Showing papers in "European Journal of Clinical Investigation in 2002"

Free fatty acids in obesity and type 2 diabetes: defining their role in the development of insulin resistance and beta-cell dysfunction.

Abstract: Plasma free fatty acids (FFA) play important physiological roles in skeletal muscle, heart, liver and pancreas. However, chronically elevated plasma FFA appear to have pathophysiological consequences. Elevated FFA concentrations are linked with the onset of peripheral and hepatic insulin resistance and, while the precise action in the liver remains unclear, a model to explain the role of raised FFA in the development of skeletal muscle insulin resistance has recently been put forward. Over 30 years ago, Randle proposed that FFA compete with glucose as the major energy substrate in cardiac muscle, leading to decreased glucose oxidation when FFA are elevated. Recent data indicate that high plasma FFA also have a significant role in contributing to insulin resistance. Elevated FFA and intracellular lipid appear to inhibit insulin signalling, leading to a reduction in insulin-stimulated muscle glucose transport that may be mediated by a decrease in GLUT-4 translocation. The resulting suppression of muscle glucose transport leads to reduced muscle glycogen synthesis and glycolysis. In the liver, elevated FFA may contribute to hyperglycaemia by antagonizing the effects of insulin on endogenous glucose production. FFA also affect insulin secretion, although the nature of this relationship remains a subject for debate. Finally, evidence is discussed that FFA represent a crucial link between insulin resistance and beta-cell dysfunction and, as such, a reduction in elevated plasma FFA should be an important therapeutic target in obesity and type 2 diabetes.

Identifying the links between obesity, insulin resistance and beta-cell function: potential role of adipocyte-derived cytokines in the pathogenesis of type 2 diabetes.

Abstract: A combination of insulin resistance and pancreatic beta-cell dysfunction underlies most cases of type 2 diabetes. While the interplay of these two impairments is believed to be important in the development and progression of type 2 diabetes, the mechanisms involved are unclear. A number of factors have been suggested as possibly linking insulin resistance and beta-cell dysfunction in the pathogenesis of type 2 diabetes mellitus. Pro-inflammatory cytokines such as tumour necrosis factor-alpha (TNF-alpha) have deleterious effects on both glucose homeostasis and beta-cell function, and can disrupt insulin signalling pathways in both pancreatic beta cells and liver and adipose tissue. The anti-inflammatory activity of the thiazolidinedione anti-diabetic agents is potentially beneficial, given the possible role of pro-inflammatory cytokines in linking insulin resistance with beta-cell dysfunction.

Iron and immunity: a double-edged sword.

Abstract: Iron is a crucial element for many central metabolic pathways of the body. Lack of iron leads to growth arrest and anaemia while increased accumulation of this metal, as it occurs in highly frequent inherited diseases such as hereditary haemochromatosis and thalassaemia, is associated with toxic radical formation and progressive tissue damage. As shown by several groups, iron also modulates immune effector mechanisms, such as cytokine activities (IFN-gamma effector pathways towards macrophages), nitric oxide (NO) formation or immune cell proliferation, and thus host immune surveillance. Therefore, gaining control over iron homeostasis is one of the central battlefields in deciding the fate of an infection with intracellular pathogens or a malignant disease. Thus, the reticulo-endothelial system has evoked sophisticated strategies to control iron metabolism in general and especially the handling of the metal within immune cells.

The evolution of beta-cell dysfunction and insulin resistance in type 2 diabetes.

Abstract: Insulin resistance and beta-cell dysfunction have important roles in the pathogenesis and evolution of type 2 diabetes. The development of precise methods to measure these factors has helped us to define the relationship between them and evidence is reviewed that changes in insulin sensitivity are compensated by inverse changes in beta-cell responsiveness such that the product of insulin sensitivity and insulin secretion (the disposition index) remains constant. While the disposition index promises to be a useful tool to predict individuals at high risk of developing type 2 diabetes, other factors that contribute to beta-cell dysfunction and mark disease onset and progression include impairments in proinsulin processing and insulin secretion, decreased beta-cell mass and islet amyloid deposition. Emerging data indicate that anti-diabetic agents, such as the thiazolidinediones that simultaneously target insulin resistance and beta-cell dysfunction, may have a beneficial impact on disease onset and progression. Several landmark clinical studies are underway to investigate if their initial promise is supported by data from large-scale trials.

Cyclooxygenase‐2‐positive macrophages infiltrate the Alzheimer’s disease brain and damage the blood–brain barrier

Abstract: Background Monocyte/macrophages are known to infiltrate the brain of patients with HIV-1 encephalitis (HIVE). In Alzheimer's disease brain, the origin of activated microglia has not been determined. Materials and methods We employed the antigen retrieval technique, immunocytochemistry, immunofluorescense, and confocal microscopy to identify macrophages and microglia in relation to amyloid-beta plaques and the blood-brain barrier in autopsy brain tissues from patients with Alzheimer's disease (AD) and HIVE. Results In both conditions, cyclooxygenase-2 positive macrophages and, to a lesser degree, T and B cells infiltrate brain perivascular spaces and neuropil. The macrophages are distinguishable from ramified microglia, and decorate the vessels at the sites of apparent of endothelial tight junction protein ZO-1 disruption. The macrophages also infiltrate amyloid-beta plaques, display intracellular amyloid-beta and are surrounded by amyloid-beta-free lacunae. Furthermore, the macrophages partially encircle the walls of amyloid-beta-containing vessels in amyloid angiopathy, and exhibit intracellular amyloid-beta but not paracellular lacunae. Significantly larger zones of fibrinogen leakage surround the microvessels in HIVE brain tissues compared with AD tissues (P = 0.034), and AD tissues have significantly greater leakage than control tissues (P = 0.0339). The AD group differs from a normal control age-matched group with respect to both the area occupied by CD68 (P = 0.03) and cyclooxygenase-2 immunoreactive cells (P = 0.004). Conclusion In both HIVE and AD, blood-borne activated monocyte/macrophages and lymphocytes appear to migrate through a disrupted blood-brain barrier. The lacunae around macrophages in amyloid-beta plaques but not in vessel walls are consistent with the ability of macrophages to phagocytize and clear amyloid-beta deposits in vitro.

