bluuebunny/crossref_metadata_2025 huggingface dataset:
Evaluation of a Fully Automated Serum Assay for C-Terminal Cross-Linking Telopeptide of Type I Collagen in Osteoporosis
Authors: Garnero Patrick , Borel Olivier , Delmas Pierre D
Published: January 2020

Background: Biochemical markers of bone turnover can provide prognostic information about the risk of osteoporotic fracture and are useful tools for monitoring efficacy of antiresorptive therapy. A serum-based automated assay may be of better clinical value than urinary markers because of lower imprecision and day-to-day within-person variability. Our aim was to evaluate the technical and clinical performances of a new, fully automated assay for serum C-terminal cross-linking telopeptide of type I collagen (CTX), a marker of bone resorption.

Methods: Serum CTX was measured on the Elecsys 2010 automated analyzer (Roche). Results were compared with those of the manual ELISA. We measured serum CTX concentrations in 728 healthy women, ages 31–89 years. We investigated the ability of this assay to predict the rate of postmenopausal forearm bone loss evaluated by four repeated bone mineral density measurements using dual-x-ray absorptiometry in 305 women followed prospectively for 4 years. Finally, in a cohort of healthy, untreated, postmenopausal women, we compared baseline serum CTX in 55 women who subsequently had a fracture (20 vertebral and 35 peripheral fractures) with values in the 380 women who did not fracture during a mean 5 years of follow-up.

Results: The within- (n = 21) and between-run (n = 21) CVs were <4.1% and 5.7%, respectively. In 728 healthy women, serum CTX concentrations (automated) correlated with those of the manual ELISA (r = 0.82; P <0.0001). The median long-term within-person variability assessed by four repeated measurements over 3 months in 18 postmenopausal women was 9.4%. Compared with 254 premenopausal women, serum CTX was 39% (P <0.0001) higher in 45 perimenopausal women and 86% (P <0.0001) higher in 429 postmenopausal women (mean age, 64 years). Baseline serum CTX correlated negatively with changes of bone mass measured at the mid (r = −0.23; P <0.0001) and distal (r = −0.27; P <0001) radius. Postmenopausal women with serum CTX greater than the mean + 2 SD values in premenopausal women accounted for 42% of the population, lost bone at the mid radius on average eightfold more rapidly than the other women (−0.27% ± 2.92% vs −2.25% ± 3.95%; P <0.0001), and had increased risk of fracture with a relative risk (95% confidence interval) of 1.8 (1.01–3.1) after adjustment for physical activity.

Conclusions: The automated assay for serum CTX is precise and predicts rate of bone loss and fracture risk in postmenopausal women. Because it is convenient to use and has high throughput, this serum bone resorption marker may be useful for the investigation of patients with osteoporosis.
The Chemical Work of Alexander and Jane Marcet
Authors: Rosenfeld Louis
Published: January 2020

Alexander Marcet was an authority on urinary calculi and their analysis when few medical practitioners appreciated the usefulness of chemistry in the explanation and treatment of disease. In An Essay on The Chemical History and Medical Treatment of Calculous Disorders, he described the discovery of an xanthine stone. He drew line illustrations of simple chemical apparatus useful for bedside analysis. His microtechnique used drops of solution and pinhead pieces of calculi; reagents were acids and alkalies and the blowpipe in conjunction with a small alcohol lamp. He reported the earliest description of a disorder later named “alcaptonuria”. Marcet’s work and that of a few others, on the chemical composition of urine and calculi, laid the foundations of our present knowledge. Between 1807 and 1820, his lectures to the medical students at Guy’s Hospital were illustrated by experiments.

Jane Haldimand Marcet wrote the very popular Conversations on Chemistry (16 editions in Great Britain). Her book dominated elementary chemical instruction during the first half of the 19th century. She followed Lavoisier’s scheme of classification and explained chemical reactions in terms of affinity, aggregation, gravitation, and repulsion. Her advocacy that experimentation accompany lecture was new. The availability of serious scientific education in the new women’s academies set the stage for increasing women’s involvement in science. She also published a series of Conversations. The topics were Political Economy, Natural Philosophy, and Vegetable Physiology.
Quantification of HER2/neu Gene Amplification by Competitive PCR Using Fluorescent Melting Curve Analysis
Authors: Lyon Elaine , Millson Alison , Lowery Mary C , Woods Rachel , Wittwer Carl T
Published: January 2020

Background: Molecular detection methods for HER2/neu gene amplification include fluorescence in situ hybridization (FISH) and competitive PCR. We designed a quantitative PCR system utilizing fluorescent hybridization probes and a competitor that differed from the HER2/neu sequence by a single base change.

Methods: Increasing twofold concentrations of competitor were coamplified with DNA from cell lines with various HER2/neu copy numbers at the HER2/neu locus. Competitor DNA was distinguished from the HER2/neu sequence by a fluorescent hybridization probe and melting curve analysis on a fluorescence-monitoring thermal cycler. The percentages of competitor to target peak areas on derivative fluorescence vs temperature curves were used to calculate copy number.

Results: Real-time monitoring of the PCR reaction showed comparable relative areas throughout the log phase and during the PCR plateau, indicating that only end-point detection is necessary. The dynamic range was over two logs (2000–250 000 competitor copies) with CVs <20%. Three cell lines (MRC-5, T-47D, and SK-BR-3) were determined to have gene doses of 1, 3, and 11, respectively. Gene amplification was detected in 3 of 13 tumor samples and was correlated with conventional real-time PCR and FISH analysis.

