Retinoid and carotenoid status in serum and liver among patients at high-risk for liver cancer.

BACKGROUND: Approximately 2.7 million Americans are chronically infected with hepatitis C virus (HCV). HCV patients with cirrhosis form the largest group of persons at high risk for hepatocellular carcinoma (HCC). Increased oxidative stress is regarded as a major mechanism of HCV-related liver disease progression. Deficiencies in retinoid and carotenoid antioxidants may represent a major modifiable risk factor for disease progression. This study aims to identify key predictors of serum antioxidant levels in patients with HCV, to examine the relationship between retinoid/carotenoid concentrations in serum and hepatic tissue, to quantify the association between systemic measures of oxidative stress and antioxidant status, and to examine the relationship between retinoids and stellate cell activation. METHODS: Patients undergoing liver biopsy (n = 69) provided fasting blood, fresh tissue, urine and completed a diet history questionnaire. Serum and questionnaire data from healthy volunteers (n = 11), normal liver tissue from public repositories and patients without liver disease (n = 11) were also collected. Urinary isoprostanes, serum and tissue retinoid concentrations were obtained by UHPLC-MS-MS. Immunohistochemistry for αSMA was performed on FFPE sections and subsequently quantified via digital image analysis. Associations between urinary isoprostanes, αSMA levels, and retinoids were assessed using Spearman correlation coefficients and non-parametric tests were utilized to test differences among disease severity groups. RESULTS: There was a significant inverse association between serum retinol, lycopene, and RBP4 concentrations with fibrosis stage. Serum ß-carotene and lycopene were strongly associated with their respective tissue concentrations. There was a weak downward trend of tissue retinyl palmitate with increasing fibrosis stage. Tissue retinyl palmitate was inversely and significantly correlated with hepatic αSMA expression, a marker for hepatic stellate cell activation (r = -0.31, P < 0.02). Urinary isoprostanes levels were inversely correlated with serum retinol, ß-carotene, and RBP4. CONCLUSIONS: A decrease in serum retinol, ß-carotene, and RBP4 is associated with early stage HCV. Retinoid and carotenoid levels decline as disease progresses, and our data suggest that this decline occurs early in the disease process, even before fibrosis is apparent. Measures of oxidative stress are associated with fibrosis stage and concurrent antioxidant depletion. Vitamin A loss is accompanied by stellate cell activation in hepatic tissue.

Waterborne cadmium and nickel impact oxidative stress responses and retinoid metabolism in yellow perch.

In this experiment, we studied the transcriptional and functional (enzymatic) responses of yellow perch (Perca flavescens) to metal stress, with a focus on oxidative stress and vitamin A metabolism. Juvenile yellow perch were exposed to two environmentally relevant concentrations of waterborne cadmium (Cd) and nickel (Ni) for a period of 6 weeks. Kidney Cd and Ni bioaccumulation significantly increased with increasing metal exposure. The major retinoid metabolites analyzed in liver and muscle decreased with metal exposure except at high Cd exposure where no variation was reported in liver. A decrease in free plasma dehydroretinol was also observed with metal exposure. In the liver of Cd-exposed fish, both epidermal retinol dehydrogenase 2 transcription level and corresponding enzyme activities retinyl ester hydrolase and lecithin dehydroretinyl acyl transferase increased. In contrast, muscle epidermal retinol dehydrogenase 2 transcription level decreased with Cd exposure. Among antioxidant defences, liver transcription levels of catalase, microsomal glutathione-S-transferase-3 and glucose-6-phosphate dehydrogenase were generally enhanced in Cd-exposed fish and this up-regulation was accompanied by an increase in the activities of corresponding enzymes, except for microsomal glutathione-S-transferase. No consistent pattern in antioxidant defence responses was observed between molecular and biochemical response when fish were exposed to Ni, suggesting a non-synchronous response of antioxidant defence in fish exposed to waterborne Ni. There was a general lack of consistency between muscle transcription level and enzyme activities analyzed. The overall findings from this investigation highlight the usefulness of transcriptional and biochemical endpoints in the identification of oxidative stress and vitamin A metabolism impairment biomarkers and the potential use of multi-level biological approaches when assessing environmental risk in fish.

Retinoids, carotenoids, and tocopherols in breast adipose tissue and serum of benign breast disease and breast cancer patients.

