bluuebunny/crossref_metadata_2025 huggingface dataset:
Abbott AxSYM random and continuous access immunoassay system for improved workflow in the clinical laboratory
Authors: Smith J , Osikowicz G
Published: January 2020

We describe a new clinical laboratory instrument, the Abbott AxSYM, which provides random- and continuous-access testing for immunoassays, 20 onboard reagents, primary tube sampling, and a throughput of 80 to 120 tests per hour. The AxSYM incorporates three separate analytical technologies for processing immunoassays: microparticle enzyme immunoassay, fluorescence polarization immunoassay, and a novel technology known as ion-capture immunoassay. The system incorporates both common and technology-specific subsystems controlled by a real-time software scheduling processor. Tests can be processed in one- or two-step sandwich or competitive formats, with variable pipetting steps, incubation periods, optical read formats, and wash sequences. Menu capabilities include tests for hepatitis, retrovirus, tumor markers, fertility markers, thyroid functions, and therapeutic drugs. The time to first result is approximately 15-25 min for most routine assays and < or = 15 min for stat assays (i.e., creatine kinase MB isoenzyme, human chorionic gonadotropin beta subunit, and therapeutic drugs). AxSYM assay performance for 23 assays was comparable with that of the Abbott IMx and TDx analyzers; specimen correlation data had correlation coefficients ranging from 0.97 to 0.99 and slopes ranging from 0.99 to 1.10. Within-run imprecision (CV) was 1.5% to 11.4%, with most assays (19 of 23) demonstrating CVs < or = 8.0%.
Simultaneous assay of alpha-fetoprotein and free beta subunit of human chorionic gonadotropin by dual-label time-resolved immunofluorometric assay
Authors: Pettersson K , Alfthan H , Stenman U H , Turpeinen U , Suonpää M , Söderholm J , Larsen S O , Nørgaard-Pedersen B
Published: January 2020

We developed a simple, rapid two-step dual-label assay for the noncompetitive determination of alpha-fetoprotein (AFP) and beta subunit of human chorionic gonadotropin (hCG beta) in serum. Monoclonal antibodies to detect AFP and hCG beta were labeled with europium (Eu) and samarium (Sm), respectively. Highly fluorescent chelates were developed by using the Delfia enhancement principle. The detection limits for AFP and hCG beta were approximately 0.02 kIU/L and approximately 0.2 IU/L, respectively. The within-run precision was < 5% over the whole range of AFP (1-500 kIU/L) and hCG beta (1-200 IU/L) concentrations tested. Cross-reaction of intact hCG was < 0.03%. The AFP concentrations determined with the dual-label assay correlated well with those obtained by Delfia AFP single-label kit. The concentrations of hCG beta were in good agreement with recently published data. Storing the serum samples for 24 h or 1 week at room temperature increased the hCG beta concentration by 4% and 26%, respectively. At 35 degrees C this dissociation of hCG increased 30-40-fold. Repeated freezing and thawing had no effect on the hCG beta concentration.
Highly sensitive enzyme immunoassay of proinsulin immunoreactivity with use of two monoclonal antibodies
Authors: Kjems L L , Røder M E , Dinesen B , Hartling S G , Jørgensen P N , Binder C
Published: January 2020

