Decreased expression of retinoid nuclear receptor (RAR alpha and RAR gamma) mRNA determined by real-time quantitative RT-PCR in peripheral blood mononuclear cells of hypothyroid patients.

In vivo assessment of the cellular impact of thyroid hormones on target tissues might be of help for physiological studies and to evaluate the consequences of various diseases of the thyroid gland in humans. Given the tenuous relationship between retinoid and tri-iodothyronine (T3) status and that retinoids have also intracellular roles, the aim of this study was to determine the effect of hypothyroidism on the expression of T3 nuclear receptors (TR) and retinoic acid nuclear receptors (RAR, RXR) in human peripheral blood mononuclear cells (PBMC). Using real time RT-PCR, we quantified the relative amount of mRNA of the thyroid (TR alpha and TR beta) and retinoid (RAR alpha, RAR gamma, and RXR alpha) nuclear receptors in PBMC of euthyroid (n = 22) compared with hypothyroid (n = 22) subjects. Classical plasma parameters (free T3 (FT3), free thyroxine (T4) (FT4), thyroid-stimulating hormone (TSH), retinol (ROH), retinol-binding protein (RBP) and transthyretin (TTR)) were also measured. In hypothyroid subjects, the concentration of TSH was elevated, and dramatically low T3 and T4 concentrations were associated with a decrease in the expression of TR beta. Expression of RAR alpha and RAR gamma significantly decreased in hypothyroid versus control subjects, while an increased concentration of ROH was emphasised by hypothyroidism. These results first indicated that primary hypothyroidism induces hypoactivation of the retinoid nuclear pathway in PBMC, which was not predicted by the plasma ROH level. Further investigations will be necessary to evaluate these parameters in very small changes in thyroid hormone production such as mild (subclinical) hypothyroidism.

Aging affects the retinoic acid and the triiodothyronine nuclear receptor mRNA expression in human peripheral blood mononuclear cells.

BACKGROUND: Inadequate retinoid status has often been described as occurring with aging. Moreover, subclinical hypothyroid status has also been evoked in the elderly. Several studies performed in animals have described the crucial incidence of age-related hypo-functioning of retinoid and thyroid signalling pathways, particularly in the brain. OBJECTIVE: The aim of the present study was to clarify whether aging modifies retinoid and thyroid signalling in humans. METHODS: Using real-time RT-PCR the relative amount of mRNA of the retinoid (RARalpha, RARgamma and RXRalpha) and thyroid (TRalpha and TRbeta) nuclear receptors in peripheral blood mononuclear cells (PBMC) of young (24-57 years old, n = 22) compared with elderly (69-90 years old, n = 24) healthy subjects was quantitated. Classical plasma parameters used to characterize the retinoid and thyroid status - retinol (ROH), retinol-binding protein (RBP), free triiodothyronine (FT3) and thyroxine (FT4), thyroid-stimulating hormone (TSH) and transthyretin (TTR) - were also assessed. RESULTS: RARgamma expression was significantly decreased in elderly versus young subjects while no modification of the retinoid-related plasma parameters ROH and RBP were emphasized by aging. Concerning thyroid criteria, the elderly exhibited an increase in TSH concentration (+39%) without significant modifications of FT3 and FT4, which indicated an age-related sub-clinical hypothyroidism. Concurrently, the amount of TR mRNA (alpha as well as beta subtypes) was significantly decreased in the elderly. CONCLUSION: These data constitute the first evidence of an age-related hypo-activation of the retinoid and thyroid nuclear pathways in PBMC. Further study of the possible association between the expression of the retinoid and thyroid nuclear receptors and age-related cognitive alterations in humans would be interesting.

Chronic ethanol administration enhances retinoic acid and triiodothyronine receptor expression in mouse liver.

Chronic alcoholism induces perturbations of storage and metabolization of retinol and related compounds. After 6 months of ethanol consumption we have observed in mouse liver an increased expression of Tri-iodothyronine receptors (TR) while the expression of retinoic acid (RA) receptors (RAR) was unaffected. After 10 months of alcoholization the TR expression was strongly increased and the RAR expression was also increased. At this time the activity of aldehyde dehydrogenase and that of alcohol dehydrogenase, two enzymes involved in biosynthesis of RA from retinol, were similar in the liver of alcoholized and pair-fed mice. Thus it can be hypothesized that (i) the change of RAR expression was, at least in part, the result of a change of TR expression (result in agreement with previous data), (ii) the increased expression of RAR could induce apoptosis and subsequently liver necrosis.

