bluuebunny/crossref_metadata_2025 huggingface dataset:
Serum transferrin receptors are decreased in the presence of iron overload
Authors: Khumalo Hlosukwazi , Gomo Zvenyika A R , Moyo Victor M , Gordeuk Victor R , Saungweme Thokozile , Rouault Tracey A , Gangaidzo Innocent T
Published: January 2020

To test the hypothesis that the quantities of circulating transferrin receptors are reduced in iron overload, we studied serum transferrin receptors and indirect measures of iron status in 150 subjects from rural Zimbabwe. We found significant inverse correlations between serum concentrations of transferrin receptors and ferritin, the ratio of ferritin to aspartate aminotransferase, and transferrin saturation (r ≥0.44; P <0.001). The mean ± SD concentration of serum transferrin receptors in 23 subjects classified as having iron overload (ferritin >300 μg/L and transferrin saturation >60%) was 1.55 ± 0.61 mg/L, significantly lower than the 2.50 ± 0.62 mg/L in 75 subjects with normal iron stores (ferritin 20–300 μg/L and transferrin saturation 15–55%; P <0.0005) and the 2.83 ± 1.14 mg/L in 8 subjects with iron deficiency (ferritin <20 μg/L; P = 0.001). In keeping with the regulation of transferrin receptor expression at the cellular level, our findings suggest that serum transferrin receptors are decreased in the presence of iron overload.
Evaluation of the lysosome-associated membrane protein LAMP-2 as a marker for lysosomal storage disorders
Authors: Hua Chi T , Hopwood John J , Carlsson Sven R , Harris Ray J , Meikle Peter J
Published: January 2020

For many lysosomal storage disorders, presymptomatic detection, before the onset of irreversible pathology, will greatly improve the efficacy of current and proposed therapies. In the absence of a family history, presymptomatic detection can be achieved only by a comprehensive newborn screening program. Recently we reported that the lysosome-associated membrane protein LAMP-1 was increased in the plasma from ∼70% of individuals with lysosomal storage disorders. Here we report on the evaluation of a second lysosome-associated membrane protein, LAMP-2, as a marker for this group of disorders. The median concentration of LAMP-2 in the plasma of healthy individuals was 1.21 mg/L, fourfold higher than the median LAMP-1 concentration (0.31 mg/L). LAMP-2 was increased in >66% of patients with lysosomal storage disorders, and the increases coincided with increased LAMP-1 concentrations. The reference intervals for LAMP-1 and LAMP-2 in blood spots taken from newborns were 0.20–0.54 mg/L (n = 1600) and 0.95–3.06 mg/L (n = 1600), respectively. A high correlation was observed between the concentrations of LAMP-1 and LAMP-2 in both control and affected individuals. The higher concentrations of LAMP-2, relative to LAMP-1, in plasma make LAMP-2 an attractive marker; however, the final selection will be dependent on the availability of new diagnostic markers and their ability to detect disorders currently not identified by LAMP-2.
Intra- and interindividual variability of carbohydrate-deficient transferrin, γ-glutamyltransferase, and mean corpuscular volume in teetotalers
Authors: Helander Anders , Vabö Erling , Levin Klas , Borg Stefan
Published: January 2020

Blood samples for determination of the biochemical alcohol markers carbohydrate-deficient transferrin (CDT) in serum, γ-glutamyltransferase (GGT) in serum, and erythrocyte mean corpuscular volume (MCV) were collected once every 1–2 weeks over ∼5 months from 10 female and 4 male teetotalers. Mean values for serum CDT (using the CDTectTM assay) ranged from 9.9 to 29.4 units/L (median, 14.2 units/L), and the highest results were obtained in the women. The mean values for serum GGT ranged from 0.15 to 0.49 μkat/L (median, 0.30 μkat/L, or 18 U/L) except for one woman with a very high mean of 3.07 μkat/L. For MCV, the mean values ranged from 79.5 to 91.5 fL. Two women showed several CDT results above the upper reference limit (mean values, 27.6 and 29.4 units/L, respectively); however, their GGT and MCV values fell within the reference intervals. One of these women exhibited an increased total transferrin concentration (mean value, 5.38 g/L), which was possibly related to the use of oral contraceptives and/or a low serum iron concentration. When the CDTect value was expressed relative to total transferrin, a ratio within the reference interval was observed for this woman but not for the other woman with increased CDTect values. The present study demonstrates a considerable variation between individuals in CDT, GGT, and MCV without drinking any alcohol. The results also show that these baseline values are fairly constant over time within the same individual.
Development of a sensitive ELISA for human leptin, using monoclonal antibodies
Authors: Imagawa Keiichi , Matsumoto Yayoi , Numata Yoshito , Morita Atsushi , Kikuoka Shino , Tamaki Mikio , Higashikubo Chie , Tsuji Tetsuo , Sasakura Kazuyuki , Teraoka Hiroshi , Masuzaki Hiroaki , Hosoda Kiminori , Ogawa Yoshihiro , Nakao Kazuwa
Published: January 2020

A new, sensitive ELISA for human leptin in plasma and cerebrospinal fluid (CSF) was developed, using monoclonal antibodies. The lower limit of detection of this ELISA was 0.78 pg/assay. Both intra- and interassay imprecision values were <7%. The dilution curves of plasma and CSF showed good linearity, and the recovery was 83.2–95.6%. There was good correlation between plasma leptin concentrations by the ELISA and a commercially available RIA (r = 0.99). Our ELISA is advantageous because it does not require radioisotopes, it produces results in hours rather than days, and more importantly, it improves on the detection limit and plasma interference of the RIA kit. The new ELISA enables measurement of low concentrations of leptin, as are seen in CSF and in plasma of patients with anorexia nervosa.
The analog free testosterone assay: are the results in men clinically useful?
Authors: Winters Stephen J , Kelley David E , Goodpaster Bret
Published: January 2020

