Validation of urinary sphingolipid metabolites as biomarker of effect for fumonisins exposure in Kenyan children.

Context: Fumonisins (FNs), a group of mycotoxins produced mainly by Fusarium species, are ubiquitous food contaminants, especially for maize. Fumonisin B1 (FB1) caused severe toxicities in farm animals, induced kidney and liver tumours in rodents and is associated with many human adverse health effects, including oesophageal cancer. International Agency for Research on Cancer (IARC) categorizes FB1 as a possible human carcinogen (Group 2B). Inhibition of ceramide synthesis and disruption of sphingolipids metabolism are well studied as the major mechanisms of FB1-induced toxicity. Increases in sphinganine (Sa) and decrease in sphingosine (So) levels and their ratio are validated biomarkers of FB1 effects. Methods: In this study, we measured urinary levels of Sa, So and Sa/So in 284 children aged 1-14 years who consume maize as a staple diet. Exfoliated cells from urine were processed and sphingolipids quantified by High Pressure Liquid Chromatography. Results and conclusions: Sa and So were detectable in 95.07% and 98.94% of samples, respectively. Creatinine adjusted mean levels and standard deviation of Sa, So and Sa/So ratio were 1.23 ± 2.18, 4.99 ± 8.3 and 0.296 ± 0.587 nM. These results further confirmed the findings in studies with human adults, i.e. urinary Sa, So levels and Sa/So ratio are good biomarkers to assess FNs exposure in children.

Urine Metabonomics Reveals Early Biomarkers in Diabetic Cognitive Dysfunction.

Recently, increasing attention has been paid to diabetic encephalopathy, which is a frequent diabetic complication and affects nearly 30% of diabetics. Because cognitive dysfunction from diabetic encephalopathy might develop into irreversible dementia, early diagnosis and detection of this disease is of great significance for its prevention and treatment. This study is to investigate the early specific metabolites biomarkers in urine prior to the onset of diabetic cognitive dysfunction (DCD) by using metabolomics technology. An ultra-high performance liquid-chromatography-quadrupole time-of-flight-mass spectrometry (UPLC-Q/TOF-MS) platform was used to analyze the urine samples from diabetic mice that were associated with mild cognitive impairment (MCI) and nonassociated with MCI in the stage of diabetes (prior to the onset of DCD). We then screened and validated the early biomarkers using OPLS-DA model and support vector machine (SVM) method. Following multivariate statistical and integration analysis, we found that seven metabolites could be accepted as early biomarkers of DCD, and the SVM results showed that the prediction accuracy is as high as 91.66%. The identities of four biomarkers were determined by mass spectrometry. The identified biomarkers were largely involved in nicotinate and nicotinamide metabolism, glutathione metabolism, tryptophan metabolism, and sphingolipid metabolism. The present study first revealed reliable biomarkers for early diagnosis of DCD. It provides new insight and strategy for the early diagnosis and treatment of DCD.

Detection of Urine Metabolites in a Rat Model of Chronic Fatigue Syndrome before and after Exercise.

Purpose. The aim of the present study was to elucidate the metabolic mechanisms associated with chronic fatigue syndrome (CFS) via an analysis of urine metabolites prior to and following exercise in a rat model. Methods. A rat model of CFS was established using restraint-stress, forced exercise, and crowded and noisy environments over a period of 4 weeks. Behavioral experiments were conducted in order to evaluate the model. Urine metabolites were analyzed via gas chromatography-mass spectrometry (GC-MS) in combination with multivariate statistical analysis before and after exercise. Results. A total of 20 metabolites were detected in CFS rats before and after exercise. Three metabolic pathways (TCA cycle; alanine, aspartate, and glutamate metabolism; steroid hormone biosynthesis) were significantly impacted before and after exercise, while sphingolipid metabolism alone exhibited significant alterations after exercise only. Conclusion. In addition to metabolic disturbances involving some energy substances, alterations in steroid hormone biosynthesis and sphingolipid metabolism were detected in CFS rats. Sphingosine and 21-hydroxypregnenolone may be key biomarkers of CFS, potentially offering evidence in support of immune dysfunction and hypothalamic-pituitary-adrenal (HPA) axis hypoactivity in patients with CFS.