Dexamethasone decreases the expression of retinoic acid receptors (RARs) in rat liver.

Although adrenalectomy was without effect on the expression of retinoic acid (RA) receptors (RARs), administration of the glucocorticoid analog dexamethasone (Dex) to both control and adrenalectomized rats reduced the expression of these receptors in rat liver. This effect can be mainly attributed to the action of Dex on 4-hydroxylation of RA. Dex, by enhancing 4-hydroxylation of RA, reduces its intracellular concentration thereby leading to a decreased expression of RARs, since RARbeta, the main type of RARs in liver, are known to be up-regulated by RA.

Chronic ethanol consumption increases the amount of mRNA for retinoic acid and triiodothyronine receptors in mouse brain.

It is known that alcohol induces disorders in the metabolism of retinoids and particularly in the biosynthetic pathways of retinoic acid (RA). Since RA has, along with other hormones and particularly triiodothyronine (T3), a physiological role in the adult brain, the effect of chronic exposure to alcohol on RA and T3 status was investigated. The amounts of RA receptor (RAR) and T3 receptor (TR) mRNAs were quantified and the activity of the 'tissue' transglutaminase (tTG; an RA-dependent enzyme) was assayed in the brain of mice following chronic ethanol consumption (CEC; 12% v/v for 6-10 months). Compared to controls, ethanol-treated mice exhibited increased amounts of RAR and TR mRNAs together with an increase in tTG activity. It is hypothesized that the enhanced cellular action of RA and T3 could play a role in the previously described brain damages induced by CEC.

Assessment of vitamin and trace element supplementation in severely burned patients undergoing long-term parenteral and enteral nutrition.

The efficacy of an oral supplement of vitamins and trace elements during a longterm artificial parenteral and enteral nutrition was investigated for 3 months in patients with extensive burns. Thirty severely burned patients (22 male, 8 female, age 41 +/- 18 years, range 23-59 years, 33 +/- 12% total body surface area burn, 22% +/- 8 full thickness burn surface area) were included. Every 10 days, from day 10 until day 90, we determined serum levels of: *vitamins B1, B12, A, E, *folic acid, *copper, zinc, iron, *transferrin, albumin, prealbumin, total proteins, *fibronectin, retinol binding protein (RBP), *calcium, *phosphorus, *triglycerides, *total cholesterol, *C reactive protein (CRP), *erythrocyte folic acid. The mean daily nutritional support was 60 Kcals and 0.4 g N per kg of body weight, 70% enterally and 30% parenterally administered, with enteral vitamin and trace element supplementation. On day 10, there was a decrease of the serum level of 19/20 parameters. For 8 parameters (vitamin A, total cholesterol, iron, transferrin, fibronectin, phosphorus, RBP, total proteins), the level was lower than usual. Between day 10 and day 20, a significant normalization of 6 of them was noted, the average levels of transferrin and iron remaining below normal values until day 50. There was a significant decrease in C-reactive protein levels, however above normal limits. No deficiency in vitamins or trace elements was found. Cyclic variations of serum levels occurred which may be more related to volemic, hydroelectrolytic, endocrine and inflammatory disorders than to nutritional problems.

Effects of a synthetic retinoid, etretinate (RO 10-9359), on the peripheral metabolism of thyroid hormones in rats.

Male Wistar rats were administered a synthetic vitamin A compound, etretinate, at various doses, including levels exceeding recommended therapeutic doses. At 2 mg/kg body weight/day given intragastrically, no effects were detected with respect to serum retinol or thyroid hormones after 15 days of treatment. At 5 mg/kg/day only a slight decrease in serum triiodothyronine was detected. In contrast, the dose of 20 mg/kg/day for 15 days resulted in relatively severe effects: body weight was decreased by 23% in comparison with the controls, serum retinol was decreased by 62% (while liver stores of vitamin A were unaffected), and serum thyroxine and triiodothyronine levels were, respectively, 24 and 29% lower than in the controls. A kinetic study demonstrated that the biological half-life of serum thyroxine decreased by 62% and that of serum triiodothyronine by 24%, suggesting increased peripheral metabolism of the thyroid hormones. The two main metabolic pathways (i.e. hepatic 5'-monodeiodination and glucuronide conjugation) were not significantly affected by etretinate treatment and do not account for the apparent metabolic loss of the thyroid hormones. This experiment demonstrates that etretinate doses beyond the therapeutic range are required to imbalance thyroid hormone homoeostasis. Possible mechanisms relating to increased hormonal clearance in rats treated with high doses of etretinate are discussed.