Effect of trefoil factors on the viscoelastic properties of mucus gels

Abstract: Background Trefoil peptides (TFFs) are expressed and secreted in a tissue-specific manner in the gastrointestinal tract. Evidence of coexpression of trefoil peptides and mucins has been demonstrated in most mucus-producing cells in the gastrointestinal tract. The expression of trefoil peptides is up-regulated in gastric ulceration and colitis. It is believed that TFF peptides interact with mucin to increase viscosity but this has never been confirmed. The aims of the present study were to elucidate the direct effect of trefoil peptides on mucus gel formation. Materials and methods The viscosity of mucin solutions was measured by means of a rotational rheometer after adding three mammalian trefoil peptides: TFF1, TFF2, and TFF3. Results Adding TFF2 (0·3%) to the mucin solutions (8%) resulted in more than a factor 10 increase in viscosity and elasticity, and the mucin solution was transformed into a gel-like structure with serpentine-like complexes between the mucin and TFF2. The dimer form of TFF3 also increased viscosity but resulted in a spider's web-like structure. The monomer forms of TFF1 and TFF3 had very little effect on the viscosity and elasticity of the mucin solutions. Conclusions The addition of TFF2 to mucin solutions results in significantly increased viscosity and elasticity, under which the mucin solutions are transformed into a gel-like state. The ability of some trefoil peptides to catalyse the formation of stable mucin complexes may be one of the ways by which these peptides exert their protective and healing functions.

Molecular insights into insulin action and secretion

Abstract: Tightly co-ordinated control of both insulin action and secretion is required in order to maintain glucose homeostasis. Gene knockout experiments have helped to define key signalling molecules that affect insulin action, including insulin and insulin-like growth factor-1 (IGF-1) receptors, insulin receptor substrate (IRS) proteins and various downstream effector proteins. beta-cell function is also a tightly regulated process, with numerous factors (including certain signalling molecules) having an impact on insulin production, insulin secretion and beta-cell mass. While signalling molecules play important roles in insulin action and secretion under normal circumstances, abnormal insulin signalling in muscle, adipose tissue, liver and pancreas leads to insulin resistance and beta-cell dysfunction. In particular, the signalling protein IRS-2 may have a central role in linking these abnormalities, although other factors are likely to be involved.

Molecular causes of colon cancer.

Abstract: Cells in a developing embryo communicate with each other through a limited number of intercellular signalling pathways, of which the Wnt signalling pathway is one. Little is known about the function of Wnt signalling beyond that in embryogenesis. However, recent insights into the molecular etiology of colon cancer have implied a central role for the Wnt signalling pathway. The malignant transformation of colorectal epithelium is well defined, leading to adenoma and sequentially carcinoma formation. Several genes that regulate the Wnt pathway are mutated in cancer of the human colon and other organs. All of these mutations lead to the inappropriate activation of the pathway, which instructs the cell to divide unrestrictedly. These insights now allow the Wnt pathway to be exploited as a new target for drug development in colon cancer.

Ferric saccharate induces oxygen radical stress and endothelial dysfunction in vivo

Abstract: Background Intravenous iron supplementation is used widely in haemodialysis patients. However, nontransferrin-bound iron (NTBI), which increases after intravenous supplementation of ferric saccharate, has been suggested to act as a catalytic agent in oxygen radical formation in vitro and may thus contribute to endothelial impairment in vivo. Materials and methods In 20 healthy volunteers the effect of 100 mg ferric saccharate infusion was investigated. Vascular ultrasound was used to assess endothelium-dependent vasodilatation at baseline, and 10 and 240 min after ferric saccharate infusion. Whole blood was collected to measure NTBI and in vivo radical formation was assessed by electron spin resonance. A time-control study was performed using saline infusion. Results Infusion of ferric saccharate induces a greater than fourfold increase in NTBI, as well as a transient, significant (P <0.01) reduction of flow-mediated dilatation 10 min after infusion of ferric saccharate, when compared with saline. The generation of superoxide in whole blood increased significantly 10 and 240 min after infusion of ferric saccharate by, respectively, 70 and 53%. Conclusions Iron infusion at a currently used therapeutic dose for intravenous iron supplementation leads to increased oxygen radical stress and acute endothelial dysfunction.

Nature of nontransferrin‐bound iron

Abstract: Nontransferrin-bound iron is now recognized to exist by many workers. It is present in the serum of patients suffering from a wide range of disease states and may be induced by certain therapeutic treatments. The chemical nature of this iron pool is unknown but almost certainly it is a multicomponent pool including a considerable proportion of protein-bound iron. Methods are required to separate and quantify these different components. The biological properties of the individual isoforms need to be established; it is possible that some forms are relatively nontoxic, while others are highly toxic. This paper reviews what is known about the nature of nontransferrin-bound iron and describes the methods currently available to quantify this important serum component.

