All-trans retinoic acid results in irregular repair of septa and fails to inhibit proinflammatory macrophages.

All-trans retinoic acid (ATRA) is controversially discussed in emphysema therapy. We re-evaluated ATRA in the elastase model and hypothesised that beneficial effects should be reflected by increased alveolar surface area, elastin expression and downregulation of inflammatory mediators and matrix metalloproteinases (MMPs). Emphysema was induced by porcine pancreatic elastase versus saline in Sprague-Dawley rats. On days 26-37, rats received daily intraperitoneal injections with ATRA (500 µg · kg(-1) body weight) versus olive oil. Lungs were removed at day 38. Rat alveolar epithelial L2 cells were incubated with/without elastase followed by ATRA- or vehicle-treatment, respectively. ATRA only partially ameliorated structural defects. Alveolar walls exhibited irregular architecture: increased arithmetic mean thickness, reduction in surface coverage by alveolar epithelial cells type II. ATRA only partially restored reduced soluble elastin. It tended to increase the ratio of ED1(+):ED2(+) macrophages. Bronchoalveolar lavage (BAL) cells exhibited a proinflammatory state and high expression of interleukin-1ß, cytokine-induced neutrophil chemoattractant-1, tumour necrosis factor-α, nuclear factor-κB, MMP-2, MMP-9, MMP-12, tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 in emphysema, with ATRA exerting only few effects. MMP-7 was highly induced by ATRA in healthy but not in emphysematous lungs. ATRA reduced both MMP-2 and TIMP-1 activity in BAL fluid of emphysematous lungs. ATRA-therapy may bear the risk of unwanted side-effects on alveolar septal architecture in emphysematous lungs.

Recent activities of EC4 in the harmonization of clinical chemistry in the European Union.

This article describes the recent activities of the European Communities Confederation of Clinical Chemistry (EC4). Main goal of EC4 is harmonization of clinical chemistry in the European Union and Europe. EC4's actions connected to that are training and registration of professionals, and accreditation of laboratories. The 35000 professionals practising clinical chemistry in the EU have different backgrounds (medical, pharmaceutical, science-oriented, veterinary, or microbiological). Thus, for the harmonization of training of clinical chemists, EC4 has published a European Syllabus for Postgraduate Training, and instituted a European Union Register for Clinical Chemists. The Syllabus is an indication of the level of requirements in postgraduate training. The EC4 initiative to implement the European Register for Clinical Chemists is based on the 8 years vocational training necessary to obtain sufficient knowledge in clinical chemistry according to the European Syllabus. A guide to the EC4 Register has been published; registration leads to the title European Clinical Chemist (EurClinChem). The accreditation of laboratories must be based on a total quality management system. EC4 has described guidelines (essential criteria) which it judges appropriate for establishing the quality of medical laboratory service; it does not wish to fulfil the role of an accrediting body. Moreover, a working group has been set up to seek to harmonize the work of national accrediting bodies. Therefore, it is logical that EC4 monitors the activities of the different standardizing bodies that might influence the practice of clinical chemistry in the EU. Finally, some aspects concerning the future strategy of EC4 are brought forward.

The European Register for Clinical Chemists. (European Communities Confederation of Clinical Chemistry, Working Group on Registration).

To ensure freedom of movement in the European Union, a limited number of professions is regulated by a so-called Sectorial Directive; all other disciplines, including clinical chemistry, fall under a General Directive. However, clinical chemists in the EU wish their specialty to be more specifically regulated; this means that common standards of education, training, experience and compliance with continuing professional developments must be guaranteed. Therefore, the European Communities Confederation of Clinical Chemistry (EC4) is about to implement the European Register for clinical chemists, and has composed a guide to this Register. The document describes the conditions for entry to specialty training, the minimum standards for registration (university education and postgraduate vocational training with a minimum total of eight years), the competencies of those qualifying for registration, and the operation of the register. Registration guarantees professional and managerial competencies; the title conferred is "European Clinical Chemist". EC4 recognises the existing national registers as far as they are based on the minimal requirements as indicated. An EC4 Register Commission (EC4RC) will maintain and control the European Register, supported by National Clinical Chemistry Registration Committees (NCCRC). An NCCRC controls the quality of the education in each country and assesses candidates. An individual (EU citizen or non-EU citizen trained in an EU country) applies privately for the European Register to EC4RC and, where applicable, the application is accompanied by a document from the NCCRC of the country of registration, stating that the applicant has the necessary qualifications. For EU citizens trained outside the EU the final decision is with EC4RC; non-EU citizens not trained in an EU country are not eligible for registration. Registration is renewed once every five years.