A highly sensitive two-site sandwich ELISA measuring total proinsulin immunoreactive material in serum or plasma was developed. The assay was based on two monoclonal antibodies, an anti-C-peptide antibody bound to a microtest plate and a biotin-labeled anti-insulin antibody. The detection limit (3 SD above zero value) in buffer was 0.05 pmol/L, corresponding to 0.25 pmol/L in human serum (diluted 1:5). The linear calibrator range was 0.05-20 pmol/L. Interassay CVs were 4.7% at a median (range) of 2.3 pmol/L (1.4-2.8 pmol/L, n = 8), 6.7% at 5.1 pmol/L (3.3-8.0 pmol/L, n = 8), and 8.7% at 10.0 pmol/L (8-12 pmol/L, n = 10). Mean analytical recovery of added human proinsulin (hPI) (2, 5, and 10 pmol/L) to serum was 84% (range 68-128%, n = 9). Human insulin and human C-peptide did not cross-react at 5000 and 10,000 pmol/L, respectively. The four major proinsulin conversion intermediates reacted 65-99%: split(32-33)hPI 74%, des-(31,32)hPI 65%, split(65-66)hPI 78%, and des(64,65)hPI 99%. All serum values from 38 fasting healthy subjects were above the detection limit: median (range) 4.0 (2.1-12.6) pmol/L.
Competitive time-resolved immunofluorometric assay for quantifying carbonic anhydrase VI in saliva
Authors: Parkkila S , Parkkila A K , Vierjoki T , Ståhlberg T , Rajaniemi H
Published: January 2020

A competitive time-resolved immunofluorometric assay sensitive and robust enough for quantifying human salivary carbonic anhydrase isoenzyme VI (HCA VI) was developed. The solid-phase immunoassay is based on competition between Eu(3+)-labeled HCA VI and salivary HCA VI for polyclonal rabbit anti-HCA VI antibodies that are attached to microtiter plate wells precoated with sheep anti-rabbit IgG. The subsequent immunoassay including the separation of free and bound HCA VI requires only one incubation step, after which the Eu3+ of the bound labeled antigen is released into an enhancement solution. The highly fluorescent Eu chelates formed in this solution are then quantified by time-resolved fluorometry (Delfia). The time-resolution principle effectively obviates possible interferences from complex biological material such as saliva. The assay detection limit was 1.5 micrograms/L. Intra- and interassay imprecisions (CVs) were 5.1% and 5.3%, respectively. The mean analytical recovery was 93%. The mean +/- SD concentration of HCA VI in paraffin-stimulated saliva was 6.8 +/- 4.3 mg/L (n = 30) and the secretion rate was 10.2 +/- 7.9 micrograms/min. The method was useful for further investigations of the role of HCA VI in difficult matrices, e.g., saliva.
"Hot spots" mutation analysis of p53 gene in gastrointestinal cancers by amplification of naturally occurring and artificially created restriction sites
Authors: Chen P H , Lin S Y , Wang C K , Chen Y J , Chen T C , Chang J G
Published: January 2020

We developed a rapid, simple method to detect "hot spot" point mutations of the p53 gene. A DNA fragment from cancer tissue of a surgical specimen was selectively amplified with specific oligonucleotide primers and then digested with restriction enzymes that recognized artificial or naturally occurring restriction sites. To detect mutations in codons 273 and 245, we created artificial Bst U1 and BgI I sites by introducing a single nucleotide mismatch into the respective mutagenesis primers. We used the naturally occurring restriction sites of Msp I, Hae III, and Hha I to detect mutations in codons 248, 249, and 175, respectively. In 74 cases of gastrointestinal cancer, 5 of 35 colorectal cancers showed mutations; 1 of 15 cases of gastric cancers showed mutation; and 1 of 24 hepatocellular carcinomas showed mutation. This nonradioactive method is an accurate and clinically useful way to detect hot-spot point mutations of the p53 gene in gastrointestinal cancers.
Prediction of alcohol-related harm by laboratory test results
Authors: Conigrave K M , Saunders J B , Reznik R B , Whitfield J B
Published: January 2020

We examined the value of laboratory markers of excessive alcohol (ethanol) intake as predictors of mortality, morbidity, and health-care utilization in a cohort of 330 patients attending an acute ambulatory care service. Among men, all four markers examined--gamma-glutamyltransferase (GGT) and aspartate aminotransferase (AST) activities, high-density lipoprotein cholesterol (HDL-C), and mean corpuscular volume (MCV)--were predictive of medical sequelae and health-care utilization over a 3-year period. In contrast, social problems were more closely related to the amount of alcohol consumption at initial assessment than to any biological marker. Serum GGT and AST activities and MCV were predictive of medical sequelae in women. The predictive value of GGT was an independent risk factor and did not merely reflect recent alcohol intake or the presence of chronic liver disease. We conclude that these readily available laboratory tests provide important prognostic information and should be an integral part of the assessment of persons with hazardous alcohol consumption.
HemoCue beta-glucose photometer evaluated for use in a neonatal intensive care unit
Authors: Vadasdi E , Jacobs E
Published: January 2020

