Blood biomarkers associated with inflammation predict poor prognosis in cerebral venous thrombosis:: a multicenter prospective observational study.

BACKGROUND AND PURPOSE: Experimental studies suggest inflammation can contribute to blood barrier disruption and brain injury in cerebral venous thrombosis (CVT). We aimed to determine whether blood biomarkers of inflammation were associated with the evolution of brain lesions, persistent venous occlusion or functional outcome in patients with CVT. METHODS: Pathophysiology of Venous Infarction-Prediction of Infarction and Recanalization in CVT (PRIORITy-CVT) was a multicenter prospective cohort study of patients with newly diagnosed CVT. Evaluation of neutrophil-to-lymphocyte ratio (NLR) and C-reactive protein (CRP) concentrations in peripheral blood samples was performed at admission in 62 patients. Additional quantification of interleukin (IL)-6 was performed at day 1, 3 and 8 in 35 patients and 22 healthy controls. Standardized magnetic resonance imaging was performed at day 1, 8 and 90. Primary outcomes were early evolution of brain lesion, early recanalization and functional outcome at 90 days. RESULTS: Interleukin-6 levels were increased in patients with CVT with a peak at baseline. IL-6, NLR and CRP levels were not related with brain lesion outcomes or early recanalization but had a significant association with unfavourable functional outcome at 90 days (IL-6: OR = 1.28, 95% CI: 1.05-1.56, P = 0.046; NLR: OR = 1.39, 95% CI: 1.4-1.87, P = 0.014; CRP: OR = 1.756, 95% CI: 1.010-3.051, P = 0.029). Baseline IL-6 had the best discriminative capacity, with an area under the receiver operating characteristic curve to predict unfavourable functional outcome of 0.74 (P = 0.031). CONCLUSIONS: Increased baseline levels of NLR, CRP and IL-6 may serve as new predictive markers of worse functional prognosis at 90 days in patients with CVT. No association was found between inflammatory markers and early evolution of brain lesion or venous recanalization.

A Single Bout of Arm-crank Exercise Promotes Positive Emotions and Post-Exercise Hypotension in Patients with Symptomatic Peripheral Artery Disease.

OBJECTIVE/BACKGROUND: The objective was to analyze the acute effects of a single bout of arm cranking exercise on affective and cardiovascular parameters in patients with symptomatic peripheral artery disease (PAD). METHODS: This was a prospective, controlled, crossover study. Eleven men with symptomatic PAD underwent two experimental sessions in a random order: control or arm crank exercise (15 × 2 minutes bouts of arm crank exercise interrupted by 2 minutes rest intervals). During exercise, ratings of perceived exertion (Borg scale) and affective responses (pleasure/displeasure) were obtained at the first, fifth, tenth, and fifteenth bouts. Before and after the experimental sessions, cardiovascular parameters (blood pressure and heart rate) were obtained. Data were analysed by a two-way repeated measure analysis of variance with significance achieved at p < .05. RESULTS: During the arm crank exercise, patients reported positive feelings of pleasure. During exercise, heart rate (HR) remained within 80-90% of peak HR. Additionally, patients performed arm crank exercise with moderate levels of perceived exertion (Borg rating of 11-13) and with pleasant affective scores (Feeling Scale of +1 to +5). Blood pressure (systolic, diastolic, and mean) increase was lower after arm crank exercise than for control (greatest net effect: -15 ± 11 mmHg [p < .001]; -9 ± 5 mmHg [p < .001]; -9 ± 6 mmHg [p < .001], respectively), while HR increased (greatest net effect: +9 ± 6 beats per minute; p < .001). CONCLUSION: A single bout of arm crank exercise promotes pleasurable feelings while reducing blood pressure in patients with symptomatic PAD.

Vascular Mechanisms of Post-exercise Blood Pressure Responses in Peripheral Artery Disease.

This study aimed to analyze the vascular mechanisms involved in post-resistance decreases in blood pressure in patients with peripheral artery disease. 17 patients underwent 2 experimental sessions conducted in random order: resistance exercise (REx-3×10 reps in 8 exercises with intensity of ~ 60% of 1 RM) and control (C- resting on the exercise machines for 50 min). Before and after each experimental session, blood pressure, reflected wave indicators, pulse wave velocity, blood flow, blood flow post-reactive hyperemia and peripheral vascular resistance responses were obtained. Both sessions increased brachial systolic, diastolic and mean blood pressure (greatest increase REx: 11 mmHg; greatest increase C: 19 mmHg; P<0.01); however, the increases were greater after the C session (P<0.01). Reflected wave indicators increased only after the C session (P<0.06), while pulse wave velocity increased similarly after both sessions (P=0.66). Individual analyses indicated a large variability between patients in vascular variables responses. A single bout of REx decreased blood pressure in peripheral artery disease patients, and these responses were followed by changes in reflected wave indicators. The other factors presented high individual variability, and thus it was not possible to identify specific factors associated with blood pressure reduction in peripheral artery disease patients.

