestinal, two bone (B1, B2), and three liver ALP isoforms. The most apparent difference compared with healthy males was observed for the bone ALP isoform B2. Patients and healthy males had a B2 activity corresponding to 75% and 35% of the total ALP activity, respectively (P <0.0001). We propose that the different bone ALP isoforms reflect different stages of osteoblast differentiation during the extracellular matrix maturation phase of osteogenesis. All bone markers except osteocalcin increased after 1 month of clodronate administration. These increases were associated with pain only in the upper part of the body. We suggest that the uptake of clodronate by the skeleton was not uniform during our treatment period.
International Federation of Clinical Chemistry standardization project for the measurement of lipoprotein(a). Phase I. Evaluation of the analytical performance of lipoprotein(a) assay systems and commercial calibrators
Authors: Tate Jillian R , Rifai Nader , Berg Kåre , Couderc Rémy , Dati Francesco , Kostner Gert M , Sakurabayashi Ikunosuke , Steinmetz Armin
Published: January 2020

A secondary reference material for lipoprotein(a) is required to standardize the measurement of lipoprotein(a) in clinical laboratories worldwide. Towards this aim, the International Federation of Clinical Chemistry Working Group for the Standardization of Lipoprotein(a) Assays has initiated a standardization project involving a total of 33 diagnostic company and clinical chemistry laboratories from 12 countries. In Phase 1, the analytical performance of 40 lipoprotein(a) assay systems was evaluated by testing sera and manufactured lipoprotein(a) calibrator materials for precision, linearity, and parallelism. Twenty test systems were nonoptimized according to the results for a pooled serum, which tested nonlinear in 16 systems and imprecise in 4. Acceptable analytical properties and harmonization of lipoprotein(a) values were shown by some commercial calibrators, suggesting their possible use as reference materials. This study highlights the problems that currently occur for lipoprotein(a) measurement in existing assay systems.
Early detection of peritonitis in continuous ambulatory peritoneal dialysis patients by use of chemiluminescence: evaluation of diagnostic accuracy by receiver-operating characteristic curve analysis
Authors: Dalaman Gönül , Haklar Goncagül , Sipahiu Armand , Özener Çetin , Akoĝlu Emel , Yalçin A Süha
Published: January 2020

Continuous ambulatory peritoneal dialysis (CAPD) is now a widely accepted treatment for end-stage renal disease. However, the high incidence of peritonitis is a major complication of CAPD. Polymorphonuclear leukocytes (PMNs) play a major role in antimicrobial response of the host. During phagocytosis, the PMNs undergo a striking increase in oxidative metabolism, known as the respiratory burst, and emit light as chemiluminescence (CL). CL is thus a sensitive measure of PMN oxidative potential and correlates well with antimicrobial activity. In view of the observation of increased susceptibility to infection in CAPD patients, we have studied lucigenin- and luminol-enhanced CL in peritoneal fluids of these patients and assessed the diagnostic accuracy of these tests by ROC curve analysis. ROC curves showed diagnostic accuracies for both tests that were superior to counts of PMNs in the dialysis fluid (P <0.001). At selected cutoff values of 150 000 cpm/vial for lucigenin CL and 600 000 cpm/vial for luminol CL, sensitivities were 100%. Specificities for lucigenin and luminol CL were 89% and 80%, respectively. Our results suggest that CL measurements can be used as an early marker for the presence of infection in CAPD patients.
Inhibition of LDL oxidation by melatonin requires supraphysiologic concentrations
Authors: Duell P Barton , Wheaton David L , Shultz Amy , Nguyen Hien
Published: January 2020

Melatonin has been suggested as a potent antioxidant that may protect against development of atherosclerosis and cancer; however, these effects are unproven and controversial. The antioxidant capacity of melatonin was tested in comparison with α-tocopherol, ascorbic acid, and the melatonin precursors tryptophan and serotonin, by measuring inhibition of metal ion-mediated and human macrophage-mediated oxidation of LDL. Melatonin had weak antioxidant activity that was detectable only at concentrations 10 000- to 100 000-fold higher than physiologic concentrations. These results were comparable with published data showing that the radical scavenging activity of melatonin requires markedly supraphysiologic concentrations. In contrast, α-tocopherol was 50- to 100-fold more potent and was efficacious at physiologic concentrations. Ascorbic acid and tryptophan also were active at physiologic concentrations and were significantly more potent than melatonin. In summary, extremely supraphysiologic concentrations of melatonin had only weak antioxidant activity, which was surpassed by α-tocopherol, ascorbic acid, and tryptophan.
What is hemoglobin A1c? An analysis of glycated hemoglobins by electrospray ionization mass spectrometry
Authors: Peterson Karen P , Pavlovich James G , Goldstein David , Little Randie , England Jack , Peterson Charles M
Published: January 2020