Exploration of the Sphingolipid Metabolite, Sphingosine-1-phosphate and Sphingosine, as Novel Biomarkers for Aspirin-exacerbated Respiratory Disease.

Sphingolipid (SL) metabolites have been suggested to be important inflammatory mediators in airway inflammation and asthma. However, little is known about SL metabolites in aspirin-exacerbated respiratory disease (AERD). We aimed to explore the potential AERD biomarkers by conducting lipidomics targeting SL metabolites. The levels of SL metabolites in serum and urine samples from 45 AERD patients and 45 aspirin-tolerant asthma (ATA) patients were quantified through mass spectrometry. During the lysine-aspirin bronchoprovocation test (ASA-BPT), the levels of serum sphingomyelin (SM) were significantly decreased in AERD (P < 0.05) but not in ATA. The serum SM levels were positively correlated with airway responsiveness to methacholine. At the basal status before the ASA-BPT, the levels of serum sphingosine-1-phosphate (S1P) and urine sphingosine were significantly higher in the AERD patients compared with that of ATA patients (P < 0.001) and were positively correlated with a greater decrease in FEV1 (%) values following the ASA-BPT test (P < 0.001 for each), and with serum periostin level (P < 0.05 for each). This study is the first to evaluate serum S1P and urine sphingosine as potential biomarkers of AERD as well as to examine the metabolic disturbance of SL in AERD patients.

Aptamer-based trapping of phytosphingosine in urine samples.

Usually, small molecules like single metabolites used in clinical diagnostic can be quantified by instrumental approaches like LC-MS or bioanalytical techniques using antibodies or aptamers as selective receptors. The present work comprises the generation of aptamers with an affinity towards the medically relevant metabolite phytosphingosine via the previously reported just in time-Selection approach (Hünniger et al., 2014). The whole approach could be seen as a proof of concept to extend the existing just in time-Selection protocol for selection towards small molecules with dissociation constants in the low nanomolar range. Moreover it is conceivable that the shown methods could be quickly adapted to further scopes. Aptamers could be applied for clean-up or concentration processes prior to further analysis. As an example, we used the selected aptamers towards phytosphingosine bound to magnetic particles for affinity enrichment in both selection buffer and urine samples. As an outcome, enrichment factors of up to 9-fold (selection buffer)/4-fold (urine samples) were achieved by this approach.

Effects of orally administered fumonisin B1 (FB1), partially hydrolysed FB1, hydrolysed FB1 and N-(1-deoxy-D-fructos-1-yl) FB1 on the sphingolipid metabolism in rats.

Fumonisin B1 (FB1) is a Fusarium mycotoxin frequently occurring in maize-based food and feed. Alkaline processing like nixtamalisation of maize generates partially and fully hydrolysed FB1 (pHFB1 and HFB1) and thermal treatment in the presence of reducing sugars leads to formation of N-(1-deoxy-D-fructos-1-yl) fumonisin B1 (NDF). The toxicity of these metabolites, in particular their effect on the sphingolipid metabolism, is either unknown or discussed controversially. We produced high purity FB1, pHFB1a+b, HFB1 and NDF and fed them to male Sprague Dawley rats for three weeks. Once a week, urine and faeces samples were collected over 24 h and analysed for fumonisin metabolites as well as for the sphinganine (Sa) to sphingosine (So) ratio by validated LC-MS/MS based methods. While the latter was significantly increased in the FB1 positive control group, the Sa/So ratios of the partially and fully hydrolysed fumonisins were indifferent from the negative control group. Although NDF was partly cleaved during digestion, the liberated amounts of FB1 did not raise the Sa/So ratio. These results show that the investigated alkaline and thermal processing products of FB1 were, at the tested concentrations, non-toxic for rats, and suggest that according food processing can reduce fumonisin toxicity for humans.