Heterologous expression and purification of a biologically active legume lectin from Cratylia mollis seeds (CRAMOLL 1).

CRAMOLL 1 is a mannose/glucose isolectin isolated from Cratylia mollis seeds. This lectin has 82% sequence identity with Con A and essentially the same quaternary structure. As with Con A, CRAMOLL 1 seems to undergo complex post-translational processing which makes it difficult to the use of traditional molecular cloning for heterologous expression. Here we report the expression and purification of functional recombinant CRAMOLL 1 (rCRAMOLL 1) in Escherichia coli. This was accomplished by introducing a chemically synthesized DNA encoding the mature CRAMOLL 1 amino acid sequence into a bacterial expression vector under T7 promoter control. Most of the recombinant lectin was found in insoluble aggregates (inclusion bodies), but we were able to recover reasonable amounts of soluble lectin in the active form by decreasing the protein induction temperature. The recombinant lectin was purified to homogeneity with one-step affinity chromatography. The plant CRAMOLL 1 (pCRAMOLL 1) and rCRAMOLL 1 share several physicochemical properties such as molecular mass, charge density and secondary and tertiary structures. However, pCRAMOLL 1 has a lower thermodynamic stability than rCRAMOLL 1 when probed by acidification, high temperature or high hydrostatic pressure, and this is probably caused by the presence of tetramers composed of fragmented monomers, which are formed in the plant cotyledon but absent from the recombinant protein. rCRAMOLL 1 behaves identically to its plant counterpart with respect to its specificity for monosaccharides, and to its agglutinating activities against rabbit erythrocytes and Trypanosoma cruzi epimastigote cells.

L-tryptophan: a common denominator of biochemical and neurological events of acute hepatic porphyria?

Hepatic porphyrias are disorders of heme synthesis characterized by genetically determined lesions of one of the key enzymes of heme synthesis. In carriers of such lesions, several factors (drugs, environmental chemicals, or diet) precipitate acute and often fatal attacks of neurologic dysfunction, which are promptly relieved by intravenous infusion of heme. However, the mechanism of such heme-induced amelioration remains elusive. To probe this mechanism, the biochemical events triggered by acute hepatic heme deficiency were examined in an animal model of chemically induced porphyria. Acute hepatic heme depletion in porphyric rats was found to impair hepatic tryptophan pyrrolase activity which, in turn, elevated tryptophan and 5-hydroxytryptamine turnover in the brain. These alterations in porphyric rats were dramatically reversed by parenteral heme administration. These findings suggest that increased tryptophan and 5-hydroxytryptamine in the nervous system may be responsible for the neurologic dysfunctions observed in humans with acute attacks of hepatic porphyria.

Effect of cannabidiol on cytochrome P-450 isozymes.

Cannabidiol (CBD) has been shown to inhibit mouse hepatic mixed-function oxidations of several drugs after acute treatment, whereas repetitive treatment resulted in the restoration of drug-metabolizing capabilities. We have found that acute CBD treatment modestly decreased cytochrome P-450 content but markedly decreased hexobarbital hydroxylase, erythromycin N-demethylase, and 6 beta-testosterone hydroxylase activities. Repetitive CBD treatment, on the other hand, resulted in the restoration of cytochrome P-450 content as well as hexobarbital hydroxylase and erythromycin N-demethylase activities. However, after such repeated treatments a fresh dose of CBD can once again inactivate erythromycin N-demethylase activity but not hexobarbital hydroxylase activity. The resistance of hexobarbital hydroxylase to re-inactivation by CBD was paralleled by stimulation of pentoxyresorufin O-dealkylase activity and the appearance of a 50 kD protein that was immunoreactive to an antibody raised against rat hepatic cytochrome P-450b. CBD metabolism in vitro by microsomes prepared from such CBD-"induced" animals, resulted in a pattern of metabolites different from that observed from comparable incubations with liver microsomes from either untreated or phenobarbital-treated animals. Thus, it appears that CBD initially inactivates at least one cytochrome P-450 isozyme, but after repetitive CBD treatment, an isozyme is induced that is resistant to further re-inactivation by CBD. This isozyme appears to be immunochemically similar to, but somewhat functionally distinct from, the isozyme induced by phenobarbital treatment in mice.