We evaluated the HemoCue beta-Glucose Photometer system for use in our neonatal intensive care unit by assaying 178 heparinized whole-blood samples obtained by heel stick. The required sample size is 5 microL. Plasma glucose was analysed by a glucose oxidase/oxygen electrode methodology. Across the glucose range of 1.28-21.87 mmol/L, the regression slope was 0.976 (r = 0.976, Sy/x = 0.475). For samples with hematocrit < or = 0.30, the regression slope was 0.981 (r = 0.950, Sy/x = 0.415); for hematocrit of 0.31-0.49, the regression slope was 0.984 (r = 0.972, Sy/x = 0.508); and for hematocrit > or = 0.50, the regression slope was 0.959 (r = 0.988, Sy/x = 0.394). Human whole blood, bovine whole blood, and bovine serum-based quality-control materials were studied. Except for assays of the low-concentration human control material, the total CV was < 3.5%. The accuracy and precision of the HemoCue system were comparable with those of conventional laboratory instrumentation.
Platelet serotonin content and free and total plasma tryptophan in healthy volunteers during 24 hours
Authors: Eynard N , Flachaire E , Lestra C , Broyer M , Zaidan R , Claustrat B , Quincy C
Published: January 2020

The determination of platelet serotonin (5-HT) and plasma tryptophan concentrations is useful in the diagnosis, investigation of etiologies, and treatment of psychiatric disorders. To determine the usual circadian variations in platelet 5-HT and free and total tryptophan concentrations, we measured these variables during 24 h at 1-h intervals and every 30 min from 2000 to 0800 in seven clinically healthy young men with an HPLC method. No common circadian rhythm for platelet serotonin concentrations was observed in our subjects; however, there was a distinct rhythm for both free and total plasma tryptophan: Concentrations were maximal in the afternoon and minimal during the night.
Clinical applications of serum and tissue markers in malignant disease: breast cancer as the paradigm
Authors: Schwartz G F , Schwarting R , Rabindranauth P , Finkel G C
Published: January 2020

Within the past few years, the measurement of serum and tissue markers, especially the latter, has assumed a more significant role influencing clinical decisions about treatment and follow-up of patients with malignant disease. Breast cancer is a useful paradigm to illustrate the types and importance of these various markers. Tissue markers, including nuclear grade, steroid hormone receptors, DNA index, ploidy, expression of oncogenes or tumor-suppressor genes, epidermal growth factors, cathepsin D, proliferating cell nuclear antigen (PCNA), Ki-67, p32, and others, may influence choices of initial treatment as well as adjuvant chemotherapy and (or) hormone administration. The serial measurement of serum markers, those currently available and those on the horizon, for example, may offer a way to monitor patients at risk for recurrent cancer. Although the current role of these markers may be controversial, as information about them is collected and refined, in the future perhaps a panel of such studies could be incorporated into forthcoming clinical staging systems for carcinoma of the breast and other malignancies to define both treatment and outcome.
Rapid, direct enzyme immunoassay of 11-keto-thromboxane B2 in urine, validated by immunoaffinity/gas chromatography-mass spectrometry
Authors: Djurup R , Chiabrando C , Jörres A , Fanelli R , Foegh M , Soerensen H U , Joergensen P N
Published: January 2020

