Magnesium Attenuates the Neutrophil Respiratory Burst in Adult Asthmatic Patients

Introduction: IV magnesium (Mg²⁺) has been proposed as an emergent treatment for acute asthma exacerbations. Recent studies have focused on the effects of Mg²⁺ on bronchial smooth muscle, yet asthma is primarily an inflammatory disease. Objective: To assess the effects of Mg²⁺ on the neutrophil respiratory burst of adult patients with asthma. Methods: A prospective, blind study of volunteer adult asthmatic patients was performed. The patients' polymorphonuclear neutrophils (PMNs) were isolated, purified, and placed into phosphate-buffered saline with the following test conditions: concentrations of magnesium chloride (MgCl₂) added: 0 mmol MgCl₂, 1 mmol MgCl₂ (low), and 10 mmol MgCl₂ (high) both with and without the calcium (Ca) ionophore A23187 (0.1 mmol). PMNs were activated using N-formyl-methionyl-leucyl-phenylalanine (fMLP) (10 pmol), and the production of superoxide (O^-₂) was measured by the spectrophotometric reduction of cytochrome c. Results: Mg²⁺ reduced activated PMN O^-₂ production compared with that for no Mg²⁺ (1.0 ± 0.1 nmol O^-₂/5 ± 10⁵ PMN/min) in both low (-0.52* ± 0.3 nmol O^-₂/5 ± 10⁵ PMN/min) and high (-0.76* ± 0.3 nmol O^-₂/5 ± 10⁵ PMN/min; *p < 0.05) concentrations. The addition of A23187 increased O^-₂ production in both the high (0.53* ± 0.02 nmol O^-₂/5 ± 10⁵ PMN/min) and the low (1.5* ± 0.6 nmol O^-₂/5 ± 10⁵ PMN/ min) Mg²⁺ groups, with no change in the control group (1.2 ± 0.2 nmol O^-₂/10⁵ PMN/min). Conclusions: In clinically relevant concentrations, Mg²⁺ attenuates the neutrophil respiratory burst in adult asthmatic patients. Mg²⁺ appears to affect PMNs by interfering with extracellular Ca²⁺ influx. Mg²⁺ may have a beneficial anti-inflammatory effect in asthmatic individuals.     

Daily variation in circulating cytokines and acute-phase proteins correlates with clinical and laboratory indices in community-acquired pneumonia

Our objective was to investigate the initial levels of circulating proinflammatory cytokines, such as interleukin 1β (IL-1β), interleukin 6 (IL-6), and tumour necrosis factor alpha (TNF-α), of certain acute-phase proteins, such as C-reactive protein (CRP), fibrinogen (FBN) and albumin, and of the glycoprotein fibronectin at presentation and their daily variation during the clinical course of community-acquired pneumonia (CAP) in relation to clinical and laboratory indices of infection. Thirty otherwise healthy hospitalized patients aged 48 ± 3 years (mean ± SEM) and with bacteriologically confirmed CAP were studied prospectively. IL-1β and IL-6 were found to be 15-fold higher on admission (122 ± 9 pg mL^?1 and 60 ± 4 pg mL^?1 respectively), whereas TNF-α was three-fold higher (102 ± 5 pg mL^?1) than those of controls, all of them showing a decline towards normal. Initial CRP levels were increased 90-fold (416 ± 1 mg L^?1), whereas fibronectin levels were reduced (242 ± 9 mg dL^?1). The presence of parapneumonic effusion was associated with a higher TNF-α serum level (127 ± 7 vs. 86 ± 4 pg mL^?1, P = 0.0002), a more rapid daily decline in TNF-α (–7.2 ± 0.7 vs. ?3.8 ± 0.5 pg mL^?1 day^?1, P = 0.0005), a slower rate of decline in CRP (?42.8 ± 3.0 vs. ?54.6 ± 3.0 mg L^?1 day^?1, P = 0.02) and a slower rate of increase in FBN (5.9 ± 1.0 vs. 11.7 ± 1.0 mg dL^?1 day^?1), P = 0.001]. Furthermore, daily progression of serum levels of cytokines and acute-phase proteins correlated strongly with pyrexia, erythrocyte sedimentation rate (ESR), neutrophil count, alveolar–arterial oxygen difference and radiographic resolution, clinically manifested by improvement in the patients' condition.     

Acute hyperinsulinaemia decreases cholesterol synthesis less in subjects with non-insulin-dependent diabetes mellitus than in non-diabetic subjects