Conclusion: Use of relative peak areas allows gene copy numbers to be quantified against an internal competitive control in <1 h.
Relationship between Triglyceride Concentrations and LDL Size Evaluated by Malondialdehyde-modified LDL
Authors: Kondo Akira , Muranaka Yoshinori , Ohta Isao , Notsu Kazuaki , Manabe Mitsuhisa , Kotani Kazuo , Saito Kazunori , Maekawa Masato , Kanno Takashi
Published: January 2020

Background: Hypertriglyceridemia is associated with decreased HDL-cholesterol (HDL-C) and increased small dense LDL. In addition, small dense LDL is known to be susceptible to oxidation.

Methods: We measured LDL particle size, using gradient gel electrophoresis, and malondialdehyde-modified LDL (MDA-LDL), using an ELISA, and investigated the association between triglyceride (TG) concentrations, LDL size, and MDA-LDL.

Results: TG concentrations correlated negatively with the predominant LDL size (r = −0.650) and HDL-C concentration (r = −0.556). The relationship between TG concentration and LDL size, evaluated by measuring MDA-LDL, distinguished subgroups derived from four subfractions of TG concentrations and four distribution ranges of LDL size. These experiments indicated that there is a threshold for oxidation susceptibility at an LDL size of 25.5 nm and a TG concentration of 1500 mg/L. To investigate the relationship between LDL size, MDA-LDL concentration, and other lipids (TGs, HDL-C, apolipoprotein B, and total cholesterol), we evaluated them in control subjects and patients with diabetes mellitus or hypertriglyceridemia. When the size range for normal LDL was postulated to be 25.5 ≤ φ (LDL diameter) < 26.5 nm, the MDA-LDL concentration was significantly higher in the subgroups of patients with LDL in the size range 24.5 ≤ φ < 25.5 nm compared with patients with normal LDL. This result also suggests that the threshold is at a LDL size of 25.5 nm.

Conclusion: The threshold for oxidation susceptibility coincided with the point of LDL size separation between the LDL subclass patterns A and B as an atherosclerotic risk.
Association between the Prevalence of Antibodies to β2-Glycoprotein I, Prothrombin, Protein C, Protein S, and Annexin V in Patients with Systemic Lupus Erythematosus and Thrombotic and Thrombocytopenic Complications
Authors: Nojima Junzo , Kuratsune Hirohiko , Suehisa Etsuji , Futsukaichi Yoshiaki , Yamanishi Hachiro , Machii Takashi , Iwatani Yoshinori , Kanakura Yuzuru
Published: January 2020

Background: Anti-phospholipid (aPL) antibodies (Abs) frequently found in the plasma of patients with systemic lupus erythematosus (SLE) have been associated with thrombotic complications. Our aim was to clarify the roles in thrombosis of aPL Abs that react with complexes of phospholipids and plasma proteins such as β2-glycoprotein I (β2-GPI), prothrombin, protein C, protein S, and annexin V.

Methods: We determined the prevalence of aPL Abs to various phospholipid-binding plasma proteins in SLE patients with arterial thrombosis (30 cases), venous thrombosis (19 cases), thrombocytopenia (14 cases), fetal loss (14 cases), and patients without complications (91 cases). The aPL Abs were measured by an ELISA system in which human plasma proteins (β2-GPI, prothrombin, protein C, protein S, and annexin V) were immobilized on γ-irradiated or plain polystyrene plates.

Results: All types of aPL Abs were frequently observed in the patients with SLE when γ-irradiated polystyrene plates were used (51 of 168 cases positive for anti-β2-GPI, 94 of 168 cases positive for anti-prothrombin, 36 of 168 cases positive for anti-protein C, 47 of 168 cases positive for anti-protein S, and 50 of 168 cases positive for anti-annexin V), whereas no Abs to these plasma proteins were detected when plain polystyrene plates were used. Multivariate analysis confirmed that both anti-β2-GPI and anti-prothrombin Abs were significant risk factors for arterial thrombosis [odds ratios (ORs), 8.8 and 14.5, respectively; 95% confidence intervals (CIs), 3.2–25 and 1.8–116, respectively] but not for venous thrombosis. The presence of anti-protein S Abs was a significant risk factor for venous thrombosis (OR, 30.4; CI, 3.3–281) but not for arterial thrombosis. The only significant risk factor for fetal loss was the presence of anti-annexin V Abs (OR, 5.9; CI, 1.4–14.8).

Conclusions: Patients with SLE frequently have some aPL Abs to β2-GPI, prothrombin, protein C, protein S, and annexin V. Thrombotic complications in SLE may depend on the antigenic specificities of these Abs, alone or in combination.
Serial Measurements of C-Reactive Protein and Interleukin-6 in the Immediate Postnatal Period: Reference Intervals and Analysis of Maternal and Perinatal Confounders
Authors: Chiesa Claudio , Signore Fabrizio , Assumma Marcello , Buffone Elsa , Tramontozzi Paola , Osborn John F , Pacifico Lucia
Published: January 2020

Background: There is a wide range of reported sensitivities and specificities for C-reactive protein (CRP) and interleukin-6 (IL-6) in the detection of early-onset neonatal infection. This prompted us to assess reference intervals for CRP and IL-6 during the 48-h period immediately after birth and to identify maternal and perinatal factors that may affect them.

