bluuebunny/crossref_metadata_2025 huggingface dataset:
False-positive and false-negative rates in meconium drug testing
Authors: Moore C , Lewis D , Leikin J
Published: January 2020

To determine the number of false-negative results produced by inefficient extraction of drugs from meconium, three published procedures were compared by using previously confirmed positive and negative meconium specimens. The methods were not equivalent in their ability to extract drugs from the matrix. To determine the number of false positives reported by the use of screen-only (unconfirmed) results, 535 screen-positive meconium specimens were subjects to confirmation by gas chromatography-mass spectrometry. Fifty-seven percent of the samples were confirmed positive for one or more of the drugs under investigation, showing that a false-positive rate as high as 43% may exist when unconfirmed screening results are used.
Application of a low-light imaging device and chemiluminescent substrates for quantitative detection of viral DNA in hybridization reactions
Authors: Girotti S , Musiani M , Pasini P , Ferri E , Gallinella G , Zerbini M L , Roda A , Gentilomi G , Venturoli S
Published: January 2020

In this quantitative dot-blot hybridization assay for detecting B19 parvovirus DNA, we used three different chemiluminescent substrates [adamantyl-1,2-dioxetane phenyl phosphates (PPD and the new PPD-Plus) and the chloro-5-substituted adamantyl-1,2-dioxetane phosphate (CSPD) plus Emerald enhancer] and a high-performance, low-intensity-light imaging luminograph apparatus. The hybridization test uses digoxigenin-labeled DNA probes, which are immunoenzymatically revealed by anti-digoxigenin Fab fragments conjugated with alkaline phosphatase. All the detection systems with the various chemiluminescent substrates gave sensitive and reproducible results for calibrators and positive or negative reference clinical samples, with high reproducibility (CV 4-17%). The signal was measured after 45 min of incubation. The luminograph apparatus could detect 10 fg of homologous DNA with the PPD-Plus substrate, whereas the detection limit with the CSPD and PPD substrates was 20 fg and 20-50 fg, respectively. Analysis of 26 samples with the three substrates showed good sensitivity and specificity for viral detection.
Enhanced detection of hematogenous circulating prostatic cells in patients with prostate adenocarcinoma by using nested reverse transcription polymerase chain reaction assay based on prostate-specific membrane antigen
Authors: Loric S , Dumas F , Eschwege P , Blanchet P , Benoit G , Jardin A , Lacour B
Published: January 2020

We report the development of a new sensitive nested reverse transcription-polymerase chain reaction (RT-PCR) assay, using primers derived from the prostate-specific membrane antigen (PSM) cDNA sequence, to detect an hematogenous spread of prostate adenocarcinoma cells. In 60 patients with a biopsy-proven prostate cancer, PSM and PSA RT-PCR detected circulating prostate cells in 40 and 20 patients, respectively. In pT4 M+ and pT3 M+ disease patients, nested PSM primers detected cells in 28 of 33 patients (85%), whereas nested PSA primers detected cells in 17 of 33 (51%). In patients with organ-confined cancer spread (pT2a and pT2b patients) before radical prostatectomy, nested PSM RT-PCR detected circulating prostatic epithelial cells in 6 of 17 patients (35%), which suggests that an hematogenous spread of prostate cells may occur early in prostate cancer history. Altogether, these results suggest that the detection of PSM-expressing cells in blood may predict the development of cancer in patients without clinically apparent prostate cancer. Nevertheless, the potential application and the clinical significance of detection of hematogenous prostate cells through the use of nested PSM primers need an extensive longitudinal study.
Automated assay of oxygen radical absorbance capacity with the COBAS FARA II
Authors: Cao G , Verdon C P , Wu A H , Wang H , Prior R L
Published: January 2020

Reactive oxygen species are of interest in biology and medicine because of evidence relating them to aging and disease processes. A relatively simple but sensitive and reliable method for quantifying the oxygen radical absorbance capacity (ORAC) of antioxidants in biological tissues has been automated for use with the COBAS FARA II centrifugal analyzer with a fluorescence-measuring attachment. In this assay, beta-phycoerythrin (beta-PE) is used as an indicator protein, 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) as a peroxyl radical generator, and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) as a calibrator for antioxidant activity. This assay is unique because the reaction goes to completion so that both inhibition time and inhibition degree are considered in quantifying ORAC (micromoles of Trolox equivalent per liter or per gram of tissue). This method can be used not only for serum but also other tissue and food samples and is suitable for application to a range of nutritional and clinical conditions.
Significance of individual sensitivity to chemicals: elucidation of host susceptibility by use of biomarkers in environmental health research
Authors: Cullen M R , Redlich C A
Published: January 2020

Biomarker research has become the predominant theme for study of human dose-host-response relations to environmental chemicals. Increasing interest has been focused on identifying markers for host susceptibility, with mixed results. Efforts to identify markers for host variability in carcinogenic risk, on the basis of theoretical knowledge of carcinogen metabolism, have been disappointing. New work in the area of acquired risk modifiers, such as nutritional status, is theoretically attractive, but results have been limited. Impressive achievements have been made in the area of immunological variability, which may elucidate the molecular basis of as well as provide practical biomarkers for several diseases. The problem of multiple chemical sensitivities, on the other hand, has proved refractory to biomarker research, reflecting inadequate knowledge of the mechanism and inappropriate application of biomarker methods.
Variability in DNA repair and individual susceptibility to genotoxins
Authors: Kyrtopoulos S A
Published: January 2020