To investigate the effect of insulin on cholesterol synthesis in vivo we measured plasma mevalonic acid (MVA) concentrations using gas chromatography–mass spectrometry in six non-obese patients with non-insulin-dependent diabetes mellitus (NIDDM) [four men, two women; age 57.5±2.2 years (mean±SEM); glycated haemoglobin (HbA1) 8.5±0.5%; total cholesterol (TC) 5.7±0.5 mmol L^?1, triglyceride (TG) 3.8±0.9 mmol L^?1] and six non-diabetic, sex- and age-matched control subjects (age 55.7±2.8 years; HbA1 6.5±0.1%; TC 5.4±0.3 mmol L^?1, TG 1.2±0.1 mmol L^?1). Subjects were studied twice: during 13-h hyperinsulinaemic (1 mu kg^?1 min^?1), euglycaemic (5 mmol L^?1) clamp and during a saline infusion. Baseline MVA concentration was significantly higher in diabetic patients than in control subjects (9.8±0.7 ng mL^?1 vs. 5.6±0.9 ng mL^?1P=0.004). At the end of each study, MVA concentration, expressed as a percentage of baseline, was significantly lower during the hyperinsulinaemic, euglycaemic clamp than during the saline study in both the diabetic (54.4±5.3% vs. 69.6±6.3%, P=0.036) and control subjects (30.5±3.4% vs. 61.7±6.0%, P=0.01). However, the decrease in MVA during the hyperinsulinaemic clamp study was more marked in the control subjects than in the diabetic subjects (P=0.03). A significant positive correlation was found between percentage decrease of MVA and non-esterified fatty acids following the insulin clamp in NIDDM (r=0.83, P=0.04). We conclude that acute hyperinsulinaemia decreases cholesterol synthesis less in subjects with NIDDM than in non-diabetic subjects and that this phenomenon, together with increased basal cholesterol synthesis in NIDDM, may in part be due to insulin resistance.     

Effect of SanOrg123781A, a synthetic hexadecasaccharide, on clot-bound thrombin and factor Xa in vitro and in vivo

Summary. Factor (F)Xa and thrombin bound to the clot during its formation contribute to the propensity of thrombi to activate the coagulation system. The aim of this work was to study the inhibition of clot‐bound FXa and clot‐bound thrombin by SanOrg123781A, a synthetic hexadecasaccharide that enhances the inhibition of thrombin and FXa by antithrombin (AT). SanOrg123781A, designed to exhibit low non‐specific binding to proteins other than AT, was compared with heparin. In buffer, heparin and SanOrg123781A inhibited FXa and thrombin at similar concentrations [concentration inhibiting 50% (IC₅₀) of Xa and IIa activity were, respectively: heparin 120 ± 7 and 3 ± 1 ng mL^?1; SanOrg123781A 77 ± 5 and 4 ± 1 ng mL^?1]. In human plasma, the activity of both compounds was reduced, although the activity of heparin was much more affected than that of SanOrg123781A (IC₅₀ values for inhibition of FXa and FIIa activity were, respectively: heparin 100 ± 5 and 800 ± 40 ng mL^?1; SanOrg123781A 10 ± 5 and 30 ± 3 ng mL^?1). We demonstrated, in agreement with our previous results, that the procoagulant activity of the clot is essentially due to clot‐bound FXa and to some extent to clot‐bound thrombin. We showed that heparin and SanOrg123781A were able to inhibit fragment F1+2 generation induced by clot‐bound FXa with IC₅₀ values of 2 ± 0.5 µg mL^?1 and 0.6 ± 0.2 µg mL^?1, respectively. Both compounds also inhibited clot‐bound thrombin activity, the IC₅₀ values of heparin and SanOrg123781A being 1 ± 0.01 µg mL^?1 and 0.1 ± 0.1 µg mL^?1, respectively. Moreover, both heparin and SanOrg123781A significantly inhibited fibrinopeptide A generated by the action of clot‐bound thrombin on fibrinogen but also by free thrombin generated from prothrombin by clot‐bound FXa with IC₅₀ values of 4 ± 0.6 and 1 ± 0.1 µg mL^?1, respectively. As with clot‐bound enzymatic activities, SanOrg123781A was three times more active than heparin in vivo on fibrinogen accretion onto a pre‐existing thrombus and as activators of recombinant tissue‐type plasminogen activator‐induced thrombolysis. In conclusion, due to the specific activities of SanOrg123781A, this compound is much more active than heparin in the presence of plasma proteins, on clot‐bound enzymes and in in vivo models of thrombosis/thrombolysis.     

Changes in free radical generation and antioxidant capacity during ultramarathon foot race

Background Exhaustive exercise has been implicated in the generation of reactive oxygen species, resulting in oxidative stress. We studied the effect of a long‐distance, endurance exercise on oxidative stress parameters in athletes who participated in the ultramarathon race Spartathlon (246 km). Materials and methods This study included 18 runners (16 men and 2 women) aged 42·8 ± 1·4 years. Blood samples were obtained 24 h before (prerace), at the end (postrace) and 48 h after the end of the race (48 h postrace). We measured oxidative stress indices, including red cell glutathione, malonyldialdehyde and 8‐iso‐prostaglandin F_2a, as well as the total antioxidant capacity. Results 8‐Iso‐prostaglandin F_2a level increased significantly at the end of the race, compared to prerace levels (up to 914·7 ± 61·4 pg mL^?1 from 197·6 ± 8·4 pg mL^?1), and remained 2·5‐fold increased over the baseline 48 h after the race (532·0 ± 54·2 pg mL^?1, P < 0·000). The total antioxidant capacity of the athletes increased from a baseline of 289·6 ± 9·0 µmol L^?1 to 358·7 ± 11·0 µmol L^?1 immediately after the race and remained elevated 48 h later (350·6 ± 7·6 µmol L^?1) (P < 0·001). Conclusions Prolonged exercise induces a marked response of oxidative stress biomarkers, which in part is compensated by serum ability to scavenge free radicals. Whether these changes have long‐term negative effects in the organism needs further investigation.     