Methods: CRP and IL-6 values were prospectively obtained for 148 healthy babies (113 term, 35 near-term) at birth and at 24 and 48 h of life, and from their mothers at delivery.

Results: Upper reference limits for CRP at each neonatal age were established. At birth, CRP was significantly lower than at 24 and 48 h of life. Rupture of membranes ≥18 h, perinatal distress, and gestational hypertension significantly affected the neonatal CRP dynamics, but at specific ages. There was no correlation between CRP concentrations in mothers and their offspring at birth. The IL-6 values observed in the delivering mothers and in their babies at all three neonatal ages were negatively associated with gestational age. In the immediate postnatal period, IL-6 dynamics for term babies were significantly different from those for near-term babies. Maternal IL-6 concentrations correlated with babies’ IL-6 concentrations only for term deliveries. Apgar score had a significant effect on babies’ IL-6 values at birth.

Conclusions: The patterns of CRP and IL-6 responses in the healthy neonate should be taken into account to optimize their use in the diagnosis of early-onset neonatal sepsis.
Novel Approach for the Determination of the Redox Status of Homocysteine and Other Aminothiols in Plasma from Healthy Subjects and Patients with Ischemic Stroke
Authors: Williams Robert H , Maggiore Jack A , Reynolds Robert D , Helgason Cathy M
Published: January 2020

Background: Plasma “redox” status can be assessed by measurements of reduced (r)-, free (f)-, oxidized (ox)-, and protein-bound (b)-homocysteine (Hcy) plus the related aminothiols cysteine, cysteinylglycine (CysGly), and glutathione (GSH), but sample collection has been complex. The redox status has not been determined in ischemic stroke patients and may provide increased understanding of its role in pathogenesis. We wished to examine the feasibility of this measurement in samples collected in readily available acidic sodium citrate.

Methods: We measured aminothiols and their stability in stabilized protein-free filtrate using acidic sodium citrate (BioPool® StabilyteTM, pH 4.3) vs EDTA whole blood. Before analysis, plasma samples were also ultrafiltered to obtain a protein-free filtrate. The concentrations of total Hcy (tHcy), fHcy, and rHcy and their related aminothiols, cysteine, cysteinylglycine, and glutathione were simultaneously determined on acidic sodium-citrated blood using reversed-phase HPLC with fluorescence detection. Bound and oxidized aminothiols were calculated by difference using the concentrations of the total, free, and reduced fractions. Using this approach, we compared the redox status in newly diagnosed ischemic stroke patients (n = 20) and healthy age- and sex-matched subjects (n = 20).

Results: tHcy, tCys, tCysGly, and tGSH concentrations in whole blood with Stabilyte were stable for 8 h; the reduced fraction of each aminothiol was stable for 4 h. Recovery in the protein-free filtrate was 90–100% for all reduced thiols in acidified sodium-citrated blood. Patients with ischemic stroke had higher plasma tHcy, fHcy, bHcy, rHcy, and oxHcy (P <0.0005) and higher plasma t-, f-, r-, and oxCys (P <0.05). t-, b-, and rCysGly concentrations were lower in the stroke patients (P <0.05), as were t-, b-, and oxGSH (P <0.005).

Conclusions: Collection of blood in acidic sodium citrate (BioPool Stabilyte) permits the determination of the redox status of Hcy and its related aminothiols, which may add to the understanding of their relationship to the etiology of cerebrovascular disease.
Quantification of Unconjugated Metanephrines in Human Plasma without Interference by Acetaminophen
Authors: Roden Michael , Raffesberg Wolfgang , Raber Wolfgang , Bernroider Elisabeth , Niederle Bruno , Waldhäusl Werner , Gasic Slobodan
Published: January 2020

Background: Pheochromocytoma is a rare cause of hypertension resulting from increased catecholamine secretion. We aimed to develop a method to measure unconjugated plasma normetanephrine (NMN) and metanephrine (MN) without interference from acetaminophen, a widely prescribed drug for headaches.

Methods: Plasma samples were obtained from 48 subjects (23 males, 25 females; mean age, 49 ± 14 years; hypertension, n = 37) under resting conditions. Following extraction on solid-phase cation-exchange columns, unconjugated metanephrines were analyzed by HPLC with electrochemical detection and with 4-hydroxy-3-methoxybenzylamine as an internal standard. Catecholamines were measured by HPLC.

Results: The assays were linear up to 2000 pg for NMN and for MN. Intraassay imprecisions (CVs) were 4.7% for NMN and 7.0% for MN, and the interassay CV was 12% for both NMN and MN. The limit of detection was 11 fmol for NMN and 17 fmol for MN. Ingestion of acetaminophen or its addition to plasma did not interfere with the MN peaks. Plasma NMN and MN were positively correlated (r = 0.52 and 0.49, respectively; P <0.01 for both) with the respective catecholamines. Plasma NMN (r = 0.27; P = 0.02) but not MN positively correlated with age, whereas only plasma catecholamines (and not metanephrines) were positively correlated (P <0.05) with diastolic blood pressure.

Conclusions: This sensitive MN assay is not affected by simultaneous acetaminophen medication, and reveals a correlation of metanephrines with plasma and urinary catecholamines and age but not with blood pressure.
Nitric Oxide Measurements during Endotoxemia
Authors: Brovkovych Viktor , Dobrucki Lawrence W , Brovkovych Svitlana , Dobrucki Iwona , Kalinowski Leszek , Kiechle Frederick , Malinski Tadeusz
Published: January 2020

Background: Excessive continuous NO release from inducible NO synthase over prolonged periods under pathological conditions, such as endotoxemia, contributes significantly to circulatory failure, hypotension, and septic shock. This NO production during endotoxemia is accompanied by superoxide release, which contributes to the fast decay of NO. Therefore, the amount of NO that diffuses to target sites may be much lower than the total amount released under pathological conditions.