Oxidative status and malondialdehyde in β-thalassaemia patients

Abstract: Background In beta-thalassaemia syndromes, decreased or impaired biosynthesis of beta-globin leads to accumulation of unpaired alpha-globin chains. Moreover, the iron overload in beta-thalassaemia patients generates oxygen-free radicals and peroxidative tissue injury. The aim of this study was to detect and correlate iron overload parameters with the oxidative stress and the antioxidant capability in beta-thalassaemia patients. Design Serum iron, transferrin saturation, serum ferritin, nontransferrin-bound iron (NTBI), levels of serum free and total (free + bound) malondialdehyde (MDA) and total peroxyl radical-trapping antioxidant parameter (TRAP) were evaluated in 21 regularly transfused beta-thalassaemia major (TM) patients, 13 untransfused beta-thalassaemia intermedia (TI) patients and 17 healthy controls. Blood from the TM patients was drawn 48 h after the last desferoxamine (20-40 mg kg(-1)) infusion and just before transfusion. Results Free and total MDA and NTBI levels were higher in the TM patients than in the TI. In the TM patients the free MDA levels correlated positively with serum iron (r = +0.3, P = 0.0006), whereas the total MDA correlated positively with NTBI (r = +0.45, P = 0.037). However, a negative correlation was observed between TRAP and NTBI (r = -0.4, P = 0.0006). In the TI patients there was no significant correlation between free or total MDA and TRAP or NTBI. Conclusions Our results confirm the peroxidative status generated by iron overload in thalassaemia patients and highlight the rapid formation of marked amounts of free MDA despite the chelation therapy in TM patients.

d-penicillamine reduces serum oxidative stress in Alzheimer's disease patients.

Abstract: Background Several lines of evidence address the emerging role for copper in Alzheimer's disease (AD) for sustaining oxidative mechanisms. Studies indicate that peripheral markers of oxidative stress in AD patients could be informative about the pathophysiology of this brain condition. Here, we present a pilot study examining the efficacy of the copper-chelating agent D-penicillamine in reducing oxidative stress in AD patients. Design Serum levels of copper sampled in AD patients and healthy controls indicate a copper homeostasis imbalance in AD. On this basis, 34 AD patients were enrolled in a 6-month, double-blind, placebo-controlled trial with the copper D-penicillamine-chelating agent. Nine patients for each group completed the trial. Oxidative stress, trace metals and clinical parameters were evaluated. Results At the start of the study (to) total peroxides and copper serum content of AD patients were higher (P < 0.0001, P < 0.0001, respectively) and antioxidants were lower (P < 0.05) than in healthy controls. Copper and peroxides were correlated in the AD population (Pearson's r = 0.61, P < 0.001). After treatment with D-penicillamine, the extent of oxidative stress (P < 0.05) was decreased, but no difference was observed in the rate of cognitive decline. Conclusion Data from this pilot study suggest that copper could play a role in the production of peroxides in AD, and that D-penicillamine has an effect in reducing oxidative damage, however, results are still inconclusive in terms of drug efficacy on the clinical progression of AD. Studies with larger cohorts are needed to elucidate the real effectiveness of D-penicillamine treatment in AD.

Factorial study of the effects of atorvastatin and fish oil on dyslipidaemia in visceral obesity

Abstract: Background Dyslipidaemia may account for increased risk of cardiovascular disease in central obesity. Pharmacotherapy is often indicated in these patients, but the optimal approach remains unclear. We investigated the effects of atorvastatin and fish oil on plasma lipid and lipoprotein levels, including remnant-like particle-cholesterol and apolipoprotein C-III, in dyslipidaemic men with visceral obesity. Methods We carried out a 6-week randomized, placebo-controlled, 2 x 2 factorial intervention study of atorvastatin (40 mg day(-1)) and fish oil (4 g day(-1)) on plasma lipids and lipoproteins in 52 obese men (age 53 +/- 1 years, BMI 33.7 +/- 0.55 kg m(-2)) with dyslipidaemia and insulin resistance. Treatment effects were analysed by general linear modelling. Results Atorvastatin had significant main effects in decreasing triglycerides (-0.38 +/- 0.02 mmol L(-1), P = 0.002), total cholesterol (-1.89 +/- 0.17 mmol L(-1), P = 0.001), LDL-cholesterol (-1.78 +/- 0.14 mmol L(-1), P = 0.001), remnant-like particle-cholesterol (-0.08 +/- 0.04 mmol L(-1), P = 0.035), apolipoprotein B (-49 +/- 4 mg dL(-1), P = 0.001), apolipoprotein C-III (-12.6 +/- 6.1 mg L(-1), P = 0.044) and in increasing HDL-cholesterol (+0.10 +/0- 0.04 mmol L(-1), P = 0.007). Fish oil had significant main effects in decreasing triglycerides (-0.38 +/- 0.11 mmol L(-1), P = 0.002) and in increasing HDL-cholesterol (+0.07 +/- 0.04 mmol L(-1), P = 0.041). There were no significant changes in weight or insulin resistance during the study. Conclusions Atorvastatin and fish oil have independent and additive effects in correcting dyslipidaemia in viscerally obese men. Improvement in abnormalities in remnant lipoproteins may occur only with use of atorvastatin. Combination treatment with statin and fish oil may, however, offer an optimal therapeutic approach for globally correcting dyslipidaemia in obesity.

Serum paraoxonase activity in patients with type 1 diabetes compared to healthy controls.

Abstract: Background The oxidation of low-density lipoprotein (LDL) is central to current theories on the initiation and progression of atherosclerosis. Type 1 diabetes is associated with an increase in oxidative stress, which may be responsible for the increased susceptibility to coronary heart disease seen in type 1 diabetes. High-density lipoprotein (HDL) associated paraoxonase (PON1) can retard the oxidation of LDL. Design Paraoxonase activity, concentration and genotype were therefore investigated in 152 people with type 1 diabetes and 282 healthy controls. These parameters were also investigated in the group with type 1 diabetes in relation to the presence of diabetic complications. Results Both PON1 activity and concentration were significantly lower by 16·7% and 19·2% (both P RQ > QQ and for the PON1-55 polymorphism LL > LM > MM. There were no differences in either the PON1 polymorphisms, PON1 activity and concentration in people with type 1 diabetes in the presence or absence of micro and macro vascular complications of diabetes. Conclusions Low PON1 activity may contribute to the increased atherosclerosis found in type 1 diabetes by reducing the ability of HDL to retard LDL oxidation despite the frequently-found increased HDL in type 1 diabetes when good glycaemic control is established.