Men with low testosterone concentrations are usually hypogonadal. However, because variations in the testosterone transport protein, sex hormone-binding globulin (SHBG), directly influence the total testosterone concentration, confirmation of a low testosterone with a measurement of free testosterone or “bioavailable” testosterone (BAT) is recommended. In the present study, we examined the relationship of SHBG with free testosterone (Coat-A-Count assay, Diagnostic Products) and with BAT in men (n = 29) and women (n = 28) who participated in a study of the metabolic determinants of body composition. As expected, total testosterone was strongly positively correlated with SHBG among men (r = 0.68; P <0.01). Although the BAT was independent of SHBG in men (r = 0.02), SHBG was an important predictor of free testosterone (r = 0. 62; P <0.01). In contrast, in women serum concentrations of total testosterone (r = −0.26; P = 0.17), free testosterone (r = −0.30; P = 0.17), and BAT (r = −0.46; P = 0.013) all tended to be lower with increasing SHBG. Free testosterone was nearly perfectly positively correlated with total testosterone (r = 0.97) in men, among whom free testosterone represented a relatively constant percentage of the total testosterone (0.5–0.65%), and the percentage of free testosterone was unrelated to SHBG. Thus the Coat-A-Count free testosterone concentration in men, like the total testosterone concentration, is determined in part by plasma SHBG. Accordingly, androgen deficiency may be misclassified with this assay in men with low SHBG. Moreover, the previous findings of reduced free testosterone concentrations with hypertension or hyperinsulinemia or as a risk factor for developing type 2 diabetes, conditions in which SHBG is reduced, may have been methodology-related.
Serum CrossLaps One Step ELISA. First application of monoclonal antibodies for measurement in serum of bone-related degradation products from C-terminal telopeptides of type I collagen
Authors: Rosenquist Christian , Fledelius Christian , Christgau Stephan , Pedersen Brian J , Bonde Martin , Qvist Per , Christiansen Claus
Published: January 2020

We have developed a two-site ELISA for measurement in serum of bone-related degradation products derived from C-terminal telopeptides of type I collagen. The assay is based on the application of two highly specific monoclonal antibodies against the amino acid sequence of AHD-β-GGR, where the aspartic acid residue (D) is β-isomerized. In a one-step incubation procedure, the degradation products containing cross-linked diisomerized EKAHD-β-GGR peptides are captured by a biotinylated antibody and a peroxidase-conjugated antibody. The generated complex is then bound to the streptavidin surface via the biotin conjugate. Desalted urinary antigens are used for standardization, and parallelism is observed with serum samples. Results are obtained in <2.5 h, and both inter- and intraassay imprecision are <8%. The serum CrossLaps™ concentration was 1748 ± 740 pmol/L (mean ± SD) in premenopausal women (n = 65) and 2952 ± 1325 pmol/L in a group of healthy postmenopausal women (n = 169). The Serum CrossLaps One Step ELISA was capable of detecting a highly significant (P <0.001) effect of hormone replacement therapy in a retrospective study involving 22 postmenopausal women.
Identification of 6-methylmercaptopurine derivative formed during acid hydrolysis of thiopurine nucleotides in erythrocytes, using liquid chromatography–mass spectrometry, infrared spectroscopy, and nuclear magnetic resonance assay
Authors: Dervieux Thierry , Boulieu Roselyne
Published: January 2020

6-Thioguanine and 6-methylmercaptopurine (Me6-MP) nucleotides are the two major thiopurine metabolites of azathioprine found in erythrocytes. During the acid hydrolysis required for the conversion of thiopurine nucleotides into their free bases, Me6-MP was converted into a compound that could be analyzed on a Purospher RP18-e column with dihydrogen phosphate-methanol buffer as eluent. The pH of the acid extract strongly influenced the conversion of Me6-MP into its derivative. The Me6-MP derivative was identified using liquid chromatography–mass spectrometry and infrared and nuclear magnetic resonance spectrometric methods. During the acid hydrolysis of thiopurine nucleotides in erythrocytes, Me6-MP undergoes degradation, leading to 4-amino-5-(methylthio)carbonyl imidazole.
Modified pentamer formation assay for measurement of tacrolimus and its active metabolites: comparison with liquid chromatography–tandem mass spectrometry and microparticle enzyme-linked immunoassay (MEIA-II)
Authors: Armstrong Victor W , Schuetz Ekkehard , Zhang Qingling , Groothuisen Stephan , Scholz Christa , Shipkova Maria , Aboleneen Hoda , Oellerich Michael
Published: January 2020

