Platelet activation in the presence of neutral protamine Hagedorn insulin: a new feature of antibodies against protamine/heparin complexes.

Essentials Protamine (PRT) is used to stabilize insulin in neutral protamine Hagedorn (NPH) insulin. The interaction between NPH-insulin, anti-PRT/heparin antibodies and platelets was investigated. Anti-PRT/heparin antibodies activate platelets in presence of NPH-insulin dependent on heparin. Cross-reactivity seems to have no major effect on the clinical outcome of medical patients. SUMMARY: Background Protamine (PRT) is used to stabilize insulin in neutral protamine Hagedorn (NPH) insulin, a commonly used therapeutic agent for diabetes mellitus. Immunization against PRT/heparin complexes is common in diabetic patients. Objectives To investigate the impact of NPH-insulin on the interaction between anti-PRT/heparin antibodies and platelets. Methods The interaction between NPH-insulin and anti-PRT/heparin antibodies was tested using in-house enzyme immunoassays. The ability of anti-PRT/heparin antibodies to activate platelets in the presence of NPH-insulin (and heparin) was investigated using flow cytometry. Results Twenty-one out of 80 sera containing anti-PRT/heparin IgG showed binding to NPH-insulin. Anti-PRT/heparin IgG from immunized patients bound to platelets in the presence of NPH-insulin, but not in the presence of native insulin. Anti-PRT/heparin antibodies induced P-selectin expression in the presence of NPH-insulin in a heparin-dependent way (median mean fluorescence intensity in the presence of NPH-insulin: 55, 95% confidence interval [CI] 18.7-100.5 vs. NPH-insulin and heparin: 204, 95% CI 106.5-372.8). The clinical relevance of platelet-activating anti-PRT/heparin antibodies was assessed by investigating a multicenter study cohort of 332 acutely ill medical patients who received heparin. None of the 21 patients with anti-PRT/heparin IgG developed thrombocytopenia or thromboembolic complications. Conclusions Anti-PRT/heparin antibodies activate platelets in the presence of NPH-insulin in a heparin-dependent way. However, results from our preliminary study indicate no major impact of these antibodies on the clinical outcome in medical patients receiving heparin, particularly on thromboembolic complications.

Current insights into the laboratory diagnosis of HIT.

Heparin-induced thrombocytopenia (HIT) is an adverse drug reaction and prothrombotic disorder caused by immunization against platelet factor 4 (PF4) after complex formation with heparin or other polyanions. After antibody binding to PF4/heparin complexes, HIT antibodies are capable of intravascular platelet activation by cross-linking Fc gamma receptor IIa (FcγRIIa) on the platelet surface leading to a platelet count decrease and/or thrombosis. In contrast to most other immune-mediated disorders, the currently available laboratory tests for anti-PF4/heparin antibodies show a high sensitivity also for clinically irrelevant antibodies. This makes the diagnosis of HIT challenging and bears the risk to substantially overdiagnose HIT. The strength of the antigen assays for HIT is in ruling out HIT when the test is negative. Functional assays have a higher specificity for clinically relevant antibodies, but they are restricted to specialized laboratories. Currently, a Bayesian approach combining the clinical likelihood estimation for HIT with laboratory tests is the most appropriate approach to diagnose HIT. In this review, we give an overview on currently available diagnostic procedures and discuss their limitations.

Anti-platelet factor 4/heparin antibodies in patients with impaired graft function after liver transplantation.

BACKGROUND: Heparin, the standard perioperative anticoagulant for the prevention of graft vessel thrombosis in patients undergoing liver transplantation (LT), binds to the chemokine platelet factor 4 (PF4). Antibodies that are formed against the resulting PF4/heparin complexes can induce heparin-induced thrombocytopenia. LT is a clinical situation that allows the study of T-cell dependency of immune responses because T-cell function is largely suppressed pharmacologically in these patients to prevent graft rejection. OBJECTIVES: To investigate the immune response against PF4/heparin complexes in patients undergoing LT. PATIENTS AND METHODS: In this prospective cohort study, 38 consecutive patients undergoing LT were systematically screened for anti-PF4/heparin antibodies (enzyme immunoassay and heparin-induced platelet aggregation assay), platelet count, liver function, and engraftment. RESULTS: At baseline, 5 (13%) of 38 patients tested positive for anti-PF4/heparin IgG (non-platelet-activating) antibodies. By day 20, an additional 5 (15%) of 33 patients seroconverted for immunoglobulin G (two platelet-activating) antibodies. No patient developed clinical heparin-induced thrombocytopenia. Two of six patients with graft function failure had anti-PF4/heparin IgG antibodies at the time of graft function failure. Graft liver biopsy samples from these patients showed thrombotic occlusions of the microcirculation. CONCLUSIONS: Anti-PF4/heparin IgG antibodies are generated despite strong pharmacologic suppression of T cells, indicating that T cells likely have a limited role in the immune response to PF4/heparin complexes in humans.