Increased serum concentration of sphingosine-1-phosphate in juvenile-onset systemic lupus erythematosus.

PURPOSE: Sphingosine-1-phosphate (S1P) is an active sphingolipid with chemotactic abilities and has been linked to inflammatory mediators and autoimmune disease. The aim of this study was to assess whether children with juvenile-onset systemic lupus erythematosus (JSLE) express increased systemic and/or urinary concentrations of S1P. METHODS: A subgroup of patients participating in the UK JSLE Cohort Study, were invited to participate. Cross sectional serum and urine samples were prospectively collected along with demographic and standard clinical data. Results were compared to a cohort of disease controls (Henoch Schonlein Purpura; HSP) and healthy controls (HC). RESULTS: The median age of JSLE patients (n = 15) was 13.6 years (7.2-16.9 years). The serum concentrations of S1P in JSLE patients (7.4 uM, IQR 6.3-12.3 uM) were statistically significantly increased when compared to patients with HSP (n = 10; 5.2 uM, IQR 4.0-7.9 uM; p = 0.016) and HCs (n = 10; 3.8 uM, IQR 2.1-5.8 uM; p = 0.003). There was a trend towards increased serum S1P concentrations between patients with active lupus nephritis (n = 8; 8.7 uM, IQR 6.2-15.3 uM) compared to lupus non-nephritis (n = 7; 6.6 uM, IQR 6.3-10.6 uM; p = 0.355). No relationship was found between disease activity markers and S1P. Urine S1P concentrations were no different between JSLE patients (56.0 nM, IQR 40.3-96.6 nM) and HCs (58.7 nM, IQR 0-241.9 nM; p = 0.889). CONCLUSIONS: We have demonstrated, for the first time, an increased serum concentration of S1P in a cohort of JSLE patients. These findings highlight a role of S1P in the pathophysiology of JSLE that warrants further investigation.

Sphinganine and sphingosine levels and ratio in urine and blood samples from a Catalonian population, Spain.

Fumonisins occur mainly in maize and they produce alterations on sphingolipid metabolism, unbalancing the sphinganine (Sa)/sphingosine (So) ratio. This alteration has been proposed as a biomarker of fumonisin exposure. The objective of this study was to establish the urinary and plasmatic levels of Sa, So as well as the ratio Sa/So from a sample of the Catalonian (Spain) population exposed to fumonisins at low levels. Firstly, plasma and urinary Sa and So levels and the ratio Sa/So were compared between two population groups, and later urinary Sa and So levels from corn food consumers and a control group were monitored for 2 weeks under controlled intake of corn foods. Sa and So levels were determined in urine and blood samples using validated methods using HPLC with fluorescence detection. Significant differences were not found in urine samples when Sa/So ratios were compared from corn food consumers and non-consumers, while significant differences were found in urine and plasma samples, but evidence of the mechanism of action of fumonisins was not apparent. Through a time-course study we have narrowed down the day in which the maximum alteration of Sa/So ratio should be expected in humans. This paper reports some useful information to improve the design of studies to validate the ratio Sa/So as a possible biomarker of fumonisin exposure.

Targeted analysis of sphingoid precursors in human biofluids by solid-phase extraction with in situ derivatization prior to µ-LC-LIF determination.