Insulin resistance affects the regulation of lipoprotein lipase in the postprandial period and in an adipose tissue-specific manner

Abstract: Aims Insulin is a potent stimulator of adipose tissue lipoprotein lipase (LPL). Logically, the postprandial period is therefore a privileged time of the day for the regulation of LPL by insulin in this tissue. It is not clear to what extent a defect such as insulin resistance could affect this regulation and contribute to postprandial, as well as fasting, hypertriglyceridaemia. The aim of the present protocol was to study the relationship between insulin resistance and LPL in adipose tissue and in plasma, in the particular context of the postprandial period. Methods For this study, 26 adult nondiabetic individuals (12 women and 14 men) with a wide range of whole-body insulin-mediated glucose uptake (as assessed with an insulin suppression test) were studied. An abdominal subcutaneous fat biopsy on one occasion, and post-heparin plasma on another occasion, were obtained 4 h into a standardized meal profile administered in the fasting state. Results Postprandial triglyceride excursions (evaluated by the incremental area under the curve during the metabolic meal profile) were inversely correlated to adipose tissue LPL mRNA levels (ρ = −0·43, P < 0·03) as well as to adipose tissue LPL heparin-releasable activity (ρ = −0·58, P < 0·01). Steady-state plasma glucose (SSPG) concentrations during the insulin suppression test, a reflection of the degree of insulin resistance, were also negatively correlated to adipose tissue LPL mRNA (ρ = −0·50, P < 0·02) and activity (ρ = −0·56, P < 0·01). There was no correlation between plasma post-heparin LPL activity/mass and postprandial triglycerides nor with insulin resistance. Conclusion Regulation of adipose tissue LPL is significantly affected in insulin-resistant individuals in the postprandial period. This presumed impaired effect of insulin on LPL postprandially could be an important contributor to the atherogenic dyslipidaemia described in insulin resistance syndrome.

Effects of high doses of glucocorticoids on free amino acids, ribosomes and protein turnover in human muscle.

Abstract: Background Treatment with glucocorticosteroids causes a negative nitrogen balance, but the kinetic mechanisms responsible for this catabolic effect are controversial. We investigated the effects of 60 mg day−1 prednisolone on protein synthesis and degradation in human skeletal muscle. Materials and methods Healthy adults (n = 9) were studied in the postabsorptive state, before and after 3 days of prednisolone treatment. The L-[ring 2,6-3H5]-phenylalanine tracer technique, concentration and size distribution of the ribosomes, mRNA content of the ubiquitin-proteasome pathway components in muscle, phenylalanine flux across the leg, and the free amino acid concentrations in skeletal muscle were used to study muscle protein metabolism. Results The concentrations of most amino acids in arterial blood increased after prednisolone. There were also increased effluxes of phenylalanine, asparagine, arginine, alanine, methionine and isoleucine from the leg. The rate of protein degradation, as measured by the appearance rate (Ra) of phenylalanine, increased by 67% (P = 0·023) which, together with a doubling of the net release of phenylalanine from the leg (P = 0·007), indicated accelerated protein degradation. The pathway was not identified but there was no significant increase in mRNAs’ encoding components of the ubiquitin-proteasome pathway. There was a 6% reduction in polyribosomes (P = 0·007), suggesting a decrease in the capacity for protein synthesis, although there was no measured decrease in the rate of protein synthesis. Conclusions These findings indicate that high doses of prednisolone lead to a sharp increase in net protein catabolism, which depends more on enhanced protein breakdown, and an uncertain effect on protein synthesis. The mechanisms stimulating proteolysis and the pathway stimulated to increase muscle protein degradation should be explored.

Myocardial fibrosis in transforming growth factor‐β1 (TGF‐β1) transgenic mice is associated with inhibition of interstitial collagenase

Abstract: BACKGROUND TGF-beta(1) mediates effects on fibroblast proliferation and collagen synthesis in the myocardium. The extracellular matrix remodeling depends on the fibrillar collagen degrading matrix metalloproteinases (MMPs) and their inhibitors (TIMPs). The in vivo effects of TGF-beta(1) on the MMP/TIMP system in TGF-beta(1) overexpressing transgenic mice were studied. METHODS Male Alb/TGF-beta(1)(cys(223,225)ser) transgenic mice (TG) and nontransgenic controls (C; 8 weeks) were examined. Protein expression of collagen type I, -III, interstitial collagenase (Int Coll), MMP-2, -9, TIMP-1, -2, -4 and TGF-beta(1) as well as enzyme activity (MMP-2, -9) were measured (Western blots, zymographic assays). mRNA expression of the interstitial collagenase and MMP-9 was studied with the Light-Cycler based real-time PCR. RESULTS Overexpression of TGF-beta(1) resulted in a 10-fold increase in plasma and a seven-fold increase in myocardial TGF-beta(1) concentrations. Relative heart weights increased (mg g(-1): 7.8 +/- 0.4 vs. 4.8 +/- 0.6, n = 6; P < 0.01) in TG compared to C. Collagen type I and III increased in TG (1.9-fold and 1.7-fold) compared to controls. Interstitial collagenase protein activity (- 91%) and mRNA expression (-75%) in TG were reduced (P < 0.05-P < 0.001). Gelatinase (MMP-2, MMP-9) expression and activity were not significantly alterated. MMP-inhibitors were increased 2.5-fold (TIMP-1, -4) and 6-fold (TIMP-2) in TG. CONCLUSIONS TGF-beta(1) produces myocardial fibrosis in vivo. This effect is not only produced by a stimulation of matrix protein formation: a complex regulation of MMP and TIMP interaction, namely decrease of expression and activity of interstitial collagenase and an enhanced inhibition by increased levels of TIMPs, are involved. These mechanisms are optional targets for therapeutic interventions in myocardial diseases.

Inflammatory imbalance between IL-10 and TNFalpha in unstable angina potential plaque stabilizing effects of IL-10.