The impact of diets with different magnesium contents on magnesium and calcium in serum and tissues of the rat.

The impact of three different magnesium diets (70, 1,000 and 9,000 ppm) on total, ionized and bound magnesium as well as ionized calcium in serum and total calcium and magnesium in femoral bone, skeletal muscle, heart and liver of male Sprague-Dawley rats was investigated. The percentage of ionized serum magnesium was unproportionally high in rats fed a low magnesium (70 ppm) diet. Femoral magnesium was correlated with ionized and total serum magnesium. In contrast, there was generally no correlation between total serum magnesium and the magnesium fractions in skeletal muscle, heart and liver. In rats fed the magnesium deficient diet, total cardiac concentration of magnesium was even significantly increased along with total calcium content, while there were no effects on total muscle and liver magnesium. Within the single groups, ionized serum calcium was never proportional to dietary magnesium, but in all three magnesium diet groups together, it was inversely correlated with dietary magnesium. Moreover, ionized serum calcium was inversely correlated with both ionized and total serum magnesium. In all 3 groups together, the concentrations of total calcium and magnesium in heart and skeletal muscle were correlated, within the single groups correlation existed only in the 1000 ppm group. Magnesium influx via calcium channels during low magnesium intake has been seen in non cardiac tissues [35,36], but nothing similar is known about non selective channels for divalent cations in the heart [33]. Thus, magnesium uptake by cardiac cells along with calcium seems to be possible, especially at low intracellular magnesium concentrations, but is still poorly investigated. We suggest that the calcium-antagonistic effect of magnesium is related to the turnover rate of magnesium rather than to its tissue concentrations.

Continuous rise of urinary iodine excretion and drop in thyroid gland size among adolescents in Mecklenburg-West-Pomerania from 1993 to 1997.

Improved legislation (1989, 1993), as well as education of the public, are likely to improve the iodine supply for the German population. Children and adolescents will be the first to profit. We investigated thyroid size and urinary iodine excretion in a total of 2906 students aged 10 to 18 in Mecklenburg-West-Pomerania in 1993, 1995, and 1997. The median urinary iodine excretion rose from 73 microg/g creatinine in 1993 to 133 microg/g in 1997. The prevalence of goiter, according to the reference range of Gutekunst, dropped from 33% to 10% over the same interval, and the median thyroid size declined from 11 ml to 6 ml. While only 6% of the test subjects excreted more than 150 microg iodine per g creatinine in 1993, this figure rose to 33% in 1997. The improved alimentary iodine supply is due to the increased use of iodine enriched salt by the food industry, food factories and in common food supply services.

[Increased dietary intake of iodine in young people in Mecklenburg-Vorpommern 1993 and 1996]. / Alimentäre Jodaufnahme bei Jugendlichen in Mecklenburg-Vorpommern zwischen 1993 und 1996 gestiegen.

We investigated the goiter prevalence and the urinary iodine excretion of 2,109 young people between 10 and 18 years in the district of Mecklenburg-Vorpommern, Germany in 1995/96. The thyroid volume was determined by ultrasound (7.5 MHz), the iodine excretion by a modified cer-arsenit method. The daily iodine excretion as related to the body surface area and the age related creatinine excretion per 24 h were measured. The results were compared with those of a similar study from 1993 in the same region and the same range of age. The goiter prevalence in 1997 amounted to 18.5%. Among the studied persons 3.6% showed one or more nodules within their thyroid gland. The iodine excretion increased from 70 micrograms in 1993 to 95 micrograms in 1997. There were no changes in the individual nutritional habits (especially iodine-containing foods, using iodized salt etc.). We believe that the raised iodine intake is the result of a higher iodine supply in the commercially produced foodstuffs and animal products due to an increased incorporation of iodine in manufacture of food products.

Metabolic fate of 4-hydroxynonenal in hepatocytes: 1,4-dihydroxynonene is not the main product.

