Reduced nitric oxide concentration in exhaled gas after exposure to hyperbaric hyperoxia.

The objective of this study was to evaluate exhaled nitric oxide concentration (FENO) and exhaled breath condensate (EBC) pH and H2O2 as biochemical markers of pulmonary oxygen toxicity in association with hyperbaric oxygen (HBO2) therapy. FENO, EBC pH and H2O2 were measured during the course of a 4 week HBO, treatment period, and the responses to a single HBO2 exposure at the start and end of the treatment period were assessed. The HBO2 exposure was at a pressure of 240 kPa for 90 min 5 days a week for 4 weeks. Eight patients undergoing HBO2 therapy and eight control subjects participated in the study. There was a reduction in FENO immediately after HBO2 exposure of 33.1 (SD = 7.8) % on Day 1 and 40.7 (SD = 8.9) % on Day 25. EBC pH was reduced after the first exposure only. Baseline F(E)NO and EBC pH and H2O2 measured before the HBO2 exposures did not change throughout the HBO2 treatment period. A single HBO2 exposure induces a significant transient decrease in FENO. Repeated exposures do not appear to induce inflammatory processes in the lung associated with an increase in FENO.

Evaluation of methods for trace-element determination with emphasis on their usability in the clinical routine laboratory.

Commonly used techniques for trace-element analysis in human biological material are flame atomic absorption spectrometry (FAAS), graphite furnace atomic absorption spectrometry (GFAAS), inductively coupled plasma atomic emission spectrometry (ICP-AES) and inductively coupled plasma mass spectrometry (ICP-MS). Elements that form volatile hydrides, first of all mercury, are analysed by hydride generation techniques. In the absorption techniques the samples are vaporized into free, neutral atoms and illuminated by a light source that emits the atomic spectrum of the element under analysis. The absorbance gives a quantitative measure of the concentration of the element. ICP-AES and ICP-MS are multi-element techniques. In ICP-AES the atoms of the sample are excited by, for example, argon plasma at very high temperatures. The emitted light is directed to a detector, and the optical signals are processed to values for the concentrations of the elements. In ICP-MS a mass spectrometer separates and detects ions produced by the ICP, according to their mass-to-charge ratio. Dilution of biological fluids is commonly needed to reduce the effect of the matrix. Digestion using acids and microwave energy in closed vessels at elevated pressure is often used. Matrix and spectral interferences may cause problems. Precautions should be taken against trace-element contamination during collection, storage and processing of samples. For clinical problems requiring the analysis of only one or a few elements, the use of FAAS may be sufficient, unless the higher sensitivity of GFAAS is required. For screening of multiple elements, however, the ICP techniques are preferable.

Analytical quality goals derived from the total deviation from patients' homeostatic set points, with a margin for analytical errors.

The deviation of test results from patients' homeostatic set points in steady-state conditions may complicate interpretation of the results and the comparison of results with clinical decision limits. In this study the total deviation from the homeostatic set point is defined as the maximum absolute deviation for 95% of measurements, and we present analytical quality requirements that prevent analytical error from increasing this deviation to more than about 12% above the value caused by biology alone. These quality requirements are: 1) The stable systematic error should be approximately 0, and 2) a systematic error that will be detected by the control program with 90% probability, should not be larger than half the value of the combined analytical and intra-individual standard deviation. As a result, when the most common control rules are used, the analytical standard deviation may be up to 0.15 times the intra-individual standard deviation. Analytical improvements beyond these requirements have little impact on the interpretability of measurement results.

Urine analysis and decision-making on cystitis in general practice.

Decision-making on cystitis in general practice was studied by addressing 1171 general practitioners of whom 909 responded. A case history was supplied with a test-strip result where leucocyte esterase was 1 + and the nitrite field either positive or negative. The respondents' estimated probability of urinary tract infection from the history had a median at 50% (10 and 90 percentiles, 30% and 90%). The test-strip results changed the estimate to 90% (50% and 100%) in the nitrite-positive group and 50% (10% and 90%) in the nitrite-negative group. Likelihood ratio was calculated from each respondent's estimated pre- and post-test probability to express the test's assumed diagnostic power. The median likelihood ratios were 4.75 (10 and 90 percentiles, 1.00 and 35.70) in the nitrite-positive group and 1.00 (0.11 and 4.00) in the nitrite-negative group. The resulting actions taken by the respondents were: no action (14 respondents, corresponding median estimated post-test probability 27.5%), await further examination (482, 50%) and prescription of antibiotics (366, 90%). Further examination included urine microscopy (13%), bacteriologic culture (21%), or both (65%). Eight-two percent of respondents who decided to prescribe antibiotics would also perform further examinations. In routine urine analysis, 79% of respondents request only test strips, 2% perform microscopy and 19% do both. The estimated probability of urinary tract infection is significantly affected by test-strip results and is important for the choice of further actions. Microscopy is performed more often than recommended in other studies.