[Optimization of a spectrophotometry assay of total and oxidized blood glutathione: comparison with a fluorimetric method]. / Optimisation du dosage spectrophotométrique du glutathion sanguin total et oxydé: comparaison avec une méthode fluorimétrique.

We developed a method for the enzymatic assay of glutathione which is easy to practice, rapid, specific, based on the reaction of the thiol group of glutathione with dithiobis-nitrobenzoic acid after the action of glutathione reductase in the presence of NADPH. This spectrophotometric technique allowed, on the one hand, the determination of total glutathione and on the other hand, that of oxidized glutathione (disulfide), after the blockage of reduced glutathione by 2-vinyl-pyridine. The improvements of the assay of blood glutathione concerned the sample preparation, the reaction sensitivity, thanks to a better definition of the optimal pH and a reduction ot the blockage time by 2-vinyl-pyridine in well defined operating conditions. We compared the performances of our technique with a fluorimetric method. We used our method for the determination of total and oxidized blood glutathione in a control population.

Efferent-mediated protection of the cochlear base from acoustic overexposure by low doses of lithium.

Many studies on anaesthetized animals and a few on awake animals have suggested that the cholinergic olivocochlear efferent feedback to outer hair cells can participate in the protection of the cochlea from acoustic overexposure. Lithium is known to stimulate acetylcholine synthesis and release in the brain and it is likely to act similarly at the level of the cochlear efferent synapses. We demonstrate here that, in the awake guinea-pig with a chronically implanted electrode on the round window of the cochlea, the temporary threshold shift induced by 1 minute exposure to different pure tones at around 90 dB sound pressure level (SPL) was reduced by as much as 40 dB, when exposure occurred after lithium treatment. The protection effect was not observed in anaesthetized animals. The effect was seen across the test frequency range of 6.4-12.5 kHz, suggesting that both 'fast' and 'slow' efferent effects are likely to be mediated by acetylcholine. Together our results provide new evidence that the olivocochlear efferents can provide a more efficient protection from acoustic overexposure when animals are awake.

Changes in glutathione status and 3,5,3'-triiodothyronine action in livers of rats given cysteine-deficient diets.

1. For a period of 32 d young rats were given a diet containing (g/kg) 220 casein, 120 casein + 1.93 L-cysteine (Cys), or 120 casein. 2. The formation of 3,5,3'-triiodothyronine (T3)-nuclear protein complexes was reduced in rats fed on the Cys-deficient diet. 3. Scatchard analysis showed that decreased formation of T3-nuclear protein complexes was due to a decreased affinity of T3 receptors; this decrease was induced, at least in part, by a reduced glutathione content. 4. In rats fed on the Cys-deficient diet there was an expected decrease in growth but an unexpected increase in the activities of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and malate dehydrogenase (oxaloacetate-decarboxylating) (NADP+) (EC 1.1.1.40). It is suggested that this increase is related to an increased oxidized glutathione:reduced glutathione ratio.

Vitamin A Deficiency Decreases the Expression of RARß and RXRß/γ in Adult Mouse Brain: Effect of RA Administration.

Recent studies have revealed a novel and unexpected role of vitamin A, via its nuclear receptors, in higher cognitive functions. We examined, in mouse brain, the impact of vitamin A status on the level of retinoic acid nuclear receptor (RAR and RXR) expression and on one of their responsive genes, that of the tissue transglutaminase (tTG). Weanling male C57B1/6 mice fed a vitamin A deficient diet developed a vitamin A deficiency which was characterized, after 26 weeks, by the depletion of serum retinol, liver retinol and retinyl palmitate, and by the decreased activity of liver tTG. After 27 weeks of the diet, the vitamin A depleted mice exhibited a significantly lower amount of brain RAR ß and RXR ß/γ mRNA relative to control mice. Vitamin A deficiency also resulted in a reduced expression of tTG. When 35-week depleted mice were subsequently administrated RA for 28 days, the expression of RA nuclear receptors and tTG was significantly induced. The vitamin A deficiency mouse model and repletion provides a physiological system for monitoring the effects of vitamin A status on gene expression and on neurobiological processes in the adult brain.

