Neuroactive Steroid Levels in a Transgenic Rat Model of CMT1A Neuropathy

Charcot–Marie–Tooth type 1A (CMT1A) represents 80% of all the demyelinating hereditary motor and sensory neuropathies. As recently suggested, neuroactive steroids may have a role in a therapeutic strategy for peripheral neuropathies, including CMT1A. To this aim, an accurate qualitative and quantitative analysis of neuroactive steroid levels in this disease could be extremely important to define effective pharmacological strategies. We here analyzed by liquid chromatography-tandem mass spectrometry the levels of neuroactive steroids present in the sciatic nerve of male and female peripheral myelin protein 22 transgenic rats (PMP22_tg rats; i.e., an experimental model of CMT1A) and of the corresponding wild-type littermates. We observed that, both in PMP22_tg rats and in the wild types, the levels of neuroactive steroids, such as progesterone, tetrahydroprogesterone (THP), isopregnanolone (3β,5α-THP), testosterone, dihydrotestosterone, and 5α-androstane-3α, 17β-diol (3α-diol) are sexually dimorphic. It is interesting to note that the levels of 3β,5α-THP and of 3α-diol, which are exclusively detectable in sciatic nerve of female and male rats, respectively, are strongly decreased in PMP22_tg rats. 3β,5α-THP and 3α-diol are modulators of gamma-amino butyric acid A receptor. Thus, the present findings may be considered an interesting background for experiments aimed to evaluate the possible therapeutic effects of modulators of this neurotransmitter receptor in male and female PMP22_tg rats.     

Involvement of Neurosteroids in the Effect of the Endogenous Benzodiazepine Receptor Ligand Octadecaneuropeptide (ODN) on Gonadotropin-Releasing Hormone Gene Expression in Rat Brain

We have recently demonstrated that different activators of the GABA_A receptor complex including reduced progesterone metabolites and the endozepine octodecaneuropeptide (ODN) exert an inhibitory influence on GnRH gene expression. In order to investigate the possible involvement of neurosteroids, especially progesterone metabolites in the effect of ODN, we have evaluated in adrenalectomized and castrated male rats the influence of pretreatment with an inhibitor of 3 β -hydroxysteroid dehydrogenase (3 β -HSD) trilostane (TRIL) and an inhibitor of 5 α -reductase MK-906 on GnRH mRNA levels in ODN-treated rats. TRIL completely prevented the inhibitory influence of ODN on GnRH mRNA. It was also found that the inhibitor of 3 β -HSD as well as pregnenolone sulfate (PREG-S), which has been shown to be increased following TRIL treatment, could induce an increase in GnRH mRNA. MK-906 could also completely reverse the negative influence of ODN. When administered alone, this antagonist of 5 α -reductase induced an increase in GnRH mRNA. These results clearly indicate that the inhibition of two key enzymes for the synthesis of reduced progesterone metabolites can completely prevent the inhibitory influence of the endozepine ODN, suggesting that the effect of this endogenous ligand might be completely or partially mediated by an activation of the synthesis of active progesterone metabolites. On the other hand, it remains possible that, as a consequence of enzymatic inhibition, an increase in some precursors known as antagonists of GABA_A may play a role in the prevention of the ODN effect by the two enzyme antagonists.     

Indomethacin improves locomotor deficit and reduces brain concentrations of neuroinhibitory steroids in rats following portacaval anastomosis

Abstract  Hepatic encephalopathy (HE) is a neuropsychiatric complication of both acute and chronic liver failure characterized by progressive neuronal inhibition. Some neurosteroids are potent positive allosteric modulators of the gamma-aminobutyric acid (GABA)-A receptor complex, and 'increased GABAergic tone' has been proposed to explain the neuroinhibition characteristics of HE. Brain levels of the neurosteroids pregnenolone, allopregnanolone and tetrahydrodesoxycorticosterone (THDOC) and the functional status of the GABA-A receptor complex were assessed in rats following portacaval anastomosis (PCA). Effects of indomethacin, an inhibitor of the 3α-hydroxysteroid dehydrogenase enzyme involved in neurosteroid synthesis, on PCA rat locomotor activity and brain neurosteroid levels were also assessed. Significant increases of the neurosteroid pregnenolone (2.6-fold), allopregnanolone (1.7-fold) and THDOC (4.7-fold) were observed in brains of PCA rats. Brain levels of these neurosteroids were in the nanomolar range, sufficient to exert positive allosteric modulatory effects at the GABA-A receptor. Indomethacin (0.1–5 mg kg⁻¹) ameliorated dose-dependently the locomotor deficit of PCA rats and concomitantly normalized brain levels of allopregnanolone and THDOC. Increased brain levels of neurosteroids with positive allosteric modulatory actions at the neuronal GABA-A receptor offer a cogent explanation for the notion of 'increased GABAergic tone' in HE. Pharmacological approaches using agents that either reduce neurosteroid synthesis or modulate the neurosteroid site on GABA-A receptor could offer new therapeutic tools for the management and treatment of HE.     