We have developed a direct enzyme immunoassay (EIA) for quantifying immunoreactive 11-keto-thromboxane B2 (iKTXB) in unprocessed human urine. Cross-reactivity with other thromboxane metabolites and prostanoids was negligible. Analytical recovery of 11-keto-TXB2 in urine specimens was 97.4% to 99.8%. Total imprecision for two clinical specimens was 8.5% and 12.2%. Intake of acetylsalicylic acid decreased the measured concentration of iKTXB. Cardiopulmonary bypass, a procedure known to activate platelets, increased the mean excretion rate of iKTXB 10-fold. Simultaneous gas chromatography-mass spectrometry analysis of 11-keto-TXB2 and 11-keto-2,3-dinor TXB2 in urine specimens (n = 17) from healthy subjects indicated that urinary iKTXB concentrations measured by EIA represented a sum of the two 11-keto metabolites. We conclude that the direct EIA is sufficiently sensitive, rapid, simple, and specific to allow screening for alterations in thromboxane biosynthesis in patients.
Improved rapid procedure for simultaneous determinations of hemoglobins A1a, A1b, A1c, F, C, and S, with indication for acetylation or carbamylation by cation-exchange liquid chromatography
Authors: Brunnekreeft J W , Eidhof H H
Published: January 2020

Although the clinical utility of glycohemoglobin (HbA1c) as an indicator of blood glucose control is generally accepted, the use of cation-exchange liquid chromatography for its quantification is controversial, given numerous studies that show no correlation between HbA1c and other indicators of blood glucose control in uremic subjects. We report a specific cation-exchange liquid-chromatographic method that measures simultaneously HbA1a, HbA1b, HbA1c, HbF, HbA0, HbC, and HbS quickly and reproducibly. Carbamylation or acetylation is indicated by extra peaks. Carbamylated hemoglobin increased the result for HbA1c by as much as 1% above the nonuremic value. The method is a fast (approximately 15 min per sample), easy to perform, reproducible, and sensitive screening method for abnormal hemoglobins. Within-assay CVs were < 0.7% in all cases and between-assay CVs were < 1.4%.
Optimized steps in fluorometric determination of thiobarbituric acid-reactive substances in serum: importance of extraction pH and influence of sample preservation and storage
Authors: Wasowicz W , Nève J , Peretz A
Published: January 2020

A simple, reliable, and reproducible fluorometric method for measuring thiobarbituric acid-reactive substances (TBARS) in serum is proposed, based on the reaction between malondialdehyde (MDA) and thiobarbituric acid. Formation of TBARS was complete at pH 2.4-2.6, but extraction with n-butanol proved complete only at lower pH, i.e., 1.6-1.7. Analytical recoveries of MDA added to serum were 94%-101%; within- and between-run CVs were 2.4-3.6% and 4.6-5.5%; and the detection limit for TBARS in serum was 0.10 mumol/L. Optimized conditions included: (a) collection of either serum or heparinized plasma, (b) preservation from in vitro autoxidation by glutathione and EDTA, and (c) storage at -20 degrees C up to 35 days. The mean (+/- SD) TBARS concentration in 47 healthy adults was 1.01 (0.21) mumol/L; no sex-related difference was observed. Higher concentrations were measured in patients with renal insufficiency undergoing hemodialysis and in patients with insulin-dependent diabetes, chronic pancreatitis, or liver cirrhosis.
Does tumor-derived human chorionic gonadotropin act as a thyroid stimulator in vivo?
Authors: Madersbacher S , Schwarz S , Mann K , Klieber R , Wick G , Berger P
Published: January 2020

To evaluate in vivo the proposed intrinsic thyroid-stimulating hormone (TSH) activity (TSA) of human chorionic gonadotropin (hCG), we monitored over 0.5-1 years the thyroid status of eight patients with hCG-producing non-seminomatous testicular cancer. The patients' sera were analyzed for concentrations of hCG, free thyroxine (fT4), hTSH, and thyroxine-binding globulin (TBG). All patients had excessively high concentrations of hCG (1 x 10(5)-5 x 10(8) ng/L, mean: 1 x 10(7) ng/L) before polychemotherapy, which decreased under successful therapy to physiological values (< 240 ng/L). Although the serum concentrations of hCG varied by more than six orders of magnitude, we saw no changes and no correlation (P > 0.05) between the concentrations of hCG and the concentrations of fT4 and hTSH. Not even when hCG concentrations were greatest (> 5 x 10(7) ng/L) were any signs of hyperthyroidism observed: fT4 (3.5-13 ng/L) and hTSH (9-700 ng/L) were in the physiological range in all patients and remained so during chemotherapy. The results of this longitudinal study were confirmed in analyzing the data for all eight patients (total: 82 samples) cross-sectionally. Again, we found no correlation (P > 0.05) between the concentrations of hCG and fT4 or hCG and hTSH. We conclude that even excessive amounts of testicular tumor-derived hCG do not display any TSH-like activity in vivo.
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