Abstract: Background The pathogenesis of atherosclerosis and acute coronary syndromes involves inflammation and immunological mechanisms. We hypothesized that patients with unstable angina may have an imbalance between inflammatory and anti-inflammatory cytokines. Design Plasma levels of tumour necrosis factor (TNF)alpha and interleukin (IL)-10 were analyzed in 44 patients with stable angina, 29 patients with unstable angina and 20 controls. mRNA levels of these cytokines were analyzed in peripheral blood mononuclear cells (PBMC). We also studied the in vitro effects of IL-10 in PBMC from unstable angina patients. Results Our main findings were: (1) the angina patients and particularly those with unstable disease had significantly raised TNFalpha in comparison with the controls, both at the protein and mRNA level; (2) in contrast, the levels of IL-10 were not different in the angina patients in comparison with the healthy controls, resulting in a markedly enhanced TNFalpha:IL-10 ratio, particularly in the unstable angina patients; (3) while exogenously added IL-10 markedly inhibited the release of TNFalpha, IL-8 and tissue factor as well as impairing the gelatinolytic activity and mRNA production of matrix metalloproteinase-9, it enhanced the tissue inhibitor of this metalloproteinase (i.e. TIMP-1) in PBMC from the unstable angina patients. Conclusion Patients with unstable angina appear to have an imbalance between TNFalpha and IL-10, possibly favouring inflammatory net effects. IL-10 may have beneficial effects on mechanisms that are important in plaque rupture and thrombus formation.

Labile iron in parenteral iron formulations and its potential for generating plasma nontransferrin-bound iron in dialysis patients

Abstract: Background Labile plasma iron (LPI) associated with iron supplementation has been implicated in complications found in dialysis patients. As LPI can potentially catalyse oxygen radical generation, we determined the presence of labile iron in the parenteral preparations and the frequency of occurrence of LPI in dialysis patients. Design The capacity to donate iron to apotransferrin (apo-) or to the chelator desferrioxamine (DFO) was measured with fluorescein-Tf (Fl-Tf) and Fl-DFO, respectively. Those probes undergo quenching upon binding to iron. Iron-catalysed generation of oxidant species was determined with dihydrorhodamine. Plasma nontransferrin-bound iron (NTBI), here termed LPI, was determined by mobilization of iron from low-affinity binding sites with oxalate, followed by its quantification with Fl-Tf in the presence of Ga(III). Results Normal individuals and most (80%) dialysis patients, analysed at least 1 week after iron supplementation showed no detectable ( 0·2 µm), in some cases weeks after iron administration. LPI levels correlated best (r2 = 0·9) with Tf saturation. The iron preparations contained 2–6% low molecular weight and redox-active iron, most of which is chelated by Tf. Conclusions Parenteral iron formulations contain a small but significant fraction of redox-active iron, most of which is scavenged by apo-Tf within <1 h. Therefore, oxidant stress associated with iron infusion is likely to be transient. The bulk of the polymeric iron is apparently inaccessible to apo-Tf. Although LPI might return to normal within 2 h of intravenous iron infusion, the long-term persistence of low-level LPI in up to 20% of end stage renal disease (ESRD) patients indicates that complete clearance of the intravenous iron may be more protracted than originally estimated.

Time-dependent effect of statins on platelet function in hypercholesterolaemia.

Abstract: Background Reduction of platelet activity induced by statins has been described as a positive effect exerted by such molecules on vascular thrombotic events. However, the relations among cholesterol (LDL-C) reduction, the timing of the antiplatelet effect, the involved mechanisms and the doses of each statin able to reduce platelet function are not actually well known. The aim of our study was to evaluate the impact of simvastatin (20 mg day−1), atorvastatin (10 mg day−1), fluvastatin (40 mg day−1) and pravastatin (40 mg day−1) on platelet function in hypercholesterolaemic subjects with relation to (LDL-C), oxidized-LDL (ox-LDL) and antiport mechanism modifications. Materials and methods Sixteen subjects were assigned to each treatment (40 males, 24 females, mean age 48·7 ± 13·4, LDL-C 5·13 ± 0,23 mmol L−1) and evaluated for platelet surface P-selectin (P-sel), lipid profile, ox-LDL, platelet-associated ox-LDL (Pox-LDL), platelet cholesterol content, antiport mechanisms, and intracellular and systemic NO synthase every 7 days for one month. Results Our data show a strong relation between enhanced P-sel and Pox-LDL (r = 0·68, P < 0·01). Simvastatin, atorvastatin, fluvastatin and pravastatin reduce platelet activity after 1, 2, 3 and 4 weeks of treatment, respectively (P < 0·001, P < 0·001, P < 0·01, P < 0·05). Pox-LDL are modulated early by simvastatin, atorvastatin and fluvastatin Pox-LDL (r = 0·66, 0·65 and 0·52; P < 0·001, 0·001 and 0·01, respectively) whereas LDL-C and ox-LDL reductions associated to modifications of antiport activity act later. Moreouer, they are the most relevant finding in pravastatin-related subjects. Conclusions Our data suggest a different impact of several statins on platelet function, which is initially related to interference with Pox-LDL rather than LDL-C reduction.

Oxysterols induce interleukin-1β production in human macrophages

Abstract: Background Oxysterols are biologically active molecules generated during the oxidation of low-density lipoprotein or formed enzymatically in vivo. In the atherosclerotic plaque newly recruited macrophages may be exposed to oxysterols present in the plaque. How these oxysterols affect the expression and secretion of inflammatory cytokines such as interleukin-1β (IL-1β) in macrophages is not known. Therefore the aim of the present study was to investigate how oxysterols regulate the expression and secretion of IL-1 β in human monocyte-derived macrophages. Methods The IL-1 β messenger RNA (mRNA) expression was analysed by reverse transcription- polymerase chain reaction, and the IL-1 β protein secretion was measured by enzyme-linked immunosorbent assay. Results A significant, dose-dependent increase in the secretion of IL-1β was given by 25-hydroxycholesterol without the addition of lipopolysaccharide (LPS). At a concentration of 2.5 μg mL -1 this increase was similar to that obtained by endotoxin (LPS, 1 aeg mL -1 ). A transient increase in IL-1β mRNA expression was found in macrophages incubated with 25-hydroxycholesterol compared with untreated controls. In addition, 25-hydroxycholesterol dramatically increased the IL-1 secretion induced by LPS. At a concentration of 5 aeg mL -1 of 25-hydroxycholesterol the LPS-induced IL-1β secretion was increased by about 25-fold. A similar tendency, but not so consistent, was found for 27-hydroxycholesterol. Conclusions Our results show that oxysterols, and 25-hydroxycholesterol in particular, may modulate the inflammatory response in human macrophages. Consequently the presence of oxysterols in atherosclerotic tissue may dramatically influence the effect of inflammation.

