bluuebunny/crossref_metadata_2025 huggingface dataset:
Quantitation of bcl-2 protein in bladder cancer tissue by enzyme immunoassay: comparison with Western blot and immunohistochemistry
Authors: Eissa Sanaa , Seada Laila S
Published: January 2020

Apoptosis (programmed cell death) and the genes regulating this process (e.g., bcl-2), have recently become a focus of interest in the study of cancer development and progression. We adapted and evaluated a new enzyme immunoassay method (EIA) for quanitifying bcl-2 in cell lysates. The range of detection of the assay was 5–400 kilounits/L with inter- and intraassay CVs of 5.5–9.2% and 5.0–8.8%, respectively. The recovery of added bcl-2 protein to cell lysates was 96–104%. The concordance rates with Western blotting and immunohistochemistry were 97.5% and 93.7%, respectively. Bcl-2 concentrations were measured in the cell lysate of bladder tumors. The amount of bcl-2 in 158 bladder cancer (mean rank, 71.3 kilounits/g protein; range, 8.4–324 kilounits/g protein), was significantly higher than in nondiseased bladder tissues distant to the tumors (mean rank, 31.5 kilounits/g protein; range, 5–54.9 kilounits/g protein), P = 0.00001. Bcl-2 expression was correlated to tumor proliferative capacity, which was measured by DNA flow cytometry as the percentage of cells in the synthetic phase of the cell cycle. The enzyme immunoassay provides a rapid, quantitative, and reliable technique for measurement of bcl-2 in tumor tissue. The detection of substantial amounts of bcl-2 in invasive tumors (compared with nondiseased tissues) suggests that the assay should be a useful tool for investigating the prognostic value of bcl-2 in bladder tumors and for selecting patients for future anti-bcl-2 therapy.
Fluorometric determination of total retinyl esters in triglyceride-rich lipoproteins
Authors: Orth Matthias , Hanisch Monika , Kröning Gerd , Porsch-Özcürümez Mustafa , Wieland Heinrich , Luley Claus
Published: January 2020

A time-consuming sample preparation and measuring procedure is required for the quantitation of retinyl palmitate by HPLC. We developed a fluorometric method for the determination of total retinyl esters in chylomicrons, chylomicron remnants, and VLDL. This method is precise, sensitive, rapid, simple, and particularly useful for large-scale studies of postprandial lipid metabolism. Because the turbidity of postprandial lipemic samples interferes with the fluorescence measurement, all samples were incubated for 10 min with a clearing buffer containing esterase and detergents. This buffer eliminates the turbidity and hydrolyzes all retinyl esters to retinol. The fluorescence signal (excitation wavelength, 330 nm; emission wavelength, 490 nm) was linear from 0.1 mg/L up to 4 mg/L retinyl palmitate, and the CVs were 3.6% within-run and 5.1% within-series. A first application studied postprandial lipoproteins, which were first separated by ultracentrifugation and then subjected to size exclusion chromatography. Fluorescence analysis revealed that the chylomicron density fraction contains large amounts of chylomicron remnants.
Sensitive enzymatic assay for erythrocyte creatine with production of methylene blue
Authors: Okumiya Toshika , Jiao Yufei , Saibara Toshiji , Miike Akira , Park Keunsik , Kageoka Takeshi , Sasaki Masahide
Published: January 2020

We developed a new, highly sensitive enzymatic method for quantifying creatine in erythrocytes, which comprises creatine amidinohydrolase, sarcosine oxidase, and peroxidase. In the present method, an N-methylcarbamoyl derivative of methylene blue, 10-N-methylcarbamoyl-3,7-bis(dimethylamino)phenothiazine (MCDP), was used as a sensitive chromogenic compound. Potassium ferrocyanide was used to prevent nonspecific oxidation of MCDP. The enzymatic method exhibited good analytical performance: precision, within-run CVs <1.0% and between-day CVs <2.0%; average analytical recovery, 99.3% ± 1.8%; detection limit, 1.0 μmol/L in hemolysate; and linearity, at least up to 500 μmol/L as creatine concentration in hemolysate. Excellent agreement was observed between the present method (y) and HPLC (x), y = 1.029x − 0.002 μmol/g hemoglobin, r = 0.9998, Sy‖x = 0.053 μmol/g hemoglobin (n = 110). No significant interference was produced by various compounds, including guanidino compounds, amino acids, and reducing materials. The reference intervals (mean ± 2 SD) for erythrocyte creatine obtained from 60 males and 60 females were (in μmol/g hemoglobin) 1.18 ± 0.52 (0.66–1.70) for males and 1.35 ± 0.49 (0.86–1.84) for females. Using this method, we documented changes in erythrocyte creatine in patients with various hemolytic conditions, including hemolytic anemia, liver cirrhosis, renal insufficiency, and chronic renal failure treated with hemodialysis with or without the administration of erythropoietin. We conclude that the use of MCDP allows sensitive measurement of erythrocyte creatine and that MCDP with potassium ferrocyanide can improve the sensitivity of assays that use peroxidase for detection of H2O2.
Transient 5-oxoprolinuria and high anion gap metabolic acidosis: clinical and biochemical findings in eleven subjects
Authors: Pitt James J , Hauser Simon
Published: January 2020