A modified pentamer formation assay (PFA) for quantification of tacrolimus and active metabolites after extraction from whole blood is described. The lower limit of detection was 2 μg/L. Intraassay precision (CV) was 5.7–13.7%, and the interassay CV was 6.1–14.9%. Tacrolimus trough concentrations in 104 whole blood specimens from liver and kidney transplant recipients were compared with results from HPLC–tandem mass spectrometry (LC/MS/MS) and microparticle enzyme immunoassay (MEIA-II). Data were analyzed by difference plots and are presented as median (95% confidence intervals) of the method differences. MEIA-II results were on average 2.00 μg/L (range, −0.08 to 5.17 μg/L) higher than LC/MS/MS, whereas PFA results were only 1.07 μg/L (range, −2.62 to 5.33 μg/L) higher. Of 104 specimens tested, 25 displayed differences ≥3 μg/L between MEIA-II and PFA: median difference, 4.65 μg/L (range, 3.01–8.79 μg/L). The corresponding median difference between PFA and LC/MS/MS was −0.91 μg/L (range, −4.11 to 0.85 μg/L), and the difference between MEIA-II and LC/MS/MS was 3.67 μg/L (range, 1.88–6.34 μg/L), suggesting the presence of inactive metabolites that caused a positive bias in the immunoassay. In contrast, similar median differences were observed for the remaining 79 specimens: MEIA-II minus LC/MS/MS, 1.78 μg/L (range, −0.45 to 4.11 μg/L); PFA minus LC/MS/MS, 1.90 μg/L (range, −1.70 to 5.50 μg/L). Active tacrolimus metabolites may have contributed to the higher apparent tacrolimus concentrations in these specimens.
When to collect blood specimens: midmorning vs fasting samples
Authors: Leppänen Esa , Dugué Benoit
Published: January 2020

This study was carried out to define the consequences of collecting blood specimens during the forenoon instead of using fasting specimens collected early in the morning. Extensive laboratory data were obtained from specimens collected from fasting participants at 0800, after breakfast at 0930, and again at 1100. The subjects were inpatients in medical and surgical wards (n = 51; 13 women and 38 men; ages, 32–87 years) and subjectively healthy volunteers corresponding to outpatients (n = 51; 31 women and 20 men; ages, 18–63 years). The coefficient of variation (CV, %) of the patient results was compared with the analytical CV. The observed CVs of the subjects’ results far exceeded the analytical CV (%), the average being 3.5-fold and up to 14-fold for some analytes. In individual results the observed change often exceeded the medically derived clinical critical difference. Laboratory data should always be interpreted in the context in which they were obtained. Clinical decisions should be based on objective data (observations) more than on experience and educated guesses. Different medical specialities and different clinical situations may require different kinds of procedures.
Improved method for measuring tartrate-resistant acid phosphatase activity in serum
Authors: Nakanishi Mamoru , Yoh Kousei , Uchida Kagehiro , Maruo Souji , Matsuoka Akira
Published: January 2020

We describe an improved method for the kinetic measurement of tartrate-resistant acid phosphatase (TrACP; EC 3.1.3.2) activity in serum. Of the TrACP derived from erythrocytes, platelets, and macrophages (osteoclasts and others), that from the first two sources is also resistant to fluoride, whereas skeletal TrACP is sensitive to fluoride. Thus, osteoclast-derived TrACP can be measured specifically by exploiting its sensitivity to fluoride. We measured the activity of tartrate-resistant and fluoride-sensitive acid phosphatase (TrFsACP) by using 2,6-dichloro-4-acetylphenyl phosphate as substrate at pH 6.2. The activity of TrFsACP in serum was increased by adding hexadimethrine bromide (Polybrene) to the reaction mixture. This method was not influenced by hemolysis with hemoglobin concentrations as great as 0.9 g/L. The mean ± SD values of TrFsACP activity by this method were 20.4 ± 2.8 and 16.4 ± 2.3 U/L for young (ages 20–29 years) men (n = 34) and women (n = 50), respectively. The highest mean TrFsACP activity was found among children younger than 15 years, followed by that in elderly subjects (older than 60).
Transient 5-oxoprolinuria (pyroglutamic aciduria) with systemic acidosis in an adult receiving antibiotic therapy
Authors: Croal Bernard L , Glen Alastair C A , Kelly Christopher J G , Logan Robert W
Published: January 2020

5-Oxoprolinuria is a recognized condition with increased urinary excretion of 5-oxoproline and is associated with a variety of inborn metabolic defects involving the series of enzyme-linked reactions known as the γ-glutamyl cycle. We report the unusual case of a 35-year-old woman who initially presented with staphylococcal pneumonia but went on to develop a transient high anion gap metabolic acidosis. The development and subsequent complete recovery from this acidosis were subsequently shown to be related in time to the intravenous administration of the antibiotics flucloxacillin and netilmicin. Analysis of the patient’s urine for organic acids revealed massively increased excretions of 5-oxoproline at the peak of her acidosis. We suggest that this patient developed a transient disturbance in the γ-glutamyl cycle involving the 5-oxoprolinase step, which resulted in accumulation of 5-oxoproline that caused a severe high anion gap metabolic acidosis. The administered antibiotics remain as possible causative agents.
Supporting pharmaceutical studies for FDA submissions: diversifying the drug monitoring laboratory
Authors: Moyer Thomas P , Oliver Lawrence K
Published: January 2020

Well-founded pharmacokinetic information is one of the cornerstones of a New Drug Application (NDA) to the Food and Drug Administration (FDA) required to introduce a new drug or a generic equivalent (ANDA) to the marketplace. The service that laboratories engaged in therapeutic drug monitoring provide to support clinical activities is also needed by the pharmaceutical industry during the evaluation and introduction of drugs to the marketplace. In considering this alternative service activity, one must be aware of and compliant with rules established by the FDA for performance of such studies. As specified in CFR 21, Parts 58, 211, and 320, good clinical and laboratory practice indicates that the laboratory should employ a Lab Study Director, who is responsible for the validation of all procedures implemented to support a study protocol, ensures that the laboratory carries out the study following these defined procedures, and personally reviews the results of all testing. The laboratory must validate each procedure by demonstrating and documenting that the procedure does what it is designed to do while meeting the analytical performance specifications required by the study. Laboratory records of all activities must be maintained and available for inspection by the FDA on request. The FDA has authority over all activities related to NDA and ANDA submissions and can bring criminal charges if results of a study are changed because a laboratory deviates from standard procedure. Competent drug monitoring laboratories are fully capable of participating in clinical trials testing activities. Laboratory staff should be fully versed in the FDA rules governing these activities, validate all procedures, and establish systems to verify the procedures are carried out as specified.
Simultaneous analysis of plasma free fatty acids and their 3-hydroxy analogs in fatty acid β-oxidation disorders
Authors: Costa Catarina G , Dorland Lambertus , Holwerda Ulbe , Tavares de Almeida Isabel , Poll-The Bwee-Tien , Jakobs Cornelis , Duran Marinus
Published: January 2020