DNA repair is an important mechanism of cellular protection from the effects of genotoxic chemicals. Although extensive evidence from studies in experimental systems indicates that variation in DNA repair can significantly influence susceptibility to genotoxins, corresponding studies in human populations are so far limited, mainly because of methodological difficulties. One system, using observations of the accumulation and repair of DNA damage in cancer patients treated with alkylating cytostatic drugs, has provided useful information for assessing the effects of interindividual variation in DNA repair activity on the induction of genotoxic effects in humans. The most detailed studies of this kind have been carried out on patients with cancer (i.e., Hodgkin disease, malignant melanoma) treated with the methylating cytostatic drugs procarbazine or dacarbazine; these studies have provided detailed information on dose-response relationships. They have also demonstrated the protective role of the repair enzyme O6-alkylguanine-DNA alkyltransferase against the accumulation of the premutagenic methylated DNA lesion O6-methylguanine in patients' DNA. Given the strong evidence that exposure of the general population to environmental methylating agents may be extensive, as indicated by the frequent discovery of methylated DNA adducts in human DNA, data on DNA damage and repair in alkylating drug-treated patients and their modulation by host factors may prove useful in efforts to assess the possible carcinogenic risks posed by exposure to environmental methylating agents.
Assessment of immunotoxic effects in humans
Authors: Descotes J , Nicolas B , Vial T
Published: January 2020

The immunotoxic effects of chemicals are varied and markedly different depending on the underlying pathogenesis, namely, direct immunotoxicity (including immunosuppression, immunodepression, and immunostimulation), hypersensitivity, and autoimmunity. A large number of immunological endpoints and functional assays have been proposed for use as biomarkers of immunotoxicity, but they often lack sensitivity or are poorly standardized, so that their relevance in assessing immunotoxic effects in humans is at best ill established. Examining sentinel immunopathological events in individuals with a defined history of chemical exposure is another approach, presumably more cost-effective at the present time. A multicenter collaboration is mandated, however, because these events are rare. We expect that progress in new technologies, e.g., molecular biology, will provide the sensitive and reliable biomarkers of immunotoxicity that are currently lacking.
Detection of prostatic cells in peripheral blood: correlation with serum concentrations of prostate-specific antigen
Authors: Jaakkola S , Vornanen T , Leinonen J , Rannikko S , Stenman U H
Published: January 2020

We have studied the expression of prostate-specific antigen (PSA) mRNA by reverse transcriptase-polymerase chain reaction in peripheral blood of 25 patients with cancer of the prostate (CAP), four with benign prostatic hyperplasia (BPH), two with renal stones, three with other types of cancer, and six healthy male and three female controls. Expression of mRNA specific for a certain tissue in peripheral blood is thought to indicate the presence of circulating cancer cells and metastatic spread of a tumor originating from this tissue. We detected PSA mRNA in 9 of 18 CAP patients with metastatic disease but in none of 7 patients without metastases. Negative results in patients with metastatic disease were associated with successful endocrine therapy and low concentrations of serum PSA, and the correlation between serum concentrations of PSA and the presence of PSA mRNA in peripheral blood was statistically significant. PSA mRNA was not found in patients with BPH, other types of cancer, or in healthy controls. Thus the occurrence of PSA mRNA in peripheral blood is associated with metastatic CAP.
Three assays for glycohemoglobin compared
Authors: Turpeinen U , Karjalainen U , Stenman U H
Published: January 2020

Using 123 specimens, we compared the concordance of three different methods for determining glycohemoglobin (GHb): the Diamat (Bio-Rad Laboratories), an automated analyzer measuring HbA1c by cation-exchange chromatography; an assay with the IMx analyzer (Abbott Laboratories), based on boronate affinity binding; and an HPLC method measuring HbA1c by cation-exchange chromatography on a PolyCAT A column (PolyLC Inc.). The Pearson's correlation coefficient between PolyCAT A and Diamat was 0.900 +/- 0.038 (mean +/- 2 SD) and between PolyCAT A and IMx, 0.857 +/- 0.042. However, up to twofold differences were seen in some samples. The proportion of GHb was consistently lower with the PolyCAT A method than with the other two assays, apparently because of better separation of HbA1c from nonglycated coeluting forms of Hb. The difference in glycation percentage between the PolyCAT A and Diamat methods is 2-3% over the whole concentration range. These results point to the limitations of Diamat as a reference method to be used to calibrate other methods for determining HbA1c. Further, a switch from one method to another is likely to cause considerable problems in the clinical follow-up of certain patients.
Two bone alkaline phosphatase assays compared with osteocalcin as a marker of bone formation in healthy elderly women
Authors: Bouman A A , Scheffer P G , Ooms M E , Lips P , Netelenbos C
Published: January 2020

Serum bone alkaline phosphatase (ALP; EC 3.1.3.1) was measured with a wheat germ agglutinin (WGA) precipitation assay and with a new IRMA in a group of healthy elderly women. Both assays were correlated with serum total ALP activity and with osteocalcin. The two bone ALP assays have comparable within- and between-run imprecisions (WGA assay within-run CVs 2.6-5.4% and between-run, 4.0-5.1%; IRMA within-run CV 5.0% and between-run, 3.2%). Comparison of the WGA precipitation assay (x) with the IRMA (y) demonstrated a correlation coefficient of 0.87 [Deming regression equation: y = (0.58 +/- 0.02)x - (4.62 +/- 0.45); n = 101; Sy/x = 1.26; P < 0.001). Correlation studies with osteocalcin and total ALP showed correlation coefficients (all P < 0.001) of 0.34 and 0.65, respectively, for the WGA precipitation assay and of 0.36 and 0.68, respectively, for the IRMA. We conclude that the two bone ALP assays have similar imprecision and that neither can be given preference over the other as a marker of bone turnover.
Prostate-specific antigen immunoreactivity in amniotic fluid
Authors: Yu H , Diamandis E P
Published: January 2020