Methods: We performed in vivo and ex vivo measurements of NO (electrochemical) and ex vivo in situ measurements of superoxide, peroxynitrite (chemiluminescence), and nitrite and nitrate (ultraviolet-visible spectroscopy). We determined the effect of lipopolysaccharide administration (20 mg/kg) on diffusible NO, total NO (diffusible plus consumed in chemical reactions), and superoxide and peroxynitrite release in the pulmonary arteries of rats.

Results: An increase in diffusible NO generated by constitutive NO synthase was observed immediately after administration of lipopolysaccharide, reaching a plateau (145 ± 18 nmol/L) after 540 ± 25 s. The plateau was followed by a decrease in NO concentration and its subsequent gradual increase after 45 min because of NO production by inducible NO synthase. The concentration of superoxide increased from 16 ± 2 nmol/L to 30 ± 3 nmol/L after 1 h and reached a plateau of 41 ± 4 nmol/L after 6 h. In contrast to the periodic changes in the concentration of diffusible NO, the total concentration of NO measured as a sum of nitrite and nitrate increased steadily during the entire period of endotoxemia, from 2.8 ± 0.2 μmol/L to 10 ± 1.8 μmol/L.

Conclusions: The direct measurement of NO concentrations in the rat pulmonary artery demonstrates dynamic changes throughout endotoxemia, which are related to the production of superoxide and the subsequent increase in peroxynitrite. Monitoring endotoxemia with total nitrate plus nitrite is not sensitive to these fluctuations in NO concentration.
Disappearance Rate of Catecholamines, Total Metanephrines, and Neuropeptide Y from the Plasma of Patients after Resection of Pheochromocytoma
Authors: Grouzmann Eric , Fathi Marc , Gillet Michel , de Torrenté Antoine , Cavadas Claudia , Brunner Hans , Buclin Thierry
Published: January 2020

Background: Plasma free metanephrines are a more reliable analyte to measure than catecholamines for the biochemical diagnosis of pheochromocytomas. We hypothesized that the long persistence of total (sulfate-conjugated plus free) metanephrines in the blood might have a significant diagnostic value.

Methods: We measured plasma concentrations of catecholamines and total metanephrines (sulfate-conjugated plus free forms) by HPLC with amperometric detection, and neuropeptide Y (NPY) by an amplified ELISA in seven patients before and after removal of their pheochromocytomas. The results for catecholamine, total metanephrines, and NPY in each patient were analyzed for up to 120 min, starting from the time of tumor vessel clamping. The persistence of analytes was quantified as the area under the concentration–time curve over 120 min.

Results: On the basis of the upper reference limit for each variable, plasma free norepinephrine (NE) and epinephrine (E) concentrations were increased preoperatively in at least one sample in seven and six patients, respectively. Total normetanephrine (NMN) and metanephrine (MN) were increased in all samples in seven and six patients, respectively. NPY was increased 2- to 465-fold. After removal of the tumor, MN and NMN showed a higher average relative increase above the upper limit of the reference interval than NE and E (P = 0.05), whereas NPY was intermediate. The persistence of increased values was significantly shorter for catecholamines than for metanephrines. The half-life estimated by nonlinear regression was 12.3 ± 7.8 min for NPY. Significant correlations were observed among NE, E, NMN, MN, and NPY concentrations, but parent markers (E and MN or NE and NMN) did not appear significantly intercorrelated.

Conclusions: A larger increase and a longer persistence of total metanephrines (reflecting predominantly sulfo-conjugated metanephrines) than catecholamines and NPY in plasma may contribute to their greater diagnostic accuracy in pheochromocytoma.
Total Homocysteine, Vitamin B12, and Total Antioxidant Status in Vegetarians
Authors: Herrmann Wolfgang , Schorr Heike , Purschwitz Kathrin , Rassoul Fausi , Richter Volker
Published: January 2020

Background: Decreasing or eliminating animal products from the diet decreases the intake of some essential nutrients, such as vitamin B12, which may lead to hyperhomocysteinemia. We investigated vitamin B12-dependent metabolism and oxidative stress in groups with various or no intake of meat or animal products.

Methods: We investigated 44 high meat eaters, 19 low meat eaters, 34 lacto-ovo/lacto vegetarians, and 7 vegan vegetarians. Homocysteine (HCY) was assayed by HPLC, methylmalonic acid (MMA) by capillary gas chromatography–mass spectrometry, serum folate and vitamin B12 with a chemiluminescence immunoassay, and total antioxidant status (TAS) by a Randox method.

Results: The mean serum HCY concentration of vegetarians was significantly increased, and in vegans the median concentration exceeded 15 μmol/L. Vegetarians had a higher serum concentration of MMA but a lower TAS. Vitamin B12 and folate did not differ significantly between vegetarian and omnivorous subjects. Overall, HCY and MMA were significantly correlated. Vitamin B12 correlated negatively with MMA, HCY, and folate, whereas the correlation with TAS was positive. Backward regression analysis revealed an independent influence of MMA on HCY, of HCY and vitamin B12 on MMA, and of vitamin B12 on TAS. The increased MMA concentration suggested a 25% frequency of functional vitamin B12 deficiency in all vegetarians. Serum vitamin B12 was below the lower reference limit in only five subjects.