Novel B and T cell epitopes of chicken ovomucoid (Gal d 1) induce T cell secretion of IL-6, IL-13, and IFN-gamma.

BACKGROUND: Chicken ovomucoid (OM, Gal d 1) has an important role in the pathogenesis of IgE-mediated allergic reactions to hen's egg white. OBJECTIVES: The purpose of this study was to clarify the mechanisms of T cell recognition of ovomucoid using intact OM and chemically modified, characterized and homogeneous solid phase synthetic peptides covering the whole molecule. METHODS: Eighteen overlapping peptides were prepared by solid phase F-moc polyamide peptide synthesis (SPPS), characterized and high-pressure liquid chromatography (HPLC) purified. The peptides, together with intact, denatured and oxidized OM, were used to stimulate patient-derived cell cultures for mapping T cell epitopes. Proliferation responses, T cell phenotype and cytokine secretion using peripheral blood mononuclear cells (PBMC) from eight individuals and T cell lines (TCL) derived from six hen's egg-allergic patients, were examined. In addition, intact, denatured, oxidized and deglycosylated OM, as well as the peptides solely or with their keyhole limpet haemocyanin (KLH) complexes, were tested. For locating IgE and IgG B cell epitopes, seven egg-allergic patient sera and three OM-polyclonal sera were used. Healthy non-allergic individuals were included as controls. RESULTS: Seven peptides were recognized by specific IgE, while OM-specific TCL recognized 10 peptides. Six of the OM peptides were commonly recognized both by patient S-IgE and blood-derived TCL. Among those, one novel epitope, peptide OM 61-74, had the ability to bind IgE. Another peptide, OM 101-114, was recognized by IgE and IgG sera, but not by any of the TCLs. In contrast, the peptides OM 41-56, OM 71-84, OM 131-144 and OM 171-186 were exclusively T cell epitopes with no affinity to specific antibodies. Abundant TCL secretion of IFN-gamma, IL-6, IL-4, IL-13, IL-10 and TNF-alpha in response to OM stimulation indicates the contribution of Th2 as well as Th1/Th0 CD4+ cell subsets. For allergic patients moderate amounts of IFN-gamma, IL-13, and high amounts of IL-6, were secreted in response to TCL stimulation by OM peptides. High amounts of IL-6 were secreted in response to all molecular forms of OM (intact-, modified-OM and the peptides 71-84 and 51-64) when TCLs from two non-allergic donors were used. CONCLUSIONS: One novel B cell epitope (OM 61-74) and 10 T cell epitopes have been identified. The most reactive epitopes of the OM molecule comprise the motifs 1-14 to 71-84, the overlapping peptide-pairs OM 121-134 and OM 131-144 and peptides OM 161-174 and 171-186. Peptides OM 1-14 and 171-186 are the only ones capable of inducing IL-4 secretion. Only one peptide (OM 11-24) induces IL-10 secretion. Those peptides recognized as both T and B cell epitopes or only T cell epitopes, have the potential to induce T cell secretion of moderate to high amounts of IL-13, IFN-gamma and particularly IL-6.

Trace element reference values in serum determined by inductively coupled plasma atomic emission spectrometry.

Serum reference values for Ba, B, Cd, Cu, Fe, Mn, Li, Se, Sr, and Zn in 141 healthy Norwegians were determined. The trace element concentrations were determined by the inductively coupled plasma atomic emission spectrometry technique that we have recently validated. The reference intervals were established according to the recommendations of the International Federation of Clinical Chemistry and Laboratory Medicine. Also coverage intervals with coverage uncertainties were calculated according to the International Union of Pure and Applied Chemistry. The population studied consisted of 69 men and 72 women of the ages 21-87 years. The effects of gender, age, smoking, and oral contraceptives on serum levels of trace elements were investigated. Median concentrations of the different trace elements in (micromol/l) were as follows: Ba (0.44), B (1.50), Cd (0.004), Cu (17.1), Fe (21.4), Li (0.06), Mn (0.003), Se (1.26), Sr (0.17), and Zn (13.3). An increase in serum Ba and Sr was detected with age. These metals and Se were also significantly higher in women over 50 years of age in comparison to younger women. Women had higher serum Cu than men and those on oral contraceptives had higher serum Cu and Fe. Serum B tended to increase with age, while it was significantly reduced with smoking.