We describe biochemical and clinical features of 11 subjects (ages, 1.2–84 years, nine females and two males) with transient 5-oxoprolinuria (0.6–23.6 mol/mol of creatinine, reference range <0.07). A variety of conditions preceded the onset of acidosis, and all had taken acetaminophen (paracetamol), although in therapeutic amounts in most subjects. Metabolic acidosis was documented in nine subjects, and all had an increased anion gap and abnormal liver functions. 5-Oxoproline was the major urinary organic acid in five subjects, whereas the rest had more complex profiles comprising 5-oxoproline and other organic acids, such as lactate, 3-hydroxybutyrate, and 4-hydroxyphenyl lactate. The 5-oxoproline was predominantly of the l-configuration. One subject died during an acidotic episode, and the rest recovered with no apparent long-term ill effects. Urinary 5-oxoproline was within the reference range in six subjects that were re-tested after the anion gap normalized. These findings suggest that acetaminophen, in association with other unidentified factors, is involved in the development of this condition through a mechanism of depletion of liver glutathione stores.
First direct assay for intact human proinsulin
Authors: Houssa Paule , Dinesen Bo , Deberg Michelle , Frank Bruce H , Van Schravendijk Chris , Sodoyez-Goffaux Françoise , Sodoyez Jean-Claude
Published: January 2020

We describe a sensitive two-site sandwich enzyme-linked immunosorbent assay for the measurement of intact human proinsulin in 100 μL of serum or plasma. The assay is based on the use of two monoclonal antibodies specific for epitopes at the C-peptide/insulin A chain junction and at the insulin B chain/C-peptide junction, respectively. Cross-reactivities with insulin, C-peptide, and the four proinsulin conversion intermediates were negligible. The detection limit in buffer was 0.2 pmol/L (3 standard deviations from zero). The working range was 0.2–100 pmol/L. The mean intra- and interassay coefficients of variation were 2.4% and 8.9%, respectively. The mean recovery of added proinsulin was 103%. Dilution curves of 40 serum samples are parallel to the proinsulin calibration curve. Proinsulin concentrations in 20 fasting healthy subjects were all above the limit of detection: median (range), 2.7 pmol/L (1.1–6.9 pmol/L). Six fasting non-insulin-dependent diabetes mellitus and five insulinoma patients had proinsulin concentrations significantly higher than healthy subjects: median (range), 7.7 pmol/L (3.2–18 pmol/L) and 153 pmol/L (98–320 pmol/L), respectively.
Monitoring ovarian function by the simultaneous time-resolved fluorescence immunoassay of two urinary steroid metabolites
Authors: Barnard Geoff , Kohen Fortune
Published: January 2020

We report the development of a novel time-resolved fluorescence immunoassay utilizing two different assay strategies for the simultaneous measurement of estrone-3-glucuronide (EG) and pregnanediol-3α-glucuronide (PG) in samples of early morning urine (EMU). The method for the measurement of EG involves the use of a labeled anti-idiotype as a surrogate antigen, whereas the other method (for the measurement of PG) is a regular competitive immunoassay using a labeled antigen. In addition, the procedure uses different lanthanide chelates as labels to monitor ovarian function in women. After washing the streptavidin-coated plate, we added 10 μL of undiluted urine or mixed standard to the coated wells, followed by the addition of 100 μL of assay buffer containing the labeled reactants (i.e., europium-labeled PG and samarium-labeled anti-idiotype recognizing the binding site of the antibody to EG). Subsequently, we added 100 μL of assay buffer containing the two biotinylated specific monoclonal anti-steroid glucuronide antibodies. After incubation for 1 h on a shaker at room temperature, we washed the plate and added 200 μL of enhancement solution to each well. We measured europium and samarium fluorescence, using a gated plate fluorometer with appropriate emission filters. The method demonstrates appropriate sensitivity and precision (all CVs, 5–8%) across the relevant working ranges for each analyte. The technique has been applied to serial EMUs collected from women with normal and stimulated menstrual cycles.
Apolipoprotein B and A-I values in 147 576 Swedish males and females, standardized according to the World Health Organization–International Federation of Clinical Chemistry First International Reference Materials
Authors: Jungner Ingmar , Marcovina Santica M , Walldius Göran , Holme Ingar , Kolar Werner , Steiner Eugen
Published: January 2020

Serum concentrations of apolipoprotein (apo) B and apo A-I were measured from 1985–1996 in a Swedish population sample of 83 112 males and 64 464 females, ages <20 to >80 years, using an automated immunoturbidimetric method calibrated against fresh pools of human serum and commercial calibrators. All values were recalculated in 1997 after calibration against the WHO-IFCC First International Reference Materials. The recalculation factor was 1.059 for apo B, whereas for apo A-I, the correction was: y = 0.989x + 0.101. The total CVs for both apo B and A-I were generally <7%. The mean value (± SD) for apo B was 1.31 ± 0.35 g/L in all males and 1.22 ± 0.36 g/L in all females. The mean apo A-I concentration was 1.36 ± 0.22 g/L in males–10% lower than in females (1.51 ± 0.24 g/L). The mean value of apo B increased up to 60 years of age in males and up to 70 years of age in females. apo A-I concentrations changed only slightly with age in both males and females. apo A-I concentrations among Swedes are nearly identical to those reported recently by two American studies and those obtained in a Finnish population sample. Mean apo B concentrations differ somewhat between the populations but mirror–as expected–differences in total cholesterol concentrations. The highest values were noted in Swedish subjects. The Swedish sample population is, to our knowledge, the largest describing the distribution of apo B and A-I in a general population of adult males and females of all ages determined with procedures standardized and traceable to the WHO-IFCC First International Reference Materials.
Simultaneous HPLC determination with light-scattering detection of lactulose and mannitol in studies of intestinal permeability in pediatrics
Authors: Marsilio Renzo , D’Antiga Lorenzo , Zancan Lucia , Dussini Noemi , Zacchello Franco
Published: January 2020