Hemoglobin A1c (HbA1c) is a stable minor Hb variant formed in vivo by posttranslational modification by glucose, originally identified by using cation exchange chromatography, and containing primarily glycated N-terminal β-chains. However, the structure(s) of the quantified species has not been elucidated, and the available methods lack a reference standard. We used electrospray ionization mass spectrometry to determine the extent of glycation of samples separated by boronate affinity and/or cation exchange chromatography. Analyses of clinical samples were consistent with the curvilinear relationship of patient glucose and HbA1c. As glycation increased, the ratio of β-chain to α-chain glycation increased, and the number of glycation sites on the β-chain increased, although these were relatively minor components. We found several glycated species that cochromatographed with HbA1c on cation exchange, including species with both glycated α- and β-chains, nonglycated α- and glycated β-chains, and multiply glycated β-chains. The combined use of affinity and cation exchange chromatography with structural confirmation by electrospray ionization mass spectrometry was found to be useful in producing samples of sufficient purity for the standardization of glycohemoglobin clinical assays.
Identification of MEN1 Mutations in Sporadic Enteropancreatic Neuroendocrine Tumors by Analysis of Paraffin-embedded Tissue
Authors: Mailman Matthew D , Muscarella Peter , Schirmer William J , Ellison E Christopher , O’Dorisio Thomas M , Prior Thomas W
Published: January 2020

Gastrinomas and other gastrointestinal neuroendocrine tumors may occur sporadically or as part of the inherited syndrome multiple endocrine neoplasia type 1 (MEN1). Mutations in the recently identified MEN1 gene have been described in sporadic gastrinomas and insulinomas. This study describes techniques used to identify mutations in the MEN1 gene in DNA extracted from paraffin-preserved tissue. Two novel mutations are identified in the MEN1 gene from nine archived paraffin-embedded neuroendocrine tumors, demonstrating that retrospective genetic analysis can be used to identify mutations in the MEN1 gene from preserved tissue. Conditions are provided by which paraffin-embedded tissue can be used as a source of genetic material for sequence information of sufficient quality for mutational studies of the MEN1 gene. It should also be possible to apply this retrospective genetic analysis of paraffin-embedded tissue to other disease models.
Validation of Double Gradient Denaturing Gradient Gel Electrophoresis through Multigenic Retrospective Analysis
Authors: Cremonesi Laura , Carrera Paola , Fumagalli Antonella , Lucchiari Sabrina , Cardillo Elena , Ferrari Maurizio , Righetti Sabina Carla , Zunino Franco , Righetti Pier Giorgio , Gelfi Cecilia
Published: January 2020

Among established techniques for the identification of either known or new mutations, denaturing gradient gel electrophoresis (DGGE) is one of the most effective. However, conventional DGGE is affected by major drawbacks that limit its routine application: the different denaturant gradient ranges and migration times required for different DNA fragments. We developed a modified version of DGGE for high-throughput mutational analysis, double gradient DGGE (DG-DGGE), by superimposing a porous gradient over the denaturant gradient, which maintains the zone-sharpening effect even during lengthy analyses. Because of this innovation, DG-DGGE achieves the double goals of retaining full effectiveness in the detection of mutations while allowing identical run time conditions for all fragments analyzed. Here we use retrospective analysis of a large number of well-characterized mutations and polymorphisms, spanning all predicted melting domains and the whole genomic sequence of three different genes—the cystic fibrosis transmembrane conductance regulator (CFTR), the β-globin, and the p53 genes—to demonstrate that DG-DGGE may be applied to the rapid scanning of any sequence variation.
A New Gas Chromatography–Mass Spectrometry Method for Simultaneous Determination of Total and Free trans-3′-Hydroxycotinine and Cotinine in the Urine of Subjects Receiving Transdermal Nicotine
Authors: Ji Allena J , Lawson George M , Anderson Rodger , Dale Lowell C , Croghan Ivana T , Hurt Richard D
Published: January 2020