Relative contribution of cysteine and methionine to glutathione content and thyroid hormone levels in the rat.

1. For a period of 24 d rats were given diets containing either casein or pea (Pisum sativum) protein at two different concentrations (180 and 120 g/kg) without or with cysteine or cysteine + methionine supplementation. 2. The effects of these diets on levels of blood and liver reduced glutathione (GSH) and serum thyroid hormones were studied. 3. When compared with the 180 g casein/kg diet, the 120 g casein/kg diet decreased liver GSH and serum thyroid hormone concentrations. These changes were related to dietary cysteine supply since supplementation induced an increase in these variables. 4. When compared with 180 g pea protein/kg diet, the 120 g pea protein/kg diet decreased liver GSH and serum thyroid hormone concentrations. These changes could not be corrected by cysteine or cysteine + methionine supplementation.

Glutathione deficiency and peripheral metabolism of thyroid hormones during dietary cysteine deprivation in rats.

1. For a period of 24 d, young rats received a diet containing 120 g casein/kg or the same basic diet supplemented with 1.93 g cysteine/kg. 2. The thyroxine (T4) turnover was decreased in rats receiving the cysteine-deficient diet compared with that of rats on the supplemented diet. Moreover, the extrathyroidal T4 pool and T4 disposal rate decreased. 3. Cysteine deprivation also decreased the peripheral metabolism of 3,5,3'-triiodothyronine (T3). The T3 distribution space, extrathyroidal pool of T3 and T3 disposal rate were diminished. 4. In vitro, deiodination of T4 in liver homogenate assayed with endogenous glutathione (GSH) demonstrated decreased T3 production rates in the case of cysteine deficiency. This difference was minimized by the addition of GSH in amounts sufficient to saturate the reaction kinetics. In the light of this finding, GSH is probably involved in the promotion of certain thyroidal problems induced by a cysteine-deficient diet.

A new apheresis system using a copolymer (polyvinyl alcohol triethylene glycol diacrylate) for removal of LDL from dog whole blood.

Studies were undertaken to determine the effectiveness of a copolymer composed of PVA-TEGDA (Poly Vinyl Alcohol TriEthylene Glycol) as a plasma-cholesterol lowering procedure. For a comparative study, five dogs underwent plasmapheresis including the transfusion bag containing gel in the plasma line, while three control dogs underwent the same plasmapheresis without gel. Numerous biological determinations were performed firstly in whole blood circulation before and after treatment over 10 days, and secondly in plasma before and after LDL binding on the gel. In the whole blood circulation, the average significant depletion of cholesterol levels was 31-51% for treated, 0-16% for control dogs and the average significant depletion of LDL cholesterol was 26-75% for treated and 0-3% for control dogs. Gel was therefore able to bind 121-217 mg of total cholesterol and 34-70 mg of LDL cholesterol per gram of gel. Lipid and lipoprotein levels rebounded 3-4 days after treatment. Adverse effects were not observed during all plasmapheresis. No significant differences between control plasmapheresis and gel-plasmapheresis were obtained for blood cell counts while lengthened coagulation times were observed during 24 h. Complement was not significantly activated by the copolymer as shown by a same decreased activity in the blood stream of all dogs: in fact, CH 50 depletion in the gel incubated plasma was due to a protein adsorption on the hydrogel. This new approach for LDL apheresis appears to be a promising new technique.

Polymorphism of alcohol dehydrogenase, alcohol and aldehyde dehydrogenase activities: implication in alcoholic cirrhosis in white patients. The French Group for Research on Alcohol and Liver.