Novel substrates for, and sources of, progestogens for reproduction

Steroid hormones, such as progesterone, are typically considered to be primarily secreted by the gonads (albeit adrenals can also be a source) and to exert their actions through cognate intracellular progestin receptors (PRs). Through its actions in the midbrain ventral tegmental Area (VTA), progesterone mediates appetitive (exploratory, anxiety, social approach) and consummatory (social, sexual) aspects of rodents' mating behaviour. However, progesterone and its natural metabolites ('progestogens') are produced in the midbrain VTA independent of peripheral sources and midbrain VTA of adult rodents is devoid of intracellular PRs. One approach that we have used to understand the effects of progesterone and mechanisms in the VTA for mating is to manipulate the actions of progesterone in the VTA and to examine effects on lordosis (the posture female rodents assume for mating to occur). This review focuses on the effects and mechanisms of progestogens to influence reproduction and related processes. The actions of progesterone and its 5α-reduced metabolite and neurosteroid, 5α-pregnan-3α-ol-20-one (3α,5α-THP; allopregnanolone) in the midbrain VTA to facilitate mating are described. The findings that 3α,5α-THP biosynthesis in the midbrain occurs with mating are discussed. Evidence for the actions of 3α,5α-THP in the midbrain VTA via nontraditional steroid targets is summarised. The broader relevance of these actions of 3α,5α-THP for aspects of reproduction, beyond lordosis, is summarised. Finally, the potential role of the pregnane xenobiotic receptor in mediating 3α,5α-THP biosynthesis in the midbrain is introduced.     

Neurosteroids: Expression of Functional 3β-Hydroxysteroid Dehydrogenase by Rat Sensory Neurons and Schwann Cells

Steroids which are synthesized within the nervous system, such as progesterone, have been termed 'neurosteroids'. Levels of progesterone are much larger in peripheral nerves of rats and mice than in plasma, and persist after removal of the steroidogenic endocrine glands. Schwann cells are a source of progesterone: when isolated from embryonic dorsal root ganglia, they can synthesize progesterone from pregnenolone, the obligate precursor of all steroids. Locally produced progesterone has been shown to play an important role in myelination of peripheral nerve. We show here that sensory neurons from embryonic dorsal root ganglia also express 3β-hydroxysteroid dehydrogenase and can convert [³H]pregnenolone to [³H]progesterone. Moreover, when cultured under different conditions and incubated for 24 h in the presence of 100 nM [³H]pregnenolone, they produce 5–10 times more [³H]progesterone than Schwann cells. The conversion of pregnenolone to progesterone by neurons is further increased by a diffusible factor produced by Schwann cells. Sensory neurons can also metabolize progesterone to 5α-dihydroprogesterone, but unlike Schwann cells, they do not produce 3α,5α-tetrahydroprogesterone, a potent positive allosteric modulator of γ-aminobutyric acid type A receptors. We also show that cells isolated from the adult nervous system still have the capacity to convert [³H]pregnenolone to progesterone and its 5α-reduced metabolites: neurons and Schwann cells purified from dorsal root ganglia of 6 week old male rats show a similar pattern of pregnenolone metabolism to cells isolated from 18 day old embryos. These findings further support the important role of progesterone in the development and regeneration of the peripheral nervous system.     