We describe a new HPLC method for the simultaneous determination of lactulose and mannitol in urine, in which cation-exchange chromatography and evaporative light-scattering detection are used. The two sugars are orally administered for the estimation of intestinal permeability in children. Samples were purified by solid phase extraction on a C18 cartridge and subsequent addition of anion-exchange resin. Cellobiose may be used as an internal standard. The chromatographic separation was carried out in 16 min at a flow rate of 0.5 mL/min, using deionized water as the mobile phase. Within-run precision (CV) measured at three concentrations was 1.6–2.3% for lactulose and 1.0–1.9% for mannitol. Between-run CVs were 2.1–4.1% and 1.3–2.7% for lactulose and mannitol, respectively. Analytical recovery of both sugar probes was 97–101%. The detection limits (signal-to-noise ratio = 3) were 0.82 mg/L for lactulose and 0.65 mg/L for mannitol. The lactulose/mannitol ratio in control subjects was 0.024 ± 0.006; in patients with Crohn’s and coeliac diseases in active phase, the ratios were 0.200 ± 0.082 and 0.072 ± 0.025, respectively. The method is rapid, simple, and sensitive, and suitable for determination of intestinal permeability in children.
Obesity and cardiovascular disease
Authors: Sowers James R
Published: January 2020

Obesity, diabetes mellitus, and hypertension are common, interrelated medical problems in Westernized, industrialized societies. These interrelated medical conditions are associated with an increased risk of cardiovascular disease and are more prevalent in several minority groups, including African-American and Hispanic populations. The associated cardiovascular risks of these problems are more thoroughly addressed in another review in this supplement. Obesity markedly enhances the development of Type 2 diabetes. Moreover, it enhances the cardiovascular risk associated with other risk factors, such as hypertension and dyslipidemia. Weight reduction in association with an aerobic exercise program improves metabolic abnormalities and reduces blood pressure in individuals with diabetes and hypertension.
Evolving lipoprotein risk factors: lipoprotein(a) and oxidized low-density lipoprotein
Authors: Jialal Ishwarlal
Published: January 2020

Cardiovascular disease is the leading cause of morbidity and mortality in Westernized populations. Evolving lipoprotein risk factors include LDL oxidation and lipoprotein(a) [lp(a)]. Several lines of evidence support a role for oxidatively modified LDL in atherogenesis and its in vivo existence. There are both direct and indirect measures of oxidative stress. The most relevant direct measure of lipid peroxidation is urinary F2 isoprostanes. The most common indirect measure of LDL oxidation is quantifying the lag phase of copper-catalyzed LDL oxidation by assaying conjugated diene formation. Lp(a) is increased in patients with cardiovascular and cerebrovascular disease. However, not all prospective studies have confirmed a positive relationship between Lp(a) and cardiovascular events. Lp(a) appears to present three major problems: standardization of the assay, establishing its role in atherogenesis, and the lack of an effective therapy that can substantially lower Lp(a) concentrations. Thus, at the present time, Lp(a) concentrations should not be recommended for the general population but be reserved for patients with coronary artery disease without established risk factors, young patients with coronary artery disease or cerebrovascular disease, or a family history of premature atherosclerosis and family members of an index patient with increased concentrations of Lp(a). Although both LDL oxidation and Lp(a) are evolving risk factors for cardiovascular disease, more data are needed before they become part of the established lipoprotein repertoire.
5-Aminolevulinic acid dehydratase deficiency porphyria: a twenty-year clinical and biochemical follow-up
Authors: Gross Ulrich , Sassa Shigeru , Jacob Karl , Deybach Jean-Charles , Nordmann Yves , Frank Margareta , Doss Manfred O
Published: January 2020

5-Aminolevulinic acid dehydratase (ALAD) activity in two patients with compound heterozygous 5-aminolevulinic acid dehydratase deficiency porphyria was studied over the last 20 years. The patients’ enzyme activity was <10% from 1977 to 1997. An acute crisis in each patient was successfully treated by infusion of glucose and heme arginate. After this therapy both urinary 5-aminolevulinic acid (ALA) and total porphyrins were diminished to 65% in patient B. In patient H, ALA was decreased to 80%, and total porphyrins were reduced to 15% after treatment with heme arginate and glucose. The patients remained free of symptoms after this therapy. Family studies of patient B showed cross-reactive immunological material (CRIM), in which the maternal mutation is CRIM(+), whereas the paternal mutation is CRIM(−). Incubation of erythrocyte lysates with ALA decreased porphyrin formation, whereas incubation with porphobilinogen produced porphyrin concentrations within reference values in both patients, confirming that ALAD activity is rate-limiting in these cells.
Capillary electrophoresis for rapid profiling of organic acidurias
Authors: García Antonia , Barbas Coral , Aguilar Rosa , Castro Mario
Published: January 2020

Organic acids analysis is a powerful technique in the diagnosis of inborn errors of metabolism. Clinically, patients present with severe symptoms, and early detection and appropriate treatment are often lifesaving. Most of the existing methods are based on gas chromatography in combination with mass spectrometry and require sophisticated equipment and complex sample pretreatment and derivatization. We propose a rapid, simple, and automated capillary electrophoretic method for routine analysis of urine to detect 27 organic acids related to metabolic diseases. With this method, direct measurements are performed on samples after initial centrifugation and dilution, if needed. Separation is performed in pH 6.0 phosphate buffer with methanol added as an organic modifier, −10 kV applied potential, and ultraviolet detection at 200 nm. The assay is completed in <15 min, and alternative separation conditions are proposed in case of overlapping peaks. The developed method allows the identification and quantitation of methylmalonic, pyroglutamic, and glutaric acids in samples of patients with diseases related to these acids.
Analytical performance and clinical application of a new rapid bedside assay for the detection of serum cardiac troponin I
Authors: Heeschen Christopher , Goldmann Britta U , Moeller Robert H , Hamm Christian W
Published: January 2020