Two types of factors can theoretically modulate alcohol metabolism toward increased acetaldehyde production. These factors are the following: (a) individual, genetically determined isoenzymes with distinct catalytic properties, and (b) modifications of enzyme activity induced by alcohol itself or liver damage. To investigate the respective roles of these factors in white individuals, we studied the alcohol dehydrogenase phenotype, together with liver alcohol dehydrogenase and aldehyde dehydrogenase activities, in 161 patients. Patients with alcoholic cirrhosis (n = 31) were compared with three types of controls: patients with nonalcoholic cirrhosis (n = 25) and excessive (n = 62) and moderate drinkers (n = 43) without liver disease. No association between alcohol dehydrogenase-3 phenotype and alcoholic cirrhosis was found. The prevalence of atypical alcohol dehydrogenase in the four groups was less than 1%. Patients with cirrhosis, regardless of its cause, had significantly lower alcohol dehydrogenase activity than the patients without cirrhosis (p less than 0.05 and p less than 0.01 vs. excessive and moderate drinkers, respectively). Among the noncirrhotic patients, alcohol dehydrogenase activity was significantly lower in the excessive drinkers than in the moderate drinkers (p less than 0.001). Aldehyde dehydrogenase activity was not different between cirrhosis-free excessive and moderate drinkers; in contrast, compared with these two groups, it was significantly lower in the two cirrhosis groups (p less than 0.01). These results suggest that no phenotypic pattern of alcohol dehydrogenase-3 associated with alcoholic cirrhosis in white patients exists, that liver alcohol dehydrogenase activity falls as a consequence of both alcohol abuse and cirrhosis and that liver aldehyde dehydrogenase activity is unaffected by alcohol abuse and only falls after the onset of cirrhosis.

Dietary vitamin A modulates the properties of retinoic acid and glucocorticoid receptors in rat liver.

Properties of retinoic acid receptors and glucocorticoid receptors of rat liver were influenced by retinol status in a nonsimilar manner. The binding of the retinoic acid receptors which was lowered in vitamin A--deficient animals relative to controls was restored by a single dose (100 micrograms) of retinoic acid; in vitamin A--overloaded animals (40-fold the control intake) the binding was greater than in controls. The binding of the glucocorticoid receptor was higher in vitamin A--deficient rats than in controls and restored by retinoic acid supplementation, but did not differ from controls in the vitamin A--overloaded rats. The cellular actions of glucocorticoid hormone and retinoic acid were investigated by assaying the activity of some related enzymes. The activity of tyrosine aminotransferase reflected glucocorticoid receptor binding in vitamin A--deficient and vitamin A--restored rats. The decreased tyrosine amino transferase activity observed in vitamin A--overloaded rats could be related to the inhibition of expression of tyrosine amino transferase gene by retinoic acid. Alcohol dehydrogenase activity was unaffected or only slightly affected by vitamin A status. The known existence of glucocorticoid hormone- and retinoic acid--sensitive elements in the alcohol dehydrogenase gene could explain such observations. Furthermore, the changes in the binding of retinoic acid receptors and glucocorticoid receptors were often in opposite directions. These results provide new evidence for the mechanisms by which the amount of dietary vitamin A modulates hormonal status.

[Effects of administration of moderate doses of etretinate (RO 10-9359) on rats deficient in vitamin A]. / Effets de l'administration de doses moderées d'étrétinate (RO 10-9359) á des rats carences en vitamine A.

Etretinate (RO 10-9359) is a synthetic retinoid used instead of Vitamin A--toxic in large dose--during treatment of some skin diseases. In this work biological properties of Etretinate were studied by administration of this drug (2 mg/kg/day) in rats in a vitamin A-deficient state. Body growth, some seric parameters and some tissue enzymatic activities were studied. Results of this restitution experiment show that Etretinate corrects all parameters affected by vitamin A-deficiency testicular growth excepted. So Etretinate exhibits properties similar to that of retinoic acid.

Improvement in lipid profiles and triglyceride removal in patients on polyamide membrane hemodialysis.

Cardiovascular morbidity and mortality in hemodialyzed patients are increased due to the frequently abnormal lipid metabolism. It has been reported that this abnormal lipid metabolism could be partially corrected by some highly permeable membranes, such as polysulfone or cellulose triacetate. We investigated the influence of 4 months of dialysis with a polyamide membrane upon the course of lipid parameters in 6 patients presenting a hypertriglyceridemia > 2 mmol/l while on bicarbonate dialysis with a cellulose membrane. Lipid parameters improved after 4 months of hemodialysis with a polyamide membrane. Serum triglyceride and cholesterol levels decreased, while HDL cholesterol and HDL levels rose significantly (p < 0.05). Apolipoprotein B decreased significantly (p < 0.05). Following heparin administration, lipoprotein lipase activity improved (p < 0.02), associated with a decrease apolipoprotein C3 (p < 0.05). The fractional clearance rate of triglycerides rose significantly (p < 0.01). The use of highly permeable polyamide membranes results in a significant improvement in lipid disturbances of dialysis patients due to an increased lipoprotein lipase activity, induced perhaps by the removal of circulating inhibitors such as apolipoprotein C3.