Regulation of platelet-activating factor production in gastric epithelial cells

The authors have previously reported that platelet-activating factor (PAF), a phospholipid mediator with potent proinflammatory activities, is produced in the gastric mucosa and stimulates gastric acid secretion in humans and animals. In the present study they used the human gastric tumour cells HGT1 (clone 6) to examine whether PAF production is regulated by neuromediators. PAF was extracted by ethanol and assayed by the washed platelet aggregation test. HGT1 cells produced PAF spontaneously (110 ± 20 pg 10⁶ cells). The addition of vasoactive intestinal peptide (VIP; 10^?9 to 10^?7 mol L^?1) or of histamine (10^?5 to 10^?3 mol L^?1) increased PAF production by three- to fivefold, while the addition of carbachol (10^?7 to 10^?4 mol L^?1) increased PAF production up to sevenfold. PAF production was also increased up to 10- to 13-fold, in a dose- and time-dependent manner, by the addition of calcium and two- to threefold by the addition of phorbol myristate acetate (PMA; 10^?7 to 10^?5 mol L^?1). However, the addition of dibutyryl cyclic AMP (dBcAMP; 10^?6 to 10^?4 mol L^?1) was without any effect. This is the first report showing PAF production by gastric epithelial cells in response to histamine, VIP and carbachol. Furthermore, the findings are consistent with a central role of calcium in this production. The results of this study, together with those of previous studies from the authors’ laboratory, support the hypothesis that PAF is a physiological mediator of gastric acid secretion.     

Influence of intravenous n-3 lipid supplementation on fatty acid profiles and lipid mediator generation in a patient with severe ulcerative colitis

Abstract. N-3 fatty acids were supplied to a 36-year-old female patient suffering from ulcerative colitis and severe steroid side-effects, in a sequence of parenteral and enteral administration. During a moderately active period of disease, 200 ml d^-1 fish oil-derived lipid emulsion (eicosapentaenoic acid [EPA], 4–2 g; docosahexaenoic acid [DHA], 4.2 g) was infused for 9 days, in parallel with rapid tapering of the steroid dose. Disease activity declined rapidly, and the patient was subsequently provided with 16 fish oil capsules per day (EPA, 2.9 g; DHA, 1.9 g) for 2 months. At the end of this period of therapy, severe colitis recurred with intestinal and extraintestinal manifestations. The n-3 lipid emulsion was then used for intravenous alimentation (29 days, maximum dose 300 ml per day); during this time, marked improvement of the inflammatory bowel disease was noted. During both periods of parenteral n-3 lipid administration, total plasma EPA and DHA contents increased several-fold, surpassing that of arachidonic acid; this plasma n-3 fatty acid enrichment was only maintained to a minor extent during the intermediate period of dietary fish oil supplementation. The intravenously administered EPA-containing triglycerides were rapidly hydrolyzed, as evidenced by the appearance of substantial quantities of EPA in the plasma free fatty acid fraction. Platelet and neutrophil total membrane content of EPA and DHA as well as n-3 fatty acid/AA membrane ratios similarly increased during the periods of intravenous n-3 lipid administration and declined during oral fish oil uptake. In contrast, erythrocyte membrane enrichment in EPA and DHA occurred only after the prolonged (2 month) period of dietary n-3 lipid supplementation. Ex vivo stimulation of neutrophils with A23187 showed progressive increase in 5-series leukotriene- and 5-HEPE-generation during both periods of n-3 lipid infusion, in parallel with the rise of plasma EPA contents. Maximum 5-series/4-series leukotriene ratios surpassed 0.25. Similarly, ratios of thromboxane B₃/B₂ liberated from ex vivo stimulated platelets surpassed 0.4 during ongoing n-3 lipid infusion. The profound changes in fatty acid profiles and lipid mediator generation may be related to the reduction in colitis activity observed during the periods of intravenous n-3 lipid supplementation.     

Detectable levels of pyridinoline are present in synovial fluid from various patients with knee effusion: preliminary results*

Abstract. There is a major interest for using biochemical markers of bone metabolism as a non-invasive tool for diagnostic purposes in the field of bone and joint diseases. Based upon the fact that the pyridinium crosslinks of collagen are markers of bone and cartilage degradation, this study was designed to assess the presence of pyridinoline in synovial fluid samples originating from various arthritic and non-arthritic knee joints. Using a sample pooling method, significant levels of pyridinoline could be measured in synovial fluid by high performance liquid chromatography. Pyridinoline levels ranged from 19.3 ± 5.8 pmol mL^-1 (mean ± SD) in osteoarthritic knee joints up to 32.4 ± 14.6 pmol mL^-1 in rheumatoid arthritis joints. Pyridinoline levels in synovial fluid were not significantly correlated to disease duration and synovial fluid cell count, but were correlated to erythrocyte sedimentation rate in osteoarthritic patients (r= 0.99, P= 0.002). This study demonstrates that synovial fluid originating from knee effusion contains significant levels of pyridinoline which can be quantified by high performance liquid chromatography and could, therefore, be a tool to investigate the metabolism of a single joint.     