Various retinoic acid (RA) isomers (all-trans, 13-cis, 11-cis, and 9-cis) as well as retinol, carotenoids, and tocopherol concentrations were determined in both serum and breast adipose tissue of 22 benign breast disease patients and 52 breast cancer patients categorized into 4 stages by malignancy. Serum RA isomers were analyzed by a newly developed sensitive method combining a high-performance liquid chromatography and a gas chromatography-mass spectrometry, and retinol, carotenoid, and tocopherol concentrations using a high-performance liquid chromatography system. The breast cancer patients showed significantly lower serum retinol, whereas significantly higher breast adipose tissue retinol concentration than those of benign breast disease patients. Although breast cancer patients showed significantly higher serum all-trans and 13-cis RA concentrations, 11-cis RA in breast adipose tissue was significantly lower in the breast cancer patients than those of benign breast disease patients and it was associated with the stage of malignancy. The current study indicates that the retinol and RA isomers in the target tissue of breast tumor patients are not reflecting their concentrations in circulation. The mechanisms of tissue specific uptake of RA isomers and their functions warrant further studies.

Quantification of retinoid concentrations in human serum and brain tumor tissues.

Retinoic acid signaling is essential for central nervous system (CNS) differentiation and appears to be impaired in tumors. Thus far, there are no established methods to quantify relevant retinoids (all-trans-retinoic acid, 9-cis-retinoic acid, 13-cis retinoic acid, and retinol) in human brain tumors. We developed a single step extraction and quantification procedure for polar and apolar retinoids in normal tissue, lipid-rich brain tumor tissues, and serum. This quantification procedure is based on high performance liquid chromatography (HPLC) with diode-array detection (DAD) using all-trans-acitretin as an internal standard and extraction by liquid-liquid partition with ethyl acetate and borate buffer at pH 9. Recovery with this extraction procedure was higher than earlier (two-step) liquid-liquid extraction procedures based on hexane, NaOH, and HCl. The overall quantification procedure was validated according to Food and Drug Administration (FDA) guidelines and fulfilled all criteria of accuracy, precision, selectivity, recovery, and stability. The overall method accuracy varied between -5.6% and +5.4% for serum and -3.8% and +6.2% for tissues, and overall precision ranged from 3.1% to 6.9% for serum and 2.1% to 8.3% for tissues (%CV batch-to-batch). The lower limit of quantification for all compounds in tumor tissue (and serum) was 3.9 ng g(-1) (ng mL(-1)). Using this assay, photodegradation of the retinoids was evaluated and endogenous polar and apolar retinoids were quantified in sera and brain tumor tissues of patients and compared with serum and tonsil tissue concentrations of controls. It may thus serve as a suitable method for the characterization of retinoid uptake and metabolism in the respective compartments.

An LC/MS method for d8-ß-carotene and d4-retinyl esters: ß-carotene absorption and its conversion to vitamin A in humans.

The intestinal absorption and metabolism of ß-carotene is of vital importance in humans, especially in populations that obtain the majority of their vitamin A from provitamin A carotenoids. MS has provided a better understanding of the absorption of ß-carotene, the most potent provitamin A carotenoid, through the use of stable isotopes of ß-carotene. We report here an HPLC-MS method that eliminates the need for complicated sample preparation and allows us to detect and quantify newly absorbed d8-ß-carotene as well as its d4-retinyl ester metabolites in human plasma and chylomicron fractions. Both retinoids and ß-carotene were recovered in a single simple extraction that did not involve saponification, thus allowing subsequent quantitation of individual fatty acyl esters of retinol. Separation of d8-ß-carotene and its d4-retinyl ester metabolites was achieved using the same C30 reversed-phase liquid chromatography followed by mass spectrometry in selected ion monitoring and negative atmospheric pressure chemical ionization modes, respectively. Total time for the two successive runs was 30 min. This HPLC-MS method allowed us to quantify the absorption of intact d8-ß-carotene as well as its extent of conversion to d4-retinyl esters in humans after consumption of a single 5 mg dose of d8-ß-carotene.

Combined α-tocopherol and ascorbic acid protects against smoke-induced lung squamous metaplasia in ferrets.