Differential regulation of brain and atrial natriuretic peptides in hemodialysis patients.

We investigated the effect of volume overload on the plasma concentrations of brain and atrial natriuretic peptides as well as cyclic GMP, using specific radioimmunoassays, in 49 patients with chronic renal failure on regular hemodialysis treatment. Markedly elevated levels of the brain (16.2 +/- 1.3 pmol/l) as well as atrial (39.0 +/- 2.8 pmol/l) natriuretic peptide in plasma were found before the dialysis session, as compared to healthy volunteers (range for brain natriuretic peptide, 0.7-7.3 pmol/l, mean level 2.55 +/- 0.32 (SEM) pmol/l). In contrast to the levels of the atrial natriuretic peptide, those of the brain natriuretic peptide were lowered less efficiently by the dialysis procedure: The mean pre-/postdialytic concentration differences were -1.5 pmol/l and -14.2 pmol/l for brain and atrial natriuretic peptide, respectively. The concentrations of the intracellular mediator of the natriuretic peptides, cyclic GMP, were found to be excessively elevated (34.8 +/- 2.8 nmol/l) and returned to near-normal values (12.4 +/- 1.6 nmol/l) at the end of the dialysis session. Concentrations of BNP in plasma of the patients were well correlated to those of ANP. Significant though less marked correlations were also observed between the plasma concentrations of cyclic GMP and BNP, or ANP, respectively. In contrast to those of ANP, pre-/postdialysis differences in plasma BNP concentrations were not correlated to the extent of volume reduction during dialysis. Our findings show that pathophysiologic states resulting in elevations of the plasma concentrations of the atrial natriuretic peptide can also lead to increased levels of the brain natriuretic peptide.(ABSTRACT TRUNCATED AT 250 WORDS)

Quantitative determination of natriuretic peptides in human biological samples with a bioassay using cultured cells.

Up to now, members of the natriuretic peptide family have usually been determined by radioimmunoassays using antibodies more or less specific for the distinct peptides so far identified. However, natriuretic peptides differing significantly in their amino acid sequence from the one against which the antibody has been raised cannot be determined by this means, and still unknown natriuretic peptides cannot be detected. We therefore developed a new bioassay system sensitive to all members of the natriuretic peptide family by taking advantage of the biological activity of these hormones, the activation of the guanylate cyclase/cyclic GMP system. In this assay, cultured cells are incubated with the natriuretic peptides, and the amount of cyclic GMP produced by the cells is determined by radioimmunoassay. From the relative stimulation of the cellular cyclic GMP production, the concentration of natriuretic peptides in the sample is determined after calibration with synthetic atrial natriuretic peptide. For a qualitative identification of the various peptides, the bioassay is combined with a reversed-phase HPLC step. Using cultured bovine aortic endothelial or bovine kidney epithelial cells for the bioassay, we achieved detection limits of 1 fmol or 50 fmol, respectively, for human atrial as well as brain natriuretic peptide. Intra-assay coefficients of variation of 4.3% (aortic endothelial cells, at 0.65 nmol/l peptide) and 5.8% (kidney epithelial cells, at 6.5 nmol/l peptide) were obtained. The total content of natriuretic peptides as well as the amounts of the individual natriuretic peptides following HPLC separation were determined in extracts of human atria obtained at aortocoronary bypass operations.(ABSTRACT TRUNCATED AT 250 WORDS)

Distribution of free and conjugated catecholamines between plasma, platelets and erythrocytes: different effects of intravenous and oral catecholamine administrations.