Differentiation-dependent progesterone synthesis and metabolism in NT2-N human neurons

Human embryonic teratocarcinoma-derived Ntera2/cl.D1 (NT2) cells recapitulate many features of embryonic neuronal progenitor cells. Upon retinoic acid (RA) treatment they terminally differentiate into post-mitotic neuron-like cells (NT2-N), akin to human fetal neurons, thus representing an in vitro model of human neuron terminal differentiation. Experimental evidence also indicate NT2-N cultures as a potential source for cell transplantation therapy. The neurosteroids progesterone and its metabolite 3α-hydroxy-5α-pregnan-20-one (3α,5α-THP) promote neurogenesis and show anti-neurodegenerative properties. This study's aim was to assess the neurosteroidogenic competence of NT2 cells during RA-induced neuronal differentiation. Radioimmunoassay measurements revealed progesterone only in NT2-N cultures (4 week RA). Accordingly, progesterone synthesis from ³H-pregnenolone was absent in NT2 cells and increased during RA exposure, being highest in NT2-N. [³H]-pregnenolone metabolism, yielding [³H]-progesterone and [³H]-5α-dihydroprogesterone ([³H]-5α-DHP), was time-dependent and inhibited by trilostane, a 3β-hydroxysteroid-dehydrogenase (3β-HSD) inhibitor. Conversely, ³H-progesterone metabolism, which yielded [³H]-5α-DHP > [³H]-3β,5α-THP > [³H]-3α,5α-THP, occurred at all time points examined, though showing a nadir in cultures treated with RA for 1 and 2 weeks. The differentiation-dependent increase of progesterone accumulation matched 3β-HSD type I mRNA expression and 3β-HSD immunoreactivity, that co-localized with Map2a/b- and GAD67 in NT2-N. Hence, in vitro differentiated human neurons, while retaining progesterone metabolic activity, also become competent in progesterone synthesis. These findings suggest an autocrine/paracrine role of neuronal progesterone, either on its own or through its 5α-reduced metabolites, in fetal brain development and allow speculation that NT2-N-produced neurosteroids may contribute to the encouraging results of NT2-N transplants in animal models of neurodegenerative diseases.     

3α,5α-THP in the raphe magnus attenuates PTZ-induced myoclonic seizures

Whether progesterone (P₄) and its metabolite, 5α-pregnan-3α-ol-20-one (3α,5α-THP) have anti-seizure effects through actions in the raphe magnus (NRM) was investigated. Ovariectomized, Long–Evans rats with unilateral implants into the NRM of P₄ or its metabolite, 5α-pregnan-3α-ol-20-one (3α,5α-THP) had a significantly lower incidence of myoclonic seizures and less EEG activity following pentylenetetrazol (PTZ; 70 mg/kg, IP) administration than did rats with control implants. Progestin implants that missed the NRM were not effective at reducing ictal activity. Following P₄ implants to the NRM levels of P₄, and following P₄ and 3α,5α-THP implants to the NRM, 3α,5α-THP levels in the ventral hindbrain were increased above those seen in rats with control implants. These data suggest that progestins’ anti-seizure effects in the NRM may be involve actions of 3α,5α-THP.     

Dehydroepiandrosterone sulphate improves cholestasis-associated fatigue in bile duct ligated rats

Abstract:  Fatigue is a common debilitating symptom in patients with primary biliary cirrhosis (PBC). The mechanism of fatigue is still poorly understood. However, it has been reported that levels of the steroid dehydroepiandrosterone sulphate (DHEAS) are reduced in plasma of patients with PBC, and substitutive therapy has been suggested to improve fatigue symptoms experienced during the course of this disease. In this study, we tested the effect of DHEAS on whole body fatigue in rats following bile duct ligation (BDL). Fatigue was estimated by the time spent on an electrified grid as a result of falling off a treadmill and by performance of rats on an infrared beam monitor which allows the assessment of travelled distance and stereotypic movement activities. On day 5 after BDL surgery, cholestatic rats exhibited increased whole body fatigue as reflected by significantly increased time spent on the electrified grid, reduced travelled distance and reduced stereotypic movements. Administration of 5 mg kg⁻¹ of DHEAS to BDL rats for three consecutive days significantly normalized their behaviour. Fatigue scores were also found to be reduced in cirrhotic rats 4 weeks after BDL surgery, and DHEAS treatment for 3 days reduced fatigue scores at this stage. Dehydroepiandrosterone sulphate treatment was sufficient to increase brain levels of DHEAS in the BDL rats in a manner that is significantly and highly correlated with those of plasma DHEAS and brain dehydroepiandrosterone (DHEA). Substitutive therapies with DHEAS or DHEA could represent novel approaches in the management of fatigue due to cholestasis-induced liver failure.     