Many epidemiological studies show the benefit of fruits and vegetables on reducing risk of lung cancer, the leading cause of cancer death in the United States. Previously, we demonstrated that cigarette smoke exposure (SM)-induced lung lesions in ferrets were prevented by a combination of low dose of ß-carotene, α-tocopherol (AT), and ascorbic acid (AA). However, the role of a combination of AT and AA alone in the protective effect on lung carcinogenesis remains to be examined. In the present study, we investigated whether the combined AT (equivalent to ∼100 mg/day in the human) and AA (equivalent to ∼210 mg/day) supplementation prevents against SM (equivalent to 1.5 packs of cigarettes/day) induced lung squamous metaplasia in ferrets. Ferrets were treated for 6 weeks in the following three groups (9 ferrets/group): (i) Control (no SM, no AT+AA), (ii) SM alone, and (iii) SM+AT+AA. Results showed that SM significantly decreased concentrations of retinoic acid, AT, and reduced form of AA, not total AA, retinol and retinyl palmitate, in the lungs of ferrets. Combined AT+AA treatment partially restored the lowered concentrations of AT, reduced AA and retinoic acid in the lungs of SM-exposed ferrets to the levels in the control group. Furthermore, the combined AT+AA supplementation prevented SM-induced squamous metaplasia [0 positive/9 total ferrets (0%) vs. 5/8 (62%); p<0.05] and cyclin D1 expression (p<0.05) in the ferret lungs, in which both were positively correlated with expression of c-Jun expression. Although there were no significant differences in lung microsomal malondialdehyde (MDA) levels among the three groups, we found a positive correlation between MDA levels and cyclin D1, as well as c-Jun expressions in the lungs of ferrets. These data indicate that the combination of antioxidant AT+AA alone exerts protective effects against SM-induced lung lesions through inhibiting cyclin D1 expression and partially restoring retinoic acid levels to normal.

Quantification of endogenous retinoids.

Numerous physiological processes require retinoids, including development, nervous system function, immune responsiveness, proliferation, differentiation, and all aspects of reproduction. Reliable retinoid quantification requires suitable handling and, in some cases, resolution of geometric isomers that have different biological activities. Here we describe procedures for reliable and accurate quantification of retinoids, including detailed descriptions for handling retinoids, preparing standard solutions, collecting samples and harvesting tissues, extracting samples, resolving isomers, and detecting with high sensitivity. Sample-specific strategies are provided for optimizing quantification. Approaches to evaluate assay performance also are provided. Retinoid assays described here for mice also are applicable to other organisms including zebrafish, rat, rabbit, and human and for cells in culture. Retinoid quantification, especially that of retinoic acid, should provide insight into many diseases, including Alzheimer's disease, type 2 diabetes, obesity, and cancer.

A DNA methyltransferase inhibitor and all-trans retinoic acid reduce oral cavity carcinogenesis induced by the carcinogen 4-nitroquinoline 1-oxide.

The transcriptional silencing of some cell cycle inhibitors and tumor suppressors, such as p16 and retinoic acid receptor beta(2), by DNA hypermethylation at CpG islands is commonly found in human oral squamous carcinoma cells. We examined the effects of the DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine (5-Aza; 0.25 mg/kg body weight), all-trans retinoic acid (RA; given at 100 microg/kg body weight and 1 mg/kg body weight), and the combination of 5-Aza and the low-dose RA on murine oral cavity carcinogenesis induced by the carcinogen 4-nitroquinoline 1-oxide (4-NQO) in a mouse model. All the drug treatments were done for 15 weeks after a 10-week 4-NQO treatment. Mice in all drug treatment groups showed decreases in the average numbers of neoplastic tongue lesions. The combination of 5-Aza and RA effectively attenuated tongue lesion severity. Although all drug treatments limited the increase in the percentage of proliferating cell nuclear antigen-positive cells and the decrease in the percentage of p16-positive cells caused by the 4-NQO treatment in mouse tongue epithelial regions without visible lesions and in the neoplastic tongue lesions, the combination of 5-Aza and RA was the most effective. Collectively, our results show that the combination of a DNA demethylating drug and RA has potential as a strategy to reduce oral cavity cancer in this 4-NQO model.

HPLC method for simultaneous determination of retinoids and tocopherols in human serum for monitoring of anticancer therapy.

