Felipe Sánchez de la Cuesta

HLA class II genotype influences the type of liver injury in drug-induced idiosyncratic liver disease.

Drug‐induced idiosyncratic liver disease (DIILD) depends largely on host susceptibility factors. Small studies support the genetic influence of human leukocyte antigen (HLA) class II molecules on the predisposition to DIILD. We sought associations between HLA‐DRB and ‐DQB alleles and DIILD considered collectively or according to the biochemical expression of liver damage. We studied a total of 140 patients with a definitive or probable diagnosis of DIILD, as assessed with the Council for International Organizations of Medical Sciences scale, with 635 volunteer bone marrow and blood donors serving as controls. HLA‐DRB1* and ‐DQB1* genotyping was performed by hybridization with sequence‐specific oligonucleotides after genomic amplification. The group with DIILD did not differ from control subjects with regard to the distribution of HLA‐DRB and ‐DQB antigens. The frequencies of alleles DRB1*15 (35.4% vs. 18.6% of controls; P = .002; odds ratio [OR] 2.31) and DQB1*06 (61.5% vs. 40.8%; P = .001; OR 2.32) were significantly increased in patients with the cholestatic/mixed type of liver damage in comparison to healthy subjects. By contrast, frequencies of alleles DRB1*07 (16.9% vs. 35.4%; P = .003; OR 0.37) and DQB1*02 (32.3% vs. 55.8%; P = .0003; OR 0.39) were significantly decreased. In conclusion, there is no association between any specific HLA allele and the propensity to develop DIILD. However, the genetic influence associated with HLA class II alleles appears to play a role in the biochemical expression of liver injury in cholestatic/mixed hepatotoxicity and may explain why a given drug may cause different patterns of liver damage. (HEPATOLOGY 2004;39:1603–1612.)

Muscarinic receptor subtypes in human and rat colon smooth muscle

Muscarinic receptor subtypes in human and rat colon smooth muscle homogenates were characterized with [3H]N-methylscopolamine ([3H]NMS) by ligand binding studies. [3H]NMS saturation experiments show the existence of a homogeneous population of non-interacting binding sites with similar affinity (KD values of 1.38 +/- 0.20 nM in human colon smooth muscle and 1.48 +/- 0.47 nM in rat colon smooth muscle) and with Hill slopes close to unity in both samples of tissue. However, a significant (P less than 0.01) increase in muscarinic receptor density (Bmax) is found in human colon (29.9 +/- 2.9 fmol/mg protein) compared with rat colon (17.2 +/- 1.5 fmol/mg protein). Inhibition of [3H]NMS binding by non-labelled compounds shows the following order in human colon: atropine greater than AF-DX 116 greater than pirenzepine. Whereas in rat colon the rank order obtained is atropine greater than pirenzepine greater than AF-DX 116. Atropine and pirenzepine bind to a homogeneous population of binding sites, although pirenzepine shows higher affinity to bind to the sites present in rat colon (Ki = 1.08 +/- 0.08 microM) than those in human colon (Ki = 1.74 +/- 0.02 microM) (P less than 0.05). Similarly, IC50 values obtained in AF-DX 116 competition experiments were significantly different (P less than 0.01) in human colon (IC50 = 1.69 +/- 0.37 microM) than in rat colon (IC50 = 3.78 +/- 0.75 microM). Unlike atropine and pirenzepine, the inhibition of [3H]NMS binding by AF-DX 116 did not yield a simple mass-action binding curve (nH less than 1, P less than 0.01) suggesting the presence of more than one subtype of muscarinic receptor in both species. Computer analysis of these curves with a two binding site model suggests the presence of two populations of receptor. The apparent Ki1 value for the high affinity binding site is 0.49 +/- 0.07 microM for human colon smooth muscle and 0.33 +/- 0.05 microM for rat colon smooth muscle. The apparent Ki2 for the low affinity binding site is 8.01 +/- 1.0 microM for human samples and 6.07 +/- 1.1 microM for rat samples. These values are close enough to suggest that the first subtype of muscarinic receptor may be considered cardiac (M2) and the second subtype glandular (M3). The relative densities of the receptor subtypes are significantly different for both species. Human colon samples show the major densities of subtype M2, 22.62 +/- 1.11 fmol/mg protein, this represents 75.66 +/- 3.73% of the total receptors.(ABSTRACT TRUNCATED AT 400 WORDS)

Antithrombotic Potential of Olive Oil Administration in Rabbits with Elevated Cholesterol