Structural and functional assembly of rat intestinal cytochrome P-450 isozymes. Effects of dietary iron and selenium.

We have reported previously that both dietary iron and selenium regulate intestinal cytochrome P-450 content by modulating the synthesis of its prosthetic heme moiety. Whether these elements are required for synthesis and/or viability of its apocytochrome moiety is unknown. We have examined the effects of intraluminal deprivation of these elements on the apocytochrome moieties of the constitutive (P-450) and the beta-naphthoflavone inducible (P-448) intestinal isozymes. The relative content of intestinal apocytochrome P-450 moieties generated by dietary deprivation of iron and/or selenium was assessed indirectly by complexing with exogenous heme in vitro, to reassemble the holocytochromes which could be monitored spectrally and catalytically. We now report that, whereas both intraluminal iron and selenium are required for maintenance of the prosthetic apocytochrome moiety of the constitutive intestinal isozyme, only intraluminal selenium is required for the viability of apocytochrome P-448. The latter apparently survives in the absence of intraluminal iron and can be assembled to the holocytochrome, with exogenously added heme. The mechanistic basis of the critical requirement of intestinal apocytochromes for intraluminal selenium is unclear. It is intriguing, however, that the deleterious effects of selenium deprivation are principally exerted in cell systems actively synthesizing protein and inexorably dependent on their extracellular milieu for their nutriment.

Induction and genetic regulation of mouse hepatic cytochrome P450 by cannabidiol.

Cannabidiol (CBD) has been shown to be a selective inactivator of cytochromes P450 (P450s) 2C and 3A in the mouse and, like many P450 inactivators, it can also induce P450s after repeated administration. The inductive effects of CBD on mouse hepatic P450s 2B, 3A, and 2C were determined using cDNA probes, polyclonal antibodies, and specific functional markers. P450 2B10 mRNA was increased markedly after repeated CBD administration and correlated well with increased P450 2B immunoquantified content and functional activity. On the other hand, although the 2-fold increase in P450 3A mRNA detected after repeated CBD administration was consistent with the increased immunoquantified P450 3A protein content, the lack of an observable increase in P450 3A-specific functional activity suggested subsequent inactivation of the induced P450 3A. Repeated CBD treatment increased P450 2C mRNA content 2-fold, but did not increase either the P450 2C immunoquantified content or its functional activity. The effect of CBD treatment on the ability of tetrahydrocannabinol (THC) to induce P450 2B was also determined. A THC dose that did not induce P450 2B significantly was administered alone or in combination with a CBD dose that markedly inactivated P450s 2C- and 3A but submaximally increased P450 2B functional activity. The combination of THC and CBD did not increase P450 2B-catalyzed activity significantly over that observed after CBD treatment alone. Thus, prior CBD-mediated P450 inactivation does not appear to increase the ability of THC to induce P450 2B. To further characterize the relationship between P450 inactivation and induction, several structurally diverse CBD analogs with varying P450 inactivating potentials were tested for their ability to induce P450 2B. At least one CBD analog that is an effective P450 inactivator failed to induce P450 2B, while at least one CBD analog that is incapable of inactivating P450 was found to be a very good P450 2B inducer. It therefore appears that inherent structural features of the CBD molecule rather than its ability to inactivate P450 determine P450 2B inducibility. The complex effects of CBD treatment on P450 inactivation and induction have the potential to influence the pharmacological action of many clinically important drugs known to be metabolized by these various P450s. The mechanism of CBD-mediated P450 induction remains to be elucidated but does not appear to be related to CBD-mediated P450 inactivation.

Characterization of cannabidiol-mediated cytochrome P450 inactivation.