We examined whether the 33-kDa serine protease prostate-specific antigen (PSA) is present in amniotic fluid and, if so, whether its concentration changes with gestational age. Analyzing 115 amniotic fluids with a highly sensitive immunofluorometric procedure, we found PSA in all the amniotic fluids examined and established that its concentration increases with increasing gestational age from 11 to 21 weeks, decreasing at delivery. PSA in amniotic fluid is present predominantly in the free (33 kDa) form; a minor fraction (< 20%) is present bound to alpha 1-antichymotrypsin. No significant correlation was seen between PSA and alpha-fetoprotein (AFP) in amniotic fluid or maternal serum in samples with high AFP. Amniotic fluid PSA was also measurable by two different established methods for PSA. Pregnant women had higher concentrations of serum PSA than nonpregnant women. The highest PSA concentration in amniotic fluid was associated with a pregnancy that was complicated by the Rhesus incompatibility syndrome but the source of the PSA was not established. From recent literature reports, and the association of PSA with prostate and breast tumors, we think PSA may serve as a growth factor regulator in cancer and in normal fetal development during pregnancy.
Coenzyme Q: potentially useful index of bioenergetic and oxidative status of spermatozoa
Authors: Angelitti A G , Colacicco L , Callà C , Arizzi M , Lippa S
Published: January 2020

The concentration of coenzyme Q10 (CoQ10), a key intermediate of the mitochondrial respiratory chain, was determined in spermatozoa of 13 fertile subjects, 8 potentially fertile patients, and 12 infertile patients. CoQ10 concentrations were significantly higher (P < 0.001) in infertile patients than in fertile and potentially fertile subjects. The difference between potentially fertile and fertile subjects was also significant (P < 0.001). We propose that a decrease in consumption of CoQ10 in both infertile and potentially fertile populations is due to an autoregulatory mechanism of ATP production.
Changes in serum lipoprotein(a) and lipids during treatment of hyperthyroidism
Authors: Kung A W , Pang R W , Lauder I , Lam K S , Janus E D
Published: January 2020

Because of suggestions that thyroid hormones modulate serum lipoprotein(a) [Lp(a)] concentration, we evaluated prospectively the serial changes of serum Lp(a), measured as apolipoprotein(a) [apo(a)], and other lipoproteins in 40 subjects with hyperthyroidism treated with radioactive iodine (RAI) therapy. Hyperthyroid patients had lower (P < 0.001) concentrations of apo(a), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and apo B, but higher apo A-I concentrations compared with age-matched controls [geometric mean (range)]; apo(a) 81 (17-614) vs 187 (17-1808 IU/L): TC 4.07 +/- 0.8 vs 5.22 +/- 1.00 mmol/L (mean +/- SD); LDL-C 2.47 +/- 0.89 vs 3.40 +/- 0.88 mmol/L; HDL-C 1.05 +/- 0.33 vs 1.24 +/- 0.34 mmol/L; apo B 0.66 +/- 0.23 vs 1.13 +/- 0.34 g/L, and apo A-I 2.07 +/- 0.42 vs 1.46 +/- 0.28 g/L, respectively. Euthyroidism was associated with normalization of serum TC, LDL-C, and apo B within 1 month of treatment. However, apo(a) required 4 months to normalize, and HDL-C and apo A-I were still abnormal 6 months after RAI. Serum apo(a), TC, LDL-C, and apo B were negatively correlated with serum thyroxine (T4), free thyroxine index, and triiodothyronine (T3) and positively correlated with thyrotropin during the transitional period from hyperthyroidism to euthyroidism. Parallel changes of these lipoproteins and thyroid hormones were also observed after treatment of hyperthyroidism. In conclusion, thyroid hormones do modulate lipoproteins, particularly Lp(a). The delay in normalization of apo(a) but not LDL suggests an effect on apo(a) production rather than on LDL removal.
Lipoprotein(a) in childhood: relation with other atherosclerosis risk factors and family history of atherosclerosis
Authors: Bailleul S , Couderc R , Rossignol C , Fermanian J , Boutouchent F , Farnier M A , Etienne J
Published: January 2020

The relation between lipoprotein(a) [Lp(a)], apolipoprotein (apo) E phenotypes, cholesterol, triglycerides, apo A-I, apo B, and a family history of atherosclerosis or risk factors was studied in 2- and 4-year-old French Caucasian children (n = 499). Lp(a) concentrations were distributed in a typical skewed manner and were found to be an independent lipid variable. The distribution of apo E phenotypes did not differ by gender. Cholesterol and apo B were under apo E phenotype control; Lp(a) was not. A significant positive relation was found between Lp(a) concentrations and the number of parental risk factors. Children whose grandparents had a history of cardiovascular disease had Lp(a) concentrations shifted towards higher values. Measurement of Lp(a) in children may help to identify those at an increased risk of atherosclerotic disease, especially when their parents have at least two relevant risk factors.
Effect of the number of apolipoprotein(a) kringle 4 domains on immunochemical measurements of lipoprotein(a)
Authors: Marcovina S M , Albers J J , Gabel B , Koschinsky M L , Gaur V P
Published: January 2020