Detection of cardiac troponin I (cTnI) in patients suspected of having an acute coronary syndrome is highly predictive for an adverse outcome. We evaluated a bedside test for cTnI that uses a polyclonal capture antibody and two monoclonal indicator antibodies. Clinical studies were performed in patients with acute coronary syndrome and patients with chest pain but no evidence of acute myocardial injury. The whole-blood, 15-minute assay had a concordance of 98.9% with an ELISA for cTnI and a detection limit of 0.14 μg/L, and the device tolerated temperatures between 4 °C and 37 °C. Diagnostic sensitivity for myocardial infarction at arrival (3.5 ± 2.7 h after onset of symptoms) was 60% [creatine kinase isoenzyme MB (CK-MB) mass, 48%; CK activity, 36%; P < 0.01], and 4 h later, diagnostic sensitivity was 98% (CK-MB mass, 91%; CK activity, 61%; P < 0.01). In 38% of the patients with unstable angina, at least one positive cTnI test was found (CK-MB mass, 4%; CK activity, 2%). No false-positive test results were found in renal failure or injury of skeletal muscle. We conclude that the diagnostic efficacy of the cTnI rapid test was comparable with the cTnI ELISA and superior to CK-MB determination. Therefore, this device could facilitate decision-making in patients with chest pain at the point of care.
False increase in C-reactive protein attributable to heterophilic antibodies in two renal transplant patients treated with rabbit antilymphocyte globulin
Authors: Benoist Jean-François , Orbach Daniel , Biou Daniel
Published: January 2020

Increased serum C-reactive protein (sCRP) is a sensitive marker of renal graft rejection. We describe the cases of two children with uncomplicated renal transplantation who had false-positive sCRP values on analyzers using rabbit anti-CRP but values within the reference range with anti-CRP from other animal species. Cross-reaction with heterophilic antibodies was suggested by clinical and biological signs of serum sickness and daily treatment with rabbit antilymphocyte globulin (ALG). The interference depended on the serum concentration of the cross-reactant and was removed by subtotal IgG adsorption to Protein A or Protein G or by immunoadsorption using rabbit ALG or total IgG in non-immune rabbit serum. Anti-rabbit IgG and IgM antibodies were detected in both patients. These are the first reported cases of cross-reaction with heterophilic antibodies in a turbidimetric CRP assay.
New Insights into the Role of Nuclear Factor-κB, a Ubiquitous Transcription Factor in the Initiation of Diseases
Authors: Chen Fei , Castranova Vince , Shi Xianglin , Demers Laurence M
Published: January 2020

Nuclear factor-κB (NF-κB) is a ubiquitous transcription factor that governs the expression of genes encoding cytokines, chemokines, growth factors, cell adhesion molecules, and some acute phase proteins in health and in various disease states. NF-κB is activated by several agents, including cytokines, oxidant free radicals, inhaled particles, ultraviolet irradiation, and bacterial or viral products. Inappropriate activation of NF-κB has been linked to inflammatory events associated with autoimmune arthritis, asthma, septic shock, lung fibrosis, glomerulonephritis, atherosclerosis, and AIDS. In contrast, complete and persistent inhibition of NF-κB has been linked directly to apoptosis, inappropriate immune cell development, and delayed cell growth. Therefore, development of modulatory strategies targeting this transcription factor may provide a novel therapeutic tool for the treatment or prevention of various diseases.
Improved Extraction Procedure and RIA for Determination of Arginine8-Vasopressin in Plasma: Role of Premeasurement Sample Treatment and Reference Values in Children
Authors: Kluge Michael , Riedl Stefan , Erhart-Hofmann Birgit , Hartmann Johannes , Waldhauser Franz
Published: January 2020

We optimized an RIA for measurement of arginine8-vasopressin (AVP) in plasma by use of 100-mg Isolute C18 columns for extraction, addition of a preincubation step, and use of maximal dilution of a commercially available antiserum. The detection limit was 0.06 ng/L when 0.5 mL of acidified plasma was extracted. The within- and between-run CVs (n = 16) at physiological concentrations were 5.8–10.2% and 6.5–11.7%, respectively. Prolonged storage of blood at 25 °C, but not at 4 °C, led to a significant increase in measured plasma AVP concentrations. When plasma samples were prepared at several centrifugation speeds, plasma AVP was significantly correlated with the platelet count (r = 0.899; P <0.001). This emphasizes the need for careful sample preparation to avoid contamination of plasma with platelet-bound AVP. Basal plasma AVP in 203 children and adolescents (105 males and 98 females; ages, 1 day to 18 years) averaged 1.1 ± 0.6 ng/L. There was no significant difference between males and females and no correlation with age. In 16 healthy adult controls, plasma AVP averaged 1.0 ± 0.5 ng/L.
Simple, Rapid, Quantitative, and Sensitive Detection of Telomere Repeats in Cell Lysate by a Hybridization Protection Assay
Authors: Nakamura Yasuhiro , Hirose Minoru , Matsuo Hajime , Tsuyama Naohiro , Kamisango Keiichi , Ide Toshinori
Published: January 2020

Background: Detection of telomere repeats by Southern hybridization of genomic DNA is time consuming, and the reading of a mean terminal restriction fragment (TRF) length from a smear pattern of an autoradiogram can be inaccurate. We developed a hybridization protection assay (HPA) for telomere repeats.

Methods: We heated 5 μL of DNA solution or 10 μL of cell or tissue lysate at 95 °C for 5 min, mixed it with 100 μL of hybridization solution containing 3 × 106 relative light units of acridinium ester-labeled probe, and incubated the mixture for 20 min at 60 °C. We then added 300 μL of selection buffer and incubated the mixture for 10 min at 60 °C to differentially hydrolyze unhybridized probe. Chemiluminescence was measured for 2 s per tube.