Olive oil is the main source of dietary fatty acids in the Mediterranean region. The objective of this study was to evaluate the effect of dietary supplementation with virgin olive oil in an experimental model with rabbits fed an atherogenic diet (saturated fat 48% of total fat). Four different groups of 10 animals each were studied: (1) normolipemic diet (NLD), (2) atherogenic diet or saturated fatty acid-enriched diet (SFAED), (3) NLD with 15% olive oil (NLD+OLIV), and (4) SFAED with 15% virgin olive oil (SFAED+OLIV). The animals were fed the experimental diets for 6 weeks, after which we determined serum lipid profile (total cholesterol, HDL-cholesterol, and triglycerides), platelet aggregation, platelet thromboxane B(2), aortic prostacyclin, and platelet and vascular lipid peroxidation. Scanning electron microscopic images of the vascular endothelium were studied, as were morphometric parameters in the arterial wall and thrombogenicity of the subendothelium (annular perfusion chamber). Animals fed the SFAED showed platelet hyperactivity and increased subendothelial thrombogenicity. Animals fed the SFAED+OLIV showed, compared with the SFAED group, an improved lipid profile with decreased platelet hyperactivity and subendothelial thrombogenicity and less severe morphological lesions of the endothelium and vascular wall. We conclude that supplementation of the SFAED with 15% olive oil reduced vascular thrombogenicity and platelet activation in rabbits. Although the percentage of olive oil in the diet was higher than the amount in the human diet, these results may be helpful in determining the effect of olive oil in the human thrombogenic system.

Effect of dipyridamole and aspirin on the platelet-neutrophil interaction via the nitric oxide pathway

This study was designed to determine the influence of the combination of aspirin and dipyridamole on the interaction in vitro between neutrophils and platelets through the nitric oxide (NO) pathway. Collagen-induced platelet aggregation (impedance method) was determined in platelet-rich plasma and in platelet-rich plasma+neutrophils, and cGMP (enzyme immunoanassay) and NO levels (electrochemical method, with a ISO-200 electrode) were also measured. The 50% inhibitory concentration (IC(50)) of aspirin was 139+/-11 microM in platelet-rich plasma, 367+/-21 microM in platelet-rich plasma+L-N(G)-nitro-arginine-methyl-ester (L-NAME), and 42+/-3 microM in platelet-rich plasma+L-arginine. The IC(50) for dipyridamole in platelet-rich plasma was not affected by L-NAME or L-arginine; the combination of aspirin with 20 microM dipyridamole (which has no effect per se) led to an IC(50) of 51+/-2 microM in platelet-rich plasma, 101+/-7 microM in platelet-rich plasma+L-NAME, and 13+/-2 microM in platelet-rich plasma+L-arginine. The cGMP levels showed the greatest increases in the aspirin plus dipyridamole group. Dipyridamole and aspirin increased the leukocyte production of NO: 50% increases were obtained at concentrations of 285+/-31 microM aspirin, 110+/-9 microM dipyridamole, and 16+/-2 microM aspirin+dipyridamole. Dipyridamole alone at a concentration of 20 microM had no significant effect on NO levels. We conclude that the combination of aspirin and dipyridamole significantly increases the antiplatelet effect of leukocytes, through an increase of NO, and that this effect is further evidence of the therapeutic benefits of this combination of drugs.

Flavonoid Inhibition of Enzymic and Nonenzymic Lipid Peroxidation in Rat Liver Differs from Its Influence on the Glutathione-Related Enzymes

Eight flavonoids were tested for their antiperoxidative activities against lipid peroxidation induced in liver cell membranes either by nonenzymic way (ascorbic acid-Fe2+ system, FeAs) or by enzymic way (arachidonic acid, AA). When lipid peroxidation is induced by FeAs, the order in the inhibitory potency for the different flavonoids assayed is: (-)-epicatechin approximately luteolin > quercetin approximately (+)-catechin > delphinidin > kaempferol >> apigenin > naringenin. However, when lipid peroxidation is induced by AA, the potency order is markedly modified: delphinidin > (-)-epicatechin > (+)-catechin > kaempferol > quercetin > luteolin > naringenin > apigenin. These flavonoids were also tested for their influence on glutathione-related enzymes, which constitute one of the aim physiological antioxidant systems. It is concluded that the antiperoxidative effect shown by most of the flavonoids is exerted without modifying these enzymes.

In vitro effects of Clopidogrel on the platelet-subendothelium interaction, platelet thromboxane and endothelial prostacyclin production, and nitric oxide synthesis

Clopidogrel is an antiplatelet drug that belongs to the group of thienopyridines. Because of its main mechanism of action most studies of clopidogrel have centered on the platelet ADP pathway. The aim of the present study was to compare the effects of clopidogrel, ticlopidine, and aspirin, on platelet activation by collagen (the main inducer of platelet activation in vivo), prostanoid, and NO production, and the effects on blood perfusion experiments. Clopidogrel inhibited platelet aggregation induced in whole blood by collagen and TxB2 production to a greater extent than did ticlopidine. Prostacyclin synthesis did not change after incubation with thienopyridines, whereas aspirin inhibited synthesis in a dose-dependent manner. Thienopyridines increased NO production to a greater extent than did aspirin. All three drugs impaired the platelet-subendothelium interaction under flow conditions. With thienopyridines, the presence of endothelium did not modify the percentage of the surface coated by platelets.