A simple and rapid HPLC method requiring small volumes (250 microL) of human serum after C18 SPE sample preparation was developed using monolithic technology for simultaneous determination of all-trans-retinoic acid, 13-cis-retinoic acid, retinol, gamma- and alpha-tocopherol. The monolithic column, Chromolith Performance RP-18e (100x4.6 mm), was operated at ambient temperature. The mobile phase consisted of a mixture of acetonitrile (ACN) and 1% ammonium acetate in water (AMC) at pH 7.0. The mobile phase started at 98:2 (v/v) ACN/AMC (column pre-treatment) at a flow rate of 2 mL/min, then changed to 95:5 (v/v) ACN/AMC for 4 min at a flow rate of 1.5 mL/min and a further 3 min at a flow rate of 3.2 mL/min. Detection and identification were performed using a photodiode array detector. Retinol, 13-cis- and all-trans-retinoic acid were monitored at 325 nm. Both alpha- and gamma-tocopherol were detected at 295 nm. The total analysis time was 7.2 min. Tocol (synthesized tocopherol, not occurring in humans) was used as internal standard. The method was linear in the range of 0.125-10.00 micromol/L for all-trans-retinoic acid, 0.125-5.00 micromol/L for 13-cis-retinoic acid, 0.25-10.00 micromol/L for retinol, 0.5-50.00 micromol/L for gamma-tocopherol, and 0.5-50.00 micromol/L for alpha-tocopherol. The present method may be useful for monitoring of retinoids and tocopherols in clinical studies.

beta-Carotene promotes the development of NNK-induced small airway-derived lung adenocarcinoma.

AIM: beta-Carotene has shown cancer-preventive effects in preclinical studies while increasing lung cancer mortality in clinical trials. We have shown that beta-carotene stimulates cAMP signalling in vitro. Here, we have tested the hypothesis that beta-carotene promotes the development of pulmonary adenocarcinoma (PAC) in vivo via cAMP signalling. METHODS: PAC was induced in hamsters with the carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), followed by beta-carotene for 1.5 years. Incidence, multiplicity and size of lung tumours were recorded, and phosphorylated CREB and ERK1/2 in tumour cells were determined by Western blots. Cyclic AMP in blood cells was analysed by immunoassays, retinoids in serum and lungs by HPLC. RESULTS: beta-Carotene increased lung tumour multiplicity, lung tumour size, blood cell cAMP, serum and lung levels of retinoids and induced p-CREB and p-ERK1/2 in lung tumours. CONCLUSIONS: Our data suggest that beta-carotene promotes the development of PAC via increased cAMP signalling.

Effects of p,p'-DDE on retinoid homeostasis and sex hormones of adult male European common frogs (Rana temporaria).

Reports of declining numbers of species and individuals of amphibians in most parts of the world have caused great concern. Several causative factors have been linked to this amphibian decline, and increased environmental pollution related to pesticide use seems to be one important factor. Persistent organic pollutants may act as endocrine disrupters, and thereby exert adverse effects on development (metamorphosis, growth and sexual differentiation) in amphibians. During periodic events, such as spring snowmelt, amphibians may be exposed to acute high levels of pesticides. 1,1,1-Trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) and its metabolite 1,1-dichloro-2,2-bis(p-chlorophenyl)ethene (p,p'-DDE) is a pesticide that is still found in wetlands and soils. In order to study the effects of p,p'-DDE on susceptible amphibian endocrine systems, adult male European common frogs (Rana temporaria) were exposed to different doses of p,p'-DDE (0, 0.01, 0.1, 1 and 10 mg p,p'-DDE/kg body mass) for 14 d and sex hormone levels (testosterone and 17beta-estradiol) in plasma and retinoid concentrations (retinol and retinyl palmitate) in liver were determined. The results showed a significant variation in the liver retinol concentration at increasing doses of p,p'-DDE, suggesting that p,p'-DDE may interfere in the hepatic metabolism of retinol in adult frogs. Lack of effects on sex hormones may indicate that after arousal from hibernation males are relatively resistant to adverse reproductive effects of p,p'-DDE.

Serum retinoids and beta-carotene as predictors of hip and other fractures in elderly women.