Results: The amount of telomere repeats was assayed by HPA within linearity from 10 to 3000 ng of purified genomic DNA or from 1000 to 100 000 cell equivalents of lysate. To normalize the amount of DNA in lysate, the amount of Alu sequence was measured by HPA. A ratio of telomere to Alu (TA ratio) = 0.01 corresponded to ∼2 kbp of mean TRF length determined by Southern blotting in cultured fibroblast and colorectal tissue samples. The TA ratio decreased from 0.06 to 0.02 with increasing division age from 30 to 90 population doubling levels of cultured human fetal fibroblasts. The assay required ∼45 min from collection of cell or tissue samples.

Conclusions: The amount of telomere repeats was quantitatively measured by HPA in 10 ng of sheared genomic DNA or in the lysate of 1000 cells. This method is simple, rapid, quantitative, sensitive, and applicable to the measurement of telomere repeats in clinical samples such as needle biopsy specimen or as few as 1000 cells in body fluid or washings.
Time-resolved Immunofluorometric Assay of Trypsin-1 Complexed with α1-Antitrypsin in Serum: Increased Immunoreactivity in Patients with Biliary Tract Cancer
Authors: Hedström Johan , Haglund Caj , Kemppainen Esko , Leinimaa Maarit , Leinonen Jari , Stenman Ulf-Håkan
Published: January 2020

Background: Increased serum concentrations of trypsin immunoreactivity occur in patients with biliary tract cancer. To characterize this trypsin, we developed a sensitive time-resolved immunofluorometric assay for trypsin-1 complexed with α1-antitrypsin (AAT) and studied the concentrations of this complex in sera from healthy individuals (n = 130) and patients with benign biliary disease (n = 32), biliary tract cancer (n = 17), pancreatic cancer (n = 27), and hepatocellular cancer (n = 12).

Methods: We used a trypsin-1-specific monoclonal antibody on the solid phase and a europium-labeled polyclonal antibody to AAT as tracer. The detection limit was 0.42 μg/L. The validity of the trypsin-1-AAT test for detection of biliary tract cancer was compared with trypsin-2-AAT and CA19-9.

Results: Increased concentrations of trypsin-1-AAT (>33 μg/L) were found in 76% of patients with biliary tract cancer, and the concentrations were significantly higher than in those with benign biliary disease (P <0.0001). The median concentration of trypsin-1-AAT in serum from patients with biliary tract cancer was 3.7-fold higher than in healthy controls, 2.6-fold higher than in patients with benign biliary tract disease, 1.7-fold higher than in patients with pancreatic cancer, and 2.0-fold higher than in patients with hepatocellular cancer.

Conclusions: Of the markers studied, trypsin-1-AAT had the largest area (0.83) under the receiver operating curve in differentiating biliary tract cancer from benign biliary tract disease. Our results suggest that trypsin-1-AAT is a new potential marker for biliary tract cancer.
Within-Person Variation of Plasma Homocysteine and Effects of Posture and Tourniquet Application
Authors: Rasmussen Karsten , Møller Jan , Lyngbak Marianne
Published: January 2020

Background: Frequently, the result of only a single determination of total homocysteine in plasma (P-Hcy) is used to distinguish between the probability of the presence or absence of risk for vascular disease. A prerequisite for the interpretation of a single P-Hcy test is knowledge of the magnitude of within-person variation and the possible effects of preanalytical variables. However, data on within-person variation are still sparse and inconsistent, and data for the effect on P-Hcy of posture and tourniquet application during venipuncture are not available.

Methods: The within-person variation of P-Hcy and the effects of posture and tourniquet application during venipuncture were studied in 24 healthy subjects. The analytical imprecision of our stable-isotope dilution assay was 3.1%.

Results: The within-person variation (CV) was 8.1%. Daily supplementation with 0.4 mg of folic acid for 2 weeks produced a small but significant decrease in P-Hcy, but there were no significant changes in within-person variation before and after supplementation. After 30 min in the horizontal posture, P-Hcy declined by 6.3%. A 3-min tourniquet application caused a 2.8% increase of P-Hcy.

Conclusions: Our value for within-person variation is consistent with results from studies reported recently in the literature. A 3-min tourniquet application does not add appreciable variation to the measurement of P-Hcy, but the posture of the subject during venipuncture contributes considerably to the within-person variation. We recommend that blood collection when the patient is in a supine position be avoided.
Neopterin Is an Independent Prognostic Variable in Females with Breast Cancer
Authors: Murr Christian , Bergant Anton , Widschwendter Martin , Heim Kurt , Schröcksnadel Hans , Fuchs Dietmar
Published: January 2020

Background: Neopterin, produced by human monocytes/macrophages upon stimulation by interferon-γ, is a sensitive marker for monitoring Th1-cell immune response in humans. In malignant diseases, the frequency of increases in neopterin in the serum and urine of patients depends on tumor stage and type.

Methods: In a retrospective study comprising 129 females with breast cancer, urinary neopterin/creatinine ratios were measured at the time of diagnosis. Tumor characteristics were determined concomitantly.

Results: Urinary neopterin was increased in 18% of the patients. It did not correlate with tumor size or lymph node status, but it was influenced by the presence of distant metastases (P <0.05) and by tumor differentiation (P = 0.01). When product-limit estimates were calculated after follow-up for up to 13 years (median follow-up, 56 months), the presence of distant metastases (P <0.001), neopterin (P <0.001), tumor size (P = 0.001), and lymph node status (P <0.01) were significant predictors of survival. By multivariate analysis, a combination of the variables presence of distant metastases (P <0.001), neopterin (P <0.01), and lymph node status (P <0.05) was found to jointly predict survival. In lymph node-negative patients without distant metastases, the relative risk of death associated with increased neopterin concentrations was 2.5 compared with patients with neopterin concentrations within the reference interval.

