ABSTRACT
Lipoprotein apheresis (LA) is currently the most powerful intervention possible to reach a maximal reduction of lipids in patients with familial hypercholesterolemia and lipoprotein(a) hyperlipidemia. Although LA is an invasive method, it has few side effects and the best results in preventing further major cardiovascular events. It has been suggested that the highly significant reduction of cardiovascular complications in patients with severe lipid disorders achieved by LA is mediated not only by the potent reduction of lipid levels but also by the removal of other proinflammatory and proatherogenic factors. Here we performed a comprehensive proteomic analysis of patients on LA treatment using intra-individually a set of differently sized apheresis filters with the INUSpheresis system. This study revealed that proteomic analysis correlates well with routine clinical chemistry in these patients. The method is eminently suited to discover new biomarkers and risk factors for cardiovascular disease in these patients. Different filters achieve reduction and removal of proatherogenic proteins in different quantities. This includes not only apolipoproteins, C-reactive protein, fibrinogen, and plasminogen but also proteins like complement factor B (CFAB), protein AMBP, afamin, and the low affinity immunoglobulin gamma Fc region receptor III-A (FcγRIIIa) among others that have been described as atherosclerosis and metabolic vascular diseases promoting factors. We therefore conclude that future trials should be designed to develop an individualized therapy approach for patients on LA based on their metabolic and vascular risk profile. Furthermore, the power of such cascade filter treatment protocols may improve the prevention of cardiometabolic disease and its complications.
Subject(s)
Blood Component Removal , Cardiovascular Diseases , Blood Component Removal/adverse effects , Blood Component Removal/methods , Cardiometabolic Risk Factors , Cardiovascular Diseases/etiology , Cardiovascular Diseases/prevention & control , Cholesterol, LDL , Humans , Lipoprotein(a) , Precision Medicine/adverse effects , Proteomics , Risk Factors , Treatment OutcomeABSTRACT
Urinary lipidomics may add new valuable biomarkers to the diagnostic armamentarium for early detection of metabolic and kidney diseases. Sources and composition of urinary lipids in healthy individuals, however, have not been investigated in detail. Shotgun lipidomics was used to quantify lipidomic profiles in native urine samples from 16 individuals (eight men, eight women) collected in five fractions over 24 h. All probands were comprehensively characterized by urinary and clinical indices. The mean total urinary lipid concentration per sample was 0.84 µM in men and 1.03 µM in women. We observed significant intra- and interindividual variations of lipid concentrations over time, but failed to detect a clear circadian pattern. Based on quantity and subclass composition it seems very unlikely that plasma serves as major source for the urinary lipidome. Considering lipid metabolites occurring in at least 20% of all samples 38 lipid species from 7 lipid classes were identified. Four phosphatidylserine and one phosphatidylethanolamine ether species (PE-O 36:5) were detectable in almost all urine samples. Sexual dimorphism has been found mainly for phosphatidylcholines and phosphatidylethanolamines. In men and in women urinary lipid species were highly correlated with urinary creatinine and albumin excretion, reflecting glomerular filtration and tubular transport processes. In women, however, lipid species deriving from urinary cells and cellular constituents of the lower genitourinary tract considerably contributed to the urinary lipidome. In conclusion, our study revealed the potential of urinary lipidomics but also the complexity of methodological challenges which have to be overcome for its implementation as a routine diagnostic tool for renal, urological and metabolic diseases.