Plasma, platelet and erythrocyte contents of free and conjugated norepinephrine, epinephrine and dopamine were determined by radioenzymatic assay in 12 resting healthy volunteers. Mean platelet/plasma concentration ratios were 533 for free norepinephrine, 502 for free epinephrine and 149 for free dopamine. Corresponding erythrocyte/plasma ratios were 1.04, 1.13 and 4.5, respectively. The presence of conjugated catecholamines in platelets and erythrocytes could be confirmed; however, their relative proportion within these cells, particularly in platelets, was lower than that in plasma. Upon intravenous infusion of dopamine for 3 hr at 5 micrograms kg-1 min-1, concentrations of free dopamine in plasma increased rapidly (280-970-fold), whereas conjugated dopamine only reached maximal values (14-19-fold increase) at 30 to 60 min after cessation of the infusion. The relative distribution of unconjugated dopamine in whole blood between plasma, platelets and erythrocytes changed from mean values of 1:0.33:3.7 at rest to 1:1.1:0.5 at the end of the infusion. As a result of the subsequent rapid decrease of dopamine in plasma and erythrocytes, this distribution was 1:17:1 shortly thereafter and remained constant up to the end of the investigation period. The relative distribution for conjugated dopamine of 1:0.001:0.5 at rest changed to about 1:0.2:0.1 at the termination of the infusion. Oral administration of norepinephrine and dopamine led to increases in the plasma concentrations of these amines in their conjugated forms only, whereas epinephrine concentrations remained constant. These elevations were not accompanied by corresponding increases in platelet and erythrocyte norepinephrine, epinephrine and dopamine contents.(ABSTRACT TRUNCATED AT 250 WORDS)

High incidence of antibodies to hepatitis C virus in alcoholic cirrhosis: fact or fiction?

An enzyme immunoassay (Ortho-HCV ELISA) for antibodies against the hepatitis C virus was used to test serum samples from 39 patients with alcoholic cirrhosis and 34 patients with alcoholic hepatitis or fatty liver. The frequency of a positive result in the cirrhotics was significantly higher than in the alcoholics without cirrhosis (38.5% vs 8.8%, P less than 0.01). However, the positive results in the cirrhotics were associated with high gammaglobulin concentrations, and optical density values in the assay correlated closely with serum globulin (r = 0.73, P less than 0.01). The findings suggest that serum from patients with alcoholic cirrhosis may contain a component that give false-positive results in the assay.

Antibodies as a source of analytical errors.

This review discusses the interference in clinical chemical analysis caused by autoantibodies and by antibodies to foreign antigens. The nature of these disturbances, their occurrence, prevalence, and detection, the different ways in which they are manifested, the clinical consequences of the failure to recognize such interference, and finally methods for avoiding these disturbances are discussed. Interference by cold agglutinins in the automatic determination of the erythrocyte count, interference by cryoglobulins in the determination of the leukocyte count, and EDTA-induced thrombocyte agglutination are well documented as sources of error in the analysis of haematological parameters. Enzyme determinations may be affected by the occurrence of macro-complexes of the measured enzyme with immunoglobulins. This type of interference, for example, may occur in the determination of amylase and creatine kinase. Immunoassays for the determination of hormones or tumour markers are sensitive to autoantibodies and heterophilic antibodies. The former are particularly important in the determination of thyroid hormones, where the interference is method-dependent. In several immunoassays, interference by heterophilic antibodies can be abolished by the addition of non-immune serum. Finally, rheumatoid factors, antibodies administered for therapeutic purposes, and monoclonal gammopathies are possible sources of interference in the determination of various analytes.

Magnitude and kinetics of alterations in plasma catecholamines and leukocyte beta-adrenergic receptors in response to anaesthesia and surgery.