Evaluation of indicators of cobalamin deficiency defined as cobalamin-induced reduction in increased serum methylmalonic acid.

BACKGROUND: Early detection of cobalamin deficiency is clinically important, and there is evidence that such deficiency occurs more frequently than previously anticipated. However, serum cobalamin and other commonly used tests have limited ability to diagnose a deficiency state. METHODS: We investigated the ability of hematological variables, serum cobalamin, plasma total homocysteine (tHcy), serum and erythrocyte folate, gastroscopy, age, and gender to predict cobalamin deficiency. Patients (n = 196; age range, 17-87 years) who had been referred from general practice for determination of serum cobalamin were studied. Cobalamin deficiency was defined as serum methylmalonic acid (MMA) >0.26 micromol/L with at least 50% reduction after cobalamin supplementation. ROC and logistic regression analyses were used. RESULTS: Serum cobalamin and tHcy were the best predictors, with areas under the ROC curve (SE) of 0. 810 (0.034) and 0.768 (0.037), respectively, but age, intrinsic factor antibodies, and gastroscopy gave additional information. CONCLUSIONS: When cobalamin deficiency is suspected in general practice, serum cobalamin should be the first diagnostic test, and the result should be interpreted in relation to the age of the patient. When a definite diagnosis cannot be reached, MMA and tHcy determination will provide additional discriminative information, but MMA, being more specific, is preferable for assessment of cobalamin status.

Tetradecylthioacetic acid and tetradecylselenoacetic acid inhibit lipid peroxidation and interact with superoxide radical.

Reactive oxygen species are thought to induce cellular damage and to play a pathological role in several human diseases. Tetradecylthioacetic acid (TTA) was previously reported to prevent the oxidative modification of low-density lipoprotein (LDL) particles and to act as an antioxidant. In this study we present a new fatty acid analogue, namely tetradecylselenoacetic acid (TSA), in which the sulfur atom of TTA is replaced by a selenium atom. TSA was more potent than TTA in increasing the lag time before the onset of LDL oxidation and this effect was dose dependent. TTA and TSA were shown to reduce the iron-ascorbate-induced microsomal lipid peroxidation, TSA being more efficient than TTA. TTA and TSA, in the presence of iron, interacted with the superoxide radical as assessed by direct and indirect testing methods. TSA like TTA failed to scavenge 1.1-diphenyl-2-picrylhydrazyl radicals. TSA bound copper ions as shown by the wavelength spectra measurement. These results suggest that TTA and TSA exert their antioxidant capacity by interaction with copper or iron ions in radical scavenging, TSA being more potent than TTA. Nevertheless, a chelating effect resulting in chemically inactive metal ions cannot be excluded.

Validation of inductively coupled plasma atomic emission spectrometry technique (ICP-AES) for multi-element analysis of trace elements in human serum.

The use of inductively coupled plasma atomic emission spectrometry (ICP-AES) for the simultaneous determination of Al, B, Ba, Be, Cd, Co, Cr, Cu, Fe, Li, Mn, Ni, Pb, Se, Sr and Zn in human serum in a clinical laboratory was validated. Samples were digested and then analysed using yttrium as an internal standard and a serum-matched calibration standard. The criteria used to assess the analytical performance of the ICP-AES were detection and quantification limits, linearity, sensitivity, recovery, interference from alkali and acid, trueness and precision. Detection limits were 0.002-0.003 micromol/L for Mn, Sr, Ba, and Cd; 0.014-0.07 micromol/L for Co, Zn, Fe, Be, Li, Pb, Cu, Ni, and Cr; and 0.2-0.9 micromol/L for B, Se, and Al. Trueness, as controlled by analysis of bovine serum certified reference material, was acceptable for Co, Cu, Se and Zn, while Fe was 5.1% and Mn 6.2% below the lowest limit of the certified material interval. We conclude that ICP-AES can be used for multi-element analysis of B, Ba, Cu, Fe, Li, Se, Sr and Zn in serum. Serum levels of Al, Be and Co were below the detection limits while serum levels of Cd, Cr, Ni and Pb were below the quantification limits of the ICP-AES. These trace metals cannot be analysed as routine by the ICP-AES. However, in cases of intoxication with elevated serum concentrations mean recovery of 100+/-10% was obtained at an addition of 2.22 micromol/L for Al, 0.11 micromol/L for Be, 0.03 micromol/L for Co, 0.39 micromol/L for Cr, 0.14 micromol/L for Ni, and 0.12 micromol/L for Pb.