Subject(s)
Lipid Metabolism/physiology , Lipids/urine , Sex Characteristics , Adult , Female , Healthy Volunteers , Humans , Male , Middle Aged , Phosphatidylcholines/urine , Phosphatidylethanolamines/urineABSTRACT
Low-density lipoprotein (LDL) is considered to be a risk factor for atherosclerosis. In the presence of hyperglycemia, LDL undergoes glycoxidative modification and this glycoxidized (glycox) LDL promotes atherosclerosis in type 2 diabetic (T2D) individuals. Moreover, because of its cholesterol content, LDL contributes to aldosterone biosynthesis, which is modulated by angiotensin II (AngII) and has been implicated in cardiovascular complications of T2D. However, the molecular mechanism of the crosstalk between glycoxLDL, AngII, and aldosterone has not been explained clearly. Therefore, this study has been aimed to investigate the impact of in vitro modified glycoxLDL on aldosterone release in an AngII-sensitized adrenocortical carcinoma cell line (NCI H295R). Native LDL (natLDL), isolated from healthy volunteers by sequential density gradient ultracentrifugation, was subjected to d-glucose (200 mmol/l), for glycoxidative modification, at 37 °C for 6 days. The AngII-sensitized H295R cells were treated with natLDL and glycoxLDL for 24 h and the supernatant was used for aldosterone measurement. The treated cells were utilized for protein isolation and mRNA quantification. Compared to natLDL, glycoxLDL produced a significantly greater effect on aldosterone release from AngII-sensitized cells. The treatment with specific pharmacological inhibitors suggests that modified LDL recruits ERK1/2 and janus kinase-2 for transcriptional regulation of aldosterone synthase. Moreover, glycoxLDL modulates aldosterone release via cAMP-dependent protein kinase A (PKA) pathway. However, glycoxLDL induces ERK phosphorylation independent of PKA activation and this novel mechanism could be targeted for therapeutic trials. In conclusion, this in vitro study emphasizes a possible causal relationship between LDL glycoxidative modification, AngII-sensitization, and adrenocortical steroid hormone release.
Subject(s)
Adrenal Cortex/metabolism , Aldosterone/metabolism , Angiotensin II/pharmacology , Lipoproteins, LDL/metabolism , Adrenal Cortex/drug effects , Cell Line , Cyclic AMP-Dependent Protein Kinases/metabolism , Cytochrome P-450 CYP11B2/genetics , Enzyme Activation/drug effects , Extracellular Signal-Regulated MAP Kinases/metabolism , Glycosylation/drug effects , Humans , Janus Kinase 2/metabolism , Oxidation-Reduction/drug effects , Phosphorylation/drug effects , Steroids/biosynthesis , Transcription, Genetic/drug effectsABSTRACT
Bariatric surgery is a well-established approach to improve metabolic disease in morbidly obese patients with high cardiovascular risk. The post-operative normalization of lipid metabolism has a central role in the prevention of future cardiovascular events. The aim of the present study therefore was to characterize changes of plasma lipidomic patterns, consisting of 229 lipid species of 13 lipid classes, 3 months after Roux-en-Y gastric bypass (RYGB) in morbidly obese patients with and without diabetes. RYGB resulted in a 15-32% decrease of body mass index, which was associated with a significant reduction of total cholesterol (TC, -28.3%; P=0.02), LDL-cholesterol (LDL-C, -26.8%; P=0.03) and triglycerides (TGs, -63.0%; P=0.05) measured by routine clinical chemistry. HDL-cholesterol remained unchanged. The effect of RYGB on the plasma lipidomic profile was characterized by significant decreases of 87 lipid species from triacylglycerides (TAGs), cholesterol esters (CholEs), lysophosphatidylcholines (LPCs), phosphatidylcholines (PCs), phosphatidylethanolamine ethers (PEOs), phosphatidylinositols (PIs) and ceramides (Cers). The total of plasma lipid components exhibited a substantial decline of 32.6% and 66 lipid species showed a decrease by over 50%. A direct correlation with HbA1C values could be demonstrated for 24 individual lipid species (10 TAG, three CholE, two LPC, one lysophosphatidylcholine ethers (LPCO) (LPC ether), one PC, two phosphatidylcholine ethers (PCO) and five Cer). Notably, two lipid species (TAG 58:5 and PEO 40:5) were inversely correlated with HbA1C. LPCO, as single whole lipid class, was directly related to HbA1C. These data indicate that RYGB-induced modulation of lipidomic profiles provides important information about post-operative metabolic adaptations and might substantially contribute to improvements of glycemic control. These striking changes in the human plasma lipidome may explain acute, weight independent and long-term effects of RYGB on the cardiovascular system, mental status and immune regulation.