We studied the response of the sympatho-adrenal system to varying intensities of different stimuli. Concentrations of norepinephrine and epinephrine in plasma as well as densities of beta 2-adrenergic receptors on mononuclear leukocytes were determined in patients subjected to operations of varying complexity and different types of anaesthesia. In patients undergoing hysterectomy (n = 9), the maximal increases in plasma norepinephrine and epinephrine were 2.7- and 2.8-fold, respectively, corresponding to a post-operative decrease of the mononuclear leukocyte beta 2-adrenergic receptors of 27% after 4 hours. Patients with coronary revascularization (n = 17) were randomly selected to receive either enflurane/N2O or neurolept anaesthesia. During intraoperative periods of stress, such as cardiopulmonary bypass and hypothermia, norepinephrine and epinephrine levels were 2-3 times higher in the neurolept patients, compared with the enflurane patients. In the former group, the respective maximal norepinephrine and epinephrine concentrations were 9.7 and 28 times the vasal values of the non-anaesthetized patients. One day postoperatively, the mononuclear leukocyte beta 2-receptor density decreased maximally by 45 +/- 11% in the enflurane patients, and by 53 +/- 6% in the neurolept patients. As early as two to five days after cardiac surgery, beta 2-receptor densities were no longer distinguishable from the preoperative values. Significant correlations between the increases in catecholamine concentrations and the decreases in beta 2-receptor densities did not exist. It is concluded that enflurane blocks the sympatho-adrenal response to surgical stress more effectively than neurolept anaesthesia.(ABSTRACT TRUNCATED AT 250 WORDS)

Dopamine infusion in healthy subjects and critically ill patients.

1. Little is known about the metabolism and the pharmacokinetics of dopamine (DA) in critically ill patients. To study the influence of the total administered DA dose on the disposition of free (i.e. unconjugated) and sulfoconjugated DA, plasma levels of free and sulfoconjugated DA were measured following infusion of 5 micrograms DA/kg per min for 0.5 and 3 h in six healthy volunteers and in eight critically ill patients receiving DA at the same infusion rate for 6.5 to 329 h. 2. In patients and volunteers steady state concentrations of free DA showing fairly large inter-individual variations (12.4-73.4 micrograms/L) were reached within 10 min of the beginning of the infusion. 3. DA sulfate was generated immediately. In volunteers peak values of the sulfoconjugate were observed 15-60 min after the termination of the DA infusion. In patients steady state concentrations of conjugated DA (63-80 micrograms/L) were reached within 5-10 h of DA infusion. 4. The initial half-life (t1/2 alpha), the terminal elimination half life (t1/2) and the distribution volume of free DA in the volunteers were significantly higher after 3 h of the DA infusion as compared to the shorter infusion. These parameters as well as the total plasma clearance of free DA were independent of the length of the DA infusion period in patients. The large distribution volumes of 19.8-75 L/kg indicate that DA has been taken up by peripheral tissues. 5. Substantial inter-individual variations in the patients' clearance of free DA (3.9-16.5 L/kg per h) may partly explain the variability in haemodynamic responses to DA infusion reported in clinical studies.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of ATP-stimulated guanylate cyclase activation in rat lung membranes.

Many of the effects of ANP are mediated through the elevation of cellular cGMP levels by the activation of particulate guanylate cyclase. While the stimulation of this enzyme is receptor-mediated, the molecular mechanism of activation remains unknown. In this study we present evidence that ATP as well as its analogues adenosine-5'-O-(3-thiotriphosphate) (ATP gamma S) and adenylylimidophosphate (AMPPNP) activates guanylate cyclase from rat lung membranes and markedly potentiates the effect of ANP on the enzyme. The order of potency is ATP gamma S greater than ATP greater than AMPPNP. The enzyme activation by adenine nucleotide and ANP together is much more than the sum of the individual activations, suggesting that ATP may be the physiological component essential for the ANP-stimulated guanylate cyclase activation. The ATP gamma S-stimulated guanylate cyclase activity diminishes in the presence of various kinds of detergents, suggesting either that the conformation of an ATP binding site in guanylate cyclase is altered by detergents or that protein-protein interaction may be involved in the activation of guanylate cyclase by ATP. Guanylate cyclase from rat lung membranes is poorly activated by ANP and/or ATP gamma S after removing the cytosolic and weakly membrane-associated proteins or factors by centrifugation. Pre-incubation of the membranes with ATP gamma S retains enzyme activation after membrane washing. These results suggest either that ATP gamma S stabilizes the conformation of nucleotide binding site in guanylate cyclase from denaturation by membrane washing, or that the stimulatory effect of ATP on guanylate cyclase activity may be mediated by accessory proteins or non-protein cofactors which are lost during membrane washing, but remain bound to membranes by ATP gamma S pretreatment.

