[Show abstract][Hide abstract] ABSTRACT: Nonalcoholic fatty liver disease is considered to be the hepatic manifestation of metabolic syndrome and is usually related to high-fat, high-cholesterol diets. With the rationale that the identification and quantification of metabolites in different metabolic pathways may facilitate the discovery of clinically accessible biomarkers, we report the use of (1)H NMR metabolomics for quantitative profiling of liver extracts from LDLr(-/-) mice, a well-documented mouse model of fatty liver disease. A total of 55 metabolites were identified, and multivariate analyses in a diet- and time-comparative strategy were performed. Dietary cholesterol increased the hepatic concentrations of cholesterol, triglycerides, and oleic acid but also decreased the [PUFA/MUFA] ratio as well as the relative amount of long-chain polyunsaturated fatty acids in the liver. This was also accompanied by variations of the hepatic concentration of taurine, glutathione, methionine, and carnitine. Heat-map correlation analyses demonstrated that hepatic inflammation and development of steatosis correlated with cholesterol and triglyceride NMR derived signals, respectively. We conclude that dietary cholesterol is a causal factor in the development of both liver steatosis and hepatic inflammation.
Journal of Proteome Research 05/2010; 9(5):2527-38. · 5.06 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Oxidative stress is associated with human immunodeficiency virus (HIV) infection. Paraoxonase-1 (PON1) is an antioxidant enzyme that is bound to high-density lipoproteins (HDLs). We evaluated whether PON1 gene haplotypes influence the metabolic disturbances, presence of subclinical atherosclerosis, and virologic outcome associated with the infection.
DNA from blood samples collected from 234 HIV-infected patients and 633 healthy control subjects had single-nucleotide polymorphisms of PON1(192), PON1(55), PON1(-162), PON1(-832), PON1(-909), PON1(-1076), and PON1(-1741) analyzed using the Iplex Gold MassArray method. Subsequently, the influence of these single-nucleotide polymorphisms on measured biochemical and clinical variables was assessed.
We observed significant differences in the haplotype distribution between the control subjects and the HIV-infected patients. Haplotype H10 (GTCCGTC) was more prevalent in the HIV-infected patients (6.41% vs 0.64%; P < .001), and haplotype H5 (GACCGTC) was less prevalent in HIV-infected patients (27.7% vs 42.9%; P = .001). In HIV-infected patients, haplotype H7 (AATTCCT) was associated with better CD4(+) cell count recovery, higher levels of HDL cholesterol (P = .048) and apolipoprotein A-I (P = .019), lower levels of triglycerides (P = .004), and lower rates of subclinical arteriosclerosis (P < .001).
PON1 haplotypes segregate with HIV infection, HDL metabolism, the presence of subclinical atherosclerosis, and CD4(+) cell recovery after treatment.
The Journal of Infectious Diseases 02/2010; 201(4):627-34. · 5.85 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: HIV infection and its treatment are associated with dyslipidaemia and increased risk of cardiovascular disease. Accurate high-density lipoprotein (HDL) cholesterol values are necessary for the management of these abnormalities, but current methods have not been properly assessed in these patients. The aim of this study was to assess in HIV-infected patients the consistency and accuracy of a synthetic polymer/detergent homogeneous assay used to measure HDL cholesterol concentrations and to evaluate the impact of storage.
HDL cholesterol was measured using a synthetic polymer/detergent homogeneous method in samples from HIV-infected patients and healthy subjects for each of the storage regimens: baseline, after 1 week at 4 degrees C, and after 12 months at -80 degrees C. The ultracentrifugation and precipitation assays were used for comparison.
Three out of every 20 samples from HIV-infected patients had discrepant HDL cholesterol values with respect to the ultracentrifugation method. Overestimation was associated with high C-reactive protein concentrations and underestimation with plasma gamma-globulin concentrations, an effect that was amplified by any of the storage conditions tested.
Caution is needed when using the synthetic polymer/detergent homogeneous method for direct measurement of HDL cholesterol concentrations in HIV-infected patients. This assay is of limited use in clinical trials in which frozen samples are analysed.
HIV Medicine 12/2009; 11(4):260-5. · 3.16 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: HIV-infected patients show an increased cardiovascular disease (CVD) risk resulting, essentially, from metabolic disturbances related to chronic infection and antiretroviral treatments. The aims of this study were: (1) to evaluate the agreement between the CVD risk estimated using the Framingham risk score (FRS) and the observed presence of subclinical atherosclerosis in HIV-infected patients; (2) to investigate the relationships between CVD and plasma biomarkers of oxidation and inflammation.
Atherosclerosis was evaluated in 187 HIV-infected patients by measuring the carotid intima-media thickness (CIMT). CVD risk was estimated using the FRS. We also measured the circulating levels of interleukin-6, monocyte chemoattractant protein-1 (MCP-1) and oxidized low-density lipoprotein (LDL), and paraoxonase-1 activity and concentration.