Lipoprotein(a) [Lp(a)] has been measured in numerous clinical and epidemiological studies by a variety of immunochemical methods. However, little, if any, consideration has been given to the confounding effect of the size heterogeneity of apolipoprotein(a) [apo(a)] on the measurement of Lp(a). We developed three direct-binding enzyme-linked immunosorbent assays (ELISAs) with detecting antibodies of different specificities to evaluate the effect of apo(a) size on Lp(a) measurement. The three assays used the same monoclonal antibody to capture the apo(a)-containing particles and were calibrated (in nanomoles per liter) with a serum containing apo(a) with 21 kringle 4 domains. Using all three ELISAs, we measured Lp(a) in a group of 723 subjects selected to have a single apo(a) band, as determined by a high-resolution phenotyping system. Essentially identical results were obtained by the two methods that measured Lp(a) by use of either a polyclonal antibody against apo B or a monoclonal antibody against apo(a) that does not recognize the kringle 4 type 2 repeats. In contrast, the ELISA using a monoclonal antibody specific for apo(a) kringle 4 type 2 repeats overestimated Lp(a) concentration in samples containing apo(a) with more than 21 kringle 4 domains and underestimated Lp(a) samples containing apo(a) with fewer than 21 kringle 4 domains. Thus, these differences in Lp(a) values varied as a function of apo(a) size. We conclude that antibody specificity and apo(a) size heterogeneity can significantly affect Lp(a) measurements.
Laboratory methods and quality assurance in the Cardiovascular Health Study
Authors: Cushman M , Cornell E S , Howard P R , Bovill E G , Tracy R P
Published: January 2020

The Cardiovascular Health Study is an observational cohort study of risk factors for cardiovascular disease in 5201 participants, ages > or = 65 years. We report the methods and quality-assurance results for blood procurement, processing, shipping, storage, and sample analysis used during the first examination period (May 1989-June 1990). The most frequent difficulty in phlebotomy and processing was the requirement of more than one venipuncture (in 2.6% of the participants). The CVs for control materials ranged from 0.93% for glucose to 10.7% for insulin; most were < 4%. In addition to standard quality-assurance methods, we applied two other methods: technical error calculation for replicates, and weighted linear regression to assess time trend in results of control materials. After outliers were excluded, technical error values ranged from 1.7 for uric acid to 18.8 for insulin. Factor VII and factor VIII had slight trends over the 12-month analysis period. Results of quality-assurance analyses used to resolve problems were successful, thereby improving the second laboratory examination.
1H NMR determination of urinary betaine in patients with premature vascular disease and mild homocysteinemia
Authors: Lundberg P , Dudman N P , Kuchel P W , Wilcken D E
Published: January 2020

Urinary N,N,N-trimethylglycine (betaine) and N,N-dimethylglycine (DMG) have been identified and quantified for clinical purposes by proton nuclear magnetic resonance (1H NMR) measurement in previous studies. We have assessed these procedures by using both one-dimensional (1-D) and 2-D NMR spectroscopy, together with pH titration of urinary extracts to help assign 1H NMR spectral peaks. The betaine calibration curve linearity was excellent (r = 0.997, P = 0.0001) over the concentration range 0.2-1.2 mmol/L, and CVs for replicate betaine analyses ranged from 7% (n = 10) at the lowest concentration to 1% (n = 9) at the highest. The detection limit for betaine was < 15 mumol/L. Urinary DMG concentrations were substantially lower than those of betaine. Urinary betaine and DMG concentrations measured by 1H NMR spectroscopy from 13 patients with premature vascular disease and 17 normal controls provided clinically pertinent data. We conclude that 1H NMR provides unique advantages as a research tool for determination of urinary betaine and DMG concentrations.
Essential and toxic element concentrations in fresh and formalin-fixed human autopsy tissues
Authors: Bush V J , Moyer T P , Batts K P , Parisi J E
Published: January 2020

The concentrations of five essential elements and six potentially toxic elements were determined in seven organs collected at autopsy from 30 human subjects. Elemental analyses were carried out by graphite furnace atomic absorption spectroscopy, and inductively coupled plasma emission spectroscopy, and inductively coupled plasma mass spectroscopy, and concentrations in fresh and formalin-fixed tissues were compared. Formalin-fixation long-term storage has little effect on most element concentrations in tissue, except for Al and Mn, which changed with prolonged storage in formalin. The kidney and liver contained the greatest concentrations of toxic elements compared with other organs, whereas the essential elements were uniformly distributed among all organs. There was no more than a 10-fold difference in the tissue concentration of the elements studied among the organs, except for the concentration of Fe in liver, and Ca and Mg in bone. We also demonstrate that these elements are homogeneously distributed in tissues.
Pharmacodynamic assessment of mycophenolic acid-induced immunosuppression by measuring IMP dehydrogenase activity
Authors: Langman L J , LeGatt D F , Yatscoff R W
Published: January 2020

Pharmacodynamic monitoring of the biological effect of immunosuppressive drugs provides an alternative to traditional therapeutic drug monitoring. We chose this method to investigate mycophenolic acid (MPA), an immunosuppressive drug that mediates its effect by inhibition of IMP dehydrogenase (IMPDH), a key enzyme in the de novo biosynthesis of purines. Using an assay developed for measuring IMPDH activity in whole blood, we found the concentration of MPA required for 50% inhibition of enzyme activity to be in the range of 2.0-5.0 mg/L for both human and rabbit blood. The amount of enzyme activity in whole blood depended on the concentration of the leukocytes, was unaffected by the type of anticoagulant used, and was stable in blood specimens stored for as long as 48 h at 4 degrees C. An inverse relationship was found between plasma MPA concentrations and IMPDH activity in rabbits administered a single dose of RS-61443, the prodrug of MPA. Maximal inhibition of IMPDH activity (by approximately 60%) occurs at peak concentrations of MPA; as the concentration of the drug decreases postdose, the enzyme activity gradually increases with little or no inhibition being observed 24 h postdose.
Analytical artifacts in hematocrit measurements by whole-blood chemistry analyzers
Authors: Stott R A , Hortin G L , Wilhite T R , Miller S B , Smith C H , Landt M
Published: January 2020

