bluuebunny/crossref_metadata_2025_split huggingface dataset:
Homocysteine and other thiols in plasma and urine: automated determination and sample stability
Authors: Fiskerstrand T , Refsum H , Kvalheim G , Ueland P M 
Published: January 2020

Abstract
We have developed a modified version of our fully automated column-switching HPLC method for determining total plasma homocysteine based on single-column (reversed-phase) separation. Homocysteine, cysteine, and cysteinylglycine in plasma (total concentrations), acid-precipitated plasma (non-protein-bound concentrations), and urine can be determined. The derivatization and chromatography were performed automatically by a sample processor. The successful separation of all thiol species (within 15 min) was accomplished by accurate adjustment of the pH of the mobile phase to 3.65 (plasma) or 3.50 (acid-precipitated plasma, urine). Maximal fluorescence yield of cysteine, cysteinylglycine, and, to a lesser degree, homocysteine was dependent on optimal concentrations of EDTA and dithioerythritol during reduction (with NaBH4) and derivatization (with monobromobimane). The method is sensitive (detection limit approximately 0.05 pmol) and has a high degree of precision (CV &lt; 5%). The sample output is approximately 70 samples in 24 h. Serum and heparin plasma can also be analyzed. Hemolysis up to approximately 2.0 g/L of hemoglobin did not interfere with the analytical recovery of homocysteine or cysteine. Collection of blood, separation of plasma from whole blood, and acid precipitation must be standardized to obtain reproducible thiol results. Our modifications and the standardization of blood-sampling procedures have substantially improved the method and broadened its applications.
Evaluation of a novel point-of-care system, the i-STAT portable clinical analyzer
Authors: Erickson K A , Wilding P 
Published: January 2020

Abstract
We evaluated a novel system designed for rapid, point-of-care measurement of sodium, potassium, chloride, urea nitrogen, glucose, and hematocrit. The i-STAT Portable Clinical Analyzer (PCA) system is composed of a hand-held analyzer and disposable cartridges. Sample analysis takes place in the cartridge, which contains a series of thin-film electrodes microfabricated on silicon chips. The PCA was evaluated for precision, accuracy, and utility in emergency department and stat laboratory settings. Precision did not differ significantly between these two locations, the CVs being as follows: sodium, 0.46-0.89%; potassium, 1.06-1.45%; chloride, 0.69-2.76%; urea nitrogen, 2.54-6.12%; and glucose, 4.39-5.19%. The assessment of accuracy was based on comparison of patients' sample values analyzed by the PCA and the Kodak Ektachem 700 (or the Coulter ST for hematocrit). Regression statistics were acceptable for all analytes except chloride, for which the regression data were influenced by the limited range of results. A difference plot of the chloride comparison showed that the bias rarely exceeded 5 mmol/L. Mean hematocrit values significantly differed between the PCA and the Coulter ST, apparently because of different calibration procedures.
Clinical and analytical evaluation of a continuous enzymatic method for measuring pancreatic lipase activity
Authors: Panteghini M , Pagani F , Bonora R 
Published: January 2020

Abstract
We report the evaluation of a new commercial kit for the determination of pancreatic lipase activity. The kit is based on the use of a 1,2-diglyceride as substrate and a specific monoglyceride lipase. The detection step is the continuous colorimetric measurement of hydrogen peroxide produced from glycerol by glycerol kinase, glycerol-3-phosphate oxidase, and peroxidase reactions. The procedure appears to be precise (between-day CV &lt; 9%) and the results show good correlation with those obtained by alternative procedures (vs turbidimetry, r = 0.965; vs ultraviolet absorbance-enzymatic method, r = 0.995; vs Ektachem, r = 0.976; vs immunometry, r = 0.970). However, the method is susceptible to interference by increased concentrations (&gt; 4.5 mmol/L) of serum triglycerides. We estimated the reference interval for healthy adults to be 8-44 U/L. When we evaluated clinical efficacy by using receiver-operating characteristic curves and the overlap index, no significant differences were found between the commercial kit and a common turbidimetric assay for diagnosing patients with acute pancreatitis; both methods performed satisfactorily.
Assessment and optimization of kinetic methods for angiotensin-converting enzyme in plasma
Authors: Buttery J E , Stuart S 
Published: January 2020