Surgical removal of insulinoma restores glucose recovery from hypoglycaemia but does not normalize insulin action

Abstract. In the present study we have evaluated the effects of chronic hyperinsulinaemia secondary to insulinoma, on insulin sensitivity and on counter-regulatory responses to hypoglycaemia. We studied six patients (M/F = 3/3; age = 40 ± years), before and 6–9 months after surgical ablation of the neoplasia, by means of an euglycaemic-hyperinsulinaemic clamp (1 mUkg^-1min^-1). Seven normal subjects (M/F = 4/ 3; age = 38 ± 6 years) underwent the same experimental study as the control subjects. In insulinoma patients after 100 min of the euglycaemic-hyperinsulinaemic clamp, glycaemia was allowed to drop to a minimum value of l.9mmol L^-1, and recovery evaluated after interrupting insulin infusion. During the entire study, 3-³H-glucose was infused to determine hepatic glucose production and glucose utilization. Surgical removal of the pancreatic adenoma was followed by a reduction in body weight (BMI=25.7 ±l.9vs. 23.0 ± 1.6 kgm^-2; P?0.005), normalization of fasting plasma levels of glucose (2.94 ±0.16 vs. 4.83± 0.11 mmol L^-1), insulin (162 ± 24 vs. 48 ±12 pmol L^-1) and of basal hepatic glucose production (7.6 ± 0.7 vs. 12.2 ± 1.11μmol kg^-1min^-1). Before the operation, insulin-mediated glucose disposal was significantly lower than in the controls (30.8 ±3.1 vs. 4.91± 3.1 μmol kg^-1min^-1). Six to nine months after surgical removal of the adenoma, glucose utilization was unchanged (30.5 ±3.3 μmol kg^-1min^-1) and still significantly lower than in controls (P<0.0). After the euglycaemic phase, the plasma glucose level dropped to the same hypoglycaemic nadir (2.0±0.1 vs. 2.2 ±0.2 mmol L^-1) in both studies. Upon withdrawal of insulin infusion, recovery from hypoglycaemia was much slower before than after removal of the insulinoma (0.66±0.16vs. 2.50 ± 0.38 μmol min^-1; P<0.01). The impaired recovery from hypoglycaemia was associated with a sluggish rise in plasma glucagon concentration (+ 49 ± 15vs. +95 ±27 ng L^-1), growth hormone (+16±6vs.+ 30±3*mu;g L^-1), and cortisol (+156 ±41 vs. +361 ±62nmol L^-1; all P<0.05–0.005). In contrast to that found after adenoma removal, hepatic glucose production in insulinoma patients remained suppressed even after induction of hypoglycaemia. Our data suggest that in hyperinsulinaemic insulinoma patients restoration of normal insulin levels (a) ameliorates the response of some parameters of the counter-regulation to acute hypoglycaemia; but (b) is not able to restore normal insulin sensitivity.     

Potentiation of clopidogrel active metabolite formation by rifampicin leads to greater P2Y12 receptor blockade and inhibition of platelet aggregation after clopidogrel

Summary. Background: The thienopyridine P2Y₁₂ receptor antagonist clopidogrel reduces the risk of arterial thrombosis and individual pharmacodynamic responses to clopidogrel are believed to reflect the levels of active metabolite (AM) generated. Rifampicin increases the inhibitory effect of clopidogrel on platelet aggregation (PA). We studied the response to clopidogrel before and during administration of rifampicin in order to study the relationship between individual AM levels and P2Y₁₂ blockade. Methods: Healthy volunteers received a 600‐mg loading dose of clopidogrel followed by 75 mg daily for 7 days and, after a washout period and treatment with rifampicin [300 mg twice a day (b.i.d.)], received the same regimen of clopidogrel. Clopidogrel AM levels were determined over 4 h after the clopidogrel loading dose and unblocked P2Y₁₂ receptor number was assessed using a ³³P‐2MeSADP binding assay. PA was measured by optical aggregometry with ADP and TRAP. Results: Rifampicin enhanced clopidogrel AM production [area‐under‐the‐curve (AUC): clopidogrel 89 ± 22 ng h mL^?1, clopidogrel + rifampicin 335 ± 86 ng h mL^?1, P < 0.0001], and P2Y₁₂ blockade (unblocked receptors: clopidogrel 48 ± 24, clopidogrel + rifampicin 4 ± 2, P < 0.0001) and reduced PA (5 μmol L^?1 ADP: clopidogrel 20 ± 4, clopidogrel + rifampicin 5 ± 2, P < 0.01). Increasing numbers of unblocked receptors were required for an aggregation response with a decreasing concentration of ADP. PA induced by ADP 2 μmol L^?1 was particularly sensitive to low levels of receptor blockade. Conclusion: Potentiation of clopidogrel AM production by rifampicin leads to greater P2Y₁₂ blockade and consequently greater inhibition of PA. PA responses to low concentrations of ADP are more sensitive to P2Y₁₂ blockade.     