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 < 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

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

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 < 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 (> 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

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

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 > 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

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

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 < 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

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

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

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

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 > 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

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

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 < 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 < 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 < 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 < 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

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

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

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

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 < 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.
Automated spectrophotometric method for determining oxidized and reduced glutathione in liver
Authors: Teare J P , Punchard N A , Powell J J , Lumb P J , Mitchell W D , Thompson R P
Published: January 2020

An enzymatic recycling method has been applied to the measurement of total and oxidized glutathione with a centrifugal analyzer. When the reduced form of glutathione (GSH) was masked with 2-vinylpyridine to measure the oxidized glutathione (GSSG), the time to ensure full derivatization was three times longer than has been reported. The method is quick, simple, accurate, and precise (1.27% for GSH, 3.3% for GSSG intraassay CV; 2.15% for GSH, 5% for GSSG interassay CV), and the automation allows large numbers of samples to be conveniently assayed.
Lipase in serum--the elusive enzyme: an overview
Authors: Tietz N W , Shuey D F
Published: January 2020

Lipase is a glycoprotein with 420-449 amino acid residues and a M(r) of 46,000-56,000 for pancreatic lipase and 32,000-39,000 for serum lipase. Lipase is present in the pancreas, intestines, and a variety of other tissues. The concentration gradient between pancreatic tissue and serum lipase is approximately 20,000-fold. Serine, as part of an Asp-His-Ser triad, is the nucleophilic residue essential for catalysis. Lipase differs from other esterases by the presence of a hydrophobic recognition site. The optimal pH is between 7.5 and 10.0, depending on the reaction condition; the pI for the various forms of the enzyme has been reported as 5.80 and 5.85; 6.4, 6.8, and 7.0; and 7.4 for a purified fraction. Several authors report the presence of two molecular forms in the pancreas and three electrophoretic bands with lipolytic activity. In normal serum two bands have been observed; in pancreatitis as many as four bands have been seen. Lipolytic activity may not always be due to lipase. Assays specific for lipase require a triglyceride as substrate as well as the presence of colipase (a water-soluble and heat-stable protein, essential for lipase action), a secondary bile salt, and Ca2+. The clinical sensitivity of all modern assays is high because of selection of a low decision limit; the clinical specificity varies greatly but can be improved by increasing the cutoff point. Lipase determinations in pancreatitis are superior to amylase determinations. The reasons for the great variability of reports regarding the clinical utility of lipase are discussed, and the clinical utility of lipase determinations is summarized.
Urinary proteins and enzymes as early indicators of renal dysfunction in chronic exposure to cadmium
Authors: Jung K , Pergande M , Graubaum H J , Fels L M , Endl U , Stolte H
Published: January 2020