Conclusions: Functional vitamin B12 deficiency in vegetarians may contribute to hyperhomocysteinemia and decreased TAS, which may partly counteract the beneficial lifestyle of vegetarians. However, increased serum HCY is most likely not responsible for the lower TAS values in vegetarians. We recommend assaying of MMA and HCY to investigate functional vitamin B12 status.
Development of a Dual Monoclonal Antibody Immunoassay for Total Human Kallikrein 2
Authors: Finlay Judith A , Day John R , Evans Cindy L , Carlson Robert , Kuus-Reichel Kristine , Millar Lisa S , Mikolajczyk Stephen D , Goodmanson Marcia , Klee George G , Rittenhouse Harry G
Published: January 2020

Background: Human kallikrein 2 (hK2) shares 80% sequence identity with prostate-specific antigen (PSA). Because both hK2 and hK2-α1-antichymotrypsin (hK2-ACT) complexes have been identified in patient sera, we devised an immunoassay for total hK2 [(thK2); hK2 and hK2-ACT] and evaluated it in healthy subjects and patients with prostate disease.

Methods: We developed monoclonal antibodies (mAbs) with high specificity for hK2 and hK2-ACT and minimal cross-reactivity to PSA. Using these mAbs, a sandwich assay was developed and its specificity for forms of hK2 was assessed. Serum samples (n = 1035) from healthy volunteers, patients with increased PSA, and men who had undergone radical prostatectomy were assayed for thK2. We also measured thK2 in samples before and after storage under common laboratory conditions.

Results: The minimum detectable concentration in the thK2 assay was 0.008 μg/L, and PSA cross-reactivity was <0.001%. The assay detected prohK2 and three different hK2–serum protease complexes. The median serum concentration of thK2 in control samples (0.013 μg/L) was significantly lower than the median in samples from patients with increased PSA concentrations (0.085 μg/L). Immunoreactive hK2 changed little in samples stored for up to 1 month at −70 °C.

Conclusions: The thK2 assay recognizes all forms of hK2 that have been found in bodily fluids to date.
Enhanced Susceptibility to Oxidation and Diminished Vitamin E Content of LDL from Patients with Stable Coronary Artery Disease
Authors: Haidari Mehran , Javadi Ebrahim , Kadkhodaee Mehry , Sanati Arashmidos
Published: January 2020

Background: Convincing evidence points to oxidative modification of LDL as an important trigger in a complex chain of events leading to atherosclerosis. We investigated the occurrence of enhanced susceptibility of LDL to oxidation and decreased vitamin E concentration in LDL as additional risk factors promoting atherosclerosis among patients with established coronary artery disease (CAD).

Methods: We examined 132 patients with angiographically confirmed CAD and compared them with 111 healthy control individuals. We measured conjugated diene production to assess susceptibility of LDL to copper-mediated oxidation. Vitamin E content of LDL was measured by HPLC.

Results: The mean lag time of LDL oxidation and LDL α-tocopherol/LDL-cholesterol ratio were lower in the patients with CAD (55 ± 14 min and 2.4 ± 1.0 mmol/mmol) than in the controls (63 ± 13 min and 2.9 ± 1.1 mmol/mmol; P <0.0001 and <0.001, respectively). Multiple stepwise regression analysis demonstrated the lag time (odds ratio, 1.96; 95% confidence interval, 1.34–2.87; P <0.0001) and concentration of vitamin E in LDL (odds ratio, 1.65; 95% confidence interval, 1.16–2.33; P <0.005) as independent determinants of CAD. Significant inverse Spearman rank correlations were found between lag time (r = −0.285; P <0.001) or concentration of vitamin E in LDL (r = −0.197; P <0.002) and severity of CAD. Lag times were not significantly correlated with serum C-reactive protein or ferritin.

Conclusions: Our data suggest that a short LDL oxidation lag time and a low concentration of vitamin E in LDL might be independent coronary risk factors for stable CAD in Iranian people.
Tumor Necrosis Factor-α in Serum of Patients with Inflammatory Bowel Disease as Measured by a Highly Sensitive Immuno-PCR
Authors: Komatsu Momoko , Kobayashi Daisuke , Saito Kaori , Furuya Daisuke , Yagihashi Atsuhito , Araake Hiroshi , Tsuji Naoki , Sakamaki Sumio , Niitsu Yoshiro , Watanabe Naoki
Published: January 2020

Background: The significance of serum concentrations of tumor necrosis factor-α (TNF-α) in the pathogenesis of inflammatory bowel disease (IBD) is uncertain. We measured TNF-α in serum from IBD patients by immuno-PCR to analyze the relationship between TNF-α and pathophysiologic state in IBD.

Methods: Serum samples were collected from 54 healthy blood donors, 29 patients with ulcerative colitis (UC; 46 samples), and 7 patients with Crohn disease (CD; 8 samples). DNA label was generated by PCR amplification using biotinylated primer and was bound with streptavidin to biotinylated third antibody. TNF-α sandwiched by antibodies was detected by PCR amplification of the DNA label.

Results: TNF-α could be measured in all samples. The median serum concentration in IBD patients overall was ∼390-fold higher than in healthy donors (median increase, 380-fold for UC, 640-fold for CD). The median serum TNF-α concentration was 1.7-fold higher in the active stage of UC than in the inactive stage (P <0.05), and this difference could be detected in individual patients.