Oral Administration of Quercitrin Modifies Intestinal Oxidative Status in Rats

1. Oral administration of quercitrin to rats for 3 days increases the mucosal glutathione contents in ileum and colon as well as inhibits non-enzymatic lipid peroxidation induced in membrane fractions from jejunal and colonic mucosa. 2. After 7 days of treatment with quercitrin, rat intestinal oxidative status trends to normalize to control rats.

Drug use for non-hepatic associated conditions in patients with liver cirrhosis

AimsTo study the prescribing patterns of practising physicians for the most frequent non-hepatic associated conditions in patients with liver cirrhosis.MethodsA multi-centre prospective observational study carried out in 25 Spanish hospitals. Inpatients admitted to gastrointestinal and liver units with a diagnosis of liver cirrhosis were included in five centrally assigned index days, between February and June 1999. Information was collected about pharmacological treatments used on admission and recommended at discharge.ResultsFive hundred and sixty-eight in-patients with a diagnosis of liver cirrhosis (44% alcoholic cirrhosis) and an average number of 2.5 co-morbid conditions were studied: diabetes mellitus (30%), infectious disorders (24%), cardiovascular disease (20%) and active alcoholism (15%)—the most common associated conditions. Chlormethiazole, amoxicillin–clavulanic acid, paracetamol, gliblenclamide, lorazepam, captopril and tiapride were the drugs used most prevalently. The average prescribed daily dose was <1 defined daily dose per day for most medication classes hepatically handled except for calcium channel blockers.ConclusionsThe present study expands current knowledge of prescribing patterns for associated conditions in patients with underlying liver cirrhosis. Drug dosing was affected in general by the influence of age and hepatic disease on the disposition of drugs, but knowledge on drug selection needs further attention.

Aminoglycoside-associated nephrotoxicity in extrahepatic obstructive jaundice

Experimental data demonstrate that biliary obstruction increases renal sensitivity to gentamicin. In the present study the incidence of and risk factors for aminoglycoside nephrotoxicity were prospectively studied in patients with extrahepatic obstructive jaundice. Two hundred and thirty-seven hospitalized adult patients were classified into three groups. Group I consisted of 84 patients with extrahepatic obstructive jaundice, who received aminoglycoside (gentamicin or tobramycin). Group II consisted of 81 patients with extrahepatic obstructive jaundice, who received either antibiotics other than aminoglycoside or no antimicrobial therapy. Group III consisted of 72 noncholestatic patients receiving aminoglycosides for different disorders. Nephrotoxicity developed in 27 patients (32%) in group I vs 9 patients (11%) in group II and 4 patients (5.6%) in group III (p < 0.00001). In group I, a comparison of patients with and without nephrotoxicity revealed significantly higher values in the former for mean serum bilirubin concentration, initial steady-state trough aminoglycoside concentration and estimated half-life. Stepwise multivariate analysis with nephrotoxicity status as the dependent variable determined that the most significant variable for predicting nephrotoxicity was serum total bilirubin level. In extrahepatic cholestasis a high serum bilirubin level is a distinct factor predisposing to aminoglycoside nephrotoxicity.

Effect of WEB 2086-BS, an antagonist of platelet-activating factor receptors, on retinal vascularity in diabetic rats

Specific antagonists of platelet-activating factor (PAF) receptors inhibit platelet aggregation and thromboxane synthesis. These two processes have been implicated in the course of diabetic retinopathy. We assessed the effect of a specific PAF receptor antagonist, WEB 2086-BS (3-(4-(2-chlorophenyl)-9-methyl-6H-thieno(3,2-f) (1,2,4 triazolo-(4,3-a(1,4)-diazepine-2-yl)-1-(4-morpholinyl)-1-propanone) on retinal vascularity in a model of experimental streptozocin-induced diabetes in rats. Rats were divided into five experimental groups (10 animals/group): group I, non-diabetic group II, untreated diabetic group III, diabetic given 1 mg/kg per day of WEB 2086-BS (p.o.) group IV, diabetic given 5 mg/kg per day (p.o.) and group V, diabetic given 10 mg/kg per day (p.o.). After 3-month treatment, platelet aggregometry, platelet synthesis of thromboxane B2, aortic production of 6-keto-prostaglandin F1alpha, platelet and vascular lipid peroxidation, and percentage of the retinal area occupied by horseradish peroxidase-labeled vessels were measured. Untreated diabetic rats showed an increase in platelet reactivity, reduced 6-keto-prostaglandin F1alpha production, increased thromboxane B2 and lipid peroxides, and a decrease in the percentage of retinal area occupied by horseradish peroxidase-labeled vessels. WEB 2086-BS produced a decrease in platelet aggregation induced by collagen in whole blood, in thromboxane B2 synthesis and lipid peroxide production, and an increase in the percentage of retinal area occupied by horseradish peroxidase-labeled vessels (13.9+/-1.1% in group II and 9.9+/-0.8% in group V). There was a statistically significant linear correlation (Y= -0.72 + 137X, r2 = 0.7247, P < 0.0007) between thromboxane B2 values and the percentages of retinal area occupied by horseradish peroxidase-labeled vessels in the groups of animals treated with WEB 2086-BS.