A method for determination of two relevant sphingoid precursors such as sphingosine and sphinganine and the corresponding conjugates sphingosine 1-phosphate and sphinganine 1-phosphate in human urine and serum is here presented. The method is characterized by a solid- phase extraction step with in situ derivatization of the sphingolipids in the eluate (o-phthaldialdehyde derivatives) to obtain fluorescent compounds. In this way, sample preparation was completely performed in a single automated step by means of a lab-on-valve system. Derivatized analytes were injected into a liquid chromatography system operating at micro regime and detected by laser-induced fluorescence. For determination of sphingoid phosphates, they were enzymatically converted to free sphingoids to obtain stable fluorescent derivatives. The detection limits were in the range 4.2-10.2 ng mL(-1) for serum and 0.56-1.36 ng mL(-1) for urine, with repeatability ranging from 3.9% to 6.2% expressed as relative standard deviation. The method was validated by direct infusion tandem mass spectrometry in multiple reaction monitoring to compare results provided by analysis of biofluids and to confirm the identity of the target compounds. Sensitivity and precision were better than or similar to those provided by the confirmatory method. The automation of sample preparation enables to scale-down this step and improves precision by minimization of human intervention, being thus suitable for clinical analysis.

Absorption and disposition of the sphingosine 1-phosphate receptor modulator fingolimod (FTY720) in healthy volunteers: a case of xenobiotic biotransformation following endogenous metabolic pathways.

Fingolimod [(FTY720), Gilenya; 2-amino-2-[2-(4-octylphenyl)ethyl]-1,3-propanediol], a new drug for the treatment of relapsing multiple sclerosis, acts through its phosphate metabolite, which modulates sphingosine 1-phosphate receptors. This represents a novel mechanism of action. In the present work, the absorption and disposition of (14)C-labeled fingolimod were investigated in healthy male volunteers after a single oral dose of 4.5 mg. Total radioactivity was determined in blood, urine, and feces. Fingolimod was quantified in blood. Metabolite profiles were determined in blood and excreta, and metabolite structures were elucidated by mass spectrometry, wet-chemical methods, and comparison with reference compounds. Fingolimod was absorbed slowly but almost completely. The biotransformation of fingolimod involved three main pathways: 1) reversible phosphorylation to fingolimod phosphate [(S)-enantiomer, active principle]; 2) ω-hydroxylation at the octyl chain, catalyzed predominantly by CYP4F enzymes, followed by further oxidation to a carboxylic acid and subsequent ß-oxidation; and 3) formation of ceramide analogs by conjugation with endogenous fatty acids. This metabolism is quite unusual because it follows metabolic pathways of structurally related endogenous compounds rather than biotransformations typical for xenobiotics. The elimination of fingolimod was slow and occurred predominantly by oxidative metabolism whereas fingolimod phosphate was eliminated mainly by dephosphorylation back to fingolimod. Drug-related material was excreted mostly in the urine in the form of oxidation products.

Distribution and metabolism of sphingosine in skin after oral administration to mice.

In this study, (3)H- or (13)C(2),D(2)-sphingosine (SPH) was orally administered to mice to assess absorption, mass balance, tissue distribution, and metabolites in the skin. The blood concentration of (3)H-SPH showed a Tmax of 10.7 hr. The radioactivity in the skin reached 763.4 ng eq./g tissue at 12 hr, and decreased to 181.7 ng eq./g tissue at 168 hr. The concentration of radioactivity at 12 hr was 577.6 and 100.7 ng eq./g tissue in the dermis and epidermis, respectively. Thereafter, the dermis concentration decreased to 158.5 ng eq./g tissue, while the epidermis concentration increased to 298.8 ng eq./g tissue, suggesting that radioactivity moves from the dermis to the epidermis. Unchanged SPH along with lipophilic metabolites was detected in the skin of mice exposed orally to (3)H- or (13)C(2),D(2)-SPH. Moreover, in an in vitro study using human skin keratinocytes, a (13)C(2),D(2)-SPH-treatment resulted in the intracellular production of glucosylceramides (GlcCer) and ceramides (Cer) containing labeled-SPH. These results indicate the followings: first, that SPH is absorbed through the digestive tract and distributed to the skin; second, it is transferred from the dermis to the epidermis; and third, SPH is partly distributed to the skin in an unchanged form, and some of the distributed compounds are converted into GlcCer and Cer by biosynthesis.