Mechanism of cyclic GMP inhibition of inositol phosphate formation in rat aorta segments and cultured bovine aortic smooth muscle cells.

In order to clarify the mechanism(s) by which cyclic GMP inhibits the generation of inositol phosphates in rat aorta segments and cultured bovine aortic smooth muscle cells, we studied phosphoinositide hydrolysis and GTPase activity in homogenates and membrane preparations of cultured bovine aortic smooth muscle cells. Pretreatment of homogenate preparations with cyclic GMP plus ATP did not inhibit [8-arginine, 3H] vasopressin (AVP) binding, but resulted in a total suppression of the AVP-induced GTPase activation. The pretreatment with cyclic GMP and ATP also inhibited the formation of inositol phosphates induced by AVP in the presence of low concentrations of guanosine 5'-(gamma-thio)triphosphate (GTP gamma S), or by high concentrations of GTP gamma S alone. However, the formation of inositol phosphates by high concentrations of Ca2+ alone was not blocked. These results suggest that the ability of cyclic GMP to inhibit phosphoinositide hydrolysis results from an inhibition of a guanine nucleotide regulatory protein activation, and the interaction between guanine nucleotide regulatory protein and phospholipase C. While the precise site of this inhibition is not presently known, the inhibition by cyclic GMP is dependent upon the addition of ATP and probably entails a phosphorylation event since adenylylimidodiphosphate can not substitute for the ATP requirement.

Brain natriuretic factor. Augmentation of cellular cyclic GMP, activation of particulate guanylate cyclase and receptor binding.

The newly discovered peptide, brain natriuretic factor (BNF), caused a concentration-dependent increase (up to 400-fold) in intracellular cyclic GMP levels in cultured endothelial, smooth muscle and fibroblast cells. The extent of cGMP augmentation was comparable to that produced by atrial natriuretic factor (ANF). The activity of the membrane-bound guanylate cyclase of different rat tissues and cultured cells was markedly stimulated by the peptide and the addition of ATP potentiated the stimulation. As opposed to tissue particulate guanylate cyclase, the enzyme in cell membranes was slightly more sensitive to activation by BNF than to stimulation by ANF. On bovine aortic smooth muscle (BASM) cells, specific high-affinity binding sites (Bmax = 398 fmol/10(6) cells, Kd = 0.52 nM) for BNF were observed for which ANF could compete with apparently equal affinity. These results suggest that activation of the cGMP pathway constitutes a common mechanism of action for both BNF and ANF.

The influence of kidney transplantation on carnitine metabolism.

In this study, we examined all three plasma carnitine fractions, free carnitine (FC), short-chain acyl- (SCC), and long-chain acylcarnitine (LCC), as well as the urinary excretion of FC and SCC in 62 patients with functioning renal allografts 1-70 months following the kidney transplantation (KT). Patients were classified into three groups according to their transplant function, as characterized by the serum creatinine concentration (CR). A comparison with normal subjects (n = 20) and with patients on chronic hemodialysis (HD, n = 46) revealed a complete normalization of all carnitine plasma fractions for group I patients (Cr less than 120 mumol/l). In contrast, significant elevations of plasma free and esterified carnitine were found in group II (Cr greater than 120, less than 200 mumol/l; with FC + 27%, SCC + 82%, and LCC + 49% and group III patients (Cr greater than 200 mumol/l; with FC + 39%, SCC + 122%, and LCC + 106%), as compared to healthy subjects (p less than 0.001). The elevations in concentrations of SCC were more pronounced than those of FC; consequently, we found a higher ratio of acylcarnitine (AC) to FC in group III than in group I patients (+ 41%, p less than 0.001). Yet even in the former group, this ratio was found to be markedly reduced when compared to HD patients (-41%, p less than 0.001). We found no significant differences in the urinary excretion of FC and SCC between the 3 groups of KT patients. It is thus to conclude that in patients with a well-functioning transplant, the pattern of carnitine fractions in plasma is fully normalized. The decrease in the ratio of AC to FC following a successful KT might suggest a better availability of FC and thereby be associated with an enhanced fatty acid oxidation.