Conclusion: Urinary neopterin provides additional prognostic information in patients with breast cancer.
Clinical Efficacy of an Automated High-Sensitivity C-Reactive Protein Assay
Authors: Rifai Nader , Tracy Russell P , Ridker Paul M
Published: January 2020

Background: Prospective studies have shown that C-reactive protein (CRP) can be used to predict risk of future cardiovascular events. High-sensitivity methods for CRP (hs-CRP) measurement are needed for this purpose.

Methods: We compared the clinical efficacy of an automated and commercially available latex-enhanced assay (Latex) for hs-CRP (Dade Behring) to a validated in-house ELISA, previously shown to predict future peripheral arterial disease (PAD) in asymptomatic populations. Using a prospective, nested, case-control design, we measured baseline hs-CRP concentrations in 144 apparently healthy men who subsequently developed symptomatic PAD and 144 age- and smoking habit-matched controls who remained free of vascular disease over the follow-up period of 60 months.

Results: The two hs-CRP assays correlated highly (r = 0.95; P <0.001), and all but two participants were classified into concordant quartiles or varied by only one quartile. The median hs-CRP of the case group was significantly higher than that of controls when measured by either the ELISA (1.34 vs 0.99 mg/L; P = 0.034) or the Latex method (1.80 vs 1.20 mg/L; P = 0.042). Furthermore, for both ELISA and the Latex method, the calculated relative risks of developing PAD increased significantly with each increasing quartile of hs-CRP. The calculated interquartile increase in relative risk of PAD was 31% (95% confidence interval, 5.2–62.2%; P = 0.01) for ELISA and 34% (95% confidence interval, 8.2–66.1%; P = 0.007) for the Latex method.

Conclusions: Our findings indicate that the Latex method is equally as efficacious as the validated ELISA in classifying patients into cutoff points established by prospective studies for risk stratification for coronary and cerebrovascular disease.
Clinical Significance of Immunoassays for Type-5 Tartrate-resistant Acid Phosphatase
Authors: Nakasato Yuri R , Janckila Anthony J , Halleen Jussi M , Vaananen H Kalervo , Walton Stephanie P , Yam Lung T
Published: January 2020

Background: Tartrate-resistant acid phosphatase (TRAP; EC 3.1.3.2) is a product of osteoclasts and a biochemical marker of bone resorption rate. However, erythrocytes and platelets contribute to total TRAP activity in serum, reducing the specificity of direct biochemical assays in serum. Osteoclast TRAP is also known as type-5 TRAP and is antigenically unique. Immunoassays are sought to improve the specificity and sensitivity of TRAP as a bone marker.

Methods: We developed two colorimetric microplate assays for type-5 TRAP: an enzyme capture immunoassay to measure antibody-bound enzymatic activity, and a two-site immunoassay to measure bound enzyme protein. Both use the same monoclonal antibody (14G6) to capture type-5 TRAP, which permits determination of specific activity of serum TRAP in health and disease.

Results: Both TRAP assays were linear from one-tenth to fivefold the mean value in 18 healthy subjects. In these subjects, the mean (SD) TRAP activity was 3.2 (0.54) U/L for the enzyme capture assay and 37 (13) μg/L for the two-site assay. Mean TRAP activity was not significantly increased in 64 patients with endstage renal disease requiring hemodialysis (HD) or 99 unselected patients with rheumatic diseases. By contrast, TRAP protein was increased in both the HD and rheumatic disease groups. The specific activity of TRAP in the 17 of 64 HD sera that had increased TRAP activity (0.088 U/μg) was similar to that in healthy subjects (0.091 U/μg). By contrast, the specific activity of TRAP in the 31 of 99 rheumatic sera with increased TRAP protein (0.035 U/μg) was significantly decreased.

Conclusions: Wide sample distributions for TRAP activity in HD patients and TRAP protein in rheumatic disease patients suggest the presence of subpopulations of HD patients with increased TRAP activity and of rheumatic patients with increased TRAP protein. Each assay for TRAP activity and protein may have its own biological significance and clinical applications in specific groups of patients.
Efficient Cleavage of Conjugates of Drugs or Poisons by Immobilized β-Glucuronidase and Arylsulfatase in Columns
Authors: Toennes Stefan W , Maurer Hans H
Published: January 2020

Background: Cleavage of conjugates is an important step in toxicological analysis, especially of urine samples. The aim of this study was to combine the advantages and to reduce the disadvantages of acid hydrolysis and conventional enzymatic hydrolysis procedures.

Methods: β-Glucuronidase (GRD; EC 3.2.1.31) and arylsulfatase (ARS; EC 3.1.6.1) were purified and coimmobilized on an agarose gel matrix and packed into columns.

Results: In columns packed with GRD and ARS, the test conjugates 4-nitrophenyl glucuronide and 4-nitrophenyl sulfate added into urine could be completely cleaved within 25 min. Even the relatively stable morphine conjugates could be completely hydrolyzed within 60 min in authentic urine samples. Therefore, an incubation time of 1 h is recommended. Enzyme inhibition by matrix or by rather high concentrations of acetaminophen conjugates was tested and found to be up to 50%. However, a large excess of GRD and ARS was used. The immobilizate columns could be reused for at least 70 incubations and had a storage stability of at least 12 weeks. Carryover of analytes in reused columns could be avoided by rinsing with 200 mL/L methanol in acetate buffer. Thus, five drugs known to be contaminants added in very high concentrations into urine could be completely removed from the columns. A study on the applicability in systematic toxicological analysis showed that 120 different drugs and/or their metabolites could be detected in 35 different authentic urine samples.