Compact analyzers suited to near-patient testing estimate hematocrit by measuring the conductivity of undiluted blood. We evaluated the accuracy of hematocrit determination of one such analyzer (Instrumentation Laboratory BGE analyzer) against an automated cell counter (EPC) and packed cell volume (PCV) microhematocrit. When specimens (n = 34) from outpatient and ward patients were analyzed with all three methods, the BGE analyzer correlated well with both EPC and PCV hematocrit determinations (BGE = 1.00 PCV + 0.3%, S(y)/x = 2.0%), suggesting that all three methods are similar in performance for most patients. However, a patient with increased plasma osmolality showed significant decreases in BGE and PCV hematocrits relative to the EPC method. The differences in hematocrit measurements could be reproduced by adding solutes to blood in vitro or by modifying the plasma osmolality of rats in vivo. Samples from patients undergoing cardiac surgery, whose blood had large changes in protein concentration, showed discrepancies between hematocrits by conductivity and other methods; similar effects could be produced by changes in protein concentration or in vitro addition of polyethylene glycol. We conclude that conductivity measurements provide accurate hematocrit results for physiologically normal subjects but not for some intensive-care and surgical patients.
Effect of thiol oxidation and thiol export from erythrocytes on determination of redox status of homocysteine and other thiols in plasma from healthy subjects and patients with cerebral infarction
Authors: Andersson A , Lindgren A , Hultberg B
Published: January 2020

Changes in concentration of reduced and oxidized low-M(r) thiols were measured in blood and plasma before and after the separation of blood cells. If centrifugation of blood was postponed, the reduced form of homocysteine in plasma increased with time at 22 degrees C; in contrast, the concentrations of other reduced thiols (cysteine, glutathione, and cysteinylglycine) decreased. In plasma the reduced forms of all thiols disappeared at a rate that followed first-order kinetics. The rates of disappearance ("half-lives") were temperature-dependent; they were about the same for glutathione and homocysteine (11.7 and 14.3 min, respectively, at 22 degrees C) and somewhat higher for cysteinylglycine and cysteine. After establishing proper sampling conditions for reduced thiols, we measured this thiol fraction as well as free (non-protein-bound) and total thiols in 10 reference subjects and 19 patients with cerebral infarction. Mild but significant hyperhomocysteinemia involving total and free homocysteine (but not reduced homocysteine) was found in the patients.
HPLC measurement, blood distribution, and pharmacokinetics of oral clotrimazole, potentially useful antisickling agent
Authors: Rifai N , Sakamoto M , Law T , Platt O , Mikati M , Armsby C C , Brugnara C
Published: January 2020

Clotrimazole (CLT) has recently been shown to be a potent and specific inhibitor of the Ca(2+)-activated K+ channel and to thereby prevent K+ loss and cellular dehydration of sickled erythrocytes. This evidence suggests that oral CLT may be a useful new therapy for sickle cell disease. Here, we describe the development of an HPLC assay to measure CLT, a method we used to study the pharmacokinetics and transport of CLT in normal volunteers. The assay's linear range extended to 10 mumol/L; the detection limit was 0.1 mumol/L, analytical recovery 97.7%, and run-to-run imprecision (CV) < 4.7%. In unaffected subjects, CLT concentration peaked within 6 h of oral administration and returned to close to baseline by 24 h. High-density lipoproteins appear to be the main carriers of this drug in both normo- and hypertriglyceridemic plasma. We conclude that the method described here is ideally suited for therapeutic monitoring of CLT concentrations.
Effect of storage at 4 degrees C and -20 degrees C on lipid, lipoprotein, and apolipoprotein concentrations
Authors: Evans K , Mitcheson J , Laker M F
Published: January 2020

We have investigated the effects on lipid, apolipoprotein, and lipoprotein measurements of storing unfractionated serum at 4 degrees C for 10 days and at -20 degrees C for 10 days or 3 months. Total serum concentrations of lipids were stable, although apolipoprotein B showed a 5.3% increase after 3 months at -20 degrees C (P < 0.001). Increases in low-density (LDL) and high-density lipoprotein (HDL) triglyceride and very-low-density lipoprotein (VLDL) esterified cholesterol concentrations and decreases in free cholesterol concentrations in LDL and HDL after storage of serum for 10 days at 4 degrees C were verified by fractionation of lipoproteins by sequential flotation ultracentrifugation. Ten days' storage of serum at -20 degrees C resulted in increases in VLDL triglyceride and phospholipid concentrations, with decreases in HDL concentrations in triglycerides and phospholipids; changes were more extensive after 3 months at -20 degrees C. We conclude that ultracentrifugation of serum for lipoprotein analysis should be performed as soon as possible after collection.
Robust nonradioactive oligonucleotide ligation assay to detect a common point mutation in the CYP2D6 gene causing abnormal drug metabolism
Authors: Hansen T S , Petersen N E , Iitiä A , Blaabjerg O , Hyltoft-Petersen P , Hørder M
Published: January 2020