We present a new derivatization procedure for the simultaneous gas chromatographic–mass spectrometric analysis of free fatty acids and 3-hydroxyfatty acids in plasma. Derivatization of target compounds involved trifluoroacetylation of hydroxyl groups and tert-butyldimethylsilylation of the carboxyl groups. This new derivatization procedure had the advantage of allowing the complete baseline separation of free fatty acids and 3-hydroxyfatty acids while the superior gas chromatographic and mass spectrometric properties of tert-butyldimethylsilyl derivatives remained unchanged, permitting a sensitive analysis of the target compounds. Thirty-nine plasma samples from control subjects and patients with known defects of mitochondrial fatty acid β-oxidation were analyzed. A characteristic increase of long-chain 3-hydroxyfatty acids was observed for all of the long-chain 3-hydroxyacyl-CoA dehydrogenase-deficient and mitochondrial trifunctional protein-deficient plasma samples. For medium-chain acyl-CoA dehydrogenase deficiency and very-long-chain acyl-CoA dehydrogenase deficiency, decenoic and tetradecenoic acids, respectively, were the main abnormal fatty acids, whereas the multiple acyl-CoA dehydrogenase-deficient patients showed variable increases of these unusual intermediates. The results showed that this selective and sensitive method is a powerful tool in the diagnosis and monitoring of mitochondrial fatty acid β-oxidation disorders.
Expression of prostate-specific antigen and prostate-specific membrane antigen transcripts in blood cells: implications for the detection of hematogenous prostate cells and standardization
Authors: Gala Jean-Luc , Heusterspreute Michel , Loric Sylvain , Hanon France , Tombal Bertrand , Van Cangh Paul , Philippe De Nayer  , Philippe Marianne
Published: January 2020

Circulating prostate cells can be detected in cancer patients by using reverse transcriptase–PCR (RT-PCR) assay for prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSM) mRNA. A quality-control study involving a conventional RT-PCR assay was performed and, surprisingly, detected both transcripts in many negative control cell lines and in normal blood samples. The existence of an illegitimate transcription of the PSA and PSM genes was evidenced by sequence analysis of several PSM and PSA-PCR products. Sequencing indeed demonstrated the presence of a PSA or PSM polymorphism in some but not all the cell lines and patient samples, as well as a heterozygous mutation (G to A; Asp to Asn) in the Jurkat cell line. Moreover, the amount of PSA transcript in MCF-7, a PSA-negative breast line, increased after incubation with cycloheximide. Interestingly, the frequency of positivity was as high as 12% in male samples if only tested once, but dropped to 3% upon multiple testing of the same cDNA. This highlights the stochastic effects in RT-PCR results at high sensitivity, hence the importance of repetitive testing in clinical samples. Decreasing the number of cycles avoided the amplification of illegitimate transcripts but also affected the limit of detection, as evidenced with PSA and PSM cDNA containing plasmids, mixing of LNCap with normal blood samples, and the PSA-PSM-negative K562 cell line. The current data raise the need for a multicentric standardization of the RT-PCR methodology used to amplify PSA and PSM transcripts.
Molecular beacons: a new approach for semiautomated mutation analysis
Authors: Giesendorf Belinda A J , Vet Jacqueline A M , Tyagi Sanjay , Mensink Ewald J M G , Trijbels Frans J M , Blom Henk J
Published: January 2020

Molecular beacons are oligonucleotide probes that become fluorescent upon hybridization. We designed molecular beacons to detect a point mutation in the methylenetetrahydrofolate reductase (MTHFR) gene, a mutation that has been related to an increased risk for cardiovascular disease and neural tube defects. The application of molecular beacons enables fast, semi- automated, accurate mutation detection. Moreover, the procedure is performed in a closed tube system, thereby avoiding carryover contamination. We believe these probes will find their way into nucleic acid research and diagnostics.
Human cardiac troponin I: precise identification of antigenic epitopes and prediction of secondary structure
Authors: Ferrieres Gaelle , Calzolari Charles , Mani Jean-Claude , Laune Daniel , Trinquier Sylvie , Laprade Michel , Larue Catherine , Pau Bernard , Granier Claude
Published: January 2020