Evaluation of fumonisin biomarkers in a cross-sectional study with two high-risk populations in China.

Levels of serum and urinary sphinganine (Sa) and sphingosine (So), the Sa/So ratio, and urinary-free fumonisin B(1) (FB(1)) were determined in a cross-sectional study consisting of 43 adults in Huaian and 34 adults in Fusui, China. Home-produced corn had 100% contamination with FB(1). There were 93.0% (40/43) of Huaian subjects and 52.9% (18/34) of Fusui subjects with daily FB(1) intakes greater than 2 microg kg(-1) body weight, which showed a significant difference (p < 0.01). Levels of sphinganine and sphingosine and the Sa/So ratio were not correlated with levels of dietary exposure. The median level of the serum Sa/So ratio in Huaian subjects (0.41, range = 0.14-0.85) was significantly lower than that in Fusui subjects (0.78, range = 0.57-1.08) (p < 0.01). The median level of the urinary Sa/So ratio was also significantly lower in Huaian subjects (0.31, range = 0.08-1.33) than in Fusui subjects (0.57, range = 0.03-2.52) (p < 0.01). Urinary-free FB(1) was detected in 83.7% (36/43) of Huaian samples and in 82.4% (28/34) of Fusui urine samples (p > 0.05). However, the median level of urinary-free FB(1) in Huaian subjects, 3.91 (range = 0.06-253.61) ng mg(-1) creatinine, was significantly higher than 0.39 (range = 0.01-3.72) ng mg(-1) creatinine found in Fusui subjects (p < 0.01). These results suggest that urinary-free FB(1) may be a potential biomarker for human fumonisin exposure, while further validation is needed in human epidemiological and intervention studies.

Toxicokinetics and toxicological effects of single oral dose of fumonisin B1 containing Fusarium verticillioides culture material in weaned piglets.

Toxicokinetics and the toxicological effects of culture material containing fumonisin B(1) (FB(1)) were studied in male weaned piglets by clinical, pathological, biochemical and sphingolipid analyses. The animals received a single oral dose of 5 mg FB(1)/kg of body weight, obtained from Fusarium verticillioides culture material. FB(1) was detected by HPLC in plasma collected at 1-h intervals up to 6h and at 12-h intervals up to 96 h. FB(1) eliminated in feces and urine was quantified over a 96-h period and in liver samples collected 96 h post-intoxication. Blood samples were obtained at the beginning and end of the experiment to determine serum enzyme activity, total bilirubin, cholesterol, sphinganine (Sa), sphingosine (So) and the Sa/So ratio. FB(1) was detected in plasma between 30 min and 36 h after administration. The highest concentration of FB(1) was observed after 2 h, with a mean concentration of 282 microg/ml. Only 0.93% of the total FB(1) was detected in urine between 75 min and 41 h after administration, the highest mean concentration (561 microg/ml) was observed during the interval after 8 at 24 h. Approximately 76.5% of FB(1) was detected in feces eliminated between 8 and 84 h after administration, with the highest levels observed between 8 and 24 h. Considering the biochemical parameters, a significant increase only occurred in cholesterol, alkaline phosphatase and aspartate aminotransferase activities. In plasma and urine, the highest Sa and Sa/So ratios were obtained at 12 and 48 h, respectively.

Sphinganine-sphingosine ratio in urine from two Portuguese populations as biomarker to fumonisins exposure.