Conclusions: Use of immobilized and column-packed GRD and ARS is an efficient alternative for the cleavage of urinary conjugates in clinical toxicology.
Dihydrofolate Reductase Enzyme Inhibition Assay for Plasma Methotrexate Determination Using a 96-Well Microplate Reader
Authors: Widemann Brigitte C , Balis Frank M , Adamson Peter C
Published: January 2020

Microplate reader assays offer several advantages over conventional spectrophotometric assays. We adapted the dihydrofolate reductase (DHFR) enzyme inhibition assay for use in a 96-well microplate reader to measure plasma methotrexate (MTX) concentrations. The assay is linear from 0.01 to 0.1 μmol/L. The within-run CVs at 0.03 μmol/L and 0.08 μmol/L MTX were 4.0% and 2.7%, respectively, and the interday (total) CVs were 7.6% and 1.8%. Cross-reactivity with the inactive MTX metabolite 2,4-diamino-N10-methylpteroic acid (DAMPA) was 3.9%, significantly less than that described with commercial immunoassays; with 7-hydroxymethotrexate cross-reactivity was 1.7%. In addition to sensitivity and specificity, the advantages of this assay are small sample volumes, simultaneous analysis of multiple samples, and rapid turnaround. Because of its greater specificity, the DHFR enzyme inhibition assay may be useful when DAMPA is present in plasma samples and HPLC is not available.
Influence of Aromatase Inhibitors on Plasma Total Homocysteine in Postmenopausal Breast Cancer Patients
Authors: Anker Gun B , Refsum Helga , Ueland Per M , Johannessen Dag C , Lien Ernst A , Lønning Per E
Published: January 2020

In this study, we evaluated the effect of estrogen suppression with three aromatase inhibitors, aminoglutethimide (n = 30), formestane (n = 12), and exemestane (n = 10), and the progestin megestrol acetate (n = 21) on plasma total homocysteine (tHcy) in patients suffering from advanced breast cancer. Treatment with 1 g/day aminoglutethimide for 2 and 3–5 months increased plasma tHcy by a mean value of 24.5% [95% confidence interval, 10.5–40.4%] at 2 months and 35.8% (95% confidence interval, 18.2–55.9%) at 3–5 months, corresponding to increases in the mean plasma tHcy of 1.90 and 3.67 μmol/L, respectively. In contrast, none of the other treatment options influenced plasma tHcy concentrations. The finding that aminoglutethimide, but none of the other aromatase inhibitors or megestrol acetate, influenced plasma tHcy suggests that this effect is achieved by mechanisms not related to suppression of plasma estrogens or to the glucocorticoids administered in concert.
Allele-specific Differences in Apolipoprotein C-III mRNA Expression in Human Liver
Authors: Esterbauer Harald , Hell Emanuel , Krempler Franz , Patsch Wolfgang
Published: January 2020

Background: Sequence variations at the apolipoprotein (apo)C-III gene locus have been associated with increased plasma triglycerides. In particular, the S2 allele of an SstI polymorphism in the 3′ untranslated region has been associated with hypertriglyceridemia in many populations. The aim of this study was to determine whether the variant S2 allele is related to increased mRNA expression in vivo.

Methods: We measured allele-specific apoC-III expression in liver biopsies of five obese subjects, using restriction isotyping and a primer extension method, both based on the SstI polymorphism.

Results: The expression of mRNA by the S1 and S2 alleles was similar in two patients, whereas the mRNA encoded by the S2 allele was 14%, 26%, and 29% more abundant than the wild-type mRNA in the remaining three patients. Because other polymorphisms at the apoC-III gene locus have been implicated in the S2-associated hypertriglyceridemia, we determined apoC-III haplotypes comprising promoter polymorphisms at −935, −641, −630, −625, −482, −455, as well as the SstI sites and a BbvI site, both located in the 3′ untranslated region. None of these polymorphisms nor any haplotype exhibited a perfect association with allele-specific expression, but variation at the T-482C site correlated in four of five subjects with the relative allele abundance.

Conclusion: These data provide preliminary evidence for allele-specific differences in apoC-III mRNA expression in vivo and suggest that such differences may contribute to associations of apoC-III gene polymorphisms with hypertriglyceridemia.
Survey of Total Error of Precipitation and Homogeneous HDL-Cholesterol Methods and Simultaneous Evaluation of Lyophilized Saccharose-containing Candidate Reference Materials for HDL-Cholesterol
Authors: Cobbaert Christa , Mulder Paul GH , Baadenhuijsen Henk , Zwang Louwerens , Weykamp Cas W , Demacker Pierre NM
Published: January 2020

Background: Standardization of HDL-cholesterol is needed for risk assessment. We assessed for the first time the accuracy of HDL-cholesterol testing in The Netherlands and evaluated 11 candidate reference materials (CRMs).

Methods: The total error (TE) of HDL-cholesterol measurements was assessed in native human sera by 25 Dutch clinical chemistry laboratories. Concomitantly, the suitability of lyophilized, saccharose-containing CRMs (n = 11) for HDL-cholesterol was evaluated.

Results: In the precipitation method group, which included 25 laboratories and four methods, the mean (minimum–maximum) TE was 11.5% (2.7–25.2%), signifying that 18 of 25 laboratories satisfied the TE goal of ≤13% issued by the National Cholesterol Education Program (NCEP). In the homogeneous HDL-cholesterol method group, which included five laboratories, each performing two different methods, the mean (minimum–maximum) TE was 9.5% (6.0–17.3%) for the Boehringer assay and 15.7% (3.3–30.7%) for the Genzyme assay. For the Boehringer homogeneous assay, one of five laboratories did not meet the TE criterion, whereas for the Genzyme homogeneous assay, three of five laboratories exceeded the 13% criterion. The biases on the HDL-cholesterol values found by various precipitation methods were highly variable in all CRMs, irrespective of the quality, whereas the biases found by the homogeneous method from Boehringer were far less than ±5% for the highest-quality CRMs (CRMs 4–6).