4-Hydroxynonenal (HNE) is a major aldehydic product of lipid peroxidation known to exert several biological and cytotoxic effects. The metabolic fate of this aldehyde was investigated in hepatocytes as a cell type with a rapid HNE degradation. The experiments were carried out in rat hepatocytes at 37 degrees C at initial HNE concentrations of 1 microM-that means in the range of physiological and pathophysiologically relevant HNE levels-, 5 microM or 100 microM, respectively. About 95% of 100 microM HNE was degraded within 3 min of incubation. At 1 microM HNE the physiological level of about 0.1 to 0.2 microM was restored already after 30 sec. As primary products of HNE in hepatocytes the glutathione-HNE- 1:1-adduct, the hydroxynonenoic acid and the corresponding alcohol of HNE, the 1,4-dihydroxynon-2-ene, were identified. In contrast to previous reports, the corresponding alcohol of the HNE, 1,4-dihydroxynon-2-ene, was not the main HNE metabolite by far. The sum of these three primary HNE products accounts for about two-thirds of the total HNE degradation after 3 min of incubation. Furthermore, the beta-oxidation of hydroxynonenoic acid including the formation of water was demonstrated. The quantitative share of HNE binding to proteins, contrary to its great functional importance, is low with about 3% of total HNE consumption after 3 min incubation. The glycine-cysteine-HNE, cysteine-HNE adducts, and the mercapturic acid from glutathione-HNE adduct are not formed. In total, almost 90% of HNE degradation could be balanced by the formation of different HNE metabolites. The fast metabolism underlines the role of HNE degrading pathways in hepatocytes as one important part of the antioxidative defense system in order to protect proteins from modification by aldehydic lipid peroxidation products.

Serum zinc levels and goitre epidemiology in Germany.

Zinc is an essential element involved in many basic biochemical reactions. Animal experiments and clinical data support the hypothesis that in the presence of inadequate iodine supply zinc deficiency may be a stimulus for the development of goitre. Within the framework of a study to record goitre prevalence and iodine supply of Germany we determined the serum zinc levels in 5932 clinically healthy persons. The mean value was 13.1 +/- 1.75 mumol/l. There were no differences neither in age, in sex nor in regional distribution. Furthermore there were no correlations between serum zinc level, thyroid volume and urinary iodide excretion. Only 1% of all cases had low zinc levels (< 10 mumol/l). Older people (> 40 years) with lowered serum zinc levels showed significantly larger thyroids. We conclude that there is no relevant undersupply of zinc and therefore the trace element is not involved in the goitre endemy of Germany.

Effect of oxidative stress by iron on 4-hydroxynonenal formation and proliferative activity in hepatomas of different degrees of differentiation.

It has been shown previously that oxidative stress by ferrous iron in vitro leads to an inhibition of proliferation of murine ascites tumour cells in vivo. This effect is associated with increased lipid peroxidation in terms of formation of the highly reactive aldehyde 4-hydroxynonenal (HNE), which has been shown to inhibit the proliferation of numerous tumours and to induce differentiation. It was the purpose of this article to study the occurrence and metabolism of HNE and its inducibility by oxidative stress in hepatomas of different degrees of differentiation to find further evidence for a possible role of HNE in proliferation and/or differentiation, because it is known that in hepatoma cells with a very low degree of differentiation basal lipid peroxidation is hardly detectable, while in normal hepatocytes the basal level of thiobarbituric acid reactive substances (TBArS) is rather high. MH1C1 hepatoma cells and Yoshida AH-130 hepatoma cells were chosen as highly differentiated and poorly differentiated tumour cells, respectively, and rat hepatocytes served as a control for normal liver phenotype. Ferrous histidinate (Fe/His) did not have a cytotoxic effect on Yoshida and MH1C1 cells, as measured by the LDH release test. In cell culture studies Fe/His revealed a dose dependent inhibition of the proliferation of Yoshida cells. The incorporation of 3H-thymidine into DNA of these cells was also inhibited by Fe/His in a dose-dependent manner, while the precursor uptake into the cytoplasm was unaffected. The basal levels of HNE were in the order: hepatocytes > MH1C1 cells > Yoshida cells. Both hepatocytes and Yoshida cells responded to the presence of Fe/His with increased formation of TBArS. Compared with hepatocytes the response of the Yoshida cells was greatly reduced. The response of cells to Fe/His with respect to HNE formation was decreased in the order: hepatocytes > MH1C1 cells > Yoshida cells, but in this case the differences were not very pronounced. The metabolic capacity of the cells to consume HNE was also decreased in the order: hepatocytes > MH1C1 cells > Yoshida cells. In this case the differences were very pronounced. These findings support the view that Yoshida cells with a low degree of differentiation and a low basal level of HNE are released from an inhibitory effect of HNE operative in hepatocytes and that HNE is causally involved in the iron induced inhibition of proliferation of poorly differentiated hepatoma cells.