trans-3′-Hydroxycotinine (THOC) has been recognized as the most abundant metabolite of nicotine. In an attempt to assess THOC and cotinine (COT) concentrations during nicotine transdermal therapy, we developed a new quantitative gas chromatography–mass spectrometry (GC–MS) method for simultaneous determination of total and free THOC and COT in human urine. The method utilizes the following: (a) hydrolysis of conjugated THOC and COT by β-glucuronidase; (b) basic extraction of THOC and COT with mixed dichloromethane and n-butyl acetate; (c) derivatization of THOC with bis(trimethylflurosilyl)acetamide; and (d) separation and identification by GC–MS with selective ion monitoring. Lower limits of quantification for the assay were 50 and 20 μg/L for THOC and COT, respectively. The intra- and interassay CVs were 4.4% and 11% for THOC, and 3.9% and 10% for COT at 1000 μg/L. The results from six consecutive 24-h urine collections in 71 subjects administered daily transdermal nicotine doses of 11, 22, and 44 mg showed that, on average, free THOC was 76% of total THOC and free COT was 48% of total COT in all subjects. THOC is the major metabolite of nicotine and constitutes 20% of total nicotine intake at steady state, whereas urinary nicotine and COT excretion were 8% and 17%, respectively. The method is useful for simultaneous determination of free and total THOCand COT and can be used to assess the urinary excretion of these metabolites during transdermal nicotine therapy.
Significance of l-Alloisoleucine in Plasma for Diagnosis of Maple Syrup Urine Disease
Authors: Schadewaldt Peter , Bodner-Leidecker Annette , Hammen Hans-Werner , Wendel Udo
Published: January 2020

Background: The significance of plasma l-alloisoleucine, which is derived from l-isoleucine in vivo, for diagnosis of maple syrup urine disease (MSUD) was examined.

Methods: Branched-chain l-amino acids were measured by automatic amino acid analysis.

Results: Alloisoleucine reference values in plasma were established in healthy adults [1.9 ± 0.6 μmol/L (mean ± SD); n = 35], children 3–11 years (1.6 ± 0.4 μmol/L; n = 17), and infants <3 years (1.3 ± 0.5 μmol/L; n = 37). The effect of dietary isoleucine was assessed in oral loading tests. In controls receiving 38 μmol (n = 6; low dose) and 1527 μmol (n = 3; high dose) of l-isoleucine per kilogram of body weight, peak increases of plasma isoleucine were 78 ± 24 and 1763 ± 133 μmol/L, respectively; the peak increase of alloisoleucine, however, was negligible for low-dose (<0.3 μmol/L) and minor for high-dose (5.5 ± 2.1 μmol/L) load. In patients with diabetes mellitus, ketotic hypoglycemia, phenylketonuria, and obligate heterozygous parents of MSUD patients, alloisoleucine was not significantly different from healthy subjects. Therefore, a plasma concentration of 5 μmol/L was used as a cutoff value. In patients with classical MSUD (n = 7), alloisoleucine was beyond the cutoff value in 2451 of 2453 unselected samples. In patients with variant MSUD (n = 9), alloisoleucine was >5 μmol/L in all samples taken for establishment of diagnosis and in 94% of the samples taken for treatment control (n = 624). With the other branched-chain amino acids, the frequency of diagnostically significant increases was <45%.

Conclusions: The present findings indicate that plasma l-alloisoleucine above the cutoff value of 5 μmol/L is the most specific and most sensitive diagnostic marker for all forms of MSUD.
Increased Fetal DNA Concentrations in the Plasma of Pregnant Women Carrying Fetuses with Trisomy 21
Authors: Lo YM Dennis , Lau Tze K , Zhang Jun , Leung Tse N , Chang Allan MZ , Hjelm N Magnus , Elmes R Sarah , Bianchi Diana W
Published: January 2020

Background: The recent discovery of the presence of circulating cell-free fetal DNA in maternal plasma opens up new prenatal diagnostic applications and provides new avenues for clinical investigation. It is of research and potential diagnostic interest to determine whether fetal trisomy 21 may be associated with quantitative abnormalities of circulating fetal DNA in maternal plasma.

Methods: Maternal plasma samples were prospectively collected from two centers situated in Hong Kong and Boston. Samples collected from Boston consisted of 7 women carrying male trisomy 21 fetuses, 19 carrying euploid male fetuses, and 13 carrying female fetuses. Samples collected from Hong Kong consisted of 6 women carrying male trisomy 21 fetuses, 18 carrying euploid male fetuses, and 10 carrying female fetuses. Male fetal DNA in maternal plasma was measured using real-time quantitative Y-chromosomal PCR.

Results: For patients recruited from Boston, the median circulating fetal DNA concentrations in women carrying trisomy 21 and euploid male fetuses were 46.0 genome-equivalents/mL and 23.3 genome-equivalents/mL, respectively (P = 0.028). For patients recruited from Hong Kong, the median circulating fetal DNA concentrations in women carrying trisomy 21 and euploid male fetuses were 48.2 genome-equivalents/mL and 16.3 genome-equivalents/mL, respectively (P = 0.026). None of the samples from women carrying female fetuses had detectable Y-chromosomal signals.