Post-polio syndrome patients treated with intravenous immunoglobulin: a double-blinded randomized controlled pilot study

Post-polio syndrome (PPS) is characterized by new muscle weakness, atrophy, fatigue and pain developing several years after the acute polio. Some studies suggest an ongoing inflammation in the spinal cord in these patients. From this perspective, intravenous immunoglobulin (IvIg) could be a therapeutic option. We performed a double-blinded randomized controlled pilot study with 20 patients to investigate the possible clinical effects of IvIg in PPS. Twenty patients were randomized to either IvIg 2 g/kg body weight or placebo. Primary endpoints were changes in pain, fatigue and muscle strength 3 months after treatment. Surrogate endpoints were changes in cerebrospinal fluid (CSF) cytokine levels. Secondary endpoints were pain, fatigue and isometric muscle strength after 6 months. Patients receiving IvIg reported a significant improvement in pain during the first 3 months, but no change was noted for subjective fatigue and muscle strength. CSF levels of tumour necrosis factor- α (TNF- α ) were increased compared with patients with non-inflammatory neurological disorders. In conclusion, in this small pilot study no effect was seen with IvIg treatment on muscle strength and fatigue, however IvIg treated PPS patients reported significantly less pain 3 months after treatment. TNF- α was increased in the CSF from PPS patients. The results are promising, but not conclusive because of the low number of patients studied.     

Exploratory, anti-anxiety, social, and sexual behaviors of rats in behavioral estrus is attenuated with inhibition of 3alpha,5alpha-THP formation in the midbrain ventral tegmental area

The progesterone (P₄) metabolite and neurosteroid, 5α-pregnan-3α-ol-20-one (3α,5α-THP) acts in the midbrain ventral tegmental area (VTA) to modulate lordosis of female rats. 3α,5α-THP also mediates exploratory, affective, and social behaviors; whether actions of 3α,5α-THP in the VTA mediate these behaviors is of interest. To elucidate the role of the VTA in mediating exploratory, affective, and social behaviors, the present study examined effects of inhibiting 3α,5α-THP formation in the VTA. Rats received intra-VTA infusions of either PK11195 (400 ng/μl, which inhibits de novo 3α,5α-THP production), indomethacin (10 μg/μl, which blocks metabolism of P₄ to 3α,5α-THP), PK11195 and indomethacin together, or β-cyclodextrin vehicle and tested on a battery of anxiety (open field and elevated plus maze), social (partner preference and social interaction), and sexual (paced mating) tasks. Compared to rats infused with vehicle to the VTA, rats infused with inhibitor(s) demonstrated significant reductions in central entries in the open field, time on open arms of an elevated plus maze, time spent interacting with a conspecific, initiation and intensity of lordosis, sexual solicitations, and midbrain 3α,5α-THP levels. These findings suggest that actions of 3α,5α-THP in the VTA are important for mediating aspects of exploration, anxiety, and social behavior related to mating.     

Finasteride Blocks the Reduction in Ictal Activity Produced by Exogenous Estrous Cyclicity

The purpose of the present study was to examine seizure activity during reduced 5α-pregnan-3α-ol-20-one (3α,5α-THP) production. Ovariectomized Long-Evans rats were stereotaxically implanted with bipolar electrodes above the perforant pathway; silastic implants filled with estradiol-17-benzoate (EB) and progesterone were inserted subcutaneously to mimic diestrus. Estrus was then induced in half of these animals by injection of EB (30 μg) and progesterone (2.5 mg), 48 and 4 h, respectively, prior to perforant pathway stimulation. Half of the estrous and diestrous rats also received a 5α-reductase inhibitor, finasteride (50 mg/kg), 6 h prior to perforant pathway stimulation. The estrous condition was associated with reduced number and duration of partial seizures, improved performance on a Morris water maze recovery of function test, reduced neuronal loss in the hilar region of the hippocampus, and elevated central and plasma 3α,5α-THP, compared to estrus+finasteride, diestrus+vehicle and diestrus+finasteride conditions, which did not differ from each another. These data suggest antiseizure effects of estrus may be caused, in part, by the action of 3α,5α-THP and that the precipitous decline in 3α,5α-THP may restore seizure threshold to control levels.     