A new nonradioactive oligonucleotide ligation assay for the detection of a common point mutation in the CYP2D6 gene is presented. The assay takes advantage of simultaneous time-resolved fluorescence measurements of lanthanide-labeled probes and the specificity of T4-DNA ligase in combination with the polymerase chain reaction. This strategy makes it possible to perform the assay using both the wild-type-specific and mutant-specific probes simultaneously, securing an internal control in each reaction. We show that the allele-specific ligation part of the assay can be performed with great accuracy over a wide range of temperatures, salt concentrations, and T4-DNA ligase concentrations. This eliminates the risk of false-positive or false-negative reactions due to variations in these factors. Because the assay is simple to perform, is very reliable, and can be partly automated, we conclude that it is well-suited for analysis in a routine laboratory.
Restriction fragment length polymorphisms of apolipoprotein B gene in Chinese population with coronary heart disease
Authors: Pan J P , Chiang A N , Tai J J , Wang S P , Chang M S
Published: January 2020

Three restriction fragment length polymorphisms (RFLPs), EcoRI (R), Xbal (X), and Mspl (M) of the apolipoprotein (apo)B gene, were studied to determine their distribution frequencies and influence on the lipid profiles in 148 Chinese patients with documented coronary heart disease (CHD) and in 153 healthy subjects. The plasma concentrations of cholesterol and apoB showed no difference between the CHD patients and controls. However, CHD patients had significantly higher concentrations of low-density lipoprotein cholesterol and triglyceride and lower concentrations of high-density lipoprotein cholesterol than the controls. The frequencies of these three apoB RFLPs did not differ between the CHD patients and controls. Compared with South Asians and Caucasians, the Chinese in Taiwan showed a much lower frequency of R-, X+, and M- alleles. There was no evidence of an association between lipid profiles and RFLPs in either CHD patients or controls. The weak association of EcoRI, Xbal, and Mspl polymorphisms of the apoB gene with CHD indicates that the three RFLPs cannot be used as a predictor for the risk of CHD in the Chinese population.
HPLC with o-phthalaldehyde precolumn derivatization to measure total, oxidized, and protein-bound glutathione in blood, plasma, and tissue
Authors: Paroni R , De Vecchi E , Cighetti G , Arcelloni C , Fermo I , Grossi A , Bonini P
Published: January 2020

This HPLC assay with o-phthalaldehyde precolumn derivatization is used to measure the total, oxidized, and protein-bound forms of glutathione in human blood, plasma, and rat tissue. Total glutathione (i.e., sum of reduced, oxidized, and protein-bound fractions) was determined after reduction with dithiothreitol and protein precipitation with perchloric acid (PCA). A preliminary selective blockage of free sulfhydryl groups with N-ethylmaleimide was necessary to evaluate the different oxidized forms. The assay showed high sensitivity (< 0.05 pmol injected) and good precision (within-day CVs of 5.5% to 6.4%), recovery (101% +/- 4%), and linearity (r > 0.999). Samples, after PCA acidification, were stable at room temperature and 4 degrees C for 3 days, and at -20 degrees C and -80 degrees C for > 1 month. The method (involving automated derivatization) not only is very rapid and simple but also allows immediate processing of many different biological samples.
Enzymatic markers of gallstone-induced pancreatitis identified by ROC curve analysis, discriminant analysis, logistic regression, likelihood ratios, and information theory
Authors: Kazmierczak S C , Catrou P G , Van Lente F
Published: January 2020

We investigated the diagnostic utility of frequent serial determinations of aspartate aminotransferase, alanine aminotransferase (ALT), lipase, amylase, and the lipase/amylase (L/A) ratio for distinguishing patients with acute pancreatitis due to biliary obstruction from those with acute pancreatitis due to other pathogenesis. Analyzed were enzyme activities obtained at admission and peak enzyme activities identified retrospectively from serial measurements in 53 patients with acute pancreatitis due to various causes. We evaluated the data with multiple statistical tools. Discriminant analysis and logistic regression revealed the diagnostic significance of ALT at initial and peak values, and the maximum information provided by peak ALT was confirmed by both logistic regression and stratum-specific likelihood ratios. Stratum-specific likelihood ratios showed peak ALT > 150 U/L was highly diagnostic of biliary pancreatitis. The L/A ratio, either at admission or at peak, was the only other significant variable for identifying patients with acute pancreatitis due to biliary obstruction. A multivariate logistic discriminant function including ALT and the L/A ratio significantly discriminated biliary acute pancreatitis from pancreatitis due to other causes. Evaluation of initial and peak enzyme data by information theory revealed that the optimal test depended on disease prevalence. Initial ALT activities were the test of choice for identifying biliary pancreatitis, up to a disease prevalence of approximately 0.75. At disease prevalence > 0.75, the initial L/A ratio provided the greatest amount of diagnostic information.
Enzyme immunoassay of immunoreactive progastrin-releasing peptide(31-98) as tumor marker for small-cell lung carcinoma: development and evaluation
Authors: Aoyagi K , Miyake Y , Urakami K , Kashiwakuma T , Hasegawa A , Kodama T , Yamaguchi K
Published: January 2020