Abstract
In the kinetic angiotensin-converting enzyme (ACE) method, a practical and optimal buffer is 80 mmol/L borate buffer at pH 8.2 (37 degrees C). A lag phase is detected in the reaction, and a 5-min incubation of substrate and plasma is suggested before the kinetic measurement. The substrate, N-[3-(2-furyl)acryloyl]-L-phenylalanylglycylglycine (FAPGG), concentration is maximized at 1.0 mmol/L and the measurement wavelength is at 345 nm to ensure linearity of measurement. The proposed procedure uses a 1:9 plasma-to-reagent volume ratio. The linear range of the assay extends to approximately 170 U/L, representing a 25% substrate hydrolysis. The FAPGG absorptivity is determined by measuring the difference in absorbance between 1.0 mmol/L FAPGG and the product solutions. The wavelength fidelity is checked by noting the expected absorbance value of the FAPGG solution, and a 1.0-nm deviation from 345 nm alters the absorbance by 15.5%. The precision of ACE assays at approximately 60 and 100 U/L is 3.5% and 2.4% within batch and 2.9% and 2.6% between batch, respectively. The reference interval (2.5th to 97.5th percentiles) is 41-139 U/L, and there is no difference between values for men and women.
Tissue-specific promoters regulate aromatase cytochrome P450 expression
Authors: Simpson E R , Mahendroo M S , Means G D , Kilgore M W , Corbin C J , Mendelson C R 
Published: January 2020

Abstract
In humans, estrogen biosynthesis occurs in several tissue sites, including ovary, placenta, adipose, and brain. Recent work from our laboratory indicates that tissue-specific expression of aromatase cytochrome P450 (P450arom), the enzyme responsible for estrogen biosynthesis, is determined, in part, by the use of tissue-specific promoters. Thus, the expression of P450arom in human ovary appears to utilize a promoter proximal to the translation start site. This promoter is not utilized in placenta; instead, the promoter used to drive aromatase expression in placenta is &gt; or = 40 kb upstream from the translational start site. In addition, a minor promoter used in the expression of a small proportion of placental transcripts is 9 kb upstream from the start of translation. Transcripts from these promoters are also expressed in other fetal tissues, including placenta-related cells such as JEG-3 choriocarcinoma cells and hydatidiform moles and other fetal tissues such as fetal liver. In adipose tissue, on the other hand, expression of P450arom may be achieved by yet another, adipose-specific promoter. The various 5'-untranslated exons unique for expression driven by each of these promoters are spliced into a common intron/exon boundary upstream from the translational start site. This means that the protein expressed in each of the various tissue-specific sites of estrogen biosynthesis is identical.
Transcriptional regulation of human genes for steroidogenic enzymes
Authors: Hum D W , Miller W L 
Published: January 2020

Abstract
Steroid hormones are synthesized from cholesterol in the adrenals, gonads, and placenta by a complex series of reactions. The human genes encoding each of these biosynthetic enzymes have been cloned, permitting study of their regulation. Tropic hormones, such as corticotropin and the gonadotropins, exert their chronic effects on steroidogenesis by increasing the amounts of steroidogenic enzymes; this in turn occurs primarily through increased gene transcription. Our studies have emphasized the cholesterol side-chain cleavage enzyme P450scc, which catalyzes the first and rate-limiting step in steroidogenesis, and P450c17, which determines what class of steroids is synthesized. By fusing the promoters of the genes for these enzymes to readily assayed reporter genes and transiently transfecting cultured cells with these constructions, we have identified the regions of each promoter that confer basal expression, induction by cAMP, and repression by activators of protein kinase C. Different segments of the P450scc promoter are used for each of these purposes in different cell types, indicating that the regulation of this gene is very complex. Transcription is not the only level at which steroidogenesis is regulated. The abundance of mRNA for adrenodoxin reductase, a flavoprotein needed for P450scc activity, is post-transcriptionally regulated by cAMP.
Production of recombinant androgen receptor in a heterologous expression system
Authors: Jänne O A , Palvimo J J , Kallio P , Mehto M , Xie Y B , Sui Y P 
Published: January 2020