Conclusions: Abnormally high concentrations of circulating fetal DNA are found in a proportion of women carrying fetuses with trisomy 21. The robustness and reproducibility of real-time PCR analysis of maternal plasma makes it a valuable tool for cross-institutional collaboration involving centers located in different parts of the world.
Common Diagnostic Test Panels for Clinical Evaluation of New Primary Care Outpatients in Japan: A Cost-Effectiveness Evaluation
Authors: Takemura Yuzuru , Ishida Haku , Inoue Yuji , Beck J Robert
Published: January 2020

Background: The Japan Society of Clinical Pathology (JSCP) has developed a guideline for common diagnostic test utilization in new primary care outpatients. To determine the scientific and economic validity of the JSCP panel testing system, we analyzed cost-effectiveness parameters of test panels advocated.

Methods: The “Essential Laboratory Tests” panel (2) [ELT(2) panel], a package of common diagnostic tests added to the ELT(1) baseline health-status screening panel, was applied to 540 new outpatients who visited the Comprehensive Medicine Clinics in an academic medical center during 1991 to 1997. A “useful result” (UR) of testing was defined as a finding that contributed to a change in a physician’s diagnosis- or decision-making, relating to a “tentative initial diagnosis” (TID) obtained from history and physical examination alone.

Results: Clinical usefulness was demonstrated in 259 patients with ELT(2), in whom 398 URs were generated. Clinical effectiveness (UR/TID) ranged from 1.65 (hematological) to 0.088 (neurological disease), with a cost disparity from ¥1251 (∼$10) to ¥23 037 (∼$200) per UR. A total of 1137 tests generated URs. We further assessed the clinical effectiveness and economic efficiency (cost/UR) of ELT(1) and restructured panels. Use of the ELT(1) alone generated 244 URs in 167 patients. The poor efficiency of the ELT(1) panel was markedly improved with the addition of certain ELT(2)-specific tests in liver/pancreatobiliary, metabolic/endocrine, and cardiovascular disease groups.

Conclusions: A wide disparity in the utility of ELT panels in different patient groups does not support the JSCP recommendation of their routine use for new outpatients. Selective test combinations should be used in selected patient groups.
Dried Plasma Spot Measurements of Ferritin and Transferrin Receptor for Assessing Iron Status
Authors: Flowers Carol H , Cook James D
Published: January 2020

Background: Efforts to reduce the high global prevalence of nutritional anemia require the use of both reliable laboratory assays to distinguish iron deficiency from other causes of anemia and cost-effective methods for collection of blood specimens under field conditions. The suitability of using small plasma samples spotted and dried on filter paper for measurements of plasma ferritin and transferrin receptor was evaluated in the present study.

Methods: Blood specimens obtained from 73 male and 83 female subjects (19–40 years) representing a wide range of iron status were used to perform parallel measurements of plasma ferritin and transferrin receptor on whole plasma and spotted plasma samples.

Results: Ratio plots, evaluating the acceptability and precision of the spot method in ferritin and transferrin receptor assays, showed the expected proportion of data points within the 95% prediction interval. In the composite group of 156 subjects, both the whole plasma and plasma spot methods gave a geometric mean transferrin receptor/ferritin ratio of 18. The regression equation for the ratio was logy = 1.045 logx − 0.05126; r = 0.986; P <0.0001. The ratio of transferrin receptor/ferritin determined from plasma spots correctly identified all 12 subjects with iron deficiency anemia compared with 11 of the 12 for whole plasma measurements.

Conclusions: Measurements of ferritin and transferrin receptor on plasma spotted and dried on filter paper are comparable to whole plasma values for the identification of iron deficiency anemia. The use of dried plasma spots will facilitate the collection, storage, and transport of samples in epidemiological studies of anemia prevalence.
Inflammatory Markers in Men with Angiographically Documented Coronary Heart Disease
Authors: Rifai Nader , Joubran Rana , Yu Harry , Asmi Mohamad , Jouma Mohidien
Published: January 2020

Background: Recent evidence suggests that atherosclerosis is a chronic inflammatory process. In this study, we examined several markers of inflammation in men with coronary heart disease (CHD) and appropriate controls.

Methods: The concentrations of C-reactive protein (CRP), serum amyloid A (SAA), interleukin-6 (IL-6), and soluble intracellular adhesion molecule (sICAM-1) were examined in 100 men with angiographically documented CHD and 100 age-, gender-, and smoking-matched controls with no history of CHD. We assessed the association of these markers with severity of disease as indicated by >50% obstruction in one vessel (n = 30), two vessels (n = 39), or three vessels (n = 31).