Characterization of urinary calculi: in vitro study of "twinkling artifact" revealed by color-flow sonography.

OBJECTIVE: The "twinkling artifact" is a color-flow sonographic artifact described behind calcifications and presenting as a random color encoding in the region where shadowing would be expected on gray-scale images. Our purpose was to study the relationship between this twinkling artifact seen behind urinary stones on color-flow sonography and the morphology or biochemical composition of these urinary stones. MATERIALS AND METHODS: Forty-seven urinary stones were studied in vitro with color-flow sonography. Transmit frequency, color gain, velocity range, color filters, focal depth, and depth of field were changed during scanning. The twinkling artifact was graded 0 when absent, 1 when present but occupying a portion of acoustic shadowing, and 2 when occupying the entire acoustic shadowing. Stones were studied under a binocular magnifying glass to characterize the surface, and infrared spectrophotometry was used to determine the chemical composition. RESULTS: Calculi of calcium oxalate dihydrate and calcium phosphate always produced a grade 1 or grade 2 twinkling artifact. Absence of artifact was noted only for calcium oxalate monohydrate and urate stones. In 100% of grade 0 calcium oxalate stones, the monohydrate compound was predominant (>93%). In 100% of grade 2 calcium oxalate stones, the dihydrate compound was predominant (>75%). For calcium oxalate stones, the surface pattern was correlated with their composition. Sensitivity and specificity for absence of artifact, as indicative of calcium oxalate monohydrate, were 60% and 83%, respectively, for all stones and 56% and 100%, respectively, only for radiopaque stones. CONCLUSION: An in vitro relationship exists between the twinkling artifact and the morphology of urinary stones. Color-flow sonography could play a role in detecting dense calcium oxalate monohydrate calculi, which in turn may help predict fragmentability.

A retinoic acid receptor antagonist suppresses brain retinoic acid receptor overexpression and reverses a working memory deficit induced by chronic ethanol consumption in mice.

BACKGROUND: Chronic ethanol consumption induces disorders in the biosynthesis of retinoic acid, an active derivative of vitamin A. Recent evidence suggests that an alteration in the retinoic acid signaling pathway leads to impairments in learning and memory in adult mice. We have previously shown that chronic ethanol consumption in mice produces an increased expression of the brain retinoic acid receptor beta (RARbeta) mRNA. These results prompted us to examine whether suppressing the overexpression of retinoid receptors in alcohol-treated mice by RAR antagonist administration would reverse their cognitive impairment. METHODS: After 10 months of ethanol consumption (12% v/v in drinking water), C57BL/6 mice were submitted to a working memory task in a T-maze. Then, mice of the control and the ethanol-treated groups received an RARbeta antagonist (CD2665 0.6 mg/kg) for 22 days. The behavioral effect of CD2665 administration was evaluated on a spontaneous alternation task and the neurochemical effect was measured by quantifying the mRNA expression of RARalpha, RARbeta, retinoid X receptor (RXRbeta/gamma) and tissue transglutaminase (tTG; a retinoic acid-target gene). RESULTS: Mice submitted to ethanol treatment exhibited a progressive decrease in spontaneous alternation rates over successive trials. Moreover, these mice displayed an increased expression of brain RARbeta and RXRbeta/gamma mRNA, together with an increased level of tTG mRNA and enzymatic activity. The administration of CD2665 to alcohol-treated mice totally reversed the working memory deficit and suppressed the overexpression of brain RARbeta, RXRbeta/gamma and tTG mRNA, whereas the same treatment in control mice decreased only the RARbeta mRNA level without affecting memory performance. CONCLUSION: These data point to the potential role of the retinoid signaling pathway in memory processes and suggest that the overexpression of brain RARbeta and RXRbeta/gamma could be responsible, at least in part, for some memory impairments observed during chronic ethanol consumption.