Abstract
To facilitate detailed studies of androgen receptor, we have produced a full-length receptor protein and some of its deletion mutants in Spodoptera frugiperda (Sf9) insect cells, using the baculovirus expression system. Recombinant baculovirus DNA-infected Sf9 cells expressed these proteins in very high quantities, which represented as much as 30-40% of total insect cell protein at 72 h after infection. Only &lt; 1% of the recombinant protein was soluble in low-salt buffers; the majority formed electron-dense cytoplasmic aggregates 30-40 nm in diameter. These aggregates could be solubilized in 6 mol/L guanidine HCl, and biologically active receptor was generated by diluting the guanidine HCl preparation 20- to 50-fold. The full-length receptor, expressed either in a soluble or aggregated form, had characteristics typical of a native receptor: it bound steroids with high affinity and specificity, interacted with DNA in a sequence-specific fashion, and was recognized by domain-specific receptor antibodies. Androgen-receptor protein purified to homogeneity in guanidine HCl required the presence of Zn2+ ions during the refolding to reconstitute its DNA-binding form; ZnCl2 was not, however, needed to restore the receptor's steroid-binding activity.
Diagnostic evaluation of creatine kinase-2 mass and creatine kinase-3 and -2 isoform ratios in early diagnosis of acute myocardial infarction
Authors: Bhayana V , Cohoe S , Leung F Y , Jablonsky G , Henderson A R 
Published: January 2020

Abstract
The diagnostic efficacy of creatine kinase (CK) isoforms (CK-3 and CK-2) was compared with measurement of CK-2 mass concentrations for the early diagnosis of myocardial infarction (MI). Serial serum samples drawn from 76 patients with confirmed MI and 55 non-MI patients were used for determining CK-2 mass concentrations and the MM3/MM1 (CK-3 isoforms) and MB2/MB1 (CK-2 isoforms) ratios. We compared the diagnostic utility of each by receiver-operating-characteristic (ROC) curve and likelihood ratio analyses. Our results indicate that all three tests were ineffective within the first 4 h after the onset of chest pain. All three were most effective at 4-18 h after onset, but both CK-3 and CK-2 isoform ratios were less effective than CK-2 mass concentrations in the next 6-h period (18-24 h). In the critical time between 3 and 6 h, the diagnostic performance of all three was comparable.
Endogenous antibodies that interfere with thyroxine fluorescence polarization assay but not with radioimmunoassay or EMIT
Authors: Ritter D , Stott R , Grant N , Nahm M H 
Published: January 2020

Abstract
We have identified an individual whose thyroxine (T4) concentration was undetectable with Abbott's fluorescence polarization immunoassay (FPIA) but within the reference range by radioimmunoassay or EMIT (Syva). The patient's thyrotropin, triiodothyronine, and T-uptake values were within the normal range. The T4 concentration measured by FPIA increased to normal when the immunoglobulin fraction was selectively removed from the serum. When the patient's immunoglobulin fraction was added to normal serum, the T4 content of the normal serum measured by FPIA became falsely low. The patient's antibody interfered with the T4 FPIA by binding to the fluorescein-T4 conjugate. The T-uptake was less affected by the patient's serum because of the low affinity of the patient's antibody to fluorescein-T4 (K = 3.5 x 10(8) L/mol). The patient's immunoglobulin bound preferentially to fluorescein-T4, in comparison with binding to fluorescein or T4 alone. We conclude that the patient's immunoglobulin bound to an epitope unique to the fluorescein-conjugated T4.
Determination of vitamin D status by radioimmunoassay with an 125I-labeled tracer
Authors: Hollis B W , Kamerud J Q , Selvaag S R , Lorenz J D , Napoli J L 
Published: January 2020

Abstract
We report here the first radioimmunoassay for a vitamin D metabolite utilizing a radioiodinated tracer. Antibodies were generated in a goat immunized with the vitamin D analog 23, 24, 25, 26, 27-pentanor-C(22)-carboxylic acid of vitamin D, coupled directly with bovine serum albumin. The 125I-labeled tracer was prepared by reacting a 3-amino-propyl derivative of vitamin D-C(22)-amide with Bolton-Hunter reagent. The primary antiserum, used at a 15,000-fold final dilution, cross-reacted equally with all cholecalciferol and ergocalciferol metabolites tested except 1,25-dihydroxycalciferol metabolites and the parent calciferols; the antiserum did not cross-react with dihydrotachysterol. Calibrators were prepared in vitamin D-stripped human serum. 25-Hydroxycholecalciferol was quantitatively extracted from serum or plasma (50 microL) with acetonitrile. The assay consists of a 90-min incubation at room temperature with primary antiserum, followed by a 20-min incubation with a second antiserum and separation of bound from free fractions by centrifugation. The detection limit of the assay was 2.8 micrograms/L for 25-hydroxycholecalciferol. Results with the present assay compared well with those from a liquid-chromatographic procedure involving specific ultraviolet detection of 25-hydroxycalciferol in plasma.
How and how long to store urine samples before albumin radioimmunoassay: a practical response
Authors: Giampietro O , Penno G , Clerico A , Cruschelli L , Cecere M 
Published: January 2020