We tested the diagnostic sensitivity of various urinary analytes for detecting cadmium-induced nephropathy at an early stage. We investigated 73 healthy persons (control group 1) and individuals exposed to cadmium, either environmentally (n = 36, risk group 2) or occupationally (n = 62, exposed group 3). All data were related to limits of the central 95% reference intervals of the control group. The serum creatinine and ribonuclease values, indicators of the glomerular filtration rate, were not different in the three groups. In the exposed persons (group 3), proximal tubular indicators (low-M(r) proteins lysozyme, ribonuclease, retinol-binding protein, and alpha 1-microglobulin) were more often increased than the glomerular indices (higher-M(r) proteins transferrin, IgG, and albumin). Both the low-M(r) proteins and tubular enzymes were differently altered in their excretion rates. Alanine aminopeptidase, alkaline phosphatase, and N-acetyl-beta-D-glucosaminidase increased even in the risk group 2. alpha 1-Microglobulin was increased in the exposed persons whose cadmium excretion was < 5 mumol/mol creatinine. The combined determination of alpha 1-microglobulin and N-acetyl-beta-D-glucosaminidase exceeded the corresponding upper reference limits in 30% of group 2 and 39% of group 3. We recommend screening for these two analytes to detect cadmium-induced renal dysfunction at an early stage.
Enzyme immunoassay of liver-type arginase and its potential clinical application
Authors: Ikemoto M , Ishida A , Tsunekawa S , Ozawa K , Kasai Y , Totani M , Ueda K
Published: January 2020

We developed an efficient enzyme-linked immunosorbent assay (ELISA) system for measurement of human liver-type arginase in serum. A conjugate of the Fab' fragment of anti-human liver (recombinant) arginase IgG and horseradish peroxidase was used as the second antibody. This assay is highly specific, sensitive, and reproducible, enabling us to detect arginase at concentrations as low as several micrograms per liter without any prior processing of serum. The reaction is linear up to 200 micrograms/L. The arginase concentration in serum, as determined by this method, increased markedly and temporarily at the time of surgical operation or later injury to the liver. The increase was accompanied or followed by increases in serum concentrations of aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase, suggesting that the arginase emerged from damaged hepatocytes. In view of a limited tissue distribution of liver-type arginase, our ELISA system may be useful in diagnosis of various hepatic disorders as well as follow-up of postoperative conditions of patients.
Separation of beta-carotene and lycopene geometrical isomers in biological samples
Authors: Stahl W , Sundquist A R , Hanusch M , Schwarz W , Sies H
Published: January 2020

The analysis of beta-carotene and lycopene, the two predominant carotenoids in human serum and tissues, was extended to the level of geometrical (cis-trans) isomers by using an improved reversed-phase HPLC methodology. We separated five geometrical isomers of beta-carotene and seven of lycopene in human serum and tissues. 13-cis-beta-Carotene was identified as the predominant cisisomer in human serum, contributing about 5% to total beta-carotene. In tissue, however, considerable amounts of 9-cis- and traces of 15-cis-beta-carotene were also detected. In contrast to beta-carotene, the lycopene isomer patterns in human serum and tissues are quite similar.
Rapid analysis of hemoglobin variants by cation-exchange HPLC
Authors: Ou C N , Rognerud C L
Published: January 2020

We investigated the use of a 3.5 x 0.46 cm HPLC column packed with 5-microns particles of porous (100 nm) silica coated with polyaspartic acid for hemoglobin analysis. A 13-min gradient was produced between two mobile phases. The method is capable of separating more than 35 commonly encountered hemoglobin variants within 12 min. Hemoglobin variants identified include Bart's, acetyl F, H, A1c, F, Camden, N-Baltimore, J-Baltimore, N-Seattle, Grady, Fannin-Lubbock, A G-Georgia, Lepore-Baltimore, P-Galveston, G-Coushatta, Lepore-Boston, E, Osu Christiansborg, A2, G-Philadelphia, Korle Bu, Russ, Richmond, D-Los Angeles, Deer Lodge, Montgomery, S, Q-Thailand, G-San Jose, A2', Hasharon, Q-India, Tampa, GS hybrid, C-Harlem, O-Arab, British Columbia, and C. Between-run precision of an in-house pooled hemoglobin control material, AFSCA2, gave CVs of 2-5% for the A, F, S, and C and 8% for the A2 over a 6-month period. The simplicity of sample preparation, high resolution of the system, and high accuracy of the method, combined with complete automation, make this an ideal methodology for the routine diagnosis of hemoglobin disorders in a clinical laboratory.