Conclusions: Sensitive measurement of serum TNF-α could provide an important pathophysiologic marker for the presence and activity of IBD.
Neonatal Screening for Galactosemia by Quantitative Analysis of Hexose Monophosphates Using Tandem Mass Spectrometry: A Retrospective Study
Authors: Jensen Ulrich Glümer , Brandt Niels Jacob , Christensen Ernst , Skovby Flemming , Nørgaard-Pedersen Bent , Simonsen Henrik
Published: January 2020

Background: Classic galactosemia (OMIM 230400) is an inherited disorder in the metabolism of galactose caused by deficiency of the enzyme galactose 1-phosphate uridyl transferase (EC 2.7.7.12). Galactosemia leads to accumulation of galactose and galactose 1-phosphate (gal-1-P) in blood and tissues and, if untreated, produces neonatal death or severe mental retardation, cirrhosis of the liver, and cataracts. Hence, the disorder is included in many neonatal screening programs.

Methods: We retrospectively analyzed filter-paper blood samples obtained 4–8 days postpartum for routine neonatal screening from 12 galactosemia patients and 2055 random controls. Total hexose monophosphates (HMPs) were used as a marker of gal-1-P and were assayed by negative-ion mode electrospray tandem mass spectrometry (tandem MS) with settings biased toward gal-1-P detection. The predominant precursor/product ion pair m/z 259/79 was used to quantify total HMPs by external standardization.

Results: Linear calibration curves were obtained in the range 0–8 mmol/L gal-1-P. The detection limit was 0.1 mmol/L HMP, and total CVs ranged from 13% at the detection limit to <8% at >1 mmol/L HMP. The method was in agreement with an alkaline phosphatase-galactose dehydrogenase method. All samples from galactosemia patients contained increased HMP concentrations (range for patients, 2.6–5.2 mmol/L; range for reference group, <0.10–0.94 mmol/L). The diagnostic sensitivity and specificity were 100% at a cutoff of 1.2 mmol/L HMP. A Duarte/classic galactosemia compound heterozygous sample could be discriminated clearly from both patient and reference samples.

Conclusion: Quantitative analysis of HMPs by tandem MS can be used in laboratory investigations of galactosemia.
Plasma Homocysteine Is Not Subject to Seasonal Variation
Authors: McKinley Michelle C , Strain J J , McPartlin Joseph , Scott John M , McNulty Helene
Published: January 2020

Background: Studies investigating the relationship between plasma total homocysteine (tHcy) and vascular disease usually rely on a single measurement. Little information is available, however, on the seasonal variability of plasma tHcy. The aim of this study was to investigate the seasonal variation in fasting plasma tHcy and related B-vitamin intake and status in a group of people who did not consume fortified foods or take B-vitamin supplements.

Methods: In this longitudinal study, a group of 22 healthy people were followed for 1 year. A fasting blood sample and dietary information were collected from each individual every 3 months, i.e., at the end of each season.

Results: There was no significant seasonal variation in plasma tHcy or in B-vitamin intake or status with the exception of red cell folate (significantly lower in spring compared with autumn or winter) and serum folate (significantly lower in spring compared with the other seasons). Although the between-person variation in plasma tHcy was high (47%), the within-person variation was low (11%). This low variation, combined with the low methodologic imprecision of 3.8%, yielded a high reliability coefficient for plasma tHcy (0.97).

Conclusions: Although there was a small seasonal variation in folate status, there was no corresponding seasonal variation in plasma tHcy. The high reliability coefficient for plasma tHcy suggests that a single measurement is reflective of an individual’s average plasma tHcy concentration, thus indicating its usefulness as a potential predictor of disease. This, however, needs to be confirmed in different subgroups of the population.
Connectivity from Source to Action
Authors: Aller Raymond D
Published: January 2020

Recent advances in electronic connectivity technology make conceivable almost instantaneous movement of data from the patient or laboratory to any other point in the healthcare system. The Internet, combined with new standards for wireless data transmission, has erased many of the previous physical barriers. Interface engines and formatting/content standards have facilitated the connection of multiple disparate systems in medicine. However, major logical barriers persist, and overcoming these barriers is key to achieving functional connectivity. These include fundamental issues of patient safety (such as reliably identifying the patient), capture of the most clinically meaningful data (patient history, physical examination findings, physician diagnostic impressions, and full range of orders), unambiguous identification of data elements, and synchronization of control files among multiple different systems within the healthcare enterprise. Fortunately, tools exist to address each of these areas. This report cites illustrative examples of such tools.
The Total Laboratory Solution: A New Laboratory E-Business Model Based on a Vertical Laboratory Meta-Network
Authors: Friedman Bruce A
Published: January 2020