Abstract
We used three study protocols to check the dependence of albumin stability, measured by an RIA, on different temperatures, durations, and materials (i.e., assay tubes) of urine storage. Albumin values obtained for samples stored in three types of assay tubes (glass, polystyrene, and polypropylene) throughout the 2 months of the first (prospective) protocol were superimposable. The 24-h storage of six urine samples at room temperature or at 4 degrees C, as well as 72-h storage at 4 degrees C, did not affect the albumin measurement by RIA. After 2 months of storage of these same six urine samples at -20 degrees C, there was still no albumin decrease. A significant albumin decrease occurred (a mean of approximately 5% per year, throughout the range of albumin concentrations tested) when samples stored at -20 degrees C were reassayed by RIA after &gt; or = 2 years (second protocol, retrospective). Finally, 3 of 21 (14.3%) urine pools stored at -20 degrees C for various periods (4-21 months) showed a significant albumin loss after storage; the time of storage as well the decrement rates of these 3 pools differed from each other (third protocol, retrospective). Short- and medium-term (2-6 months) freezing of urine samples at -20 degrees C does not significantly affect the stability of immunoreactive albumin. For longer preservation periods, storage of urine samples at -70 degrees C may be preferable.
Enzyme immunoassay for intact human insulin in serum or plasma
Authors: Andersen L , Dinesen B , Jørgensen P N , Poulsen F , Røder M E 
Published: January 2020

Abstract
We describe an enzyme-linked two-site immunoassay for quantitation of intact insulin in human serum and plasma. The method uses two murine monoclonal antibodies that bind to two different epitopes on the insulin molecule. The immunoassay is specific. Human proinsulin is not bound by the antibodies, and the binding of partially processed proinsulin intermediates is believed to be of minor clinical importance. The relative response of human, bovine, and porcine insulin is 1, 1, and 3, respectively. The assay is sensitive (detection limit 5 pmol/L), accurate (101% recovery with 50 pmol/L insulin added to samples, 95% with 100 pmol/L, and 89% with 300 pmol/L), and fast (results within 3 h), and has a high analytical capacity (done in microtiter plates). The working assay range selected is 5-600 pmol/L, corresponding to a clinically useful range. Because of its specificity, this two-site immunoassay gives results that are lower than those obtained by using a competitive radioimmunoassay, both in normal individuals and in non-insulin-dependent diabetics.
Carnitine measurements in liver, muscle tissue, and blood in normal subjects
Authors: Harper P , Wadström C , Cederblad G 
Published: January 2020

Abstract
We determined carnitine concentrations in blood and in liver and abdominal muscle biopsy specimens in 13 men and 16 women undergoing elective surgery (mostly gallbladder removal). The data suggest that the carnitine pools of plasma and erythrocytes are different. The erythrocytes show a higher acylcarnitine concentration than does plasma (P &lt; 0.001). Several reference bases for values in tissues have been used--dry weight, noncollagen protein (NCP), and DNA--because these may be differently influenced by disease. In liver specimens, the quotient NCP (g)/DNA (g) was significantly higher in men, 54.4 +/- 6.3 (mean +/- SD), than in women, 47.7 +/- 7.0 (P &lt; 0.01). Liver total carnitine content in relation to DNA was significantly higher in men than in women: 0.29 +/- 0.06 vs 0.22 +/- 0.08 mmol/g DNA (P &lt; 0.01). Free carnitine content was significantly higher in men than in women independently of the reference base, e.g., 3.7 +/- 1.0 mumol/g NCP for men vs 2.9 +/- 1.0 for women (P &lt; 0.05). No difference was found between the sexes in the abdominal muscle carnitine content, 20.6 +/- 6.7 mumol/g NCP for men vs 17.9 +/- 5.0 for women. Our study establishes control ranges, thereby providing an important basis for studies of patients with abnormal carnitine metabolism.
Specific glycation of albumin depends on its half-life
Authors: Schleicher E D , Olgemöller B , Wiedenmann E , Gerbitz K D 
Published: January 2020