Effects of nitrovasodilators, endothelium-dependent vasodilators, and atrial peptides on cGMP.

The interactions of the nitrovasodilators, endothelium-dependent vasodilators, and atrial peptides with cGMP synthesis have proved to be useful and important leads to the functions of cGMP and signal transduction mechanisms. Although considerable progress has been made, many important questions remain to be answered. Nevertheless, the guanylate cyclase-cGMP system clearly represents an important second-messenger system that mediates the effects of numerous agents.

Alterations of beta-adrenoceptors on human leukocyte subsets induced by dynamic exercise: effect of prednisone.

1. In order to investigate exercise-induced changes of beta 2-adrenoceptors on human leukocyte subsets, beta-adrenoceptor density was determined as specific binding of [125I]-iodocyanopindolol to granulocytes, monocytes, B and T lymphocytes of six subjects immediately before and after exercise and after 30 min of rest. 2. A 10 min graded bicycle exercise with a workload of 50-250 W caused a transient increase of granulocytes, monocytes, B and T cells of about 32, 43, 120 and 25%, respectively. 3. While the number of beta 2-adrenoceptors in granulocytes remained unchanged, beta-adrenoceptor densities in B cells, T cells and monocytes increased from pre-exercise mean values of 2730, 870 and 2400 binding sites/cell to 3500, 1230 and 3220 binding sites/cell, respectively, under physical stress. The rise in receptor numbers was accompanied by an enhanced isoprenaline-stimulated cyclic AMP formation in unfractionated mononuclear leukocytes (MNL) of about 26% as well as by a 2-3-fold increase in plasma catecholamine levels. Cell concentrations, beta 2-adrenoceptor numbers and adrenergic responsiveness returned to normal after 30 min rest. 4. Administration of 60 mg prednisone 2 h before exercise resulted in granulocytosis and lymphopenia with a preponderant effect on the exercise-induced rise in B cells and monocytes. Corticosteroids showed no effect on stress-induced changes of beta 2-adrenoceptors and responsiveness. 5. It is concluded that exercise-induced increases in beta 2-adrenoceptor density and adrenergic responsiveness of unfractionated MNL are caused by a release of receptor-enriched cells into the circulation, particularly of B lymphocytes and monocytes which carry the highest beta 2-adrenoceptor density.

Quantitative monitoring by high-performance liquid chromatography of the dissociation of human serum cholinesterase by limited proteolysis.

Proteolytic action on human serum cholinesterase, a tetrameric enzyme, results in a partial disintegration which can be recorded only qualitatively by time-consuming electrophoretic techniques. In this study, a rapid high-performance liquid chromatographic method was used for the separation and determination of the active dissociation products. Separation of the cholinesterase subunits was accomplished by high-performance gel permeation chromatography on a combination of DIOL columns (Zorbax GF 450/GF 250) in 0.2 M phosphate buffer (pH 7.0). Detection and quantification of enzyme activity in the fractionated eluate were carried out using a Flexigem analyser (substrate, butyrylthiocholine). On limited tryptic digestion of partially purified human ChE, up to three peaks of enzyme activity could be identified. Their elution volumes corresponded to apparent molecular masses of 480,000, 270,000 and 120,000, indicating, in addition to the tetrameric holoenzyme, a dimeric and a monomeric form. Quantification of the relative amounts of individual enzyme activity peaks revealed that in the course of degradation, the dimer appeared first, followed by the monomer. This suggests that the first step in the sequence of dissociation is cleavage of the tetramer into a pair of dimers, then further into the monomeric subunit. During the incubation with trypsin, a significant change in the pattern of the different peaks had already occurred when the total enzyme activity was only slightly reduced.