Previously, using recombinant human progastrin-releasing peptide (ProGRP)(31-98), we developed a RIA for ProGRP(31-98) and demonstrated that the determination of serum ProGRP(31-98) was a reliable marker for small-cell lung carcinoma (SCLC) (Miyake et al., Cancer Res 1994;54:2136-40). Aiming for a more convenient assay system, we have now developed and evaluated a highly sensitive and specific ELISA for ProGRP(31-98). Only 50 microL of nonextracted serum is needed, and results are obtained in only 2 h. Intraassay and between-day CVs were 1.7-4.6% and 4.2-6.8%, respectively. The log-log calibration curve was linear to 1000 ng/L, and analytical recovery was 91.5-108.7%. The detection limit of this assay, 1.9 ng/L, means that basal concentrations of ProGRP(31-98) were detectable in all healthy subjects. The cutoff value, based on the mean + 3 SD of concentrations in 247 healthy subjects, was set to 45.1 ng/L. Serum concentrations exceeded this value in 18 of 25 SCLC patients, similar to the frequency of increased values found by RIA previously. In contrast, the frequency of increased serum ProGRP(31-98) in patients with nonmalignant pulmonary diseases or non-SCLC was quite low: 0% and 5.0%, respectively. Such results may justify a clinical trial for evaluating this ELISA for the diagnosis and monitoring of SCLC patients.
Electrochemical determination of low blood lead concentrations with a disposable carbon microarray electrode
Authors: Feldman B J , D'Alessandro A , Osterloh J D , Hata B H
Published: January 2020

Lead concentrations in blood samples typical of unexposed individuals are determined by using square-wave anodic stripping voltammetry at a disposable 287-element (15-35-microns diameter) carbon microarray electrode. Analysis of a series of low Pb-containing samples (blood [Pb] = 17 to 92 micrograms/L) by standard addition gives good accuracy (average bias vs graphite furnace atomic absorption spectroscopy = -4 micrograms/L) and precision (pooled SD = 7 micrograms/L). The signal/noise ratio is improved by about an order of magnitude over that found at a single carbon disk (10 microns diameter), resulting in a detection limit (for the microarray) of 5 micrograms/L for a 60-s deposition of sample.
Determination of pyridinoline and deoxypyridinoline in urine, with special attention to retaining their stability
Authors: Gerrits M I , Thijssen J H , van Rijn H J
Published: January 2020

Urinary excretion of the pyridinium crosslinks pyridinoline (Pyr) and deoxypyridinoline (Dpyr) is used as a biochemical marker of bone resorption. The present study was undertaken to determine the long-term stability of these compounds in stored urine, using the HPLC method. Systematic investigation of their chemical stability in urine demonstrated that both the free and conjugated forms of Pyr and Dpyr are extremely stable: No significant changes were observed after 6 weeks at -20 degrees C storage (e.g., free Pyr 9.6 +/- 1.2 mumol/mol creatinine (before) and 10.6 +/- 3.2 (after); free Dpyr 2.3 +/- 0.2 mumol/mol creatinine (before) and 2.5 +/- 1.2 (after)). These results predict stability of urines stored for 10-20 years at -20 degrees C in the dark. Also, freezing and thawing as many as 10 times had no effect on the concentrations of the crosslinks. Study of the stability of the excretion pattern in healthy women showed substantially higher variations in excretions of free and total Dpyr (18% and 13%, respectively) than of Pyr (10% for both forms).
Automated measurement of urinary iodine with use of ultraviolet irradiation
Authors: Tsuda K , Namba H , Nomura T , Yokoyama N , Yamashita S , Izumi M , Nagataki S
Published: January 2020

We have modified an automated measurement system of urinary iodine (UI) and established a sensitive UI assay system by using ultraviolet (UV) digestion. The automated system is sensitive enough to detect concentrations of UI < 0.78 mumol/L (< 10 micrograms/dL) in a small volume of urine (500 microL). Sample throughput is > 30/h, including a water washing. The within-assay imprecision (CV) was < or = 10% in the UI range of 0.10-3.00 mumol/L; the between-assay CV was usually < or = 15% in the same range. Analytical recovery of iodine added to urine samples was consistently > 90%. The theoretical values were recovered when UV irradiation was used but not in its absence. High (supraphysiological) doses of thiocyanate or ascorbic acid, which are major interfering substances to the ceric-arsenious acid reaction, did not interfere with this system. The correlation between UI determined by this method and by the acid digestion method was linear (r = 0.994). For samples containing iodine at < 1.00 mumol/L, the correlation between values by both methods was still significant (r = 0.937). UI in an iodine-deficient area in Ukraine, measured by this system, ranged from 0.06 to 1.83 mumol/L (median 0.44 mumol/L, n = 95), significantly lower than in Japan (range 0.23-50.70 mumol/L, median 4.70 mumol/L, n = 84) and consistent with mild iodine deficiency. This modified automated assay system, therefore, is useful and applicable for screening UI in inhabitants of iodine-deficient areas.
Automated homogeneous liposome-based assay system for total complement activity
Authors: Yamamoto S , Kubotsu K , Kida M , Kondo K , Matsuura S , Uchiyama S , Yonekawa O , Kanno T
Published: January 2020

We developed an automated homogeneous immunoassay, based on immune lysis of dinitrophenyl (DNP)-labeled liposomes, for measuring total complement activity. Liposome lysis caused by complement activity was detected spectrophotometrically from entrapped glucose-6-phosphate dehydrogenase activity. Complement activity in human sera was quantified by comparison with a calibration curve. For ease of application to fully automated routine clinical analyzers, we adopted a two-reagent system, one reagent containing a homogeneous population of small DNP-labeled liposomes and one containing antibody/substrate. This system required calibration only once a week. Within-run and between-run CVs were 0.4-1.3% (n = 10) and 1.8-4.7% (n = 10), respectively. Serum results were linear upon dilution (with saline) over a twofold range. Bilirubin, hemoglobin, Intrafat, and serum proteins such as rheumatoid factor, M protein, IgG, and IgA did not affect the assay results. The results (y) correlated well with those from a hemolytic complement activity test (x): y = 1.05x - 1.14, r = 0.92, on 66 samples in the range < 10- > 50 kU/L. This method should therefore be of great use for the determination of complement activity.
Elimination of paraprotein interference in determination of plasma inorganic phosphate by ammonium molybdate method
Authors: Zaman Z , Sneyers L , Van Orshoven A , Blanckaert N , Mariën G
Published: January 2020