Type 1 5α-reductase may be required for estrous cycle changes in affective behaviors of female mice

There are estrous cycle differences in affective behaviors of rodents that are generally attributed to cyclic variations in estradiol, progesterone (P) and its metabolites. A question is the role of the steroid metabolism enzyme, 5α-reductase, for these estrous cycle differences. To address the requirement of 5α-reductase, estrous cycle variations in the behavior of wildtype mice and their littermates that are deficient in the 5α-reductase type 1 enzyme (5αRKO mice) were examined. The hypothesis was that if some of the estrous cycle differences in exploratory (open field) and anxiety (elevated plus maze) are due to P's 5α-reduction to 5α-pregnan-3α-ol-20-one (3α,5α-THP), then wildtype mice will have estrous cycle differences in the expression of these behaviors, but 5αRKO mice will not. Mice were tested in these tasks and then had plasma and brains collected so that steroid levels (estradiol, P, 3α,5α-THP, corticosterone) could be measured in these tissues. Results supported this hypothesis. There were estrous cycle differences among wildtype, but not 5αRKO, mice. Proestrous wildtype mice made more central entries in the open field and spent more time on the open arms of the plus maze, coincident with higher 3α,5α-THP levels in plasma and brain regions important for these behaviors, such as the hippocampus and cortex, compared to their diestrous counterparts. Variability in the open field and elevated plus maze could be explained by circulating and hippocampus levels of 3α,5α-THP, respectively. Thus, 5α-reductase may be required for the estrous cycle variations in affective behavior and 3α,5α-THP levels of female mice.     

Subunit- and brain region-specific reduction of GABAA receptor subunit mRNAs during chronic treatment of rats with diazepam

The mRNA levels for several GABA_A receptor subunits were measured by Northern blot analysis. Rats were treated for 3 wk by continuous release of diazepam (DZP) from subcutaneous reservoirs, and then sacrificed immediately or 48 h after removing the reservoirs. Poly(A)⁺ RNAs, isolated from cerebral cortex, cerebellum, and hippocampus, were hybridized with oligonucleotide probes for GABA_A receptor subunits and a cDNA probe for β-actin. Subunit mRNAs were expressed relative to the corresponding β-actin mRNA. DZP treatment decreased the α₁ subunit mRNA level 40% in hippocampus, but it was not changed in cortex or cerebellum. The α₅ subunit mRNA level was decreased in cerebral cortex (28%) and hippocampus (15%). The γ₂ subunit mRNA level was decreased (40%) only in cortex. DZP treatment did not affect α₂, α₃, α₄, β₂, or β₃ subunit mRNA levels. Decreases in mRNA levels had reversed within 48 h after stopping chronic treatment. Acute DZP did not change α₁, α₅, or γ₂ subunit mRNA levels. The decreases in GABA_A receptor subunit mRNA levels were specific to subunit and brain region. These results, coupled with those after chronic flurazepam treatment, also indicated that the effects on GABA_A receptor subunit mRNA levels are specific to the benzodiazepine (BZ) used for chronic treatment.     

Inhibition of 5α-reductase activity in late pregnancy decreases gestational length and fecundity and impairs object memory and central progestogen milieu of juvenile rat offspring

Psychological, physical and/or immune stressors during pregnancy are associated with negative birth outcomes, such as preterm birth and developmental abnormalities. In rodents, prenatal stressors can alter the expression of 5α-reductase enzymes in the brain and may influence cognitive function and anxiety-type behaviour in the offspring. Progesterone plays a critical role in maintaining gestation. In the present study, it was hypothesised that 5α-reduced progesterone metabolites influence birth outcomes and/or the cognitive and neuroendocrine function of the offspring. 5α-Reduced steroids were manipulated in pregnant Long-Evans rats via the administration of vehicle, the 5α-reduced, neuroactive metabolite of progesterone, 5α-pregnan-3α-ol-20-one (3α,5α-THP, allopregnanolone; 10 mg/kg/ml, s.c.), or the 5α-reductase inhibitor, finasteride (50 mg/kg/ml, s.c.), daily from gestational days 17-21. Compared to vehicle or 3α,5α-THP treatment, finasteride, significantly reduced the length of gestation and the number of pups per litter found in the dams' nests after parturition. The behaviour of the offspring in hippocampus-dependent tasks (i.e. object recognition, open field) was examined on post-natal days 28-30. Compared to vehicle-exposed controls, prenatal 3α,5α-THP treatment significantly increased motor behaviour in females compared to males, decreased progesterone content in the medial prefrontal cortex (mPFC) and diencephalon, increased 3α,5α-THP and 17β-oestradiol content in the hippocampus, mPFC and diencephalon, and significantly increased serum corticosterone concentrations in males and females. Prenatal finasteride treatment significantly reduced object recognition, decreased hippocampal 3α,5α-THP content, increased progesterone concentration in the mPFC and diencephalon, and increased serum corticosterone concentration in female (but not male) juvenile offspring, compared to vehicle-exposed controls. Thus, inhibiting the formation of 5α-reduced steroids during late gestation in rats reduces gestational length, the number of viable pups per litter, and impairs cognitive and neuroendocrine function in the juvenile offspring.     