The presence of human cardiac troponin I (hcTnI) in serum is considered to be a highly specific biochemical marker of acute myocardial infarction. To better understand the antigenic properties of hcTnI, a set of 68 overlapping peptides covering the complete amino acid sequence of hcTnI was prepared and used in epitope mapping experiments. All 16 anti-hcTnI monoclonal antibodies tested were found to recognize a peptide epitope, indicating that recognition by anti-hcTnI monoclonal antibodies was not dependent on the tertiary structure of the protein. Furthermore, the peptide reactivity with anti-hcTnI polyclonal antibodies indicated that most of the sequence of the protein was antigenic; in particular, the N- and C-terminal extremities were found to be the strongest antigenic regions. By using accurate secondary structure prediction methods, hcTnI was found to be an all-alpha type protein, with five regions predicted as helices. Matching the results of the epitope analysis with the structural prediction led us to the view that hcTnI is not a globular protein but probably adopts an extended conformation, allowing a large part of the amino acid sequence of this molecule to be recognized by the immune system. This improved knowledge of the antigenic and structural properties of hcTnI may help in developing new antibodies and immunoassays for use in diagnosing myocardial infarction.
Chondrex: new marker of joint disease
Authors: Harvey Sheryl , Weisman Michael , O’Dell James , Scott Tonya , Visor Mindy Krusemeier Jill , Swindlehurst Cathy
Published: January 2020

Chondrex, a major secretory protein of human chondrocytes and synovial fibroblasts, is increased in serum of patients with joint and cartilage disease. We have developed a sandwich-type ELISA for quantifying chondrex in serum. The interassay CVs were 2.8–3.7% and the average within-run and total CVs were 3.6% and 5.4%, respectively. The limit of detectability by linear dilution was 20 μg/L, recovery upon dilution was 102% ± 5%, and analytical recovery (of added analyte) was 98% ± 11%. The reference interval (central 90% interval) for chondrex in healthy adults was 25–95 μg/L. Chondrex values for patients with active rheumatoid arthritis or osteoarthritis were significantly greater than in healthy adults, inactive rheumatoid arthritis patients, and diabetes patients (P <0.05). In patients treated with disease-modifying antirheumatic drug therapy, decreasing chondrex values reflected the clinical improvement observed in responders, whereas the values were maintained or increased in nonresponders. In conclusion, chondrex may be a useful marker in the clinical investigation of arthritis.
Effect of arsenosugar ingestion on urinary arsenic speciation
Authors: Ma Mingsheng , Le X Chris
Published: January 2020

We developed and evaluated a method for the determination of μg/L concentrations of individual arsenic species in urine samples. We have mainly studied arsenite [As(III)], arsenate [As(V)], monomethylarsonic acid (MMAA), and dimethylarsinic acid (DMAA) because these are the most commonly used biomarkers of exposure by the general population to inorganic arsenic and because of concerns over these arsenic species on their toxicity and carcinogenicity. We have also detected five unidentified urinary arsenic species resulting from the metabolism of arsenosugars. We combined ion pair liquid chromatography with on-line hydride generation and subsequent atomic fluorescence detection (HPLC/HGAFS). Detection limits, determined as three times the standard deviation of the baseline noise, are 0.8, 1.2, 0.7, and 1.0 μ/L arsenic for arsenite, arsenate, MMAA, and DMAA, respectively. These correspond to 16, 24, 14, and 20 pg of arsenic, respectively, for a 20-μL sample injected for analysis. The excellent detection limit enabled us to determine trace concentrations of arsenic species in urine samples from healthy subjects who did not have excess exposure to arsenic. There was no need for any sample pretreatment step. We used Standard Reference Materials, containing both normal and increased concentrations of arsenic, to validate the method. Interlaboratory studies with independent techniques also confirmed the results obtained with the HPLC/HGAFS method. We demonstrated an application of the method to the determination of arsenic species in urine samples after the ingestion of seaweed by four volunteers. We observed substantial increases of DMAA concentrations in the samples collected from the volunteers after the consumption of seaweed. The increase of urinary DMAA concentration is due to the metabolism of arsenosugars that are present in the seaweed. Our results suggest that the commonly used biomarkers of exposure to inorganic arsenic, based on the measurement of arsenite, arsenate, MMAA, and DMAA, are not reliable when arsenosugars are ingested from the diet.
Radioimmunoassay of rat leptin: sexual dimorphism reversed from humans
Authors: Landt Michael , Gingerich Ronald L , Havel Peter J , Mueller Wendy M , Schoner Bridgette , Hale John E , Heiman Mark L
Published: January 2020

Adipose tissue secretes leptin, which interacts with receptors in the hypothalamus. In rodent models of obesity, leptin increases metabolism and decreases food intake, which helps to maintain normal body composition. Accurate and precise methods to quantitate circulating leptin concentrations are needed for physiological studies. We developed an RIA to measure leptin in rat plasma, serum, or adipocyte culture fluids. The working range of the assay, defined by the detection limit and the highest calibrator, was 0.5–50 μg/L. Recovery of 1.6–11.6 μg/L leptin added to serum was 92–103%. The rat leptin RIA correlated well with a previously developed mouse RIA when rat plasma was assayed with both methods (r = 0.94), but the mouse leptin assay underestimated rat leptin in plasma. Within- and between-run CVs were 2.4% to 5.7%. Plasma leptin concentrations correlated directly with percentage of body fat, and correlation improved when the results were separated by gender (r = 0.796, P <0.001 for males; r = 0.710, P <0.001 for females). Leptin concentrations were generally higher in male rats than in females; plasma leptin increased 0.60 μg/L for each percentage of increase in body fat for males but only 0.22 μg/L for females. We conclude that rat serum/plasma leptin concentrations are accurately and precisely measured with this new RIA.
Multicenter evaluation of the Paragon CZETM 2000 capillary zone electrophoresis system for serum protein electrophoresis and monoclonal component typing
Authors: Bienvenu Jacques , Graziani Maria Stella , Arpin François , Bernon Hélène , Blessum Cynthia , Marchetti Cristina , Righetti Gabriella , Somenzini Manuela , Verga Gioacchino , Aguzzi Francesco
Published: January 2020