Ibuprofen does not impair renal function in patients undergoing infrarenal aortic surgery with epidural anaesthesia

Objective: To investigate the effect of preoperative ibuprofen administration on renal function during and after infrarenal aortic surgery under thoracolumbar epidural anaesthesia (EPA). Design: A prospective randomised, double-blinded clinical study. Setting: Operation room and intensive care unit in a university hospital. Patients: Twenty-six consecutive patients scheduled for elective infrarenal aortic surgery. Interventions: The patients were prospectively randomised to receive 400 mg ibuprofen intravenously (i. v.) or a placebo aliquot before surgery. Measurements and results: We assessed renal function by calculating creatinine clearance, and fractional sodium excretion before surgery (baseline), 1 h after cross-clamping (intraoperative), 6 h after cross-clamping (postoperative) and 24 h after cross-clamping (on the 1 st postoperative day). At each point in time, we additionally registered haemodynamics and determined the plasma concentration of 6-keto-PGF₁α (stable metabolite of prostacyclin, PGI₂), bicyclic PGE₂ (stable metabolite of PGE₁ E₂), active renin, aldosterone and vasopressin by radioimmunoassays. Throughout the observation period the renal function parameters mostly remained within the normal range without a significant difference between ibuprofen- and placebo-treated patients (creatinine clearance: baseline 41 ± 3 vs 38 ± 6, intraoperative 57 ± 8 vs 64 ± 11, postoperative 64 ± 9 vs 56 ± 9, first postoperative day 43 ± 5 vs 47 ± 6 ml · min · m^{− 2}, means ± SEM). The plasma levels of 6-keto-PGF₁α (68 ± 8 vs 380 ± 71^* ng · l^{− 1}), bicyclic PGE₂ (57 ± 5 vs 88 ± 9^* ng · l^{− 1}) and vasopressin (14 ± 7 vs 45 ± 10^* ng · l^{− 1}, p < 0.0125), however, were significantly higher during the intraoperative period in the placebo-treated patients. Conclusion: The inhibition of endogenous prostaglandin release by ibuprofen does not substantially impair renal function during infrarenal aortic surgery under EPA. Received: 24 June 1997 Accepted: 26 January 1998     

Monitoring of plasma levels of activated protein C using a clinically applicable oligonucleotide‐based enzyme capture assay

Summary. Background: Human‐activated protein C (APC) is a serine protease with anticoagulant, anti‐inflammatory and cytoprotective functions. This feature renders APC to be a promising vascular‐inflammatory biomarker.Objective: The aim of the present study was the development and validation of a technique that allows the measurement of APC plasma levels under practical laboratory conditions.Methods/patients: Based on the APC‐binding ssDNA aptamer HS02‐52G we developed an oligonucleotide‐based enzyme capture assay (OECA) that quantifies aptamer‐captured APC through hydrolysis rates of a fluorogenic peptide substrate. After optimization of pre‐analytical conditions, plasma APC levels were measured in healthy individuals and patients undergoing hip replacement surgery.Results and conclusion: A combination of APC–OECA with an aprotinin‐based quenching strategy allowed APC analysis with a limit of detection as low as 0.022 ± 0.005 ng mL^?1 (0.39 ± 0.10 pmol L^?1) and a limit of quantification of 0.116 ± 0.055 ng mL^?1 (2.06 ± 0.98 pmol L^?1). While APC plasma levels in healthy individuals fell below the quantifiable range of the APC–OECA platform, levels substantially increased in patients undergoing hip replacement surgery reaching peak values of up to 12 ng mL^?1 (214 pmol L^?1). When normalized to the amount of thrombin generated, interindividual variabilities in the APC generating capacity were observed. In general, with a turn‐around time from blood sampling to generation of test results of < 7 h, the APC–OECA platform allows sensitive and rapid determination of circulating APC levels under pathological conditions.     

Four week administration of an ACE inhibitor and a cardioselective β-blocker in healthy volunteers: no influence on insulin sensitivity

Abstract. In most, but not all, studies antihypertensive treatment with angiotensin converting enzyme inhibitors (ACE inhibitors) improves insulin sensitivity, whereas β-blockers decrease insulin sensitivity. However, there was a significant increase in body weight with β-blockers and changes in the body potassium homeostasis with ACE inhibitors. In order to compare the drug specific metabolic effects of an ACE inhibitor and a cardioselective β-blocker controlling these factors, we measured insulin sensitivity in a randomized, double-blind cross-over study in 22 healthy volunteers (age 27 ± 3 years; BMI 22.0± 1.5kg m^-2 (mean ± SD)) during euglycaemic glucose clamps before and after 4 weeks' administration of 5mg Lisinopril or 5mg Bisoprolol. Both drug phases were separated by 4 weeks of no drug administration. During the insulin sensitivity measurements potassium concentrations were clamped at basal levels by means of a variable i.v. potassium infusion. Body weight was monitored at weekly intervals and kept constant within ± 1 kg of the subjects' baseline weight throughout the entire study period. Insulin sensitivity did not change significantly during either drug administration period. The insulin sensitivity index of the 22 volunteers after administration of the ACE inhibitor was 7.9±2.4 mL min^-1 m²μU^-1 mL^-1 (basal index 8.3 ± l.9mL min^-1 m²μ^-1 mL^-1, and 7.5 ± 2.1 mL min^-1 m²μU^-1 mL^-1 after administration of the β-blocker (basal index 8.2 ± l.9mL min^-1 m²μ^=-1 mL^-1; NS). In order to maintain blood potassium concentrations during the glucose c lamps at basal levels of 3.9 ± 0.3 mmol l^-1 a mean of 7.9±4.2 mval of potassium had to be infused during the low insulin step and a mean of 24.1 ±9.7mval during the high insulin step. Systolic and diastolic blood pressure were lowered significantly by Lisinopril (5±8 mmHg and 7 ± 9 mmHg) and Bisoprolol (7 ± 7 mmHg and 4 ± 8 mmHg; NS between both drugs), respectively. In healthy normo-tensive subjects administration of Lisinopril or Bisoprolol for 4 weeks does not influence insulin sensitivity, when appropriate experimental conditions are maintained.     