Subject(s)
Diabetes Mellitus, Type 2/blood , Gastric Bypass , Lipids/blood , Obesity, Morbid/surgery , Chromatography, High Pressure Liquid , Diabetes Mellitus, Type 2/complications , Humans , Lipids/classification , Obesity, Morbid/blood , Obesity, Morbid/complicationsABSTRACT
OBJECTIVE AND METHODS: The acute changes of circulating oxidative stress parameters were compared in 16 patients undergoing two different apheresis techniques: plasma lipidfiltration (LF) or whole blood dextran sulfate adsorption (DSA). RESULTS: Immediately after apheresis LF was associated with an increase in systemic phagocyte count, enhanced formation of reactive oxygen species and decreased activity of the antioxidant enzyme paraoxonase. After DSA, circulating phagocyte oxidant generating activity was significantly lower. Compared to LF, the systemic level of oxidized LDL and antioxLDL antibodies showed a larger decrease in DSA. All measured oxidative stress parameters returned to nearly pre-apheresis level at day three after apheresis, CONCLUSION: The data show a more pronounced leukocyte activation immediately after LF in contrast to DSA, possibly as a consequence of necessity of prior separation of blood plasma. The pathophysiological importance of the short-term oxidative burden after a single apheresis session remains to be determined.
Subject(s)
Blood Component Removal/methods , Dextran Sulfate/therapeutic use , Hyperlipidemias/therapy , Lipoproteins/blood , Oxidative Stress , Adsorption , Aged , Analysis of Variance , Aryldialkylphosphatase/blood , Autoantibodies/blood , Biomarkers/blood , Blood Component Removal/adverse effects , Cholesterol, HDL/blood , Cholesterol, LDL/blood , Dextran Sulfate/adverse effects , Female , Humans , Hyperlipidemias/blood , Hyperlipidemias/diagnosis , Hyperlipidemias/immunology , Lipoprotein(a)/blood , Lipoproteins/immunology , Lipoproteins, LDL/blood , Lipoproteins, LDL/immunology , Male , Middle Aged , Phagocytes/metabolism , Reactive Oxygen Species/blood , Time Factors , Treatment Outcome , Triglycerides/bloodABSTRACT
Roux-en-Y gastric bypass (RYGB) has become a prominent therapeutic option for long-term treatment of morbid obesity and type 2 diabetes mellitus (T2D). Cross talk and pathogenetic consequences of RYGB-induced profound effects on metabolism and gut microbiome are poorly understood. The aim of the present study therefore was to characterize intra-individual changes of gut microbial composition before and 3 months after RYGB by metagenomic sequencing in morbidly obese patients (body mass index (BMI)>40 kg m(-)(2)) with T2D. Subsequently, metagenomic data were correlated with clinical indices. Based on gene relative abundance profile, 1061 species, 729 genera, 44 phyla and 5127 KO (KEGG Orthology) were identified. Despite high diversity, bacteria could mostly be assigned to seven bacterial divisions. The overall metagenomic RYGB-induced shift was characterized by a reduction of Firmicutes and Bacteroidetes and an increase of Proteobacteria. Twenty-two microbial species and 11 genera were significantly altered by RYGB. Using principal component analysis, highly correlated species were assembled into two common components. Component 1 consisted of species that were mainly associated with BMI and C-reactive protein. This component was characterized by increased numbers of Proteobacterium Enterobacter cancerogenus and decreased Firmicutes Faecalibacterium prausnitzii and Coprococcus comes. Functional analysis of carbohydrate metabolism by KO revealed significant effects in 13 KOs assigned to phosphotransferase system. Spearmen's Rank correlation indicated an association of 10 species with plasma total- or low-density lipoprotein cholesterol, and 5 species with triglycerides. F. prausnitzii was directly correlated to fasting blood glucose. This is the first clinical demonstration of a profound and specific intra-individual modification of gut microbial composition by full metagenomic sequencing. A clear correlation exists of microbiome composition and gene function with an improvement in metabolic and inflammatory parameters. This will allow to develop new diagnostic and therapeutic strategies based on metagenomic sequencing of the human gut microbiome.