Fumonisins (FBs), mycotoxins specially found in maize, interfere with sphingolipid metabolism, altering the sphinganine (Sa) to sphingosine (So) ratio. In humans, FBs have been epidemiologically associated with esophageal cancer. The exposure evaluation of different populations is of primordial interest for public health maintenance. This might be accomplished through the determination of biomarkers, such as the Sa/So ratio, in biological samples. This paper presents the optimization of an analytical methodology for the determination of the Sa/So ratio in human urine. Isolation of exfoliated cells from urine, followed by extraction with ethyl acetate, derivatisation with naphthalene-2,3-dicarboxaldehyde (NDA), and detection and quantification by liquid chromatography (LC) with fluorescence detection (FD) allowed adequate sensitivity, accuracy, and precision. The application of this analytical procedure to a rural and an urban population from the Central zone of Portugal demonstrated that the Sa/So ratio was 0.43+/-0.22 and 0.42+/-0.17, respectively. Regarding the rural population, the Sa/So ratio presented mean levels of 0.48+/-0.22 for females, and 0.30+/-0.16 for males. Concerning the urban population, the Sa/So ratio presented a mean of 0.44+/-0.18 for females, and was 0.29 in the only male sample where its establishment was possible. The statistical analysis showed that no significant differences were found between the different studied populations.

Evaluation of sphingolipids in Wistar rats treated to prolonged and single oral doses of fumonisin b1.

The objective of the present study was to evaluate sphingolipid levels (sphingosine-So and sphinganine-Sa) and to compare the Sa/So ratio in liver, serum and urine of Wistar rats after prolonged administration (21 days) of fumonisin B(1) (FB(1)). In parallel, the kinetics of sphingolipid elimination in urine was studied in animals receiving a single dose of FB(1). Prolonged exposure to FB(1) caused an increase in Sa levels in urine, serum and liver. The most marked effect on sphingolipid biosynthesis was observed in animals treated with the highest dose of FB(1). Animals receiving a single dose of FB(1) presented variations in Sa and So levels and in the Sa/So ratio.

Urine ochratoxin a and sphinganine/sphingosine ratio in residents of the endemic nephropathy area in Croatia.

The most plausible theory of the aetiology of endemic nephropathy links it with exposure to nephrotoxic mycotoxin ochratoxin A (OTA). In this study, the concentration of OTA and sphinganine/sphingosine (Sa/So) ratio, the biomarker of another nephrotoxic mycotoxin fumonisin B1 exposure, were analysed in 45 human urine samples collected in the endemic village of Kaniza in Croatia and in 18 samples from control village. Samples were collected twice from the same persons in 2000 and 2005. In both years the frequency of OTA-positive samples was higher in Kaniza (43 % and 18 %, respectively) than in the control village (28 % and 6 %, respectively). OTA concentrations in samples collected in Kaniza were higher in 2000 than in 2005 (p<0.005). Although in both years Sa/So ratio was higher in Kaniza, the difference from the control group was not statistically significant. No control sample contained OTA and had the Sa/So ratio >1 at the same time, while in Kaniza four such samples were collected in 2000 and one in 2005.

Sphingoid base levels in humans consuming fumonisin-contaminated maize in rural areas of the former Transkei, South Africa: a cross-sectional study.

High incidences of oesophageal cancer are associated with the consumption of subsistence-grown maize by rural populations in the former Transkei region of Eastern Cape Province, South Africa. This cross-sectional study was conducted in the north-eastern magisterial area of Bizana (a previously low oesophageal cancer incidence area) and the south-eastern area of Centane (a previously high incidence area). Plasma and urine samples of male and female participants were analysed for the sphingoid bases, sphinganine and sphingosine. Good home-grown and visibly mouldy maize samples, collected from the households of the participants, were analysed for fumonisin B(1), B(2) and B(3). Plasma sphinganine/sphingosine ratios in males and females were significantly lower (p < 0.05) due to lower sphinganine levels in Bizana compared to Centane. In contrast, the urinary female and combined (males + females) sphinganine/sphingosine ratios were significantly higher (p < 0.05) in Bizana due to the significantly lower (p < 0.05) urinary sphingosine levels. Interestingly, urinary sphingoid base levels were significantly lower (p < 0.05) in males than females within each area. Based on the mean total fumonisin levels in good maize, the estimated mean probable daily intake (PDI) was 5.8 microg kg(-1) body weight day(-1) in Bizana during 2000 and 4.4 and 6.7 5.8 microg kg(-1) body weight day(-1) in Centane during 1997 and 2000, respectively, exceeding the maximum tolerable daily intake proposed by JECFA. However, there was no significant difference in the mean total fumonisin levels in the maize between the magisterial areas. The observed differences in plasma and urinary sphingoid base levels could not be ascribed as a biomarker of fumonisin exposure and further studies at an individual level are required.