UNLABELLED: There is debate about the possible deleterious effect of excessive vitamin A exposure on fracture risk. In this nested case control study in older women (312 cases and 934 controls), serum retinol, retinyl palmitate, and beta-carotene were not associated with fracture risk, and there was no evidence of excess risk with multivitamin or cod liver oil supplementation. INTRODUCTION: Recent studies have suggested that higher vitamin A intake may account for a component of fracture risk within the general population and that supplemental vitamin A may be harmful even within recommended limits. No studies have examined the relationship between biochemical retinol status and fracture in older women. MATERIALS AND METHODS: We examined serum retinol, retinyl palmitate, and beta-carotene as predictors of incident hip and other fractures in a large prospective study of British women over the age of 75 years (n = 2606, 312 incident osteoporotic fractures, 92 incident hip fractures; mean follow-up duration, 3.7 years). Fasting blood samples (9:00-11:00 a.m.) were collected at baseline. Using a case-control design (three controls per case), serum retinol, retinyl palmitate, and beta-carotene were assessed as univariate predictors of incident osteoporotic fracture or hip fracture. Baseline BMD at the total hip, age, 25(OH)D, serum beta Crosslaps, bone-specific alkaline phosphatase, weight, height, and smoking were considered as covariates in a multivariate model. RESULTS: Serum retinol, retinyl palmitate, and beta-carotene were not significant univariate predictors of either hip fracture or any fracture (all p > 0.05; Cox proportional hazards regression). For all osteoporotic fractures, the hazard ratio (HR) was 0.92 (95% CI, 0.81-1.05) per 1 SD increase in serum retinol. Risk of any osteoporotic fracture was slightly less in the highest quartile of serum retinol compared with the lowest quartile (HR, 0.85; 95% CI, 0.69-1.05; p = 0.132) There was a tendency for increased serum retinol to predict benefit rather than harm in terms of BMD (r = 0.09, p = 0.002). Multivitamin or cod liver oil supplementation was associated with a significantly lower risk of any fracture (HR, 0.76; 95% CI, 0.60-0.96; p = 0.021). In multivariate analysis, only age, total hip BMD, and weight were associated with fracture risk (p < 0.05). CONCLUSIONS: We found no evidence to support any skeletal harm associated with increased serum indices of retinol exposure or modest retinol supplementation in this population.

Altered retinoid metabolism in female Long-Evans and Han/Wistar rats following long-term 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-treatment.

This study investigated the effects of long-term low-dose 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure on retinoid, thyroid hormone, and vitamin D homeostasis in Long-Evans and Han/Wistar rats using a tumor promotion exposure protocol. Female rats (ten/group) were partially hepatectomized, initiated with nitrosodiethylamine (NDEA), and given TCDD once per week by sc injection for 20 weeks at calculated daily doses of 0, 1, 10, 100, or 1000 ng/kg bw/day. Groups of nonhepatectomized/uninitiated rats (five/group) were identically maintained. After 20 weeks, the rats were killed, and apolar retinoid levels were determined in the liver and kidneys. No consistent differences were seen between partially hepatectomized/initiated and nonhepatectomized/uninitiated animals with respect to apolar retinoid levels or hepatic TCDD concentration. Further analyses of polar and apolar retinoid levels in liver, plasma, and kidney, as well as free thyroxine (FT4) and vitamin D (25-OH-D(3)) concentrations were carried out in partially hepatectomized/inititated animals. In Long-Evans rats, TCDD exposure dose-dependently decreased hepatic retinyl ester concentrations at doses of 1-100 ng/kg bw/day. Likewise, hepatic all-trans-retinoic acid (all-trans-RA) concentration was decreased 39 and 54% at 10 and 100 ng/kg bw/day respectively, whereas 9-cis-4-oxo-13,14-dihydro-retinoic acid (9-cis-4-oxo-13,14-dihydro-RA), a recently discovered retinoic acid metabolite, was decreased approximately 60% in the liver at 1 ng/kg bw/day. TCDD dose-dependently increased plasma retinol and kidney retinol concentrations, whereas all-trans-RA concentration was also increased in the plasma and kidney at 10 and 100 ng/kg bw/day. Plasma 9-cis-4-oxo-13,14-dihydro-RA was decreased to below detection limits from doses of 1 ng/kg bw/day TCDD. A qualitatively similar pattern of retinoid disruption was observed in the Han/Wistar rat strain following TCDD exposure. FT4 was decreased to a similar extent in both strains, whereas 25-OH-D(3) was decreased only at 100 ng/kg bw/day in Long-Evans rats. Together these results show that TCDD disrupts both retinoid storage and metabolism of retinoic acid and retinoic acid metabolites in liver, kidney, and plasma from doses as low as 1 ng/kg bw/day. Furthermore, 9-cis-4-oxo-13,14-dihydro-RA was identified as a novel and sensitive indicator of TCDD exposure, in a resistant and sensitive rat strain, thereby extending the database of low-dose TCDD effects.

Plasma retinoid profile in bullfrogs, Rana catesbeiana, in relation to agricultural intensity of sub-watersheds in the Yamaska River drainage basin, Québec, Canada.