Serum protein electrophoresis and typing of monoclonal components (MCs) are routine but time-consuming and technically demanding assays. We evaluated capillary electrophoresis (Paragon CZETM 2000) for automation of the two assays. CZE and cellulose acetate electrophoresis gave similar data on 794 samples. Within-run and between-run CVs were <2% for albumin and γ-globulins and 4–7% for α1-, α2-, and β-globulins. Bilirubin, hemoglobin, triglycerides, and fibrinogen were found not to interfere. No carryover by capillaries was detected. The detection limit for MC was <0.5 g/L. MC assessment by immunosubtraction on 403 samples identified the monoclonal type in all samples with peak concentrations >10 g/L; only 50% of MCs that could not be quantified by densitometric scan were typed.
Improved protamine-sensitive membrane electrode for monitoring heparin concentrations in whole blood via protamine titration§
Authors: Ramamurthy Narayanan , Baliga Narayan , Wahr Joyce A , Schaller Ulrich , Yang Victor C , Meyerhoff Mark E
Published: January 2020

An improved protamine-sensitive electrode based on a polymeric membrane doped with the charged ion exchanger dinonylnaphthalenesulfonate (DNNS) is used for monitoring heparin concentrations in whole blood. The electrode exhibits significant nonequilibrium potentiometric response to polycationic protamine over the concentration range of 0.5–20 mg/L in undiluted whole-blood samples. The sensor can serve as a simple end point detector for the determination of heparin via potentiometric titrations with protamine. Whole-blood heparin concentrations determined by the electrode method (n ≥157) correlate well with other protamine titration-based methods, including the commercial Hepcon HMS assay (r = 0.934) and a previously reported potentiometric heparin sensor-based method (r = 0.973). Reasonable correlation was also found with a commercial chromogenic anti-Xa heparin assay (r = 0.891) with corresponding plasma samples and appropriate correction for whole-blood hematocrit levels. Whereas a significant positive bias (0.62 kU/L; P <0.001) is observed between the anti-Xa assay and the protamine sensor methods, insignificant bias is observed between the protamine sensor and the Hepcon HMS tests (0.08 kU/L; P = 0.02). The possibility of fully automating these titrations offers a potentially simple, inexpensive, and accurate method for monitoring heparin concentrations in whole blood.
Multicenter harmonization of common enzyme results by fresh patient-pool sera
Authors: Franck Paul F H , Steen Gerard , Lombarts Arnold J P F , Souverijn John H M , van Wermeskerken Robert K A
Published: January 2020

A region consisting of 19 clinical laboratories harmonized their calibration of seven common enzymes by using fresh patient-pool sera. One of the laboratories was chosen to act as Regional Reference Laboratory (RRL). This laboratory used internationally accepted (mostly IFCC) methods at 37 °C, with an intralaboratory CV ≤2.5%. First, the reference ranges of the RRL were verified by analysis of a reference population and calculation of the results by a parametric method. Next, all laboratories, including the RRL, received six patient-pool sera and analyzed them at the same time on the same date. Enzyme calibration factors at each laboratory were converted on the basis of the slope, and occasionally the intercept, of regression analysis with the RRL and the individual laboratory. Before harmonization, the interlaboratory CVs varied from 16.9% to 61.6%. After harmonization, CVs decreased to between 5.0% and 9.5%. These results proved to be reproducible over a period of more than a year. Using internationally accepted inaccuracy and imprecision criteria, the achieved interlaboratory CVs permit the use of one set of reference ranges by all participating laboratories. Certified Reference Materials were analyzed, resulting in interlaboratory CVs as low as those achieved with patient-pool sera. These materials can act as commutable reference preparations, except for creatine kinase.
Proposal for standardizing direct-reading biosensors for blood glucose
Authors: Fogh-Andersen Niels , D’Orazio Paul
Published: January 2020

Direct-reading glucose biosensors sense molality (glucose per unit water mass) in the sample. With aqueous calibration, a direct-reading glucose biosensor produces higher results in blood and plasma than methods measuring concentration, theoretically by the ratio of water concentrations in calibrator and sample. To confirm this, we measured glucose in 140 blood and 40 plasma samples with the direct-reading glucose sensor in the Chiron Model 860 Blood Gas and Critical Analyte System and with our routine method (ESAT 6660; Eppendorf). The Chiron instrument is calibrated with a 10 mmol/L (180 mg/dL) glucose calibrator (mass concentration of water = 0.99 kg/L). Assuming normal water concentrations of 0.84 and 0.93 kg/L in blood and plasma, respectively, we multiplied results from the Chiron sensor by 0.84/0.99 and 0.93/0.99 to obtain concentrations in blood and plasma. This conversion resulted in agreement of results with our routine method. An individual conversion based on hematocrit in each whole-blood sample was less satisfactory. To avoid confusion over variously measured and reported glucose results and reference values, we suggest standardization and reporting of whole-blood glucose results as equivalent plasma concentrations. This proposal may be conveniently achieved by using a commercially available reference material for glucose, NIST SRM 965.
Determination of gene dosage at the PMP22 and androgen receptor loci by quantitative PCR
Authors: Poropat Renee A , Nicholson Garth A
Published: January 2020