There was a weak, albeit statistically significant, agreement between FRS and CIMT (kappa=0.229, P<0.001). A high proportion of patients with an estimated low risk had subclinical atherosclerosis (n=66; 56.4%). In a multivariate analysis, the presence of subclinical atherosclerosis in this subgroup of patients was associated with age [odds ratio (OR) 1.285; 95% confidence interval (CI) 1.084-1.524; P=0.004], body mass index (OR 0.799; 95% CI 0.642-0.994; P=0.044), MCP-1 (OR 1.027; 95% CI 1.004-1.050; P=0.020) and oxidized LDL (OR 1.026; 95% CI 1.001-1.051; P=0.041).
FRS underestimated the presence of subclinical atherosclerosis in HIV-infected patients. The increased CVD risk was related, in part, to the chronic oxidative stress and inflammatory status associated with this patient population.
HIV Medicine 10/2009; 11(4):225-31. · 3.16 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To assess the role of monocyte chemoattractant protein-1 (MCP-1/CCL2) in the development of fatty liver, we have used LDLr(-/-) mice as an animal model of high-fat, high-cholesterol diet-induced liver steatosis. The rapid dietary induction of hepatic mRNA MCP-1 expression was paralleled by a concomitant increase in plasma MCP-1 that was strongly associated with the degree of liver steatosis. Hepatocytes showed an intense immunoreactivity for MCP-1 that was mainly located surrounding the hepatic lipid droplets. The intake of cholesterol also increased the concentration of MCP-1 in liver homogenates. This was accompanied by a differential expression of members of the PPAR family. Additionally, complete MCP-1 deficiency prevents the development of liver steatosis in LDLr(-/-) mice and partial deficiency is accompanied by a certain protective effect. Our data also suggest that MCP-1 may be important in the regulation of hepatic insulin resistance and may represent a link between inflammation and metabolic diseases. We conclude that dietary cholesterol upregulation of hepatic MCP-1 may help to understand the role of circulating MCP-1 in conditions where liver derangements are clinically important and in the association of liver steatosis with the metabolic syndrome.
[Show abstract][Hide abstract] ABSTRACT: Monocyte chemoattractant protein-1 (MCP-1) plays a relevant role in macrophage migration but recent findings suggest an additional role in lipid and glucose metabolism. We report the use of (1)H NMR spectroscopy as a useful complementary method to assess the metabolic function of this gene in a comparative strategy. This metabonomic analysis was rapid, simple, quantitative and reproducible, and revealed a suggestive relationship between the expression of the MCP-1 gene and hepatic glucose and taurine concentrations. This approach should be considered in genetically modified mice when a metabolic alteration is suspected, or in routine assessment of metabolic phenotype.
[Show abstract][Hide abstract] ABSTRACT: Paraoxonase-1 (PON1) is an antioxidant enzyme synthesized by the liver. It protects against liver impairment and attenuates the production of the pro-inflammatory monocyte chemoattractant protein-1 (MCP-1). We investigated the relationships between hepatic PON1 and MCP-1 expression in rats with liver disease and explored the possible molecular mechanisms involved.
CCl4 was administered for up to 12 weeks to induce liver damage. Serum and hepatic levels of PON1 and MCP-1, their gene and protein expression, nuclear transcription factors, and histological and biochemical markers of liver impairment were measured.
High levels of PON1 and MCP-1 expression were observed at 12th week in the hepatocytes surrounding the fibrous septa and inflammatory areas. CCl4-administered rats had an increased hepatic PON1 concentration that was related to decreased gene transcription and inhibited protein degradation. Decreased PON1 gene transcription was associated with PPARdelta expression. These changes were accompanied by increased hepatic MCP-1 concentration and gene expression. There were significant direct relationships between hepatic PON1 and MCP-1 concentrations (P = 0.005) and between PON1 and the amount of activated stellate cells (P = 0.001).
Our results from this experimental model suggest a hepato-protective role for PON1 against inflammation, fibrosis and liver disease mediated by MCP-1.
[Show abstract][Hide abstract] ABSTRACT: Fatty acid synthase (FASN) is an enzyme synthesized by the liver and plays an important role in lipogenesis. The present study aimed to assess whether serum FASN concentrations are altered in patients with chronic liver disease, and to investigate whether its measurement may be a useful tool in the clinical evaluation of this derangement.
We investigated 93 patients with chronic liver disease (14 minimal change disease, 79 steatohepatitis) and 100 control subjects. Serum FASN concentrations were measured using ELISA.
Patients had a significant increase in serum FASN concentration (p<0.001), which was specifically associated with the hepatic Knodell sub-index III of portal inflammation (p=0.019). In addition, serum FASN concentrations were significantly correlated with the circulating levels of the monocyte chemoattractant protein-1 (MCP-1) (Spearman rho=0.375; p<0.001) and type III procollagen-N-peptide (P-III-P) (Spearman rho=0.297; p<0.001).