Amphibian populations are decreasing globally and the causes are presently unclear. Retinoids have been extensively studied in other vertebrate classes where they are associated with pleiotropic effects such as susceptibility to disease (including cancer and parasitic infections), deformities and reproduction. To investigate the hypothesis that retinoid homeostasis is influenced by agricultural activities, blood samples were collected from adult bullfrogs, Rana catesbeiana, at each of six sub-watersheds chosen to represent a gradient of agricultural intensity within the Yamaska River drainage basin. Samples of surface water were collected at each of the study sites approximately 1 month after spraying and analyzed for 53 pesticides. Male body weight was significantly different (p<0.001) between study sites with the smallest bullfrogs captured from the Rivière à la Barbue sub-watershed associated with high agricultural intensity. A significant linear regression (p<0.001; R2=0.176) was obtained between plasma retinol and body weight. Plasma retinol concentrations were significantly different between study sites (p<0.001) being lowest at both Rivière Noire and Rivière à la Barbue. More than 60% of the land area in these sub-watersheds is under intensive corn-soya cultivation and surface water contained the highest concentrations of the herbicides atrazine, deethyl-atrazine, simazine, metolachlor, dimethenamide, chlopyralide, dicamba and bentazone. Plasma 13-cis-4-oxo-retinoic acid was significantly different (p<0.001) between sub-watersheds, however this effect was apparently unrelated to agricultural intensity. Plasma retinol was negatively correlated (p=0.026; r=-0.237) with plasma 13-cis-4-oxo-retinoic acid. These results suggest that retinoid homeostasis in bullfrogs may be influenced by agricultural practices.

Involvement of serum retinoids and Leiden mutation in patients with esophageal, gastric, liver, pancreatic, and colorectal cancers in Hungary.

AIM: To analyze the serum levels of retinoids and Leiden mutation in patients with esophageal, gastric, liver, pancreatic, and colorectal cancers. METHODS: The changes in serum levels of retinoids (vitamin A, alpha- and beta-carotene, alpha- and beta-cryptoxanthin, zeaxanthin, lutein) and Leiden mutation were measured by high liquid performance chromatography (HPLC) and polymerase chain reaction (PCR) in 107 patients (70 males/37 females) with esophageal (0/8), gastric (16/5), liver (8/7), pancreatic (6/4), and colorectal (30/21 including 9 patients suffering from in situ colon cancer) cancer. Fifty-seven healthy subjects (in matched groups) for controls of serum retinoids and 600 healthy blood donors for Leiden mutation were used. RESULTS: The serum levels of vitamin A and zeaxanthin were decreased significantly in all groups of patients with gastrointestinal (GI) tumors except for vitamin A in patients with pancreatic cancer. No changes were obtained in the serum levels of alpha- and beta-carotene, alpha- and beta-cryptoxanthin, zeaxanthin, lutein in patients with GI cancer. The prevalence of Leiden mutation significantly increased in all groups of patients with GI cancer. CONCLUSION: Retinoids (as environmental factors) are decreased significantly with increased prevalence of Leiden mutation (as a genetic factor) in patients before the clinical manifestation of histologically different (planocellular and hepatocellular carcinoma, and adenocarcinoma) GI cancer.

Quantification of a novel retinoic acid metabolism inhibitor, 4-(1H-imidazol-1-yl)retinoic acid (VN/14-1RA) and other retinoids in rat plasma by liquid chromatography with diode-array detection.

A simple reversed phase high performance liquid chromatographic (HPLC) method was developed for the separation and quantification of a novel retinoic acid metabolism inhibitor, 4-(1H-imidazol-1-yl)retinoic acid (VN/14-1RA), and other retinoids in rat plasma. VN/14-1RA, alone or in combination with ATRA, is effective at inhibiting the proliferation of prostate and breast cancer cell lines in vitro. Aliquots of rat plasma were spiked with the retinoids followed by addition of acetonitrile for precipitation of plasma proteins. The decanted supernatant was evaporated under a stream of nitrogen and reconstituted in acetonitrile. Analysis was accomplished by injection of an aliquot of the reconstituted sample into an HPLC system consisting of a Zorbax Rx-C18 column and a diode array detector. A mobile phase composed of ammonium acetate (0.1 M), acetic acid solution (2% (v/v)) and methanol at a flow rate of 1.0 mL/min was used for gradient elution. The recoveries for all compounds ranged from 65 to 85% regardless of the concentrations examined. The HPLC assay was linear over the range 0.10-5.0 microg/mL (CV < 10%) with a limit of quantification of 100 ng/mL for VN/14-1RA. A one-compartment model with apparent first-order elimination was used to describe the plasma concentration-time profile for VN/14-1RA after intravenous administration. The mean terminal elimination half-life (t(1/2)) was 19.0 +/- 3.2 min. This HPLC method is useful for the analysis and evaluation of the pharmacokinetics of VN/14-1RA in rats.