[Analytical uncertainty--how wrong can a laboratory result be?]. / Analytisk usikkerhet--hvor stor feil kan laboratoriesvaret ha?

Some uncertainty encumbers the outcome of all laboratory tests. Patient conditions and handling of specimens, as well as analytical variation and systematic error will affect the results. In order not to confuse the interpretation of test results, the analytical standard deviation should not exceed one half of the intraindividual biological standard deviation, and systematic error should not exceed 1/16 of the reference interval. However, these goals cannot always be achieved. Moreover, analytical control procedures have limited ability to detect errors in the analytical process. After one analysis of control material, using +/- 2 standard deviations (analytical) from the mean as acceptance limits, the magnitude of a systematic error must be 3.3 times the analytical standard deviation in order to be detected with 90% probability. As a result of such error, patients' results can be released with a total error up to five times the analytical standard deviation. More complicated control procedures may give smaller total error. Practising physicians should be familiar with the variations in lab results, and interpret the results accordingly. Lab results that conflict with results of other investigations should be used with caution.

[How do antioxidants work?]. / Hvordan virker antioksidanter?

Reactive oxygen species, formed by incomplete reduction of molecular oxygen, may cause oxidative stress in the organism and be involved in the pathogenesis of many diseases. A number of anti-oxidants are necessary to counteract the harmful effects. Some antioxidants are enzymes which catalyze the breakdown of reactive oxygen species, some act by chelating transition metals, which makes them non-reactive, while chain-breaking antioxidants act by halting the cascade of free radical reactions. The effect of redox active antioxidants depends on their redox status. In some circumstances antioxidants can have pro-oxidant effects. The mechanisms may be reductive release of metals, accumulation of the oxidized form of redox active antioxidants, or merely an unfavourable equilibrium between various antioxidants. At present there is no basis for recommending the prophylactic use of commercially produced antioxidants. In particular, the optimal combinations of antioxidants are not yet known. If the aim is to improve the antioxidant defence of the body, our advice is still a mixed diet and regular physical activity.

[Quality assurance of laboratories outside hospitals. Use of internal control]. / Kvalitetssikring av laboratorievirksomhet i legepraksis utenfor sykehus. Bruk av internkontroll.

Norway has a national programme for quality assurance of laboratory analyses performed in general practice. A central laboratory distributes control material, and a local team gives practical assistance in each county. To perform laboratory tests properly, the use of quality control material ("internal control") is essential. The users' control results provide a basis for finding both systematic error and the analytical coefficient of variation. The analytical and biological coefficients of variation can be used to calculate the confidence intervals around laboratory test results, and around medical decision thresholds. It is also possible to calculate the magnitude of significant changes in serial results ("critical difference"). In Rogaland county, 11-14 general practitioners showed an analytical coefficient of variation ranging from 0.5 to 5.0% on haemoglobin determination, from 1.2 to 9.9% on glucose, and from 3.1 to 10.5% on prothrombin time. Five general practitioners showed an analytical coefficient of variation from 3.3 to 9.7% for cholesterol. This gives a critical difference ranging from 7.8 to 15.8% for haemoglobin and 18.8 to 31.6% for cholesterol. We present a diagram that can be used to find the critical difference for any test. Such an evaluation of laboratory analyses should become routine in all near-patient testing.

Determination of manganese superoxide dismutase activity by direct spectrophotometry.

A method to determine Mn-superoxide dismutase activity by measuring directly the rate of decay of O2- in a spectrophotometer, is described. Decay of O2- generated by KO2 at pH 9.5, was monitored as the fall in absorbance (A250nm-A360nm). Mn-superoxide dismutase was determined as the activity of cyanide-resistant superoxide dismutase, calculated from the rate of O2- dismutation. Mn-superoxide dismutase could be determined in the presence of a 700 times higher Cu,Zn-superoxide dismutase activity. The alkaline pH did not cause analytical problems. The assay was used to measure both Mn- and Cu,Zn-superoxide dismutase activity in mitochondrial preparations. The assay had a detection limit of 2.8 ng/ml when Mn-superoxide dismutase from E. coli was used, and the between-day CV was 5.8%. The assay is an alternative to indirect methods for detecting superoxide dismutase activity.

Stimulated decay of superoxide caused by ferritin-bound copper.