Cocaine administration enhances platelet reactivity to subendothelial components: studies in a pig model

Myocardial infarction in cocaine abusers may be related to a direct platelet-activating effect. We analysed this possibility in an experimental model. Studies were carried out in eight normal, anaesthetized pigs with a weight of 30.7 ± 3.7 kg. Blood samples were withdrawn before and 20 min after i.v. administration of cocaine (10 mg kg^?1; at 1 mg kg^?1 every 2 min). Modifications in platelet responses to arachidonic acid (AA; 1.4 mmol L^?1), ADP (1–4 μM), synthetic thromboxane endoperoxide analogue (U46619; 1 μM), collagen (2.5–5 μg mL^?1), adrenaline (10 μM) and ristocetin (0.8–1 mg mL^?1) were tested by conventional aggregometry. Changes in the capacity of platelets to form aggregates on damaged subendothelium were assessed by means of an ex vivo perfusion system in which blood was circulated for 10 min at 800 s^?1, a shear rate similar to that found in normal coronary arteries. The interaction of platelets with perfused denuded arterial segments was morphometrically quantified and expressed as a percentage of damaged vessel surface covered by platelets (%CS). Cocaine administration did not influence platelet aggregation patterns in pigs. However, there was a significant increase in the interaction of pig platelets with subendothelial structures after cocaine infusion (%CS = 40 ± 17% vs. 27 ± 16% baseline; mean ± SD; P < 0.01). Cocaine administration in this animal model increases the reactivity of platelets exposed to subendothelium. These results support the concept that the administration of cocaine to pigs has a prothrombotic effect by facilitating the interaction of platelets with damaged arteries.     

The prognostic value of the soluble urokinase-type plasminogen activator receptor (s-uPAR) in plasma of breast cancer patients with and without metastatic disease

Summary. Elevated levels of soluble uPAR (s‐uPAR) and other fibrinolytic parameters functionally related to the urokinase‐type plasminogen activator system might indicate the presence of cancer cells. In 25 breast cancer patients with metastases s‐uPAR was significantly increased compared with 25 patients without metastases and with 25 healthy controls: 420 pg mL^?1 vs. 145 pg mL^?1 (P = 0.005) and 190 pg mL^?1 (P = 0.003). Plasmin–α₂‐antiplasmin (PAP) complexes and d ‐dimers were significantly increased in breast cancer patients with metastases compared with patients without metastases and with healthy controls. The levels of plasminogen activator inhibitor (PAI)‐1 activity, uPA antigen and factor (F)XIIa did not significantly differ between the patient groups and healthy controls. PAP complexes (529 µg L^?1 vs. 420 µg L^?1; P = 0.03), d ‐dimers (278.5 ng mL^?1 vs. 79.0 ng mL^?1; P = 0.005) and FXIIa (1.64 ng mL^?1 vs. 1.19 ng mL^?1; P = 0.01) were significantly higher in patients with metastases not surviving compared with patients with metastases surviving the 3‐year follow‐up period. Plasma s‐uPAR levels in the patients with metastases did not discriminate between patients surviving and patients not surviving after 3‐year follow‐up. No significant differences in s‐uPAR or any of the other parameters were found in the five patients developing metastases during follow‐up. A single value of s‐uPAR is of limited value in the follow‐up of breast cancer patients with and without metastatic disease and does not predict survival or future metastases.     

Increased soluble CD40 ligand levels in cystic fibrosis

Summary. Chronic inflammation represents a key pathogeneric event in the progression of lung disease in cystic fibrosis (CF). To identify novel mechanisms of the inflammatory reaction in CF and analyze its relation with coagulative activation, we carried‐out a cross‐sectional study to evaluate circulating levels of the inflammatory mediators soluble (s) CD40L, C‐reactive protein (CRP), interleukin (IL)‐1β, the coagulation markers activated factor VII (FVIIa) and prothrombin fragment (F) 1+2, as well as urinary 11‐dehydro‐thromboxane (TX)B₂, an index of in vivo platelet activation, in 34 CF patients and 34 matched healthy subjects. We observed that CF patients displayed significantly increased circulating levels of sCD40L compared to controls [2.8 (0.4–15.6) vs 1.1 (0.2–2.7) ng mL^?1 ,P = 0.0003]. sCD40L levels inversely correlated with forced expiratory volume at 1 second (FEV₁) (ρ = ?0.788, P = 0.0001), whereas it directly correlated with CRP and IL‐1β levels (ρ = 0.621, P = 0.0004; and ρ = 0.745, P = 0.0001, respectively), which were also elevated in CF patients. CF patients had also enhanced levels of FVIIa and F1+2 compared to controls [39.2 (22.6–69.8) vs 22.3 (16.2–32.4) mU mL^?1, P = 0.0001; 0.60 (0.30–1.80) vs 0.17 (0.10–0.40) nmol L^?1, P = 0.0001, respectively]. A direct correlation was observed between sCD40L and both plasma FVIIa (ρ = 0.691, P = 0.0001) and F1+2 (ρ = 0.545, P = 0.0017) as well as between sCD40L and urinary 11‐dehydro‐TXB₂ (ρ = 0.433, P = 0.0129). Our findings suggest that in CF patients, sCD40L could represent a biochemical link between the inflammatory state, and endothelial damage and coagulative activation, leading to progressive impairment of pulmonary function.     