Infusion of 3α,5α-THP to the pontine reticular formation attenuates PTZ-induced seizures

Whether progesterone (P₄) and its metabolite, 5α-pregnan-3α-ol-20-one (3α,5α-THP) have anti-seizure effects through actions in the pontine reticular formation (PRF) was investigated. Concentrations of P₄ and 3α,5α-THP in the PRF were greater in proestrous and hormone-primed rats, that are typically more resistant to seizure-induction, than diestrous and males rats. Ovx, Long-Evans rats with unilateral microinjections into the PRF of 3α,5α-THP (5 μg/0.2 μl), but not P₄ (11 μg/0.2 μl) or vehicle (β-cyclodextrin), had a greater latency and lower incidence of tonic-clonic seizures induced by pentylenetetrazol (PTZ; 70 mg/kg, IP) administration. Infusions that missed the PRF were not effective. These data suggest 3α,5α-THP has anti-seizure effects in part through actions in the PRF.     

The neurosteroids, progesterone and 3α,5α-THP, enhance sexual motivation, receptivity, and proceptivity in female rats

The effects of progesterone (P) and the neurosteroid and P metabolite, 3α-hydroxy-5α-pregnan-20-one (3α,5α-THP) on ovariectomized (ovx), estradiol-3-benzoate (EB)-primed rats on sexual motivation, receptivity, and proceptivity were examined. Changes in central P and 3α,5α-THP were measured following administration of EB, EB+P, EB+3α,5α-THP, or EB+inhibitor of 5α-reductase or P metabolism (epostane and finasteride)+P (Expt. 1). Partner preference was measured as the duration of time females in these different hormonal treatments spent in proximity to a male vs. female conspecific (Expt. 2). Receptivity (lordosis quotients and ratings) and proceptivity (darting, hopping, ear wiggling, and pacing), for different hormone treatments were assessed (Expt. 3 and Expt. 4, respectively). Conditioned place preference following hormone treatments and paced mating enabled assessment of sexual motivation (Expt. 5). Central P and 3α,5α-THP were measured in various combinations of hormone/mating conditions (Expt. 6). Studies revealed that 3α,5α-THP has a significant role in these reproductive measures. Brain concentrations of 3α,5α-THP were significantly higher in animals receiving EB+P or EB+3α,5α-THP compared to animals receiving EB alone, or EB+P in conjunction with an inhibitor of P metabolism. EB+P and EB+3α,5α-THP significantly increased time spent in proximity to the male, receptivity and proceptivity. When administered to ovx, EB-primed rats, the progestin metabolite, 3α,5α-THP, had effects on these behaviors similar to P. Epostane, an inhibitor of P and 3α,5α-THP biosynthesis, and finasteride, an inhibitor of P metabolism to 3α,5α-THP, administered to EB+P animals reduced male partner preference, proceptive, and receptive behaviors to levels seen in EB+vehicle animals. Notably, whole brain 3α,5α-THP levels were significantly increased and whole brain P levels were significantly reduced in paced mated rats compared to standard mated, and receptive non-mated animals. These studies suggest that P and 3α,5α-THP may have some common effects on reproductive behavior, e.g., sexual motivation, receptivity, and proceptivity.     