Major forces are now reshaping all businesses on a global basis, including the healthcare and clinical laboratory industries. One of the major forces at work is information technology (IT), which now provides the opportunity to create a new economic and business model for the clinical laboratory industry based on the creation of an integrated vertical meta-network, referred to here as the “total laboratory solution” (TLS). Participants at the most basic level of such a network would include a hospital-based laboratory, a reference laboratory, a laboratory information system/application service provider/laboratory portal vendor, an in vitro diagnostic manufacturer, and a pharmaceutical/biotechnology manufacturer. It is suggested that each of these participants would add value to the network primarily in its area of core competency. Subvariants of such a network have evolved over recent years, but a TLS comprising all or most of these participants does not exist at this time. Although the TLS, enabled by IT and closely akin to the various e-businesses that are now taking shape, offers many advantages from a theoretical perspective over the current laboratory business model, its success will depend largely on (a) market forces, (b) how the collaborative networks are organized and managed, and (c) whether the network can offer healthcare organizations higher quality testing services at lower cost. If the concept is successful, new demands will be placed on hospital-based laboratory professionals to shift the range of professional services that they offer toward clinical consulting, integration of laboratory information from multiple sources, and laboratory information management. These information management and integration tasks can only increase in complexity in the future as new genomic and proteomics testing modalities are developed and come on-line in clinical laboratories.
Guidelines and Algorithms: Perceptions of Why and When They Are Successful and How to Improve Them
Authors: Keffer Joseph H
Published: January 2020

Medicine is increasingly complex, a reality created by the explosion of knowledge during the last 50 years. The cost of applying this knowledge creates a daunting economic challenge. As a result, there has been a profusion of guidelines intended to influence medical practice. This report explores the interrelated issues and concepts that impact the value and success of guidelines. These include medical quality and error, compliance, and the impact on outcomes in an evidence-based medicine context. Lessons learned from previous guidelines must be understood in relation to human behavior. Legal implications of the guidelines must be considered because both an increase and a decrease in liability can be anticipated. Many products have been labeled “advocacy guidelines” with a negative context. They are believed to express motivation rather than optimizing care. The ideal of professionalism is challenged, and there is potential for the growing use of guidelines in enforcing punitive actions. Constructive experience has emphasized the appropriate required elements for practice guidelines: a systematic review of the literature, an assessment of the volume and level of the evidence, and development of a review process by an appropriate multidisciplinary group for consistency, clinical impact, and resource implications leading to clearly stated and reasonable recommendations. The dissemination of guidelines, beyond conventional publication in a journal, will impact the success of the intended outcomes. The exploitation of electronic avenues, including the Internet and the evolving interactive electronic medical record, seems to be essential for future success in these endeavors.
Mitochondrial Gene Mutations in the tRNALeu(UUR) Region and Diabetes: Prevalence and Clinical Phenotypes in Japan
Authors: Ohkubo Kumiko , Yamano Akemi , Nagashima Mariko , Mori Yumiko , Anzai Keizo , Akehi Yuko , Nomiyama Riku , Asano Takashi , Urae Akinori , Ono Junko
Published: January 2020

Background: Mitochondrial gene mutations play a role in the development of diabetes mellitus. We have assessed the frequency of the A3243G and other mitochondrial mutations in Japan and in the relationship to clinical features of diabetes.

Methods: DNA was obtained from peripheral leukocytes of 240 patients with diabetes mellitus (39 with type 1; 188 with type 2; 13 with gestational diabetes) and 125 control subjects. We used PCR-restriction fragment length polymorphism analysis (ApaI) for A3243G and PCR-single-strand conformation polymorphism analysis to determine the mutations in the mitochondrial gene including nucleotide position 3243.

Results: The A3243G mutation was found in seven patients, and an inverse relationship was observed between the degree of heteroplasmy and the age at onset of diabetes. A3156G, G3357A, C3375A, and T3394C were detected in addition. Those who shared the same mutation showed similar clinical characteristics, thus representing a putative clinical subtype. The patients with A3156G had a sudden onset of hyperglycemia and showed a rapid progression to an insulin-dependent state with positive anti-glutamic acid decarboxylase antibody. Those with T3394C showed a mild defect in glucose-stimulated insulin secretion, and hyperglycemia appeared after adding such factors as aging or obesity.

Conclusions: The identification of mitochondrial gene mutations allows preclinical diagnosis of diabetes and prediction of the age at onset by evaluating the degree of heteroplasmy in cases with A3243G. Mutation detection may also be important for patient management and identification of affected family members.
Multivariate Discriminant Function Based on Six Biochemical Markers in Blood Can Predict the Cirrhotic Evolution of Chronic Hepatitis
Authors: Fortunato Giuliana , Castaldo Giuseppe , Oriani Giovannangelo , Cerini Raimondo , Intrieri Mariano , Molinaro Eugenia , Gentile Ivan , Borgia Guglielmo , Piazza Marcello , Salvatore Francesco , Sacchetti Lucia
Published: January 2020

Background: Serologic markers have been proposed for monitoring hepatic fibrosis in chronic active liver disease. Because none of these markers, when used singly, is totally satisfactory, we developed and evaluated a multivariate approach.

Methods: We studied two cohorts of chronic hepatitis (54 patients) and cirrhosis patients (49 patients) to identify a panel of biochemical markers that discriminates between the two diseases. Using multivariate discriminant analysis, we selected a function, based on the concentrations of six biochemical markers (fibronectin, prothrombin, pseudocholinesterase, alanine aminotransferase, manganese superoxide dismutase, and N-acetyl-β-glucosaminidase). We then prospectively validated this function on a second temporal cohort of patients.

Results: Multivariate discriminant analysis correctly classified 93.7% of patients (94.3% of chronic hepatitis and 92.9% of cirrhosis patients) in the first cohort and 85% of patients (89.5% of chronic hepatitis patients and 81% of cirrhosis patients) in the second cohort.