Pharmacology of all-trans-retinoic acid in children with acute promyelocytic leukemia.

BACKGROUND: Due to severe side effects in virtually all children treated with a standard dose of 45 mg/m(2)/day all-trans-retinoic acid (ATRA) for acute promyelocytic leukemia (APL) the AML-BFM study group reduced the dosage to 25 mg/m(2)/day. For the lack of data on the use of ATRA at this dosage in children with APL, the study group further decided to evaluate the pharmacokinetics and metabolism of ATRA in children. PROCEDURE: Twenty-three pharmacokinetic and metabolic profiles of ATRA were studied in 14 children (aged 0.9-18.4 years) with APL. Eleven plasma samples were collected over a period of 8 hr and analyzed for ATRA and its metabolites by high-performance liquid chromatography. RESULTS: Peak plasma concentrations of ATRA were characterized by wide interpatient variability (range: 28.6-513.0 nM). Compared to adults the same metabolic pathways were observed in children. Even though peak plasma concentrations were in the lower range of those considered effective in vitro, ATRA side effects, notably neurotoxicity, still required dose reduction, treatment break, or drug withdrawal in eight patients. In this small number of patients, neurotoxicity could not be related to age or any specific level of ATRA or metabolites in the plasma. Plasma concentrations of vitamin A, however, were significantly higher in those patients, who developed signs of neurotoxicity (P = 0.03, Mann-Whitney Rank Sum test). CONCLUSIONS: Considering the low plasma concentrations and the persistence of toxicity in spite of dose reduction intermittent dosing schedules might be considered as an alternative to further dose reduction of ATRA in the treatment of APL especially in children, who might be at risk of ATRA-induced neurotoxicity.

Prospective study of carotenoids, tocopherols, and retinoid concentrations and the risk of breast cancer.

Previous prospective studies have raised the possibility that the antioxidantproperties of carotenoids and vitamin E (alpha-tocopherol) and the role of vitamin A (retinol) in cellular differentiation may be associated with a reduced risk of subsequent breast cancer. To investigate the association between serum and plasma concentrations of retinol, retinyl palmitate, alpha-carotene, beta-carotene, beta-cryptoxanthin, lutein, lycopene, total-carotenoids, alpha-tocopherol, and gamma-tocopherol with subsequent development of breast cancer, a nested case control study was conducted among female residents of Washington County, Maryland, who had donated blood for a serum bank in 1974 or 1989. Cases (n = 295) and controls (n = 295) were matched on age, race, menopausal status, and date of blood donation, and the analyses were stratified by cohort participation. Median concentrations of beta-carotene, lycopene, and total carotene were significantly lower in cases compared with controls in the 1974 cohort (13.1, 12.5, and 7.9% difference; P = 0.01, 0.04, and 0.04, respectively) and for lutein in the 1989 cohort (6.7% difference; P = 0.02). The risk of developing breast cancer in the highest fifth was approximately half of that of women in the lowest fifth for beta-carotene [odds ratio (OR) = 0.41; 95% confidence interval (CI) 0.22-0.79; P trend = 0.007], lycopene (OR = 0.55; 95% CI 0.29-1.06; P trend = 0.04), and total carotene (OR = 0.55; 95% CI 0.29-1.03; P trend = 0.02) in the 1974 cohort. There was generally a protective association for other micronutrients in both cohorts, although none reached statistical significance. The results suggest that carotenoids may protect against the development of breast cancer.

Suppression of mammary tumorigenesis in transgenic mice by the RXR-selective retinoid, LGD1069.