Cannibidiol (CBD) has been shown to impair hepatic drug metabolism in several animal species and to markedly inhibit mouse hepatic microsomal delta 1-tetrahydrocannabinol (THC) metabolism by inactivating specific cytochrome P450s (P450) belonging to the 2C and 3A subfamilies. Elucidation of the mechanism of CBD-mediated P450 inhibition would be clinically very important for predicting its effect on metabolism of THC and the many other clinically important drugs known to be metabolized by P450s 2C and 3A. CBD-mediated inactivation of mouse hepatic microsomal P450s did not decrease hepatic microsomal heme content. However, [14C]CBD was found covalently bound to microsomal protein in an approximately equimolar ratio to P450 loss. Immunoprecipitation of microsomal protein with antibodies raised against either P450 2C or 3A revealed that approximately equal amounts of [14C]-CBD were bound to each of these P450s after CBD-mediated inactivation. Furthermore, this specific P450 binding was equivalent to P450 loss and accounted for nearly all of the microsomal [14C]CBD irreversible binding. Although > 80% of the enzyme activities attributed to P450s 2C and 3A were inactivated by CBD at the anticonvulsant dose of 120 mg/kg, P450 2C was approximately 3-fold more sensitive than P450 3A at the lower CBD doses tested. CBD analogs were synthesized in order to elucidate the chemical pathways for CBD-mediated P450 inactivation in vivo. Oxidations at allylic carbon positions or saturation of either the exocyclic double bond or both double bonds of the terpene moiety did not markedly affect the inhibitory properties of the analogs. Methylation of both phenolic groups of the resorcinol moiety completely blocked the P450-inhibitory properties of this analog, revealing the involvement of a free hydroxyl group in the inactivation process. Rotation of the resorcinol moiety in abnormal-CBD did not impair the inhibitory properties of the analog, suggesting that the position of the hydroxyl group relative to the terpene ring is unimportant. Further studies are required to fully understand the chemical basis of CBD-mediated P450 inactivation.

Microsomal cytochrome P450-mediated liver and brain anandamide metabolism.

Anandamide (AN) is an arachidonic acid congener, found in the brain, that binds to the cannabinoid receptor and elicits cannabinoid-like pharmacological activity. Cytochromes P450 (P450s) are known to oxidize arachidonic acid to a wide variety of metabolites, yielding many physiologically potent compounds. To determine if AN could be similarly oxidized by P450s, its metabolism by mouse liver and brain microsomes was examined. Mouse hepatic microsomal incubation of AN with NADPH resulted in the generation of at least 20 metabolites, determined after HPLC separation by increased UV-absorbance at 205 nm. Pretreatment of mice with various P450 inducers resulted in increased hepatic microsomal formation of several AN metabolites, with dexamethasone being the most effective inducer. Phenobarbital pretreatment resulted in a metabolic profile similar to that observed after dexamethasone pretreatment, whereas 3-methylcholanthrene pretreatment selectively increased the formation of several other metabolites. Clofibrate pretreatment had no effect on hepatic AN metabolism. Polyclonal antibodies prepared against mouse hepatic P450 3A inhibited the formation of several AN metabolites by hepatic microsomes from untreated mice as well as the formation of those metabolites found to be increased after dexamethasone pretreatment. AN metabolism by brain microsomes resulted in the formation of two NADPH- and protein-dependent metabolites. Hepatic P450 3A antibody partially inhibited the formation of only one of these metabolites. Thus, P450 3A is a major contributor to AN metabolism in the liver but not in the brain. The physiological consequences of P450-mediated AN metabolism remain to be determined.

Regulation of intestinal cytochrome P-450 and heme by dietary nutrients. Critical role of selenium.

The intestinal cytochrome P-450 (I-P-450)-dependent mixed function oxidase (MFO) system is regulated to a remarkable extent by various ingested xenobiotics, including drugs and carcinogens, as well as dietary nutrients. Accordingly, acute dietary iron deprivation is found to result in a marked decrease in I-P-450 content and activity. This decrease is most pronounced in the villous tip cells, the very cells committed to absorption of ingested materials. We investigated the mechanistic basis for such acute reduction and report that iron was not only required as a co-substrate for I-P-450 heme formation, but also as a regulator of two key heme-synthetic enzymes, delta-aminolevulinic acid synthetase and ferrochelatase. In addition, our studies revealed that dietary deprivation of selenium for a single day dramatically reduced I-P-450-dependent MFO activity. This prompt reduction apparently reflects impaired I-P-450 formation resulting from lowered ferrochelatase activity and consequently decreased intestinal heme availability, and was not a consequence of intracellular peroxidation presumably enhanced by concomitant lowering of the seleno-dependent glutathione peroxidase. Thus, we report the novel observation that dietary selenium also appears to be a critical modulator of intestinal cytochrome P-450-dependent metabolism of ingested drugs, carcinogens, and toxins that are absorbed by the intestinal mucosa.

Drug-induced modulations of hepatic heme metabolism. Neurological consequences.

Administration of the porphyrinogenic agent DDEP to PB-pretreated rats results in acute hepatic heme depletion, which is a characteristic of acute hepatic porphyria. Such acute heme depletion is associated with impaired hepatic tryptophan degradation and enhanced serotonergic tone in the CNS. We showed that intestinal motility in these rats is also significantly decreased, indicating that the serotonergic tone of the enteric nervous system may also be enhanced. In addition, the marked hepatic accumulation of glucogenic precursors, observed in parallel, indicates that the elevated tryptophan levels may also block hepatic glucogenesis. The clinical implications of these findings to acute heme-deficient states, such as the acute hepatic porphyrias, was discussed.