Conclusions: Discriminant analysis of results of six inexpensive biochemical markers provides a high predictive value for differentiation between liver cirrhosis and chronic hepatitis. Consequently, these biochemical markers condensed into a multivariate discriminant analysis value for each patient provide information that can be contributory for subsequent options during the evolution of the natural history of chronic hepatitis.
Differential DNA Methylation between Fetus and Mother as a Strategy for Detecting Fetal DNA in Maternal Plasma
Authors: Poon Leo LM , Leung Tse N , Lau Tze K , Chow Katherine CK , Lo YM Dennis
Published: January 2020

Background: Fetal DNA has been detected in maternal plasma by the use of genetic differences between mother and fetus. We explore the possibility of using epigenetic markers for the specific detection of fetal DNA in maternal plasma.

Methods: A differentially methylated region in the human IGF2-H19 locus and a single-nucleotide polymorphism in this region were chosen for the study. The methylation status in this region is maintained in such a way that the paternal allele is methylated and the maternal allele is unmethylated. The single-nucleotide polymorphism was typed by direct sequencing of PCR products. The methylation status of this region was ascertained by bisulfite conversion and methylation-specific PCR. Differentially methylated fetal alleles were detected in maternal plasma by direct sequencing and a primer-extension assay.

Results: Women in the second (n = 21; 17–21 weeks) and third (n = 18; 37–42 weeks) trimesters of pregnancy were recruited. Among these 39 volunteers, the 16 who were heterozygous for the single-nucleotide polymorphism were chosen for further analysis. In 11 of these 16 cases, paternally inherited methylated fetal alleles were different from the methylated alleles of the respective mothers. Using direct sequencing, we detected paternally inherited methylated fetal DNA in 6 of 11 (55%) cases. In 8 of the 16 heterozygous cases, the fetuses possessed an unmethylated maternally inherited allele that was different from the unmethylated allele of the mother. Using a primer-extension assay, we detected fetal-derived maternally inherited alleles in maternal plasma of four of eight (50%) cases.

Conclusions: These results represent the first use of fetal epigenetic markers in noninvasive prenatal analysis. These data may also have implications for the investigation of other types of chimerism.
The History and Future of the Fluorescence Activated Cell Sorter and Flow Cytometry: A View from Stanford
Authors: Herzenberg Leonard A , Parks David , Sahaf Bita , Perez Omar , Roederer Mario , Herzenberg Leonore A
Published: January 2020

The Fluorescence Activated Cell Sorter (FACS) was invented in the late 1960s by Bonner, Sweet, Hulett, Herzenberg, and others to do flow cytometry and cell sorting of viable cells. Becton Dickinson Immunocytometry Systems introduced the commercial machines in the early 1970s, using the Stanford patent and expertise supplied by the Herzenberg Laboratory and a Becton Dickinson engineering group under Bernie Shoor. Over the years, we have increased the number of measured FACS dimensions (parameters) and the speed of sorting to where we now simultaneously measure 12 fluorescent colors plus 2 scatter parameters. In this history, I illustrate the great utility of this state-of-the-art instrument, which allows us to simultaneously stain, analyze, and then sort cells from small samples of human blood cells from AIDS patients, infants, stem cell transplant patients, and others. I also illustrate analysis and sorting of multiple subpopulations of lymphocytes by use of 8–12 colors. In addition, I review single cell sorting used to clone and analyze hybridomas and discuss other applications of FACS developed over the past 30 years, as well as give our ideas on the future of FACS. These ideas are currently being implemented in new programs using the internet for data storage and analysis as well as developing new fluorochromes, e.g., green fluorescent protein and tandem dyes, with applications in such areas as apoptosis, gene expression, cytokine expression, cell biochemistry, redox regulation, and AIDS. Finally, I describe new FACS methods for measuring activated kinases and phosphatases and redox active enzymes in individual cells simultaneously with cell surface phenotyping. Thus, key functions can be studied in various subsets of cells without the need for prior sorting.
Screening for Primary Aldosteronism: Implications of an Increased Plasma Aldosterone/Renin Ratio
Authors: Schwartz Gary L , Chapman Arlene B , Boerwinkle Eric , Kisabeth Robert M , Turner Stephen T
Published: January 2020

Background: The value of the ratio of plasma aldosterone concentration (aldosterone) to plasma renin activity (renin) to identify patients at risk for primary aldosteronism is controversial. We determined the sensitivity, specificity, and predictive value of the ratio to identify combinations of renin and aldosterone compatible with primary aldosteronism.

Methods: The ratio was calculated in 505 adults with essential hypertension (143 black women, 82 black men, 122 white women, and 158 white men). We used a conventional cutpoint for an increased ratio (i.e., 20 mL/dL · h). The primary combination of renin and aldosterone considered compatible with primary aldosteronism was increased aldosterone for the concomitant renin, defined as aldosterone in the highest quartile predicted by linear regression on renin. Renin was considered low if it was in the lowest quartile of the sample distribution.

Results: The sensitivity of the ratio to identify individuals with increased aldosterone for the concomitant renin was 66% (80% in blacks and 56% in whites; P = 0.009), and the specificity of the ratio was 67% (46% in blacks and 84% in whites; P <0.0001). In 36% of instances of an increased ratio, it was a measure of low renin alone without increased aldosterone for renin (32% in blacks and 45% in whites; P = 0.072). The positive predictive value of the ratio to identify individuals with increased aldosterone for the concomitant renin was 34% (49% in whites and 27% in blacks; P <0.002).

Conclusion: The aldosterone/renin ratio lacks sensitivity and specificity and has only a modest predictive value for combinations of renin and aldosterone that are compatible with primary aldosteronism.