Measurement procedure for glucose in serum using liquid chromatography isotope dilution--tandem mass spectrometry (LC-ID-MS/MS).

BACKGROUND: We developed and validated a measurement procedure for glucose using liquid chromatography-isotope dilution tandem mass spectrometry. The proposed method is intended to be used for setting target values in EQAS samples and for certification of glucose reference materials, including those in biological matrices. METHODS: Serum samples were spiked with internal standard 13C6 D-glucose. Protein precipitation was performed with ethanol. The samples were vortexed and centrifuged. An aliquot of the supernatant was evaporated to dryness, the residue dissolved in elution buffer and injected into the LC-MS/MS system. Measurements were performed in the positive ion mode monitoring the Cs+ adducts for glucose at m/z 313 --> 132.9 and m/z 319 --> 132.9 for the internal standard. RESULTS: The coefficient of variation (CV) of the measurement procedure for lyophilized, liquid, and fresh serum samples was between 0.27 and 1.77%. The bias from certified target values for NIST reference materials was < or = 0.62%. A Deming regression comparison demonstrated a good correlation of results obtained with the proposed LC-ID-MS/MS method and target values obtained in the internationally accepted IFCC-RELA ring trial using JCTLM-recognized reference measurement procedures. CONCLUSIONS: The proposed LC-ID-MS/MS measurement procedure with traceability to SI units shows excellent accuracy and precision and is suitable for use as reference measurement procedure for certification of target values in lyophilized and fresh serum samples.

A R59W mutation in human protoporphyrinogen oxidase results in decreased enzyme activity and is prevalent in South Africans with variegate porphyria.

Variegate porphyria (VP), a low-penetrant autosomal dominant inherited disorder of haem metabolism, is characterised by photosensitivity (Fig. 1) and a propensity to develop acute neuropsychiatric attacks with abdominal pain, vomiting, constipation, tachycardia, hypertension, psychiatric symptoms and, in the worst cases, quadriplegia. Acute attacks, often precipitated by inappropriate drug therapy, are potentially fatal. While earlier workers thought the distal haem biosynthetic enzyme ferrochelatase may be involved in the genesis of VP, it was shown in the early 1980's, and is now accepted, that VP is associated with decreased protoporphyrinogen oxidase activity (PPO) (E.C.1.3.3.4). VP prevalence is much higher in South Africa than elsewhere; probably due to a founder effect with patients descending from a 17th century Dutch immigrant. PPO cDNAs from Bacillus subtilis, Myxococcus xanthus, human placenta and mouse liver have been cloned, sequenced and expressed. Human and mouse cDNAs consist of open reading frames 1431 nucleotides long, encoding a 477 amino acid protein. The human PPO gene contains thirteen exons, spanning approximately 4.5 kb. We have identified a C to T transition in codon 59 (in exon 3) resulting in an arginine to tryptophan substitution (R59W). A protein expressed from an in vitro-mutagenized PPO construct exhibits substantially less activity than the wild type. The R59W mutation was present in 43 of 45 patients with VP from 26 of 27 South African families investigated, but not in 34 unaffected relatives or 9 unrelated British patients with PPO deficiency. Since at least one of these families is descended from the founder of South African VP, this defect may represent the founder gene defect associated causally with VP in South Africa.

[The White man's burden - a case study caught between bipolar affective disorder and Huntington's disease]. / White Man's Burden - Im Grenzgebiet zwischen bipolarer affektiver Störung und Huntington-Erkrankung.

We report upon a case of a 55 year old patient with a bipolar affective disorder, presenting herself with a depressive symptomatology in addition to a severe motor perturbation. The main emphasis upon admittance was perfecting and improving her latest medication. Four weeks prior to her stay at our clinic a thorough neurological examination had taken place in terms of an invalidity pension trial which did not result in any diagnostic findings. Therefore a neurological disease seemed at first highly unlikely. Even though the prior testing was negative, the ensuing neurological examination at our clinic resulted in movement disorders very much indicative of Huntington's Disease. A detailed investigation in regards to the particular family history of the patient was positive for Huntington's Disease. However, whether the patient's mother had also been a genetic carrier of Huntington's Disease was still unknown at the time the patient was admitted to our clinic. It was nevertheless discovered that her mother had also suffered from a bipolar affective disorder. A genetic testing that followed the neurological examination of the patient proved positive for Huntington's Disease. Neuro-imaging resulted in a bicaudate-index of 2.4 (the critical value is 1.8). In a clinical psychological test battery the ensuing results were highly uncommon for patients with solely a bipolar affective disorder people. Under the medical regimen of Quetiapine, Citalopram and Tiaprid the patient's mood could be stabilized and there was some improvement of her motor pertubation.