Retinoids have been used in the clinic for the prevention and treatment of human cancers. They regulate several cellular processes including growth, differentiation, and apoptosis. Previously, we demonstrated that a pan-agonist retinoid 9-cis retinoic acid was able to suppress mammary tumorigenesis in the C3(1)-SV40 T-antigen (Tag) transgenic mouse model. However, significant toxicity was seen with this naturally occurring retinoid. We hypothesized that the cancer preventive effects of retinoids could be dissected from the toxic effects by using receptor-selective retinoids. In this study, we used TTNPB, an retinoic acid receptor-selective retinoid, and LGD1069, an retinoid X receptor-selective retinoid, to preferentially activate retinoic acid receptors and retinoid X receptors. In vitro, both compounds were able to inhibit the growth of T47D breast cancer cells. We then determined whether these retinoids prevented mammary tumorigenesis. C3(1)-SV40 Tag mice were treated daily by gastric gavage with vehicle, two different doses of TTNPB (0.3 or 3.0 microg/kg), or two different doses of LGD1069 (10 or 100 mg/kg). Mice were treated from approximately 6-8 weeks to 7-8 months of age. Tumor size and number were measured twice each week, and toxicities were recorded daily. Our data show that LGD1069 suppresses mammary tumorigenesis in C3(1)-SV40 Tag transgenic mice with no observable toxicity, whereas TTNPB had a modest chemopreventive effect, yet was very toxic. Median time to tumor development was 129 days in vehicle-treated mice versus 156 days in mice treated with 100 mg/kg LGD1069 (P = 0.05). In addition, tumor multiplicity was reduced by approximately 50% in mice treated with LGD1069 (2.9 for vehicle, 2.4 for 10 mg/kg LGD1069, and 1.4 for 100 mg/kg, P < or = 0.03). TTNPB-treated mice showed a delayed median time to tumor development (131 days for vehicle versus 154 days for 3.0 microg/kg TTNPB; P < or = 0.05), but no changes were seen in tumor multiplicity. However, toxicity (skin erythema, hair loss) was seen in all of the mice treated with TTNPB. These data demonstrate that receptor-selective retinoids suppress mammary tumorigenesis in transgenic mice and that preventive effects of retinoids can be separated from their toxicity, demonstrating that receptor-selective retinoids are promising agents for the prevention of breast cancer.

Leiden mutation (as genetic) and environmental (retinoids) sequences in the acute and chronic inflammatory and premalignant colon disease in human gastrointestinal tract.

BACKGROUND: Tumor, calor, dolor, pallor and functio laesa are together involved in the different acute and chronic inflammatory processes. The processes involved in the inflammation are determined by differently acquired and hereditary factors. Recently the presence of a new genetic marker (Leiden point mutation) was found in Crohn's disease and ulcerative colitis. On the other hand, the GI mucosal integrity was proven on gastrointestinal mucosal damage to be produced by different chemicals, xenobiotics, drugs. In human observations, the serum level of retinoids (vitamin A, lutein, zeaxanthin, alpha-, beta-carotene) was proven in patients with chronic gastrointestinal inflammatory bowel disease. The aims of this study were (1) to measure the prevalence of Leiden mutation; (2) to identify the changes in the serum retinoid level in patients with Helicobacter pylori infection of the stomach (n=24), hepatitis C infection (n=75), ileitis terminalis (Crohn's disease; n=49), ulcerative colitis (n=35), colon polyposis (n=59) and adenocarcinoma in colon polyps (n=9), and 57 healthy persons were used in the control group; (3) to compare the directions of the changes in the measured parameters in the acute (H. pylori and hepatitis C infections), chronic (ileitis terminalis, ulcerative colitis) GI inflammatory diseases and in colon polyposis without and with malignisation. METHODS: The Leiden mutation was measured by the method of polymerase chain reaction, the retinoid level in the patient's serum was measured by high liquid cromathografic method (HPCL). RESULTS: (1) It has been found that the prevalence of Leiden mutation increased significantly in patients with ileitis terminalis (P<0.001), ulcerative colitis (P<0.001), colon polyposis (P<0.001) and with colon polyps with malignisation (P<0.01). (2) Serum level of vitamin A and zeaxantin were decreased significantly in all group of patients except for the group with H. pylori infections. (3) alpha- and beta-carotenes were found to be practically at the same level as those in the control groups, except in patients of colon polyps with malignisation. (4) The vitamin A, lutein, zeaxantin, alpha- and beta-carotenes were decreased in patients with ileitis terminalis. CONCLUSIONS: (1) The essential role of retinoids (carotenoids) as environmental factors are suggested for keeping GI mucosal integrity in human healthy subjects and patients. (2) Leiden mutation, as a genetic marker, can be used in the screening of patients with ileitis terminalis, ulcerative colitis and colon polyposis (without and with malignisation). (3) An opposite direction can be found between the increased prevalence of Leiden mutation and decrease of serum levels of retinoids in group of patients with ileitis terminalis, ulcerative colitis and colon polyposis (without and with malignisation).