Serum FASN concentrations are increased in patients with chronic liver impairment, and are associated with specific histological alterations and biochemical markers of portal inflammation. These data suggest that FASN measurement may contribute significantly to the evaluation of these patients.
Clinical Chemistry and Laboratory Medicine 01/2009; 47(9):1097-9. · 3.01 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We investigated the analytical performance of a new assay of the lactonase activity of paraoxonase-1 and its efficacy in the assessment of liver damage.
Serum lactonase activity was determined by the hydrolysis of 5-thiobutyl butyrolactone in 633 healthy individuals and 369 patients with chronic liver disease. Paraoxonase-1, 2, and 3 gene polymorphisms were analyzed by the MassArray method.
Linearity was up to 10 U/L. Detection limit was 0.12 U/L. Imprecision was < or = 17.7%. Lactonase values in our normal population were 5.99 (3.29-13.61) U/L. Lactonase activity showed a lower influence of genetic polymorphisms than the classical assay using paraoxon. Both measurements showed a similar efficiency in testing for liver dysfunction.
We report a reliable assay using a non-toxic substrate for the measurement of serum lactonase activity. The influence of genetic variability is low. The assay could be a useful addition to tests evaluating liver impairment.
[Show abstract][Hide abstract] ABSTRACT: A method for selective extraction using SPE, electrophoretic separation at basic condition and the identification by using exact masses and fragmentation patterns has been developed in order to know the anthocyanins in dried calyces of Hibiscus sabdariffa L. A detailed and comparative study of several extraction procedures has been carried out to obtain the maximum number of anthocyanidins from the calyces and then a CE-TOF-MS method in positive mode using ESI has been developed for the separation and rapid identification of anthocyanins in H. sabdariffa L. Delphinidin-3-sambubioside, cyanidin-3-sambubioside have been detected as main components and cyanidin-3-O-rutinoside, delphinidin-3-O-glucoside and cyanidin-3,5-diglucoside, and chlorogenic acid as minor constituents. The confirmation of the anthocyanidins and chlorogenic acid was carried out using fragmentation ions with the IT-mass spectrometer (IT-MS).
[Show abstract][Hide abstract] ABSTRACT: The paraoxonase (PON) enzyme family, comprising PON1, PON2, and PON3, are antioxidant enzymes that degrade oxidised phospholipids. We describe the immunohistochemical localisation of the PON proteins in the normal mouse. Antibodies were obtained by inoculating rabbits with peptides derived from specific sequences of mature PONs. PON1 and PON3 were detected in the skin external epithelium, acini of the sebaceous glands, tongue epithelium, acini of the submandibular gland, surface epithelia of the stomach and the intestine, hepatocytes, exocrine pancreas acini, fibre tracts of the encephalon and the spinal cord, skeletal and cardiac muscle, eye lens epithelium and retinal layers, adipocytes, chondrocytes, epithelial cells of the trachea and bronchiole, ovary follicular fluid, seminiferous tubules, spermatozoa, and kidney proximal tubules. PON2 expression was weaker than that of PON1 and PON3, and was absent in some of the tissues studied, such as submandibular gland, nerve cells, and adipocytes. In muscle cells, PON2 expression was restricted to the endomysium. Apolipoprotein A-I did not colocalise with PONs, suggesting local synthesis. This study provides an experimental model to investigate the role played by these enzymes as antioxidants and their relationship with the development of a variety of diseases.
Free Radical Biology and Medicine 08/2008; 45(2):146-57. · 5.27 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Chronic liver diseases are accompanied by changes in the biochemical pathways related to the regulation of apoptosis and extra-cellular matrix deposition. The present study was designed to investigate, using low density arrays, changes in the hepatic gene expression together with hepatic biochemical and histological alterations in rats that had liver impairment induced by chronic exposure to CCl(4). Further, we examined the possible recovery of genetic and pathological changes following the cessation of the hepatotoxic injury. Experimental fibrosis was induced in male Wistar rats by CCl(4) administration. Animals were subdivided into two groups. One group was given CCl(4 )and animals were killed at 8 and 12 weeks of treatment. The other group was treated with CCl(4) for 6 weeks, the CCl(4 )was then stopped and, subsequently, subgroups of animals were killed after 1 and 2 weeks of recovery. CCl(4) administration over 12 weeks was associated with significant changes in B-cell leukemia/lymphoma 2, procollagen type I alpha 2, matrix metalloproteinases 3 and 8, tissue inhibitors of metalloproteinases 1, 2, and 3 and the inhibitor of apoptosis 4 gene expressions. Recovery after CCl(4) cessation was associated with changes in procollagen type I alpha 2, matrix metalloproteinase 7, tissue inhibitors of metalloproteinases 1 and 2, inhibitor of apoptosis 4, and survivin gene expressions. This study shows an association between changes in the expression of several genes regulating hepatic cell apoptosis, the fibrosis process, and the recovery of the hepatic function after removal of the toxic injury.
Molecular and Cellular Biochemistry 02/2008; 308(1-2):101-9. · 2.33 Impact Factor