Effects of deoxycholate on human colon cancer cells: apoptosis or proliferation.

Abstract: Background Deoxycholic acid has long been attributed as a tumour promoter in the colon. It exerts its growth-related actions in a phorbol ester-like manner, by stimulating protein kinase C. The aim of this study was to investigate the effect of deoxycholic acid on proliferation and apoptosis in the colon, by exposing colon cancer cells to it in increasing concentrations. Methods Human colon cancer cells (Caco-2 and HT-29) were treated with deoxycholate or its two structural isomers, 3-beta-12-alpha-dihydroxy-5-beta-cholan-24-oic acid and 3alpha-12-beta-dihydroxy-5-beta-cholan-24-oic acid. Proliferation was evaluated by cell counting, and apoptosis by estimating percentage cell survival and assessment of nuclear morphology. Results Within the concentration range of up to 20 aeM, deoxycholate stimulated growth of both human colon cancer cell lines. Its growth-promoting effect was abolished after inhibition of protein kinase C. At concentrations above 100 aeM, deoxycholate induced apoptosis in both cell lines. Epimers of deoxycholate were significantly less potent in stimulating growth. Conclusion Low-dose deoxycholate stimulates colon cancer cell proliferation while > 100 aemol L -1 of this secondary bile acid induces apoptosis in colon cancer cells. Deoxycholate might promote the likelihood of malignant transformation by increasing epithelial cell turnover in the colon.

Nontransferrin-bound iron in the plasma of haemodialysis patients after intravenous iron saccharate infusion

Abstract: BACKGROUND Many haemodialysis patients treated with recombinant human erythropoietin (r-HuEPO) receive intravenous iron supplementation on a regular basis. It has been shown previously that this may result in a transient "oversaturation" of transferrin. METHODS Ten stable haemodialysis patients on r-HuEPO treatment received 100 mg iron saccharate in 60 min, and 1 week later 100 mg in 6 min. Conventional iron metabolism parameters and nontransferrin-bond iron, detected with HPLC after addition of nitrilotriacetate and pretreatment with cobalt, were measured. Also, iron was measured in dialysate. RESULTS Serum iron increased from 9.6 +/- 6.2 to 213.7 +/- 49.4 micromol L(-1) (P < 0.001) when iron was given in 60 min, and from 11.1 +/- 4.7 to 219.3 +/- 43.7 micromol L(-1) (P < 0.001) when iron was given in 6 min. Transferrin saturation increased from 0.22 +/- 0.18 to 4.75 +/- 1.35 in protocol 1 and 0.26 +/- 0.16 to 4.91 +/- 1.38 in protocol 2. Nontransferrin-bound iron increased from 0.74 +/- 0.69 to 3.79 +/- 1.41 micromol L(-1) in protocol 1, and from 0.90 +/- 0.92 to 2.90 +/- 0.96 micromol L(-1) in protocol 2. No significant iron concentrations were found in dialysate before or during the iron saccharate infusion. CONCLUSION Nontransferrin-bound iron exists in plasma of dialysis patients after infusion of iron saccharate. There was no difference when 100 mg iron was given in 60 min or in 6 min. Before iron infusion, appreciable concentrations of nontransferrin-bound iron could already be detected. The clinical significance is not clear, but the findings may be important since nontransferrin-bound iron can act as a catalytic agent in the formation of hydroxyl radicals, thus potentially inducing cell damage and atherosclerosis.

The effects of acetylsalicylic acid on proliferation, apoptosis, and invasion of cyclooxygenase-2 negative colon cancer cells.

Abstract: Summary Background Acetylsalicylic acid (ASA, aspirin), the most common nonsteroidal anti-inflammatory drug (NSAID), has been shown to have a protective effect against the incidence and mortality of colorectal cancer. However, the mechanism of its anticancer function remains unclear. The aim of this study was to determine the effects of acetylsalicylic acid on proliferation, apoptosis, and invasion in human cyclooxygenase-2 (COX-2) negative colorectal cancer cell lines. Materials and Methods After treatment with various concentrations of ASA, cell proliferation was measured in the human colon cancer cell line SW480. Apoptotic cells were identified by transmission electron microscopy, acridine orange staining, and flow cytometry. The invasive potential of SW480 cells was detected using an in vitro invasion assay. The production of carcinoembryonic antigen was measured by microparticle enzyme immunoassay. Expression of Bcl2, Bax, CD44v6, and nm23 were evaluated by immunocytochemistry. Results ASA significantly inhibited the proliferation of SW480 cells and stimulated apoptosis. Production of carcinoembryonic antigen and the invasive potential of SW480 cells were also inhibited by ASA. After treatment with ASA, down-regulation of Bcl2 and CD44v6 expression and up-regulation of nm23 expression were observed in SW480 cells. No obvious effect of ASA was found on Bax expression. Conclusion Our findings reveal that ASA inhibits the proliferation and promotes apoptosis in the human colon cancer cell line SW480. Down-regulation of Bcl2 expression might represent a potential mechanism by which ASA induces apoptosis in this COX-2 negative colon cancer cell line. Our results also suggest that ASA decreases the invasive potential of these colon cancer cells. Decreased CEA content and CD44v6 expression and elevated nm23 expression may contribute to the effect of ASA on invasive potential of SW480 colon cancer cells.