Effect of platelet turnover on whole blood platelet aggregation in patients with coronary artery disease

Summary. Background: Previous studies have demonstrated considerable variation in the antiplatelet effect of aspirin. Objectives: To investigate the impact of platelet turnover on the antiplatelet effect of aspirin in patients with stable coronary artery disease (CAD) and to identify determinants of platelet turnover. Methods: Platelet turnover was evaluated by measurements of immature platelets and thrombopoietin in 177 stable CAD patients on aspirin monotherapy, including 85 type 2 diabetics and 92 non‐diabetics. Whole blood platelet aggregation was determined using the VerifyNow^® Aspirin test and multiple electrode aggregometry (MEA, Multiplate^®) induced by arachidonic acid (AA) (1.0 mm ), adenosine diphosphate (ADP) (10 μm ) and collagen (1.0 μg mL^?1). Results: Immature platelet levels significantly correlated with MEA (r = 0.31–0.36, P‐values < 0.0001) and the platelet activation marker sP‐selectin (r = 0.19, P = 0.014). Contrary to the VerifyNow^® test, MEA significantly correlated with variations in platelet count (r = 0.45–0.68, P‐values < 0.0001). Among patients with residual platelet reactivity according to AA, there were significantly more diabetics (61% vs. 41%, P = 0.027) and higher levels of sP‐selectin (77.7 ± 29 vs. 70.2 ± 25 ng mL^?1, P = 0.070) and serum thromboxane B₂ (0.81 [0.46; 1.70] vs. 0.56 [0.31; 1.12] ng mL^?1, P = 0.034). In a multivariate regression analysis, immature platelet levels were determined by thrombopoietin levels (P < 0.001), smoking (P = 0.020) and type 2 diabetes (P = 0.042). Conclusions: The antiplatelet effect of aspirin was reduced in CAD patients with an increased platelet turnover. Once‐daily dosing of aspirin might not suffice to adequately inhibit platelet aggregation in patients with an increased platelet turnover.     

Effect of glibenclamide on insulin release at moderate and high blood glucose levels in normal man

Insulin release occurs in two phases; sulphonylurea derivatives may have different potencies in stimulating first- and second-phase insulin release. We studied the effect of glibenclamide on insulin secretion at submaximally and maximally stimulating blood glucose levels with a primed hyperglycaemic glucose clamp. Twelve healthy male subjects, age (mean ± SEM) 22.5 ± 0.5 years, body mass index (BMI) 21.7 ± 0.6 kg m^?2, were studied in a randomized, double-blind study design. Glibenclamide 10 mg or placebo was taken before a 4-h hyperglycaemic clamp (blood glucose 8 mmol L^?1 during the first 2 h and 32 mmol L^?1 during the next 2 h). During hyperglycaemic clamp at 8 mmol L^?1, the areas under the Δinsulin curve (AUC_Δinsulin , mean ± SEM) from 0 to 10 min (first phase) were not different: 1007 ± 235 vs. 1059 ± 261 pmol L^?1 × 10 min (with and without glibenclamide, P = 0.81). However, glibenclamide led to a significantly larger increase in AUC_Δinsulin from 30 to 120 min (second phase): 16 087 ± 4489 vs. 7107 ± 1533 pmol L^?1 × 90 min (with and without glibenclamide respectively, P < 0.03). The same was true for AUC_ΔC-peptide: no difference from 0 to 10 min but a significantly higher AUC_ΔC-peptide from 30 to 120 min on the glibenclamide day (P < 0.01). The M/I ratio (mean glucose infusion rate divided by mean plasma insulin concentration) from 60 to 120 min, a measure of insulin sensitivity, did not change: 0.26 ± 0.05 vs. 0.22 ± 0.03 μmol kg^?1 min^?1 pmol L^?1 (with and without glibenclamide, P = 0.64). During hyperglycaemic clamp at 32 mmol L^?1, the AUC_Δinsulin from 120 to 130 min (first phase) was not different on both study days: 2411 ± 640 vs. 3193 ± 866 pmol L^?1 × 10 min (with and without glibenclamide, P = 0.29). AUC_Δinsulin from 150 to 240 min (second phase) also showed no difference: 59 623 ± 8735 vs. 77389 ± 15161 pmol L^?1 × 90 min (with and without glibenclamide, P = 0.24). AUC_ΔC-peptide from 120 to 130 min and from 150 to 240 min were slightly lower on the glibenclamide study day (both P < 0.04). The M/I ratio from 180 to 240 min did not change: 0.24 ± 0.04 vs. 0.30 ± 0.07 μmol kg^?1 min^?1 pmol L^?1 (with and without glibenclamide, P = 0.25). In conclusion, glibenclamide increases second-phase insulin secretion only at a submaximally stimulating blood glucose level without enhancement of first-phase insulin release and has no additive effect on insulin secretion at maximally stimulating blood glucose levels. Glibenclamide did not change insulin sensitivity in this acute experiment.     