[Current state of alimentary iodine deficiency in Germany]. / Aktueller Stand der alimentären lodversorgung in Deutschland.

Germany finds itself among the most iodine deficient countries of Europe. Voluntary use of iodized salt constitutes the only goiter prophylaxis. In the last few years, measures such as the opening up of European internal markets, abolition of lac pertaining to the alimentary iodine consumption. Random samples of urine collected from 5932 persons without thyroid ailment, distributed over 32 regions of Germany, were measured for iodine excretion. The median value of iodine excretion was 72.4 micrograms I/g creatinine. Children under 10 years (76.9) and persons over 70 years (80.7) showed a slightly higher iodine elimination than those between 11-70 years (71.9). No differences between the former East Germany and West Germany as well as North, South and middle regions of unified Germany were observed. 55% of the study population presented with iodine values between 50 and 100 micrograms, 19% with lower than 50 micrograms. Only 9% showed sufficient iodine state. The results expose the inadequacy of the voluntary measures to tackle the problem of alimentary iodine deficiency.

Metabolism of 4-hydroxynonenal, a cytotoxic lipid peroxidation product, in Ehrlich mouse ascites cells at different proliferation stages.

The aldehydic lipid peroxidation product 4-hydroxynonenal (HNE) is cytotoxic at high concentrations (in the range of 100 microM); at low concentrations, it disturbs cell proliferation and exhibits genotoxic effects, and in the submicromolar range, HNE is chemotactic and stimulates phospholipase C. HNE is rapidly metabolized in eukaryotic cells. Here the metabolism of HNE was studied in suspensions of Ehrlich mouse ascites cells at different periods of the tumor age. The main products of HNE which were identified in the Ehrlich ascites cells, were glutathione-HNE-conjugate, hydroxynonenoic acid, and 1,4-dihydroxynonene. The formation of glutathione conjugates following the addition of HNE was higher in early phase cells when compared with cells in the late phase of tumor growth. That was in accordance with the increased consumption of the reduced form of glutathione. Ehrlich ascites tumor cells at the proliferation phase were able to reduce a higher amount of exogenous-added HNE, compared with cells at the stationary phase.

[Capillary blood as diagnostic sample in pediatric laboratory diagnosis. Arterio-capillary-venous difference and blood collection technique]. / Kapillarblut als Prüfmaterial in der pädiatrischen Laboratoriumsdiagnostik. Arterio-kapillär-venöse Differenz und Entnahmetechnik.

It is preferable to use capillary blood as diagnostic material in paediatric laboratory tests rather than venous blood. The collection of capillary blood makes less demands on the child and is easy in principle. The objection to capillary blood primarily concerns differences in the analyte concentration in arterial, capillary and venous blood. Comparative studies in the same child under standardised conditions are not available for a number of parameters. However, according to the results available at present, for almost all routine clinical chemical and haematological examinations clinically usable results can be obtained. Using the example of total protein analysis for the same child we did not find any differences between arterial and venous blood. It is highly probably that the same would apply to many other components. Suspected differences are probably a result of short-comings in the collection technique.

The metabolism of 4-hydroxynonenal, a lipid peroxidation product, is dependent on tumor age in Ehrlich mouse ascites cells.

4-Hydroxynonenal is a major product formed by lipid peroxidation from omega 6-polyunsaturated fatty acids as linoleic acid and arachidonic acid. This aldehyde is cytotoxic at high concentrations (in the range of 100 microM), disturbs cell proliferation at low concentrations and exhibits genotoxic effects. Furthermore, in the submicromolar range 4-hydroxynonenal is chemotactic and stimulates phospholipase C. 4-Hydroxynonenal is rapidly metabolized in eucaryotic cells. Here the metabolism of 4-hydroxynonenal was studied in suspensions of Ehrlich mouse ascites cells at different periods of the tumor age. The Ehrlich ascites tumor is a convenient biological model for the investigation of tumor cells in different age and proliferation phases of the tumor. The main products of 4-hydroxynonenal which were identified in the Ehrlich ascites cells were glutathione-HNE-conjugate, hydroxynonenoic acid and 1,4-dihydroxynonene. The formation of glutathione conjugates following the addition of 4-hydroxynonenal was higher in cells of the early phase in comparison with cells of the late phase of tumor growth. That was in accordance with the increased consumption of the reduced form of glutathione during 4-hydroxynonenal utilization. The degradation of 4-hydroxynonenal and other aldehydic products of lipid peroxidation is postulated to be an important part of the intracellular antioxidative defense system.