Soluble leptin receptors and serum leptin in end-stage renal disease: relationship with inflammation and body composition.

Abstract: BACKGROUND Elevated serum leptin (S-leptin) levels have been reported in patients with end-stage renal disease (ESRD). Apart from the decreased glomerular filtration rate (GFR), body composition and inflammation may affect leptin levels in ESRD. Leptin circulates both free of and bound to soluble leptin receptors (sOB-R), which are the main determinants of leptin activity and have not been described in ESRD until now. DESIGN To analyze the association between S-leptin, sOB-R, and inflammation and body composition, we studied 149 (62% males) normal weight (BMI 24.7 +/- 0.4 kg m(-2)) ESRD patients (51 +/- 1 years old) shortly before the start of dialysis (GFR 7.0 +/- 0.2 mL min(-1)). sOB-R and plasma interleukin-6 (IL-6; n= 113) levels were evaluated using ELISA, S-leptin using RIA, and body composition was assessed by X-ray absorptiometry (n = 139). Forty-one healthy subjects age (51 +/- 1 years), BMI (23.6 +/- 0.5 kg m(-2)) and gender-matched (59% males) were used as controls. RESULTS Median S-leptin was higher in the ESRD patients (10.0 ng mL(-1)) compared with the controls (3.9 ng mL(-1)) (P < 0.001). The median sOB-R did not differ significantly between the ESRD patients (44 U mL-1) and the controls (37 U mL-1). Thus, the sOB-R/S-leptin ratio was lower in the ESRD patients (9.5 +/- 1.2 vs. 12.3 +/- 1.8; P < 0.01) than the controls. A negative correlation was observed between S-leptin and sOB-R (Rho = -0.42; P < 0.0001) in the ESRD patients, a positive correlation was observed between lean body mass and the sOB-R/S-leptin ratio (Rho = 0.33, P = 0.0001) whereas fat mass was negatively correlated to both sOB-R (Rho = -0.26, P = 0.002), and the sOB-R/S-leptin ratio (Rho = -0.62, P < 0.0001). Positive correlations were observed between IL-6 and S-leptin (Rho = 0.19; P < 0.05) and weak but significant body fat mass (Rho = 0.20; P < 0.05), respectively. CONCLUSIONS This study demonstrates that despite markedly elevated S-leptin levels in the ESRD patients, sOB-R did not differ from the controls. In view of the anorexigenic and pro-atherogenic effects of leptin, further elucidation of the consequences of free bioactive leptin in the development of complications such as malnutrition and cardiovascular disease in ESRD patients is required.

Selectin-P and interleukin-8 plasma levels in coronary heart disease patients.

Abstract: BACKGROUND Alterations of the immune system are now believed to play crucial role in the pathogenesis of atherosclerosis. The aim of this study was analysis of soluble forms of selectin-P and interleukin-8 levels in patients with different form of coronary heart disease. MATERIALS AND METHODS In the study took part 18 patients with stable coronary heart disease, 20 patients with unstable coronary heart disease and 15 healthy persons from control group. Soluble selectin-P and interleukin-8 levels were measured in EDTA plasma with the use of enzyme immunoassay ELISA. RESULTS The level of soluble selectin-P was significantly higher in unstable coronary heart disease patients in comparison to the stable coronary heart disease patients (P < or = 0.01) and nonsignificantly higher in comparison to the control group. The level of interleukin-8 were significantly higher in unstable coronary heart disease patients in comparison to the stable coronary heart disease patients (P < or = 0.01) and in comparison to the control group (P < or = 0.02). CONCLUSION Our findings suggest that soluble form of selectin-P and interleukin-8 may be useful clinical predictors of unstable coronary heart disease. The assessment of the risk for the development of coronary heart disease requires further serial investigation.

A proposal to redefine familial combined hyperlipidaemia - Third workshop on FCHL held in Barcelona from 3 to 5 May 2001, during the Scientific Sessions of the European Society for Clinical Investigation

Abstract: Familial combined hyperlipidaemia (FCHL) was described in 1973 by three separate groups as a common familial disorder characterized by multiple lipoprotein phenotypes and an increased risk of premature coronary artery disease [1– 3]. No metabolic explanation was offered for the variable lipid phenotypes and opinion differed as to whether this was likely to be a monogenic or polygenic disorder. In 1986, the first FCHL workshop was held in Seattle and at that meeting an elevated plasma apolipoprotein B (apoB) was added to the list of characteristics. However, it was not made an essential feature, nor was any change to the fundamental approach to phenotypic classification suggested, notwithstanding that complexity of diagnosis severely limits clinical application and comparison of research results [4]. In 1998, investigators met in Helsinki for the second FCHL workshop and heard of the newest efforts to identify the genetic and metabolic bases for FCHL. All of the analyses were presented within the context of the original diagnostic approach. At the most recent meeting of the European Society for Clinical Investigation in Barcelona, the third workshop on FCHL was organized by Dr J. Ribalta (Reus, Spain) and Dr M. Castro Cabezas (Utrecht, the Netherlands) to reconsider this most common, but least well characterized, familial atherogenic dyslipoproteinaemia. Our objective became to search for the most important pathophysiological features. From the outset, as outlined by Professor M-R. Taskinen (Helsinki, Finland) , the two most well-documented features are increased very low-density lipoprotein (VLDL) secretion and impaired clearance of postprandial lipoproteins [5,6]. The increased VLDL2 secretion results in hypertriglyceridaemia and an elevated plasma apoB. Long residence time of VLDL1 particles favour the formation of small dense low-density lipoprotein (LDL). Based on this, we considered the hypothesis that the phenotype of FCHL might not be multiple but unitary – namely, hypertriglyceridaemic (hyperTg) hyperapoB. If so, FCHL phenotype could be defined more simply and consistently as follows. The phenotype of hyperTg hyperapoB would have to be present in more than one family member and at least one individual in the family must have premature symptomatic coronary artery disease. Other genetic disorders and secondary causes of dyslipidaemia, including type 1 and type 2 diabetes would, of course, have to be excluded [4,5]. It was emphasized that such a change only represents an evolution in diagnosis based on the advances in knowledge and technology that have occurred since the disorder was Mike Rosenbloom Laboratory for Cardiovascular Research, McGill University Health Centre, McGill University, Montreal, Quebec, Canada (A. D. Sniderman); Departments of Internal Medicine and Endocrinology, University Medical Centre, Utrecht, the Netherlands (M. Castro Cabezas, D. W. Erkelens); Unitat de Recerca de Lípids i Arteriosclerosi, Facultat de Medicina, Hospital Universitari de Sant Joan, Universitat Rovira i Virgili, Reus, Spain (J. Ribalta, L. Masana); Servicio de Endocrinología, Departamento de Medicina, Universidad de Valencia, Valencia, Spain (R. Carmena, J. T. Real); Departments of Medicine and Endocrinology, Academic Hospital, 6202 AZ Maastricht, the Netherlands (T. W. A. de Bruin); Department General Internal Medicine 541, UMC Nijmegen, the Netherlands (J. de Graaf); Division of Cardiovascular Genetics, Department of Medicine, Royal Free and University College Medical School, London WC1E 6JJ, UK (S. E. Humphries, P. J. Talmud); Department of Medicine, University of Helsinki, Helsinki, Finland (M. R. Taskinen).