Abstract
Some suggest that measurements of plasma fructosamine concentration should be corrected for the plasma protein (or albumin) concentration because the extent of glycation per volume depends on both protein and glucose concentrations. Several reports, however, demonstrate a poor correlation between plasma fructosamine and albumin concentrations in diabetic patients. In vitro kinetic and in vivo studies have shown that glycation is also dependent on the half-lives of plasma proteins. Because a decrease in plasma albumin diminishes its catabolism, we speculated that low albumin concentrations are associated with a greater extent of glycation on a molar basis (specific glycation) and vice versa. To test this hypothesis, we studied plasma albumin, total protein, and fructosamine in 63 nondiabetic subjects with normal plasma fasting glucose concentrations and hemoglobin A1c between 5.1% and 5.9%. Plasma fructosamine was poorly correlated with albumin concentration (r = 0.348) but a logarithmic plot of the specific glycation of albumin vs albumin concentration showed a better correlation (r = -0.842), suggesting that the kinetic considerations were operating in vivo. Therefore, because lower specific glycation of plasma albumin "compensates" for higher concentration, correction of fructosamine for albumin content in patients will overestimate mean blood glucose when albumin is low and underestimate it when albumin is high.
Measuring L-dopa in plasma and urine to monitor therapy of elderly patients with Parkinson disease treated with L-dopa and a dopa decarboxylase inhibitor
Authors: Dutton J , Copeland L G , Playfer J R , Roberts N B 
Published: January 2020

Abstract
We have established a method for measuring L-dopa in plasma and urine, including the metabolites dopamine and L-dopac, using separation by ion-pair reversed-phase HPLC and quantification with an electrochemical detector. The assay was applied to the therapeutic monitoring of elderly patients with established Parkinson disease being treated with L-dopa plus a dopa decarboxylase inhibitor. Plasma L-dopa was evaluated in relation to dosage and postdose sampling time in 71 outpatients with Parkinson disease. L-Dopa concentrations were greatest in the patients taking the highest dosages prescribed and decreased significantly with increasing time after postdose sampling. Comparison of plasma L-dopa concentrations with a published therapeutic range established by intravenous administration of L-dopa was helpful in assessing the suitability of each patient's drug dosage, assessing patients' compliance, and avoiding overdosage but was not useful in the overall clinical assessment of progression of disease or of the long-term therapeutic response. Urine measurements confirmed the plasma concentrations but showed no further advantage. The recommended time for sample collection is between 1.5 and 3 h after the first morning dose. Plasma is the preferred matrix but if blood sampling is difficult, particularly from elderly/infirm individuals, an untimed urine collection could be used.
Comparison of lipid fatty acids on a concentration basis vs weight percentage basis in patients with and without coronary artery disease or diabetes
Authors: Schwertner H A , Mosser E L 
Published: January 2020

Abstract
Lipid fatty acid compositions are almost always expressed on a weight percentage basis rather than as weight or molar concentrations. With the former method of calculation, each fatty acid has an effect on the composition of the other fatty acids, an interdependence that can potentially lead to inaccurate and misleading results. Here we compare the concentrations per liter and the weight percentage compositions of cholesteryl ester and phospholipid fatty acids in individuals with and without coronary artery disease as well as free fatty acid compositions in individuals with and without diabetes. A new method for quantifying lipid fatty acids is also presented. Fatty acid results expressed as concentrations and those calculated on a weight percentage basis may lead to different conclusions, because significant differences between patient groups can be lost with the latter approach. We conclude that serum concentrations of lipid fatty acids are more accurate reflections of changes in lipid fatty acids than are weight percentage calculations, and are more easily interpreted in metabolic and therapeutic terms.
Respiratory chain enzyme activities in lymphocytes from untreated patients with Parkinson disease
Authors: Barroso N , Campos Y , Huertas R , Esteban J , Molina J A , Alonso A , Gutierrez-Rivas E , Arenas J 
Published: January 2020

Abstract
Respiratory chain enzyme activities were studied in lymphocytes from patients with Parkinson disease (PD) (n = 16) and age-matched control subjects (n = 15). The patients had received no therapy before the study was conducted. Complex I, III, and IV activities were significantly lower (P &lt; 0.05) in patients than in control subjects. A complex I defect was found in one patient, whereas complex IV was defective in another. Two patients had combined defects of both complexes. The use of lymphocytes for investigating the respiratory chain enzymes provides an easy, noninvasive method to assess mitochondrial function in patients with PD. Furthermore, our study supports the hypothesis that a biochemical defect in the respiratory chain may be involved in the pathogenesis of PD.