Although many genetic diseases are caused by the presence of point mutations in respective genes, an increasing number of diseases are known to be caused by gene copy number changes. We report the development of a rapid and reliable PCR-based method for quantitation of gene copy number with sufficient sensitivity to detect single copy changes without the use of radioactive or fluorescent labeling. The sensitivity of this technique has been demonstrated by the detection of the DNA duplication or deletion occurring in two inherited peripheral neuropathies, Charcot-Marie-Tooth type 1A (CMT1A) and hereditary neuropathy with liability to pressure palsies (HNPP), that are caused by a reciprocal duplication or deletion event on chromosome 17p11.2–12. This method relies on the comparison of the amount of PCR product generated from a potentially duplicated or deleted target sequence with the amount of product generated from a disomic reference gene. The value of this ratio (target PCR product:reference PCR product) indicates whether the target sequence is duplicated, deleted, or unchanged. Using primers from within a duplicated or deleted region (PMP22 gene and EW401) and from within a reference region (NF1 gene), we tested 50 CMT1A, 30 HNPP, and 50 unaffected individuals for the presence of a DNA duplication or deletion. Target:reference ratios of 1.58, 1.02, and 0.56 were detected for the CMT1A, unaffected, and HNPP groups, respectively. Thus, differentiation of the three groups of individuals was on the basis of gene copy number. This technique was successfully used to detect the difference in the X chromosome copy number between males and females (target:reference ratios of 1.1 and 2.3, respectively). This approach to the detection of DNA duplications and deletions is sensitive, accurate, and has potential applications in the quantitation of changes in gene copy number associated with diseases characterized by such chromosomal alterations.
Serum protein electrophoresis by CZE 2000 clinical capillary electrophoresis system
Authors: Bossuyt Xavier , Schiettekatte Gilberte , Bogaerts Ann , Blanckaert Norbert
Published: January 2020

We compared the automated Paragon 2000 clinical capillary zone electrophoresis (CZE) system with two manual methods, agarose electrophoresis (AGE) and cellulose acetate electrophoresis (CAE). Reference intervals in healthy adults were determined for each method. When compared with AGE and CAE, CZE gave substantially higher reference values for the α1-globulin fraction. With CZE, within-run precision for fraction quantitation was between 0.5% (albumin) and 4.1% (α1-globulin). Total precision was between 0.8% (albumin) and 5.3% (β-globulin). Data obtained from CZE showed poor linear correlation with results obtained by AGE but good linear correlation with data from CAE. Analysis of serum from patients with inter alia inflammation, nephrotic syndrome, or polyclonal gammopathy showed that clinical information obtained by CZE is comparable with information obtained by AGE and CAE. We conclude that CZE offers a clinically reliable alternative to AGE and CAE and has the advantages of automation, higher precision, and faster turnaround time.
Standards of laboratory practice: cardiac drug monitoring
Authors: Valdes Roland , Jortani Saeed A , Gheorghiade Mihai
Published: January 2020

In this Standard of Laboratory Practice we recommend guidelines for therapeutic monitoring of cardiac drugs. Cardiac drugs are primarily used for treatment of angina, arrhythmias, and congestive heart failure. Digoxin, used in congestive heart failure, is widely prescribed and therapeutically monitored. Monitoring and use of antiarrhythmics such as disopyramide and lidocaine have been steadily declining. Immunoassay techniques are currently the most popular methods for measuring cardiac drugs. Several reasons make measurement of cardiac drugs in serum important: their narrow therapeutic index, similarity in clinical complications and presentation of under- and overmedicated patients, need for dosage adjustments, and confirmation of patient compliance. Monitoring may also be necessary in other circumstances, such as assessment of acetylator phenotypes. We present recommendations for measuring digoxin, quinidine, procainamide (and N-acetylprocainamide), lidocaine, and flecainide. We discuss guidelines for measuring unbound digoxin in the presence of an antidote (Fab fragments), for characterizing the impact of digoxin-like immunoreactive factor (DLIF) and other cross-reactants on immunoassays, and for monitoring the unbound (free fraction) of drugs that bind to α1-acid glycoprotein. We also discuss logistic, clinical, hospital, and laboratory practice guidelines needed for implementation of a successful therapeutic drug monitoring service for cardiac drugs.
Cardiac troponin T in patients with end-stage renal disease: absence of expression in truncal skeletal muscle
Authors: Haller Christlieb , Zehelein Jörg , Remppis Andrew , Müller-Bardorff Margit , Katus Hugo A
Published: January 2020

In patients with end-stage renal disease (ESRD), the serum concentration of cardiac troponin T (cTnT) may be increased without cardiac ischemia. One reason for this unexplained increase could be the extracardiac expression of cTnT. However, truncal skeletal muscle biopsies of five patients with ESRD showed no evidence of the expression of either cTnT mRNA (reverse transcription-PCR) or protein (immunoblot, immunofluorescence). We also measured the serum concentration of cTnT in 97 patients with ESRD. The serum cTnT concentration determined in both first and second generation cTnT assays was significantly lower P <0.01 in patients with a low cardiac risk than in patients with positive indicators of coronary artery disease. The correlation between cTnT and indicators of coronary artery disease is consistent with the hypothesis that cTnT in the serum of patients with ESRD originates from the heart.
Serum creatine kinase activity is not a reliable marker for muscle damage in conditions associated with low extracellular glutathione concentration
Authors: Gunst Johan J , Langlois Michel R , Delanghe Joris R , De Buyzere Marc L , Leroux-Roels Geert G
Published: January 2020