A chatbot-based intervention with ELME to improve stress and health-related parameters in a stressed sample: Study protocol of a randomised controlled trial.

Background: Stress levels in the general population had already been increasing in recent years, and have subsequently been exacerbated by the global pandemic. One approach for innovative online-based interventions are "chatbots" - computer programs that can simulate a text-based interaction with human users via a conversational interface. Research on the efficacy of chatbot-based interventions in the context of mental health is sparse. The present study is designed to investigate the effects of a three-week chatbot-based intervention with the chatbot ELME, aiming to reduce stress and to improve various health-related parameters in a stressed sample. Methods: In this multicenter, two-armed randomised controlled trial with a parallel design, a three-week chatbot-based intervention group including two daily interactive intervention sessions via smartphone (á 10-20 min.) is compared to a treatment-as-usual control group. A total of 130 adult participants with a medium to high stress levels will be recruited in Germany. Assessments will take place pre-intervention, post-intervention (after three weeks), and follow-up (after six weeks). The primary outcome is perceived stress. Secondary outcomes include self-reported interoceptive accuracy, mindfulness, anxiety, depression, personality, emotion regulation, psychological well-being, stress mindset, intervention credibility and expectancies, affinity for technology, and attitudes towards artificial intelligence. During the intervention, participants undergo ecological momentary assessments. Furthermore, satisfaction with the intervention, the usability of the chatbot, potential negative effects of the intervention, adherence, potential dropout reasons, and open feedback questions regarding the chatbot are assessed post-intervention. Discussion: To the best of our knowledge, this is the first chatbot-based intervention addressing interoception, as well as in the context with the target variables stress and mindfulness. The design of the present study and the usability of the chatbot were successfully tested in a previous feasibility study. To counteract a low adherence of the chatbot-based intervention, a high guidance by the chatbot, short sessions, individual and flexible time points of the intervention units and the ecological momentary assessments, reminder messages, and the opportunity to postpone single units were implemented. Trial registration: The trial is registered at the WHO International Clinical Trials Registry Platform via the German Clinical Trials Register (DRKS00027560; date of registration: 06 January 2022). This is protocol version No. 1. In case of important protocol modifications, trial registration will be updated.

Editing of GluR2 RNA in the gerbil hippocampus after global cerebral ischemia.

Postischemic delayed neuronal death (DND) in CA1 of the gerbil hippocampus is thought to be caused by an abnormal increase of Ca2+ influx into the cell, mediated by excessive activation of glutamate receptors. One subtype of glutamate receptors, the AMPA receptor, is not permeable to calcium ions as long as an edited form of its GluR2 subunit is present. It is possible that global ischemia interferes with the posttranscriptional editing of the GluR2 mRNA and thus leads to calcium influx via the AMPA receptor. In order to test this hypothesis, we examined the extent of GluR2 RNA editing in CA1 and CA3 microdissected from gerbil hippocampus after 5 min of global ischemia and various recirculation intervals. At each interval tested, quality and quantity of mRNA editing in the vulnerable CA1 region were the same as in CA3. Furthermore, postischemic mRNA editing in both hippocampal regions was indistinguishable from editing in untreated control animals. Our results clearly demonstrate that global ischemia does not cause impairment of GluR2 RNA editing, which is thus not responsible for the abnormal calcium permeability of the postischemic cell membrane.

Impact of concentrations of glycated hemoglobin, alpha-tocopherol, copper, and manganese on oxidation of low-density lipoproteins in patients with type I diabetes, type II diabetes and control subjects.