Relation of endothelins to volume regulating neurohumoral systems in patients with cirrhosis of the liver

Abstract. The aim of the present study was to investigate the relationship between ET plasma concentrations and other hormonal systems in acute volume regulation of patients with cirrhosis. Ten healthy controls and 10 cirrhotic patients, five without and five with ascites were studied after 1 h in a sitting posture and subsequently subjected to 1 h head-out water immersion. Blood was collected for determinations of ET-1, ET-3, ANF, aldosterone, renin activity and noradrenaline. In addition, in 10 patients with compensated cirrhosis the effect of loop diuretics on ET-3, aldosterone and renin was studied. ETs in cirrhosis were significantly (P<0.01) higher than in controls both before (ET-1, 19.6 ± 1.3pgmL^-1 vs. 11.8 ± 0.4pgmL^-1; ET-3, 18.5±1.4pgmL^-1 vs. 9.5 ± 0.5 pgmL^-1) and after water immersion (ET-1, 18.6± 1.2 pgmL^-1 vs. 12.4±0.3 pgmL^-1; ET-3,18.7 ± 1.7pgmL^-1 vs. 10.0±0.5pg mL^-1). In cirrhotic patients, basal and immersion concentrations of ET-1 were significantly correlated to noradrenaline plasma concentrations (r= 0.79, P<0.05). ET-3 plasma concentrations in cirrhosis were correlated to renin activity (r= 0.65, P<0.05). Furthermore, ET-3 in cirrhosis was inversely correlated to systolic and mean arterial blood pressure (r= -0.55, P<0.01 and r= -0.50, P<0.05, respectively). To investigate the effect of hypovolaemia in compensated cirrhosis, 10 patients without ascites were studied before and after treatment with loop diuretics. In compensated cirrhosis ET-3 was significantly increased 6h after oral diuretic treatment (17.9 ± 1.0 pgmL^-1 vs. 15.5±0.4pg mL^-1, P< 0.001). The presented data demonstrate relations of endothelins, particularly of ET-3 to neurohumoral systems in patients with cirrhosis of the liver.     

Defects of β2-adrenergic signal transduction in chronic lymphocytic leukaemia: relationship to disease progression

The second messenger 3′:5′-cyclic adenosine monophosphate (cAMP) inhibits the proliferation of human B lymphocytes. In lymphoid malignancies, cAMP levels or the number of β₂-adrenergic receptors seem to be decreased. In order to explore this phenomenon further, the function of the β₂-adrenergic receptor complex was examined in mononuclear leucocytes (MNLs) from patients with B-cell chronic lymphocytic leukaemia (CLL). Peripheral blood MNLs from 25 CLL patients (16 male, nine female; aged 62 ± 9 years) and 10 healthy volunteers (seven male, three female; aged 47 ± 19 years) were used. The binding characteristics of β₂-adrenergic receptors (β₂-AR) on MNLs were determined by radioligand binding assays with [¹²⁵I]-cyanopindolol ([¹²⁵I]-CYP). The number of high-affinity binding sites for [¹²⁵I]-CYP was significantly lower in CLL patients (313 ± 300 sites per cell; mean ± SD) than in control subjects (1479 ± 1268 sites per cell). Moreover, the density of β₂-AR decreased with disease progression, from Binet stage A (371 ± 236, n = 13) to B (236 ± 136, n = 7) and C (141 ± 59, n = 5) (P < 0.05; Kruskal–Wallis analysis). Functional analyses of the β₂-AR complex were performed by measuring the cellular cAMP content of MNLs in response to different stimulators. The cAMP production of MNLs upon isoprenaline stimulation (ISO; 10 min, 10^?4 mol L^?1) was slightly lower in CLL patients (12.5 ± 7.04 pmol 10^?6 cells) than in control subjects (15.91 ± 10.08 pmol 10^?6 cells), and decreased with CLL progression (stage A 14 ± 7; stage B 13.66 ± 3.91; stage C 3.07 ± 0.79 pmol 10^?6 cells). In contrast, cAMP accumulation in response to cholera toxin (CHO; 10^?4 g ml^?1, 120 min) was not different in control subjects (70.07 ± 31.30 pmol 10^?6 cells) and CLL patients (stage A 95.24 ± 123.07 , stage B 70.76 ± 57.37, stage C 33.21 ± 33.73 pmol 10^?6 cells). When stimulated with forskolin (100 μmol L^?1, 15 min), control MNLs produced about ten-fold more cAMP than CLL MNLs (188.56 ± 92.53 vs. 17.88 ± 10.32 pmol 10^?6 cells); this response was not stage dependent. Taken together, the results show that the β₂-AR transmembrane signalling is impaired in CLL patients. The correlation of some β₂-AR signalling defects with disease progression suggests that they may contribute to the disease progression of CLL patients.