Conclusions: The NCEP goal was met by 24 of 35 laboratories assessed by use of native human sera. Selectively pooled, lyophilized CRMs that are cryoprotected with 200 g/L saccharose have ample potential for use in the standardization of homogeneous HDL-cholesterol methods.
Assessment of Serum Thyroxine Binding Capacity-dependent Biases in Free Thyroxine Assays
Authors: Christofides Nicos D , Wilkinson Ewan , Stoddart Mary , Ray David C , Beckett Geoff J
Published: January 2020

Background: Free thyroxine (FT4) assays may exhibit biases that are related to serum T4 binding capacity (sBC). We describe two tests that can be used to assess the presence and magnitude of sBC-dependent biases in FT4 assays.

Methods: We used a direct equilibrium dialysis FT4 assay as the reference method and compared the results obtained with those of the FT4 assays under investigation, in patient sera having a wide range of sBC. We then compared the expected and observed FT4 results for sera diluted with an inert buffer. Because serum dilution causes a predictable decrease in sBC, an increasingly negative bias on progressive dilution is indicative of a sBC-dependent bias.

Results: The automated FT4 assay investigated (Vitros FT4) showed no demonstrable sBC-dependent bias by either test.

Conclusion: These two tests can be used to screen for sBC-dependent biases in FT4 assays.
Within- and Between-Subject Variation in Commonly Measured Anthropometric and Biochemical Variables
Authors: Widjaja Adji , Morris Richard J , Levy Jonathan C , Frayn Keith N , Manley Susan E , Turner Robert C
Published: January 2020

Background: The biological variation of some commonly assessed metabolic variables in healthy subjects has not been studied extensively. The aim of the study was to assess, in 12 healthy subjects (6 male and 6 female; mean (SD) age; 22.7 (1.5) years) following an overnight fast, the day-to-day variation of body fat (impedance method), triglycerides, nonesterified fatty acid (NEFAs), glycerol, 3-hydroxybutyrate (3-OHB), lactate, glucose, insulin (RIA), C-peptide, and glucagon on 12 consecutive days.

Methods: Between- and within-subject coefficients of variation (CVG and CVW) were estimated using a random effects analysis of variance, and assay variation was subtracted to give the coefficient of within-subject biological variation (CVI). Individuality indices were calculated as CVW/CVG.

Results: The overall means, CVI, and individuality indices were as follows: for body fat, 24.2%, 10%, and 0.3; for triglycerides, 0.61 mmol/L, 21%, and 1.1; for NEFAs, 376 μmol/L, 45%, and 1.4; for glycerol, 48 μmol/L, 36%, and 0.8; for 3-OHB, 43 μmol/L, 61%, and 1.5; for lactate, 0.88 mmol/L, 31%, and 1.1; for glucose, 4.9 mmol/L, 4.8%, and 0.7; for insulin, 52 pmol/L, 26%, and 1.0; for C-peptide, 0.39 nmol/L, 24%, and 0.9; and for glucagon, 53 ng/L, 19%, and 0.8.

Conclusions: The data presented here are necessary for the evaluation of several important metabolic variables in individual and group studies. The biological variation of some metabolites makes it difficult to characterize the status of healthy subjects with a single measurement.
Measurement of Plasma Renin Activity with Use of HPLC-Electrospray-Tandem Mass Spectrometry
Authors: Fredline Victoria F , Kovacs Eva M , Taylor Paul J , Johnson Anthony G
Published: January 2020

Background: The measurement of renin activity is complicated by difficulties in the quantification of angiotensin 1 (Ang1), the product of the renin-catalyzed reaction. We report an HPLC-electrospray-tandem mass spectrometry (HPLC-ESI-MS/MS) method for the quantification of Ang1 as a measure of plasma renin activity (PRA).

Methods: After incubation (37 °C for 3 or 18 h), samples were prepared using C18 solid-phase extraction. [Val]5Ang1 was used as the internal standard (IS). Chromatography was performed on a C18 column, using 200 mL/L ammonium acetate buffer–800 mL/L methanol as the mobile phase. The flow rate was 150 μL/min, with a chromatographic run time of 5 min/sample. Mass spectrometric detection was in the positive ionization mode with selected reaction monitoring (Ang1 m/z 649.0→784.0; IS m/z 641.9→770.4).

Results: The assay was linear over the range 2.5–500 ng Ang1/mL, which corresponded to a limit of detection (signal-to-noise ratio of 3:1) of PRA of 0.14 ng Ang1 · mL−1 · h−1. The imprecision (CV) of the assay at PRA values of 26.1, 13.5, 3.2, and 0.78 ng Ang1 · mL−1 · h−1 was 7.0%, 7.0%, 15%, and 11%, respectively. Absolute recoveries were 92.3% (Ang1) and 87.4% (IS). Incubation times of 3 h vs 18 h in the PRA assay gave good agreement at PRA <2 ng Ang1 · mL−1 · h−1, but samples with a PRA of 2–5 ng Ang1 · mL−1 · h−1 gave lower PRA results after incubation for 18 h than after 3 h. We compared the HPLC-ESI-MS/MS assay and an RIA for the determination of PRA, with PRA incubation times of 3 h and 1.5 h, respectively. The mean PRA based on RIA of Ang1 was higher than that obtained using HPLC-ESI-MS/MS.

Conclusion: The HPLC-ESI-MS/MS method allows sensitive and specific measurement of PRA. The higher activities measured with the RIA method highlight its potential for overestimation of PRA.