The late organ complications in diabetic patients are associated with enhanced oxidation of low-density lipoproteins (LDL). The role of vitamin and trace metal concentrations in this process is not clear. Therefore, we compared the oxidative susceptibility and alpha-tocopherol concentration of LDL with the levels of glycated hemoglobin (HbA1c), copper and manganese. Sixty-three diabetic patients (23 female and 40 male; 53 of type II, 10 of type I) and 35 control subjects (17 female and 18 male) were investigated. The in vitro-formation of conjugated dienes in purified LDL preparations in the presence of copper was followed as absorbance at 234 nm. LDL exhibited a shorter lagtime (44.5 +/- 10.1 vs. 67.8 +/- 16.0 and 50.1 +/- 14.3 vs. 68.8 +/- 14.6 min) for type I and type II diabetic patients vs. sex and age-matched controls, P < 0.001. For all subjects together the lagtime was inversely correlated to HbA1c (r = -0.230, P = 0.023) and positively correlated to LDL alpha-tocopherol/LDL (mol/mol). This ratio was lower in diabetic patients (P < 0.01 for type II) than in control subjects. The copper and manganese plasma levels were not different between diabetic and nondiabetic groups. However, parameters of LDL oxidizability (amount and rate of oxidation) were positively correlated with both copper and manganese concentrations. We conclude that in diabetes the resistance of LDL against oxidation is diminished in relation to the quality of glucose control.

Changes in salivary [K+], [Na+] and [K+]/[Na+] with varied test demands.

It was hypothesised that choice reaction-time (CRT) testing would cause salivary [K+]/[Na+] to increase. Relative contributions of [K+] and [Na+] to ratio changes were investigated in 23 hypertensives and ten hospital staff. Changes in post-rest and post-test ionic concentrations and [K+]/[Na+], replicated earlier studies. Phasic [K+]/[Na+] changes were mainly due to [K+] changes. Significant increases in [K+] and decreases in [Na+] from a relaxed session, the day before CRT testing, to the testing session per se indicated test anticipation effects. In both groups, changes from pre-test "rest" to "on test" were significant only for [K+]. [K+] discriminated well between conditions in hypertensives. This was interpreted in terms of adaptive changes in sympathetic activation. Results show the robustness of salivary ion indices (especially of [K+]) as indicators of within-subject response to mental task demands.

The determination of free and protein-bound haemoglobin in plasma using a combination of HPLC and absorption spectrometry.

The aim of the study was to develop a method for the determination of haemoglobin in plasma suitable for use to set target values for external quality assessment schemes for this analyte using commercially available test kits and equipment. In the early phase of the method development it became clear that the use of a single method, namely HPLC, would not be possible. However, by combining HPLC and absorption spectrophotometry, both qualitative and quantitative rapid determinations of protein-bound and free haemoglobin were able to be performed on equipment present in most routine clinical chemistry laboratories. The separation of protein-bound and free haemoglobin could be carried out using commercial HPLC equipment for the determination of haemoglobin A1c (HbA1c) without modification of the conditions used. Instead of haemolysed blood, the same volume of plasma (10 microl) was injected. The eluate was not discarded, but collected in 1-minute fractions so that the void volume (protein-bound Hb) and the haemoglobin peaks (free Hb) were available for the colorimetric determination of haemoglobin using the pseudoperoxidase activity of the haem moiety on hydrogen peroxide and a chromogen (3,3',5,5'-tetramethylbenzidine) in concentrated acetic acid and optimal determination at 600 nm. (In this publication at 578 nm due to the use of a spectrophotometer with Hg-discharge lamp and filter). The appearance of a blue colour in the reaction tube or cuvette indicated the presence of haemoglobin. The use of the above chromogen, with its absorption maximum around 600 nm excluded interference from serum components such as bilirubin, which may interfere in the conventional method often used to determine plasma haemoglobin. The method can be used quantitatively by including an aqueous human haemoglobin standard in the run. This elutes from the HPLC column only as free haemoglobin in the concentration range from 0.1 to 10 g/l. Addition of human haemoglobin to haemoglobin-free plasma resulted in the binding of all Hb to plasma proteins up to a concentration between 2 and 3 g/l (void-volume fraction). At higher concentrations free Hb appeared in the 3-5 minute fractions. These observations agree with published data on the scavenging capacity of plasma for Hb released from erythrocytes. The method is rapid, (HPLC-run maximally 6 min, quantitative colorimetric results 5-10 min) precise (inter-assay coefficients of variation < 8%) and suitable for answering the question as to whether the protein-binding (scavenging) system which prevents the nephro- and cerebrotoxic effects of haemoglobin has been saturated or not, an important question in patients with acute haemolysis problems. A qualitative result is obtainable within 10 minutes of injecting the sample into the HPLC-system. The use of this assay in controlling blood transfusion and haemolytic events arising from surgery, intravascular haemolytic bacteria or artificial heart valves can help in rapid corrective action, if needed.