Morphine metabolism revisited: I. Metabolic activation of morphine to a reactive species in rats.

Treatment of fasted rats with relatively high doses of morphine rapidly results in depletion of hepatic glutathione (GSH) content and marked elevation of serum transaminase activity. Such morphine-induced response has been generally attributed to central nervous system mediated effects of the drug. We now report that this response might be due to a direct effect of the drug in the liver. That is, its metabolic activation to reactive electrophilic metabolite(s), by the hepatic cytochrome P-450-dependent mixed function oxidase system. Structure-activity relationships of morphine and its congeners indicate that the (-)-3-hydroxy-N- methylmorphinan moiety is linked with the potential of these opioids to deplete hepatic GSH and to raise serum transaminases in rats.

Morphine metabolism revisited. III. Confirmation of a novel metabolic pathway.

Previous studies have led to the partial structural characterization of a glutathionylmorphine adduct which was isolated from rat liver microsomal incubations. The formation of this adduct was shown to be catalyzed by cytochrome P-450. As an extension of this work, we have carried out similar studies with N-acetylcysteine in an attempt to obtain a product amenable to complete NMR analysis and unambiguous structure assignment. Incubation of [3H]morphine and N-acetylcysteine with microsomal preparations isolated from human and from phenobarbital-treated rats led to the isolation by HPLC of a labeled species displaying a fast atom bombardment mass spectrum consistent with the expected N-acetylcysteinyl adduct of morphine. Data obtained from the high resolution 1H-NMR spectrum of the adduct and that of synthetic 10 alpha-hydroxycodeine established the structure of the metabolite as 10 alpha-S-(N-acetylcysteinyl)morphine. The results are consistent with the biotransformation of morphine involving oxidation at the benzylic C-10 position to form an electrophilic species capable of reacting with nucleophilic thiols such as N-acetylcysteine and glutathione.

Morphine metabolism revisited. II. Isolation and chemical characterization of a glutathionylmorphine adduct from rat liver microsomal preparations.

Incubation of tritium-labeled morphine and cold glutathione (GSH) or cold morphine and tritiated GSH with liver microsomal preparations obtained from phenobarbital-treated rats led to the identification by high performance liquid chromatography (HPLC) of a glutathionylmorphine adduct. Liquid secondary ion mass spectral analysis established the molecular weight of the metabolite to be 590 which corresponds to the mass of a mono-GSH-morphine adduct. High resolution (360 and 500 MHz) 1H-NMR experiments have led to the tentative assignment of the structure of this metabolite as 10-alpha-S-glutathionylmorphine. Based on both in vivo and in vitro data, the formation of this product appears to be mediated by cytochrome P-450 and to involve a reactive intermediate that may be responsible for the observed covalent binding of radiolabeled morphine to proteins and, at least in part, for the morphine-induced depletion of GSH in the rat.

[Drug-addicted mothers]. / Mães toxicodependentes.

Drug addiction in Portuguese women has greatly increased recently and affects women of child-bearing age. The lack of scientific knowledge of the influence of drug addiction on pregnancy led us to create a model to approach the problem. With that purpose, a Clinic for Pregnant Drug Addicts was opened in the Alfredo da Costa Maternity Hospital in 1989, intended to set up a special permanent team to provide personalized pre-natal care. This clinic should be considered an integral part of multi-disciplinary action covering obstetrics, pediatrics, anesthesiology, nursing, clinical psychology and social assistance. The evolution of 164 pregnant women was monitored from October 1989 to December 1992, urine and amniotic fluid was analysed in 51 women. Due to the difficulty in applying standard criteria to the pregnancies observed, three levels of pre-natal care for the aforementioned 51 pregnant women, who are the object of this study, are proposed. The pre-natal observation of 164 pregnant drug addicts revealed that 74% were aged from 20 to 29 years, 49% had completed compulsory education, 59% were unemployed, 61% were unmarried and 82% had not planned their baby and had attended their first pre-natal clinic in the 19th week of pregnancy. The women's partners were drug addicts in 80% of cases. Their toxicological history revealed that 29% of them began taking drugs between the ages of 11 and 15, cannabis-based products being the first drug in 67% and opium-based in 28% of cases.(ABSTRACT TRUNCATED AT 250 WORDS)