Subject(s)
Diabetes Mellitus, Type 2/complications , Gastric Bypass , Inflammation/complications , Metagenome , Microbiota , Obesity/surgery , Adult , Diabetes Mellitus, Type 2/metabolism , Female , Humans , Male , Middle Aged , Obesity/complications , Obesity/metabolism , Postoperative PeriodABSTRACT
Apart from their role in cardiovascular homeostasis and immunomodulation, aldosterone and cortisol are also implicated in the pathogenesis of insulin resistance and type 2 diabetes mellitus (T2DM). Furthermore, glycoxidative modifications of lipoproteins are increasingly recognized as an etiological factor for increased cardiovascular morbidity and mortality in prediabetic individuals. The causative relationship between in vivo lipoprotein modifications and steroidogenesis in subjects with impaired glucose tolerance (IGT), however, is not well defined. Therefore, we aimed to investigate the impact of in vivo modified lipoproteins on aldosterone and cortisol release from human adrenocortical H295R cells. Following an oral glucose tolerance test, 20 individuals with normal glucose tolerance (NGT) and 20 IGT subjects were randomly selected from the ongoing PRAEDIAS prevention study in our department. Cells were incubated for 24 h with lipoproteins isolated from NGT and IGT individuals and aldosterone and cortisol release was measured in the supernatants. VLDL induced a greater stimulating effect on adrenocortical aldosterone and cortisol release compared to HDL and LDL. Moreover, IGT-VLDL evoked a significantly higher effect (p<0.05) on hormone release than NGT-VLDL. Incubation of cells with in vitro modified lipoproteins and specific pharmacological inhibitors suggests that VLDL presumably recruits ERK1/2 as one of the downstream effectors of Jak-2. In summary, in vivo modified VLDL are able to promote prediabetic hormonal dysregulation by modulating adrenocortical steroidogenesis via Jak-2-ERK dependent pathway.
Subject(s)
Adrenal Cortex/metabolism , Aldosterone/metabolism , Glucose Intolerance/metabolism , Hydrocortisone/metabolism , Lipoproteins, VLDL/metabolism , Up-Regulation , Adrenal Cortex/drug effects , Aged , Cell Line , Glucose Intolerance/blood , Glucose Intolerance/physiopathology , Glycated Hemoglobin/analysis , Humans , Hyperglycemia/etiology , Lipoproteins, HDL/blood , Lipoproteins, HDL/metabolism , Lipoproteins, LDL/blood , Lipoproteins, LDL/metabolism , Lipoproteins, VLDL/blood , MAP Kinase Signaling System/drug effects , Male , Middle Aged , Phosphorylation/drug effects , Prediabetic State/blood , Prediabetic State/metabolism , Prediabetic State/physiopathology , Protein Kinase Inhibitors/pharmacology , Protein Processing, Post-Translational/drug effects , Up-Regulation/drug effectsABSTRACT
An increased prevalence of diabetes mellitus (DM) has been reported in patients with primary aldosteronism (PA). DM is associated with abnormal structure and metabolism of circulating lipoproteins, which normally serve as a major source of cholesterol for adrenocortical steroidogenesis. The present study has been designed to investigate the effect of diabetically modified lipoproteins on adrenocortical aldosterone synthesis. Lipoproteins (VLDL, LDL, HDL) isolated from healthy volunteers, were subjected to oxidation or glycoxidation in the presence of sodium hypochlorite (3 mmol/l) or glucose (200 mmol/l), and aldosterone synthesis in human adrenocortical cells (H295R) was examined. Native and glycoxidized VLDL had greatest stimulatory effect on aldosterone production by 15-fold and 14-fold, respectively. At the molecular level, these VLDL produced maximum increases in Cyp11B2 mRNA level up to 17-fold. Experiments with the highly selective scavenger receptor class B type I (SR-BI) inhibitor BLT-1 revealed that cholesterol uptake from native and glycoxidized HDL and VLDL for hormone production is considerably mediated by SR-BI. Western blot analysis of extracellular signal-regulated kinase (ERK 1/2) phosphorylation and experiments with the MEK inhibitor U0126 indicated a specific mechanistic role of the ERK cascade in lipoprotein-mediated steroid hormone release. In summary, diabetic dyslipidemia and modification of circulating lipoproteins may promote adrenocortical aldosterone synthesis.