Evaluation of the LIAISON thyroid chemiluminescence immunoassays.

The LIAISON thyroid hormone assays TSH, FT4, FT3, T4 and T3 were evaluated by determining the imprecision, the reference ranges, the functional sensitivity (TSH), the dilution characteristics (accuracy) (FT4, FT3), and the recovery after spiking (TSH, T4, T3). Furthermore, inter-method comparisons were performed with following methods: Elecsys (Roche Diagnostics; TSH), AxSYM (Abbott Diagnostics; TSH, FT4, FT3, T4), ACS:180 (Bayer Diagnostics; all analytes), Amerlex-M (Johnson & Johnson; T4) and LISO-Phase (Techno Genetics; FT4). The fully automated LIAISON random access analyser is based on microparticle immunoassays and chemiluminescence. The coefficients of variation (CV) of intra-assay imprecision were between 0.2-6.0%, except for the control sample with extremely low TSH concentrations and low T3 concentrations. Inter-assay imprecision was performed by measuring controls covering the measuring range over a period of 9 to 20 days, with CVs ranging from 2.3-16.0%. The suitability of the sample material was determined by analysing serum and samples treated with EDTA, citrate or heparin in parallel. The results showed good correlations of the thyroid hormone concentrations between serum and plasma samples except for LIAISON FT3, for which lower results were observed with EDTA-plasma. The regression analysis of correlation studies gave slopes from 0.849 to 0.957 for TSH, from 1.023 to 1.375 for FT4, from 0.670 to 0.911 for FT3, from 0.917 to 1.166 for T4 and 1.00 for T3 depending on the concentration range and the method of comparison. The LIAISON FT4 assay showed a trend towards higher values in the high concentration range when compared with the ACS:180. The ranges of thyroid hormone concentrations determined in serum taken from apparently healthy subjects were found to be in accordance with published data. The clinical sample study confirmed that the LIAISON thyroid hormone assays are sensitive methods for the differentiation of euthyroid subjects and patients with hyper- and hypothyroidism. In conclusion, the automated thyroid hormone immunoassays on the random-access LIAISON immunoassay analyser proved to be very satisfactory, both from the analytical and the clinical point of view.

Influence of cyanoacrylate on the efficiency of forensic PCRs.

Cyanoacrylate ester (CA) is commonly used by criminalists to detect latent fingerprints on smooth surfaces. We investigated whether this treatment has an influence on a subsequent DNA typing of biological stains, and on the efficiency of three different forensic PCRs (mtDNA, Y-STR determination and the Profiler Plus kit). Using fluorescence labeled primers and an automated detection system, we could show that the presence of CA led to weaker PCR products. Depending on the DNA extraction method the amplification results were significantly weaker compared to untreated controls. To simulate forensic cases we prepared blood and saliva stains on glass slides, extracted the DNA using two different methods and compared the signal intensities of the amplified DNA fragments. Depending on the extraction methods, the presence of CA significantly hampered the amplification of DNA from small stains whereas there was virtually no difference comparing the amplification results of DNA extracted from bigger stains.

[Reference values for blood and serum selenium in the Dresden area]. / Referenzwerte von Selen in Blut und Serum im Raum Dresden.

BACKGROUND: To ensure a correct interpretation of patient's data the regional differences in the supply with selenium have to be taken into consideration. PATIENTS AND RESULTS: In 256 healthy women and men from the area of Dresden aged 20 to 62 years the selenium reference values were examined in blood serum by 1.09 +/- 0.17 (0.75 to 1.43) and in whole blood by 1.29 +/- 0.21 (0.87 to 1.71) mumol/l. There was no dependence upon age and sex and no influence of alcohol, tobacco and vegetarian diet was found. Consumption of beer yeast and frequent fish meals caused improvement of the selenium status. CONCLUSION: In area of Dresden, similar to the whole of Germany, a marginal selenium supply exists. Therefore it is of high importance to consider a balanced nutrition and to control the selenium status especially in serious acute diseases and in intensive care.