Creatine kinase (CK, EC 2.7.3.2) assays usually contain thiol-reducing compounds to restore the enzyme activity. In this study, we investigated the effect of endogenous extracellular glutathione on serum CK activity. We examined CK activity and glutathione concentrations in serum from 200 healthy subjects (107 males, 93 females) and 38 patients with multiple organ failure, muscle wasting, and low serum CK activity (<50 U/L) (24 males, 14 females). Muscle damage was further evaluated using serum myoglobin concentrations and aldolase activity. In the overall group, serum glutathione concentrations correlated with serum CK activity (r = 0.791) but not with myoglobin concentrations and aldolase activity. In patients with multiple organ failure, low serum CK activities were accompanied by extremely low serum glutathione concentrations (<0.5 μmol/L, P <0.001). Endogenous glutathione can be regarded as a CK-preserving agent during the lifetime of the enzyme in the circulation (22 h on average). Serum CK activity should be interpreted with caution in patients with liver disease and multiple organ failure. In these conditions, the loss of CK activity due to extracellular glutathione depletion cannot be restored by the presence of thiol-reducing compounds in the CK assays.
Remnant lipoprotein cholesterol and triglyceride reference ranges from the Framingham Heart Study
Authors: McNamara Judith R , Shah Paulesh K , Nakajima Katsuyuki , Cupples L Adrienne , Wilson Peter W F , Ordovas Jose M , Schaefer Ernst J
Published: January 2020

Remnants of triglyceride-rich lipoproteins of both intestinal and liver origin are considered atherogenic, but they have been difficult to isolate and measure. An assay has been developed that allows the measurement of remnant-like particle cholesterol (RLP-C) and triglyceride (RLP-TG). RLP-C and RLP-TG concentrations were measured in >3000 fasting plasma samples obtained from participants in exam cycle 4 of the Framingham Offspring Study and stored at −80 °C. After exclusions, comparisons were made for 2821 samples (1385 women, 1436 men; mean age, 52 years). For women, the mean RLP-C and RLP-TG values were 0.176 ± 0.058 mmol/L (6.8 ± 2.3 mg/dL) and 0.204 ± 0.159 mmol/L (18.1 ± 14.1 mg/dL), respectively; for men, the mean values were 0.208 ± 0.096 mmol/L (8.0 ± 3.7 mg/dL) and 0.301 ± 0.261 mmol/L (26.7 ± 23.1 mg/dL), respectively. Women had significantly lower RLP-C and RLP-TG values (P <0.0001) than men; premenopausal women had significantly lower values than postmenopausal women (P <0.0001); and younger subjects (<50 years) had significantly lower values than older individuals (P <0.0001). The 75th percentile values for RLP-C and RLP-TG were 0.186 mmol/L (7.2 mg/dL) and 0.225 mmol/L (19.9 mg/dL), respectively, for women, and 0.225 mmol/L (8.7 mg/dL) and 0.346 mmol/L (30.6 mg/dL) for men. These data provide reference ranges for use in the evaluation of RLP-C and RLP-TG as potential indicators of risk for coronary heart disease.
Automated simultaneous quantification of the immunosuppressants 40-O-(2-hydroxyethyl)rapamycin and cyclosporine in blood with electrospray-mass spectrometric detection
Authors: Vidal Christian , Kirchner Gabriele I , Wünsch Gerold , Sewing Karl-Friedrich
Published: January 2020

A new analytical method to quantify 40-O-(2-hydroxyethyl)rapamycin (SDZ RAD) and cyclosporine (Cs) simultaneously in blood is presented. The combination of an on-line solid-phase extraction step with an HPLC system coupled to an electrospray mass spectrometer gave excellent specificity, sensitivity, and reproducibility. Aliquots of deproteinized blood samples were injected into the HPLC system and extracted on-line, using a conventional C18 guard column. The extract was eluted from the guard column in the backflush mode and injected into the liquid chromatography–mass spectrometry system. The calibration functions for SDZ RAD and Cs extracted from blood with added analyte were linear from 0.15 to 30 μg/L (r2 = 0.999) and from 1.5 to 1000 μg/L (r2 = 0.999), respectively. The CVs of peak areas were 6.2% at 10 μg/L SDZ RAD (n = 6) and 6.2% at 100 μg/L Cs (n = 6). Recovery ranged from 84.3% to 102.3% for SDZ RAD and from 81.7% to 92.2% for Cs. The lower limit of detection for both drugs was 0.05 μg/L. A rate of four samples per hour was maintained during the consecutive analysis of SDZ RAD and Cs in >500 blood samples with one single extraction and analytical column. The method described is a powerful tool for the simultaneous determination of SDZ RAD and Cs in blood. It works without time-consuming sample preparation steps and with excellent reproducibility. Because of the detection performance of electrospray mass spectrometry, this system offers flexibility in the working range, which is essential for therapeutic drug monitoring under different conditions.
Cardiac troponin T in hemodialyzed patients
Authors: Ooi Daylily S , House Andrew A
Published: January 2020

We studied the extent and pattern of increased cardiac troponin T (cTnT) concentrations in 174 hemodialyzed patients. cTnT concentrations were above 0.10 and 0.20 μg/L in 29% and 10% of patients, respectively. In patients without acute coronary disease, the highest value observed was 3.2 μg/L. cTnT increased after dialysis in 10 of 12 patients, with a mean increase of 0.14 μg/L. In 125 patients with samples taken at 1-month intervals, 34% of patients showed differences <20%, but 16% of patients had differences greater than twofold. Serum creatinine and urea, adequacy of dialysis, and duration on dialysis did not explain increased concentrations. Sixty percent of 57 diabetic patients had increased concentrations; the patients with multiple diabetic complications had the highest positivity. cTnT was increased in all eight patients with complications of neuropathy, retinopathy, coronary, and peripheral vascular disease; in 80% of patients with neuropathy; in 77% with peripheral vascular disease; in 73% with retinopathy; and in 70% with coronary artery disease.