Subject(s)
Adrenal Cortex/metabolism , Aldosterone/biosynthesis , Diabetes Complications/metabolism , Hyperaldosteronism/metabolism , Lipoproteins/metabolism , Cell Line, Tumor , Cytochrome P-450 CYP11B2/genetics , Cytochrome P-450 CYP11B2/metabolism , Diabetes Complications/enzymology , Diabetes Complications/genetics , Humans , Hyperaldosteronism/complications , Hyperaldosteronism/enzymology , Hyperaldosteronism/genetics , Oxidation-ReductionABSTRACT
AIMS/HYPOTHESIS: Glycoxidised LDL has been implicated in the pathogenesis of atherosclerosis, a major complication of diabetes. Since atherogenesis may occur at an early stage of diabetes, we investigated whether circulating LDL isolated from subjects with IGT (n = 20) showed an increased glycoxidation status and explored the proatherogenic effects of LDL samples on macrophages. SUBJECTS AND METHODS: We investigated LDL modifications using GC-MS. Murine macrophages were incubated with LDL samples for 1 h, and then mRNA expression rates of the scavenger receptors CD36 and scavenger receptor class B type 1 (SCARB1, formerly known as SR-BI) and transcription factor peroxisome proliferator-activator receptor gamma (PPARgamma) were quantified by real-time RT-PCR. RESULTS: The GC-MS experiments revealed that oxidative modifications of proline, arginine, lysine and tyrosine residues in apolipoprotein B100 were three- to fivefold higher in LDL samples from IGT subjects compared with those from NGT subjects (n = 20). Moreover, LDL glycoxidation estimated by both Nepsilon-(carboxymethyl)lysine (CML) and Nepsilon-(carboxyethyl)lysine (CEL) residues was increased more than ninefold in LDL from IGT subjects compared with samples from NGT subjects. Compared with NGT LDL, IGT LDL elicited a significantly higher CD36 (p < 0.05) and PPARG (p < 0.05) gene expression, whereas SCARB1 mRNA expression was not affected. CONCLUSIONS/INTERPRETATION: These data suggest that IGT is associated with increased glycoxidation of circulating LDL, which might contribute to the conversion of macrophages into a proatherogenic phenotype.
Subject(s)
CD36 Antigens/genetics , Gene Expression Regulation , Glucose Intolerance/blood , Lipoproteins, LDL/blood , Macrophages/physiology , PPAR gamma/genetics , Adult , Antigens, CD/genetics , Blood Glucose/metabolism , Female , Humans , Lipoproteins, LDL/isolation & purification , Male , Middle Aged , Reference ValuesABSTRACT
Mutations in the hepatocyte nuclear factor - 1 beta (HNF-1 beta) gene cause maturity onset diabetes of the young type 5 (MODY 5). A clinical feature of the resulting phenotype besides impaired glucose tolerance is a variety of renal abnormalities, ranging from renal cysts to end-stage renal failure. Using a candidate gene approach we investigated the prevalence of mutations in the HNF-1 beta gene in a group of 63 patients from two different European populations (33 Germans, 30 Czechs) with type 2 diabetes mellitus and diabetic nephropathy diagnosed by increased albuminuria (39 patients) or end-stage renal failure (24 patients). No mutations were found in any of the 9 exons or in a minimal promoter region. Three intronic variants (single nucleotide polymorphisms - SNPs) were detected. The frequencies of these variants showed no difference between the two studied populations and were comparable to data reported from healthy subjects. No association between SNPs or formed haplotypes and any clinical parameters (like age of disease onset, BMI and severity of renal failure) was found. The results confirm that the genetic variations in the HNF-1 beta gene would be a very uncommon cause of progressive nephropathy in patients with type 2 diabetes mellitus.