[Effect of selenium administration on various laboratory parameters in patients with acute pancreatitis]. / Einfluss einer Selensubstitution auf verschiedene Laborparameter bei Patienten mit akuter Pankreatitis.

BACKGROUND: Recent studies presented evidence that activation of oxygen derived free radicals occurs in patients with acute pancreatitis. The purpose of this study was to evaluate the effect of sodium selenite as a possible antioxidant therapy in acute pancreatitis. PATIENTS AND METHOD: 16 patients with moderate form of acute pancreatitis received a high dose of sodium selenite. Selenium in serum and whole blood, zinc, copper, manganese, superoxid dismutase (SOD), glutathione peroxidase (Gpx) and malondialdehyde (MDA) were determined (before selenium substitution, 3 days later, 8 days later, before demission). No selenium deficiency could be detected before selenium substitution. RESULT: The selenium therapy caused a significant increase in selenium, a moderate increase in activity of Gpx, a significant decrease in activity of MDA, whereas SOD remained unchanged. CONCLUSION: Concerning the particular point of view of "deficiency management", there is no need of selenium substitution in patients with a moderate form of acute pancreatitis in our region. The highly normal selenium concentration we established by our therapy is possibly connected with a decrease of the oxidative stress in acute pancreatitis. More clinical follow-up studies with more patients, who have different grades of severity of the acute pancreatitis, and besides that a control group of patients without selenium substitution, are necessary for evaluating the clinical relevance of our results.

[Effect of preventive selenium administration on development of ERCP-induced acute pancreatitis]. / Einfluss einer prophylaktischen Selensubstitution auf die Entwicklung der ERCP-bedingten akuten Pankreatitis.

BACKGROUND: Sodium selenite may play a role in reduction of enhanced oxygen free radicals in the early phase of experimental acute pancreatitis. The aim of the study was to determine whether ERCP induced pancreatitis can be used as a human model for early acute pancreatitis and if a prophylactic antioxidant therapy with sodium selenite or a prophylactic antibiotic therapy has a beneficial effect on the clinical outcome in patients with ERCP. PATIENTS AND METHODS: Sixty patients were randomly allocated in 3 groups: A (n = 20, sodium selenite i.v. 1 day before ERCP 1 mg bolus/2 x 1 mg infusion); B (n = 20, metronidazole 0.5 g/ofloxacin 0.2 g before and 6 hours after ERCP); C (n = 20, controls, no prophylaxis). Various labor parameters and physical complaints after ERCP were determined. RESULTS: Patients with an antibiotic prophylaxis or with a sodium selenite substitution had no less physical complaints or pancreatitis than the controls. Seven patients (= 11.7%) developed a pancreatitis after ERCP. Concentration of zinc, copper, manganese, vitamin A and E did not differ significantly between the various groups. CONCLUSION: A prophylactic substitution with sodium selenite or prophylactic antibiotic therapy with metronidazole/ofloxacin has no beneficial effect on the clinical outcome in patients with ERCP.

How to design magneto-based total analysis systems for biomedical applications.

This article reviews recent developments on magnetoresistive detection of magnetic beads or nanoparticles by nanoscale sized sensors. Sensors are analyzed from an experimental and a numerical point of view in respect to their capability to either localize the position of a single magnetic particle or to detect the number of particles in a certain range. Guidelines are shown up on how to extend single sensors to sensor arrays with very high spatial resolution and how to modify the sensor shape in order to provide long distance measurements. Further, sensors in biological lab-on-a-chip environments are discussed. The magnetic ratchet and a gravitation based microfluidic component are reviewed as important tools to position and, therefore, detect biological components in continuous-flow devices.

[Manganese and arteriosclerosis]. / Mangan und Arteriosklerose.

The relations between manganese and atherosclerosis were examined on rabbits with experimental atherosclerosis, on patients with atherosclerosis and on workers exposed to manganese. Here a favourable influence of manganese was shown which effected a decrease of the cholesterol content of serum, liver and aorta and inhibited the entry of lipids into the aorta. The influence of the manganese on various enzymes as well as a manganese-copper interaction are discussed as possible causes.