The redox interaction between O2.- and ferritin cannot solely be regarded as as a Fe(II) release reaction. We demonstrate that native copper bound to horse spleen ferritin and apoferritin, stimulated the decay of O2.- in a catalytic reaction. Copper was determined by atomic absorption spectrophotometry. Decay of O2.- was monitored spectrophotometrically as the decrease in (A250-A360) at pH 9.5. The catalytic effect was linearly related to the copper content of the protein. Ferritin copper was less efficient than equimolar CuCl2, and iron-poor ferritin was more efficient than iron-rich ferritin. The results support a direct antioxidant function of ferritin.

Decay of superoxide catalyzed by ferritin.

Ferritin iron can be reduced by O2.-, released, and form a Fe(II)-chelator complex. However, the thermodynamic influence of the chelator may disturb the reaction balance. We therefore excluded the chelator and measured instead the effect of ferritin on the decay of O.2-, monitored by direct spectrophotometry at pH 9.5. Ferritin, but not apoferritin, accelerated the decay of O.2-. Ferritin iron was apparently the responsible agent. The effect of ferritin was maintained after several bursts of O.2-, and the ratio degraded O.2-/released Fe(II) greatly exceeded one, consistent with a catalytic reaction.

Normothermic liver ischemia in rats: xanthine oxidase is not the main source of oxygen free radicals.

We studied the effect of allopurinol (ALL) on the activity of xanthine dehydrogenase (XDH), xanthine oxidase (XOX), superoxide dismutase (SOD), and catalase (CAT) in rat liver during ischemia followed by 60 min of reperfusion. We induced 60-min ischemia in the median and left lobes by clamping the hepatic artery and portal branches. The percentage XOX relative to total oxidase activity increased significantly in the control group, from 10% during the stabilization period to 18% after 60 min of reperfusion. The XDH activity decreased during reperfusion. Activity of both XDH and XOX was almost completely blocked by ALL. The activity of SOD and CAT did not differ significantly between the ALL group and controls after 60 min of reperfusion. ALL treatment did not affect liver injury parameters, as concentrations of lactate dehydrogenase (LDH) and alanine transferase (ALT) increased in plasma after ischemia, both in controls and in the ALL-treated group. We concluded that ischemia promotes conversion of XDH to XOX during reperfusion. XOX may not be the main source of free radical production, since intracellular scavengers (SOD and CAT) did not differ significantly between controls and the ALL-treated group, despite the fact that ALL blocked XOX activity completely.

Decay kinetics of O2.- studied by direct spectrophotometry. Interaction with catalytic and non-catalytic substances.

The kinetic behaviour of O2.- during spontaneous dismutation and in the presence of Cu,Zn-superoxide dismutase and other compounds, was studied by monitoring the decrease in absorbance (A250nm-A360nm) on a time-scale of > or = 1 min, at pH 9.5. O2.- was generated from KO2, and calculations were performed between 25 and 4 microM of O2.-. An algorithm for the simultaneous calculation of the 1st and 2nd-order rate constants from the decay curve, was evolved. The respective fractions of O2.- which interacted with catalysts or disappeared spontaneously, in various experimental situations, could be estimated. Substances could be classified as inert, catalysts or scavengers. The high assay pH excluded examination of the effect of alkali sensitive substances, e.g., Mn-superoxide dismutase. However, the high pH minimized the interfering effect of trace amounts of Cu(II). Therefore a metal chelator was superfluous and even the effect of metals and metal complexes could be tested. The extremely high sensitivity of the method allowed minute concentrations of reagents to be used, including proteins absorbing in the UV-region. The rate constants found by this simple method, agreed with those obtained by more sophisticated and inaccessible techniques like pulse radiolysis and stopped-flow spectrophotometry.

Improvement of a direct spectrophotometric assay for routine determination of superoxide dismutase activity.

The growing interest in measuring superoxide dismutase (EC 1.15.1.1) in many diseases calls for useful routine assays. For this purpose, the direct spectrophotometric method of Marklund (J Biol Chem 1976;251:7504-7) was improved to offer an alternative to the imprecise, indirect assays currently used. The decay of O2.- (from KO2) at pH 9.5 was monitored as the decrease in delta A (delta A = A250nm-A360nm). Superoxide dismutase was determined from the pseudo-first-order rate constant of O2.- dismutation. The precision of the assay was improved by increasing the concentration of O2.- and expanding the interval for measurements of O2.- concentrations to 4-16 mumol/L. Other assay characteristics, including temperature, were also optimized. In hemolysate the assay had a within-day CV of 5.5-13% and a between-day CV of 4%. Mn-superoxide dismutase and some superoxide dismutase mimics are inhibited at alkaline pH. Therefore, the method is primarily recommended for Cu,Zn-superoxide dismutase.