Subject(s)
DNA-Binding Proteins/genetics , Diabetes Mellitus, Type 2/genetics , Diabetic Neuropathies/genetics , Genetic Variation , Introns , Mutation , Transcription Factors/genetics , White People/genetics , Age of Onset , Albuminuria , Body Mass Index , Czech Republic , Diabetic Nephropathies/genetics , Germany , Hepatocyte Nuclear Factor 1-beta , Humans , Kidney Failure, Chronic/genetics , Middle AgedABSTRACT
To characterize the possible multiple implications of galectin 9, described as eosinophil chemoattractant protein as well as urate transporter/channel, the porcine homologue of galectin 9, Gal9p, was cloned from LLC-PK(1) cells and stably expressed in human embryonic kidney 293 cells. Significant Gal9p-mediated transport could be demonstrated for [(14)C]-uric acid and for [(14)C]-PAH(1). Transport was dependent on imposed changes of membrane potential of the host cells, but did not follow the classical carrier criteria. Gal9p-mRNA (1573 bp) as well as a 96 bp-elongated isoform show highest abundance in organs of the gastrointestinal tract, to a lesser extent in the aorta and the liver. RT-PCR on human tissue let to the identification of galectin 9 mRNA in human kidney, in HUVEC and in prototype cells of the monocyte/macrophage system (U-937, HL60). In HUVEC, three constitutively expressed galectin 9 mRNA-isoforms were identified. On the basis of the functional characteristics together with a detailed sequence analysis along the galectin family, different domains of galectin 9 are discussed. The data of the present study sustain the idea of the involvement of galectin 9 in immune/inflammation processes and in potential-sensitive uric acid translocation.
Subject(s)
Galectins , Lectins/genetics , Organic Anion Transporters , Protein Isoforms/chemistry , RNA, Messenger/chemistry , Animals , Base Sequence , Carrier Proteins/metabolism , Cells, Cultured , Chemotactic Factors/chemistry , Endothelium, Vascular/cytology , Humans , Kidney/cytology , LLC-PK1 Cells , Lectins/chemistry , Molecular Sequence Data , Organic Anion Transport Protein 1/metabolism , Organic Cation Transport Proteins , Plasmids , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Structure, Tertiary , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Sequence Homology, Amino Acid , Swine , Tissue Distribution , TransfectionABSTRACT
Hypercholesterolemia and oxidative stress are major risk factors in atherogenesis. In the last years, lipid apheresis has been established as an effective clinical therapy by lowering not only elevated plasma low-density lipoprotein (LDL) levels but also by reducing the incidence of cardiovascular events. The aim of the present study was to investigate peripheral leukocyte oxidant generation in patients with familial hypercholesterolemia (FH) undergoing regular LDL apheresis. The activity state of leukocytes was estimated prior to, immediately after, and 2 days after LDL apheresis carried out by two distinct techniques: hemoperfusion with the DALI system and heparin-induced extracorporeal LDL precipitation (HELP). Oxidant generating activity was measured by chemiluminescence (CL) in whole blood and isolated polymorphonuclear leukocytes (PMNL). The results of our study show increased baseline respiratory burst activities in FH patients as compared to healthy controls. Apheresis with the HELP system was followed by increases in leukocyte count, zymosan-induced whole blood CL, and plasma PMNL elastase levels. The DALI technique caused no changes in leukocyte count and elastase levels and decreased whole blood CL activity. Two days after lipid removal the observed changes returned to pre-apheresis levels. Leukocyte activity parameters before and after apheresis did not correlate with the corresponding plasma levels of triglycerides, total cholesterol, and LDL cholesterol, suggesting that different handling in the framework of both apheresis techniques rather than lipid profile changes during therapy accounted for leukocyte activity modulation.