The Refugee Act of 1980: what have we learned?

"This article discusses the objectives of legislation enacted in 1980 that incorporated into U.S. law the international definition of a refugee. It examines the political climate that existed when the legislation was debated and how sudden, unforseen events affected the implementation of the new law. Particular attention is directed at understanding the difficulty the U.S. has experienced in achieving a nationality-neutral refugee program and in establishing a political asylum system that is fair but resistant to abuse." (SUMMARY IN FRE AND SPA)

[The determination of cortisol in plasma with a direct luminescence enzyme immunoassay]. / Die Bestimmung von Cortisol im Plasma mit einem direkten Lumineszenzenzymimmunoassay.

The assay is based on the luminescent measurement of horseradish peroxidase. The IgG fraction of a cortisol antiserum is coated on suitable polystyrene tubes (in use for the measurement of luminescence). The enzyme activity of bound label was determined using a p-iodophenol enhanced chemiluminescent reaction. The assay was sensitive and precise for a wide cortisol concentration range and showed a good agreement with conventional ELISA-technique. There was no extraction necessary for cortisol from plasma. The diluted sample was heated to 60 degrees C or 8-aniline-1-naphthalene sulfonic acid was added in order to release cortisol from protein binding in plasma.

Improved cryoprotection and freeze-substitution of embryonic quail retina: a TEM study on ultrastructural preservation.

Conditions for cryofixation and freeze-substitution crucial to the ultrastructural preservation of embryonic quail retina were improved. As freeze-substitution makes gentle dehydration and chemical fixation of tissue possible, the suitability of different cryoprotectants were tested in the preceding cryofixation. Additionally, different conditions for chemical prefixation were studied. In cryofixation, all of the "classic" cryoprotectants caused more or less severe tissue destruction. Only dimethylformamide (DMF) and--with certain reservations--dimethylsulfoxide (DMSO) yielded improved structure preservation. Perfusion fixation with a mixture of formaldehyde/glutaraldehyde (FA/GA) was superior to GA alone. In comparison to conventional fixation and dehydration methods, freeze-substitution yielded better ultrastructural preservation of the embryos with fewer artifacts.

New results concerning the behavior of cellulose acetate in solutions and films by means of CD measurements.

Cellulose acetate (DS = 2.45) was extensively investigated by Circular Dichroism (CD) in acetonitrile and dioxane. We found great differences between the CD spectra of a 1 wt % acetonitrile solution and the corresponding dioxane solution of cellulose acetate (CA) indicating that the macromolecules exist in those solutions in different molecular arrangements (e.g., persistence length, solvatation shell). The resulting morphologies could be transformed reversibly into each other, as we found by measuring the CA in mixtures of both solvents. Solid CA films show discernible CD spectra depending on the solvent they were evaporated from. In this way, we prepared solid films of the same polysaccharide owning different chiral properties. Furthermore, changes in the spectra occurred with increasing CA concentration. Basing upon our findings, some general statements concerning the polymer behavior of CA are possible.

An improved solid-phase enzyme and luminescent immunoassay system for steroid hormones and digoxin.

A uniform solid-phase system has been developed for enzyme (ELISA) or luminescent (LIA) immunoassays for steroids. These assays were improved by (a) irradiating microtiter plates or polystyrene tubes before coating with antibody or Protein A, (b) coating the plastic trays with nonspecific anti-gamma-globulin or Protein A instead of the steroid-specific first antibody, and (c) partial denaturation of the second antibody before coating the plates or tubes with it. Specific antibodies were raised against cortisol, aldosterone, 17-hydroxyprogesterone, and digoxin. Horseradish peroxidase was used as label for the ELISA and aminoethylisoluminol for the LIA. In comparison with the first (specific) antibody coating method we observed some advantages: From 10- to 33-fold lower concentrations of first antibodies were necessary to bind the same amount of steroids; precision was better (CV, 3.8-7.5% vs 6.9-15.5%). The high sensitivity of these assays (0.5-2.0 pg per tube for the steroids) also allows determination of the steroids and digoxin in plasma and saliva.