Phosphate concentrations were determined in 52 cases of paraproteinemia. The unmodified acidic ammonium molybdate method produced 19% spuriously high results. The false increase of phosphate concentration was attributable to formation of precipitate in the reaction mixture. The precipitate was formed by interaction between immunoglobulins and the unmodified acidic ammonium molybdate reagent. The magnitude of interference bore no relation to the type, concentrations, or isoelectric point of the paraproteins or to the presence or absence of free light chains. Diluting the sample to approximately 40 g/L total protein reduced but did not always eliminate the interference. In some cases paraprotein concentration as low as 8 g/L falsely increased plasma phosphate results. Apparently, only IgG and IgM but not IgA paraproteins produced the interference. Deproteination by ultrafiltration or by treatment with trichloroacetic acid removed the interference. The Kodak slide method and the new modified Boehringer Mannheim phosphate test were found to be interference-free. However, in some cases the latter new formulation is sensitive to substantial changes in ionic concentration of the reaction mixture.
Pseudo-pseudohypertriglyceridemia: a case of increased free glycerol without evidence for glycerol kinase deficiency
Authors: Nauck M , Winkler K , Siekmeier R , Marangos N , Richter B , März W , Wieland H
Published: January 2020

High concentrations of glycerol in plasma may result in overestimation of triglycerides in chemical and enzymatic assays. Known causes of increased glycerol concentrations in blood include glycerol-contaminated blood-collector tubes and pseudohypertriglyceridemia, a condition caused by genetic defects in the gene for glycerol kinase. We report a patient with sudden hearing loss who presented with an increased blood glycerol concentration after undergoing an oral glycerol test.
Bone alkaline phosphatase and height velocity in short normal children undergoing growth-promoting treatments: longitudinal study
Authors: Crofton P M , Stirling H F , Kelnar C J
Published: January 2020

We studied the temporal and quantitative relation between bone alkaline phosphatase (ALP) and height velocity in 62 short normal children as part of a prospective randomized study to compare placebo, growth hormone, oxandralone, and testosterone, singly and in combination, in promoting short-term growth acceleration and increased final height. The pretreatment cross-sectional correlation between bone ALP and height velocity was poor (P > or = 0.25), but was much higher (P = 0.0001) 3 months after treatment started. In each treatment group, there was a parallel relation between bone ALP and height velocity through time. Individual children showed a variety of growth responses over 12-42 months, but in almost all cases bone ALP paralleled height velocity. Within individual children, bone ALP was strongly correlated with 6-month height velocity (r > 0.9 in 30% of the children, r > 0.7 in 70%). We conclude that bone ALP is a useful short-term marker of growth in short normal children treated with growth hormone.
Validation of allele-specific polymerase chain reaction for DNA typing of HLA-B27
Authors: Steffens-Nakken H M , Zwart G , van den Bergh F A
Published: January 2020

To find a specific method for HLA-B27 typing for the diagnosis of rheumatic disorders, we extensively tested the single-step B27-specific polymerase chain reaction (PCR) described by Dominguez et al. (Immunogenetics 1992;36:277-82). This method, which relies on specific primer recognition of a sequence in the third exon (unique to the B27-allele), was used for screening of 270 characterized blood samples, 57 of which were B27-positive. The method proved to be both sensitive and specific: It unambiguously identified all B27-positive samples and produced no false-positive results. For approximately 1% of the samples, we had to repeat DNA isolation and PCR to obtain a clear control amplification signal. In contrast to the specificity of the PCR method, parallel-performed flow cytometry gave ambiguous results in 3% of the samples because of antibody cross-reactivity. Flow cytometry and the PCR method described were similar in labor and costs. Therefore, we conclude that the proposed single-step PCR is feasible in a routine laboratory and would improve the reliability of HLA-B27 typing.
Prevalence of serum antithyroid peroxidase antibodies in 85-year-old women and men
Authors: Sundbeck G , Edén S , Jagenburg R , Lundberg P A , Lindstedt G
Published: January 2020

Antithyroid peroxidase antibodies (TPOAb) were determined by competitive radioassay in serum samples obtained from a representative population of 85-year-olds (601 women and 285 men). The prevalence of increased TPOAb concentration was significantly higher in the women. In individuals without previously known thyroid dysfunction, 16% of the women and 9% of the men had TPOAb concentration > 100 kilo-units/L, the upper decision limit. We found a direct relationship between TPOAb and thyrotropin (TSH) concentrations and an inverse relationship between TPOAb and free thyroxine (T4) concentrations, but no relationship between TPOAb and defined disorders or treatment with pharmaceutical drugs. Exclusion of individuals with increased TPOAb concentration and conditions influencing TSH and free T4 concentrations resulted in a decrease of the upper reference limit for TSH concentration and an increase of the lower limit for free T4 concentration, indicating that high TPOAb concentration should be taken into account when evaluating normal reference intervals for thyroid-function tests in the elderly.