Subject(s)
Blood Component Removal , Hemoperfusion , Heparin/pharmacology , Hyperlipoproteinemia Type II/therapy , Leukocytes/metabolism , Lipoproteins, LDL/isolation & purification , Oxidative Stress , Adult , Aged , Chemical Precipitation , Female , Humans , Hyperlipoproteinemia Type II/metabolism , Lipoproteins, LDL/blood , Male , Middle Aged , Respiratory BurstSubject(s)
Carrier Proteins/genetics , Galectins , Membrane Potentials/physiology , Organic Anion Transport Protein 1/genetics , Organic Anion Transporters , Uric Acid/metabolism , p-Aminohippuric Acid/metabolism , Amino Acids/analysis , Animals , Carrier Proteins/physiology , Cell Line, Transformed , Epithelial Cells/cytology , Epithelial Cells/metabolism , Gene Expression , Humans , Kidney/cytology , LLC-PK1 Cells , Lectins/analysis , Organic Anion Transport Protein 1/physiology , Organic Cation Transport Proteins , Rats , Swine , Tissue DistributionSubject(s)
Galectins , Lectins/genetics , Exons , Humans , Introns , Mutation, Missense , Point MutationABSTRACT
The chemiluminescence (CL) technique with scavengers for superoxide anion (superoxide dismutase) and hydrogen peroxide (catalase) was used to characterize the generation of reactive oxygen species (ROS) inside and outside the human neutrophil after stimulation with both soluble (formyl-methionyl-leucyl-phenylalanine, FMLP) and particulate (urate crystals, zymosan, oxidized LDL) stimuli. Depending on the stimulus used, ROS generation differed in composition and absolute amounts. The ratio between extracellularly and intracellularly produced ROS ranged from 0.3 (zymosan) to 4.2 (FMLP). While enhancing substantially FMLP-stimulated CL, horseradish peroxidase inhibited CL induced by particulate stimuli by 40-80%. Furthermore, an azide-insensitive and therefore peroxidase-independent part of CL was found in FMLP-, LDL- and zymosan-stimulated cells. The results indicate that different agonists may lead through distinct chemical pathways to neutrophil luminol-amplified light generation.
Subject(s)
Horseradish Peroxidase , Luminol , Neutrophils/physiology , Reactive Oxygen Species/metabolism , Superoxides/blood , Autoanalysis , Humans , In Vitro Techniques , Kinetics , Lipoproteins, LDL/pharmacology , Luminescent Measurements , N-Formylmethionine Leucyl-Phenylalanine/pharmacology , Neutrophils/drug effects , Zymosan/pharmacologyABSTRACT
Evaluation of catecholamine modulation of PMNL extracellular and intracellular oxidant production may reflect beneficial and harmful effects of beta-adrenergic agonists in various disease states. We investigated the kinetics and potency of adrenaline-mediated inhibition of oxidant generation in FMLP- and zymosan-stimulated PMNLs. In FMLP-stimulated cells, the short-term burst of oxidant generation was inhibited by adrenaline in a dose-dependent fashion. Intra- and extracellular chemiluminescence and extracellular superoxide anion and hydrogen peroxide generation showed similar IC50 values for adrenaline (1.3-3.0 x 10(-8) M) indicating that both extracellular and intracellular events were inhibited with the same potency. In contrast, intracellular oxidant production evoked by the phagocytosis of zymosan was only minimally affected by 3 x 10(-5) -3 x 10(-12) M adrenaline. Extracellular inhibition of oxidant production was also apparent in zymosan-stimulated cells. In conclusion, adrenaline's ability to depress extracellular generation of oxygen metabolites while retaining prolonged intracellular oxidant production for phagocytosis supports its beneficial role as selectively targeted physiological protector.
Subject(s)
Adrenergic beta-Agonists/pharmacology , N-Formylmethionine Leucyl-Phenylalanine/pharmacology , Neutrophils/drug effects , Oxidants/metabolism , Zymosan/pharmacology , Humans , Neutrophils/metabolism , Reactive Oxygen Species , Respiratory Burst/drug effectsABSTRACT
The generation of reactive oxygen species (ROS) was measured by chemiluminescence (CL) in whole blood and isolated polymorphonuclear leukocytes (PMNLs) in 113 subjects. There were no differences in ROS production in males and females, smokers and non-smokers and no age dependency. A significant correlation was found between whole blood CL parameters and between parameters derived from isolated cells, but there was no correlation when these parameters were compared using isolated PMNLs and whole blood. Variations in serum lipid levels did not account for the variability of CL parameters in blood or PMNLs. Both methodologies had the same validity in demonstrating priming of phagocytes. Zymosan-induced CL was more sensitive in detecting distinct functional states of phagocytes than FMLP-induced CL.