[Relative parathyroid insufficiency in hypomagnesemia]. / Relative Nebenschilddrüseninsuffizienz bei Hypomagnesiämie.

In patients with normocalcaemic tetany and hyperventilation syndrome (functional breathing syndrome), respectively, the parathyroid function was studied by means of the EDTA test according to Kaiser and Ponsold. In agreement with experimental results on inhibition of the parathyroid hormone release in magnesium deficiency patients with low serum magnesium concentrations show a pathological EDTA test signalling a relative parathyroid insufficiency.

Identification of metabolic pathways of the lipid peroxidation product 4-hydroxynonenal by enterocytes of rat small intestine.

The cytotoxic lipid peroxidation product 4-hydroxynonenal (HNE1) is rapidly metabolized in enterocytes. The degradation of HNE and other aldehydic products of lipid peroxidation processes seems to be an antioxidative defense system. The metabolism of HNE was studied in suspensions of rat enterocytes at 37 degrees C, pH 7.4 and at initial HNE concentration of 100 microM. About 70% of the HNE were degraded within three minutes of incubation. Main products of HNE which were identified in enterocytes were the glutathione-HNE-1:1-conjugate, the hydroxynonenoic acid and the 1,4-dihydroxynonene. Furthermore, the formation of metabolites of the tricarboxylic acid cycle is suggested. The quantitative share of HNE binding to proteins was low with about 1% of total HNE consumption after three minutes of incubation.

Chemistry and biochemistry of 4-hydroxynonenal, malonaldehyde and related aldehydes.

Lipid peroxidation often occurs in response to oxidative stress, and a great diversity of aldehydes are formed when lipid hydroperoxides break down in biological systems. Some of these aldehydes are highly reactive and may be considered as second toxic messengers which disseminate and augment initial free radical events. The aldehydes most intensively studied so far are 4-hydroxynonenal, 4-hydroxyhexenal, and malonaldehyde. The purpose of this review is to provide a comprehensive summary on the chemical properties of these aldehydes, the mechanisms of their formation and their occurrence in biological systems and methods for their determination. We will also review the reactions of 4-hydroxyalkenals and malonaldehyde with biomolecules (amino acids, proteins, nucleic acid bases), their metabolism in isolated cells and excretion in whole animals, as well as the many types of biological activities described so far, including cytotoxicity, genotoxicity, chemotactic activity, and effects on cell proliferation and gene expression. Structurally related compounds, such as acrolein, crotonaldehyde, and other 2-alkenals are also briefly discussed, since they have some properties in common with 4-hydroxyalkenals.

[Laboratory diagnostic monitoring of blood gas parameters in children during bronchologic studies]. / Labordiagnostische Uberwachung der Blutgasparameter bei Kindern während bronchologischer Untersuchungen.

Changes in PaO2 and parameters of acid-base-status during bronchological investigations were studied. 20 patients of both sexes with chronic nonspecific respiratory diseases have been investigated. The most important results were a respiratory acidosis after intubation and simultaneous hyperoxigenation during controlled (manual) ventilation. A respiratory acidosis with a small metabolic part was observed during extubation and change to normal respiration. In some cases acidosis was decomposated. These changes were found to be more extensive after bronchography than after bronchoscopy. Acidosis was normalized latest 60 minutes after extubation in all cases. The used method of bronchological examination has caused a favourable influence on the conditions of ventilation.

Measuring the impedance of erythrocytes in patients with cystic fibrosis.

A study of the dielectric properties of fresh, washed erythrocytes from three groups of 13 children, one with cystic fibrosis, and one each of healthy children of comparable age and comparable weight, was conducted after resuspension in their own plasma or in sodium phosphate buffer. The erythrocytes of the patients with cystic fibrosis showed significant variations in impedance, especially compared to the control group of comparable age. Further studies are required to ascertain whether these results are of diagnostic significance.