Current and adolescent levels of cardiopulmonary fitness are related to large artery properties at age 36: the Amsterdam Growth and Health Longitudinal Study

Abstract: BackgroundHigh levels of cardiopulmonary fitness (VO2max) are associated with reduced risk of cardiovascular morbidity and mortality, but little is known to what extent this is related to the effects of cardiopulmonary fitness on atherosclerosis and arterial stiffness. Moreover, the time course of these relationships needs to be elucidated. We sought to investigate (i) the cross-sectional relationship between VO2max and carotid atherosclerosis and carotid and femoral arterial stiffness at age 36, as well as (ii) the relationship between VO2max during adolescence (13–16 years) and the same arterial properties at age 36 (prospective analyses). DesignCross-sectional analyses consisted of 351 subjects (183 women) and the prospective analyses of a subpopulation of 154 subjects (79 girls). Arterial properties were assessed noninvasively by ultrasound imaging; VO2max was measured with a maximal running text on a treadmill with direct measurements of oxygen uptake. ResultsAfter adjustment for confounding by other known risk factors, current and adolescent levels of VO2max were independently associated with carotid intima-media thickness (β = −0·288, P = 0·004 and β = −0·381, P = 0·012) in men, and with the diameter of the femoral artery (β = 0·375, P < 0·001 and β = 0·252, P = 0·026, respectively) in both sexes. Current levels of VO2max were positively associated with the compliance of the carotid and the femoral arteries (β = 0·186, P = 0·023 and β = 0·183, P = 0·033, respectively), and with the distensibility of the carotid (β = 0·162, P = 0·047) but not the femoral artery. Conclusion We conclude that cardiopulmonary fitness is associated with large artery properties at age 36, and that the roots of this association may already be present in adolescence.

Statins decrease bone turnover in postmenopausal women: a cross-sectional study.

Abstract: Background Statins have been suggested as potential agents in the management of osteoporosis. Reviews of medical records have shown an increased bone mass and some studies have shown a reduced occurrence of fractures in subjects on long-term treatment with statins. We studied the effects of treatment with statins on calcium homeostasis, bone turnover and bone mineral density. Design In a cross-sectional design, plasma levels of parathyroid hormone (PTH) and biochemical markers of bone turnover, bone mineral density (BMD) and body composition (fat- and lean tissue-mass) were measured in 140 postmenopausal women who had been treated with a statin for more than 2 years (median 4 years) and compared to 140 age- and gender-matched, population-based controls. Results Plasma levels of bone turnover markers were lower in the statin-treated subjects than in the controls: osteocalcin (−9%, P = 0·03), bone-specific alkaline phosphatase (−14%, P < 0·01), and C-terminal telopeptide of type I collagen (−11%, P < 0·01). On the other hand, plasma PTH levels were 16% higher in the statin-treated subjects than in the controls (P < 0·01). However, body composition and BMD at the lumbar spine, hip, forearm and whole body did not differ between the two groups. No correlation could be demonstrated between changes in biochemical quantities and dose or duration of statin use. Conclusion Our data show that statins affect the function of bone cells. Most likely, the effect is antiresorptive.

Effect of homocysteine on arachidonic acid release in human platelets.

Abstract: Background It has been suggested that homocysteine is implicated in the risk of atherosclerosis and thrombosis. The pathogenic mechanism has not been clarified, but oxygen-free species produced by the homocysteine metabolism and auto-oxidation could have a role. Design We have studied the effect of homocysteine on arachidonic acid release in human platelets. Two important products of arachidonic acid metabolism – thromboxane B2 (TXB2) and reactive oxygen species (ROS) – have been assayed. Results Results indicate that homocysteine induces arachidonic acid release that is partially inhibited by 5,8,11,14-eicosatetraynoic acid (ETYA). Platelet incubation with homocysteine significantly increases basal levels of TXB2 and ROS. The effect is time- and dose-dependent. The TXB2 formation is strictly correlated with the arachidonic acid release. Moreover, ROS accumulation is largely inhibited by ETYA and partially reduced by diphenyleneiodonium (DPI), suggesting the involvement both of enzymes metabolising arachidonic acid (cyclooxygenase, lipooxygenase, cytochrome P450 monooxygenase) and of NAD(P)H oxidase. Conclusion Homocysteine induces oxidative stress in human platelets in vitro. The unbalance in platelet redox-state and the increased TXB2 formation may generate hyperactivation, contributing to a thrombogenic state leading to cardiovascular diseases.