Valproate Metabolites in High-Dose Valproate Plus Phenytoin Therapy

Summary: Purpose: We wished to determine the relation between liver function, β-, and ω-, and ω-1-oxidation metabolites and 4-en-valproate (VPA).
Methods: We measured the serum levels of VPA and its metabolites in children and adolescent receiving high-dose VPA plus phenytoin (PHT) therapy using gas chromatography-mass spectrometry with selected ion monitoring (GC/MS/SIM).
Results: In high-dose VPA plus PHT polytherapy, the total VPA serum concentration was distinctly low, the concentrations of total β -oxidation metabolites were decreased, the percentage values of VPA (percent of VPA) of total β -oxidation metabolites were increased, and the E-2-en-VPM3-keto-VPA ratios were decreased, as compared with those in high-dose VPA monotherapy. In high-dose VPA plus PHT polytherapy, 4-en-VPA (μ M ) was decreased and the concentrations of (ω+ (ω - 1)]-oxidation metabolites (μ M) were decreased as compared with those in high-dose VPA monotherapy. In high-dose VPA plus PHT, serum glutamic-oxaloacetic transarninase (GOT), glutarnic-pyruvic transaminase (GPT) and lactic dehy-drogenase (LDH) did not correlate significantly with the (β/ω+ (ω - 1) metabolites ratio and 4-en-VPA levels, but serum GOT, GPT, and LDH were increased as compared with those in high-dose VPA therapy. We were not able to establish a significant relation between the formation of metabolites of VPA metabolites and liver dysfunction in patients receiving high-dose VPA and PHT concurrently.
Conclusions: Metabolic levels do not appear to be a reliable predictor of hepatotoxicity in children receiving pharrnacological antiepileptic drug (AED) therapy.     

Pharmacogenetics of low dose clonidine in irritable bowel syndrome

Abstract  Adrenergic and serotonergic (ADR-SER) mechanisms alter gut (gastrointestinal, GI) sensorimotor functions. We aimed to determine whether candidate ADR-SER genes affect GI responses to low dose clonidine (CLO) in humans. Forty healthy and 120 irritable bowel syndrome (IBS) participants received CLO, 0.1 mg or 0.15 mg b.i.d., for 6 days. At baseline and post-CLO, we measured: gastric volume (GV); satiation volume; rectal compliance, sensation thresholds and ratings with distensions. Genetic variations tested were: α2A (C-1291G), α2C (Del 322-325), GNβ3 (C825T) and solute carrier family 6 ( neurotransmitter transporter, serotonin ) , member 4 ( SLC6A4 ) (serotonin transporter linked polymorphic region). CLO reduced volume to satiation ( P  = 0.002), postprandial GV ( P  < 0.001), sensation threshold for pain (<0.001); CLO increased rectal compliance ( P  = 0.024). There were significant associations between post-CLO responses and gene variations for ΔGV ( α2A and SLC6A4 ), rectal sensation of gas ( α2A , GNβ3 ), urgency ( α2A ); and pain ( GNβ3 and SLC6A4 ); and rectal compliance ( SLC6A4 ). α2A , GNβ3 and SLC6A4 genotypes significantly modify responses to CLO on sensory and motor GI functions in health and IBS.     

The Ability of Oestradiol Administration to Regulate Protein Levels of Oestrogen Receptor Alpha in the Hippocampus and Prefrontal Cortex of Middle-Aged Rats is Altered Following Long-Term Ovarian Hormone Deprivation

Beneficial effects of oestrogen administration on cognition are attenuated if treatment is initiated following long-term ovarian hormone deprivation. The mechanisms underlying this attenuation are unknown. The present study aimed to assess the effects of long-term ovarian hormone deprivation on the ability of subsequent oestradiol treatment to regulate oestrogen receptor (ER) α and ERβ, and steroid receptor coactivator (SRC)-1 in the hippocampus and prefrontal cortex of middle-aged rats. In an initial experiment to assess oestradiol regulation of these proteins, 2-month-old rats were ovariectomised and immediately implanted with capsules containing cholesterol or oestradiol. Brains were collected 10 days later. In a second experiment, middle-aged (10-month-old) rats were ovariectomised or underwent sham surgeries. Five months later, sham-operated rats were ovariectomised and received oestradiol implants. Previously ovariectomised rats underwent sham surgeries and received oestradiol or cholesterol implants. Protein levels of ERα, ERβ, and SRC-1 were measured following 10 days of oestradiol treatment using western blotting. In young animals, oestradiol treatment significantly increased ERα in the hippocampus and prefrontal cortex relative to control treatment. In middle-aged animals, immediate oestradiol treatment significantly increased ERα in hippocampus, but not the prefrontal cortex. However, delayed oestradiol treatment failed to significantly increase ERα protein levels in hippocampus, but did so in prefrontal cortex. Levels of ERβ and SRC-1 were unaffected by oestradiol treatment in either brain area in either of the age groups. These data indicate that prolonged ovarian hormone deprivation alters the ability of subsequent oestradiol replacement to regulate ERα protein levels in brain areas important for cognition.