Results: Significant increases were noted in serum CRP (median for cases vs controls, 3.4 vs 1.5 mg/L; P <0.0001), SAA (5.9 vs 3.7 mg/L; P <0.005), and IL-6 (2.3 vs 1.7 ng/L; P <0.013) in patients with CHD compared with controls. These differences remained significant after correction for age, smoking, hypertension, diabetes, and lipid and homocysteine concentrations. Plasma sICAM-1 was not significantly different between the two groups (335 vs 339 μg/L). No significant correlation was seen between these markers and the severity of coronary disease.

Conclusions: Concentrations of CRP, SAA, and IL-6 were increased in patients with CHD but failed to correlate with severity of coronary disease. These markers might reflect the diffuse atherosclerotic process in the vascular system rather than the degree of localized obstruction from coronary lesions.
Sample Preparation and Storage Can Change Arsenic Speciation in Human Urine
Authors: Feldmann Jörg , Lai Vivian W-M , Cullen William R , Ma Mingsheng , Lu Xiufen , Le X Chris
Published: January 2020

Background: Stability of chemical speciation during sample handling and storage is a prerequisite to obtaining reliable results of trace element speciation analysis. There is no comprehensive information on the stability of common arsenic species, such as inorganic arsenite [As(III)], arsenate [As(V)], monomethylarsonic acid, dimethylarsinic acid, and arsenobetaine, in human urine.

Methods: We compared the effects of the following storage conditions on the stability of these arsenic species: temperature (25, 4, and −20 °C), storage time (1, 2, 4, and 8 months), and the use of additives (HCl, sodium azide, benzoic acid, benzyltrimethylammonium chloride, and cetylpyridinium chloride). HPLC with both inductively coupled plasma mass spectrometry and hydride generation atomic fluorescence detection techniques were used for the speciation of arsenic.

Results: We found that all five of the arsenic species were stable for up to 2 months when urine samples were stored at 4 and −20 °C without any additives. For longer period of storage (4 and 8 months), the stability of arsenic species was dependent on urine matrices. Whereas the arsenic speciation in some urine samples was stable for the entire 8 months at both 4 and −20 °C, other urine samples stored under identical conditions showed substantial changes in the concentration of As(III), As(V), monomethylarsonic acid, and dimethylarsinic acid. The use of additives did not improve the stability of arsenic speciation in urine. The addition of 0.1 mol/L HCl (final concentration) to urine samples produced relative changes in inorganic As(III) and As(V) concentrations.

Conclusions: Low temperature (4 and −20 °C) conditions are suitable for the storage of urine samples for up to 2 months. Untreated samples maintain their concentration of arsenic species, and additives have no particular benefit. Strong acidification is not appropriate for speciation analysis.
Zinc Protoporphyrin: A Metabolite with a Mission
Authors: Labbé Robert F , Vreman Hendrik J , Stevenson David K
Published: January 2020

Zinc protoporphyrin (ZnPP) is a normal metabolite that is formed in trace amounts during heme biosynthesis. The final reaction in the biosynthetic pathway of heme is the chelation of iron with protoporphyrin. During periods of iron insufficiency or impaired iron utilization, zinc becomes an alternative metal substrate for ferrochelatase, leading to increased ZnPP formation. Evidence suggests that this metal substitution is one of the first biochemical responses to iron depletion, causing increased ZnPP to appear in circulating erythrocytes. Because this zinc-for-iron substitution occurs predominantly within the bone marrow, the ZnPP/heme ratio in erythrocytes reflects iron status in the bone marrow. In addition, ZnPP may regulate heme catabolism through competitive inhibition of heme oxygenase, the rate-limiting enzyme in the heme degradation pathway that produces bilirubin and carbon monoxide. Physiological roles, especially relating to carbon monoxide and possibly nitric oxide production, have been suggested for ZnPP. Clinically, ZnPP quantification is valuable as a sensitive and specific tool for evaluating iron nutrition and metabolism. Diagnostic determinations are applicable in a variety of clinical settings, including pediatrics, obstetrics, and blood banking. ZnPP analytical methodologies for clinical studies are discussed. In addition to diagnostic tests and metabolic studies, ZnPP has a potential therapeutic application in controlling bilirubin formation in neonates as a preventive measure for hyperbilirubinemia. Biochemical research techniques, both in vivo and in vitro, are described for further studies into the role of ZnPP in metabolism and physiology.