Ethnic sensitivity study of fingolimod in white and Asian subjects.

OBJECTIVE: The authors compared the pharmacokinetics and pharmacological effects of the immunomodulator fingolimod in healthy white and Asian subjects for potential ethnic differences. METHODS: White and Asian (Japanese) healthy subjects were demographically matched for sex, age and weight. Subjects received single 1.25 mg doses of fingolimod (6 ethnic pairs), 2.5 mg (7 pairs), 5 mg (6 pairs) or 5 mg/day for 7 days (6 pairs). The pharmacokinetics of fingolimod, major metabolites, peripheral blood lymphocyte counts and heart rate were characterized over 1 month after single-dose and 2 months after multiple-dose administration. RESULTS: There were no clinically relevant differences in the fingolimod dose Cmax or dose AUC relationships between Asian subjects (slopes 0.84 and 1.05) versus white subjects (slopes 1.13 and 1.26) after single-dose administration. During multiple-dose administration, there were no clinically relevant interethnic differences in fingolimod accumulation ratios (6.6 +/- 0.4 for whites, 7.0 +/- 0.7 for Asians), area under the concentration-time curve (390 +/- 73 versus 382 +/- 106 ng x h/ml), or elimination half-life (7.4 +/- 0.8 versus 7.9 +/- 2.0 days). The acute decrease in lymphocyte counts after single- and multiple-dose fingolimod were similar in the two ethnic groups. The lymphocyte recovery rate to baseline after a 5 mg single dose and 5 mg/day multiple dose was reduced by 36 and 15% in Asian subjects compared with white subjects. The transient, acute decrease in heart rate after the first dose of fingolimod and the subsequent return to baseline was similar in the two ethnic groups. CONCLUSION: There were no marked differences between healthy white and Asian subjects in fingolimod single-dose and multiple-dose pharmacokinetics, lymphocyte trafficking and heart rate responses.

Sphingoid bases as possible diagnostic parameters.

AIM: To determine the concentrations and ratios of sphingoid bases, sphinganine and sphingosine, in the serum and urine of healthy individuals, as a basis for the normal value range, which may be useful in the diagnosis of diseases characterized by sphingolipid metabolism impairment. Possible sex differences were also investigated, as well as effects of hormonal changes on sphingoid base concentrations during pregnancy or menopause. METHOD: Sphingolipids were extracted from the serum and urine and hydrolyzed. Sphinganine and sphingosine were determined by high performance liquid chromatography. The analysis included serum and urine samples of 20 men and 20 women, and urine samples of 5 healthy postmenopausal and 5 healthy pregnant women. RESULTS: Serum concentrations of free and total sphingoid bases showed no major variations in healthy individuals of both sexes: total sphingosine 28.28-/+8.96 x 10(3) pmol/mL in men and 22.52-/+10.19 x 10(3) pmol/mL in women (p=0.080); total sphinganine 0.61-/+0.15 x 10(3) pmol/mL in men and 0.58-/+0.25 x 10(3) pmol/mL in women (p=0.574). Urine concentrations showed greater variability. Hormonal changes associated with menopause or pregnancy significantly decreased the urinary concentrations of total sphinganine in postmenopausal women, and increased free sphinganine/sphingozine ratio. CONCLUSION: Serum but not urine concentrations of sphingoid bases could be used as a sensitive indicator in the diagnosis of the diseases associated with sphingolipid metabolism impairment.

In vitro and in vivo studies to assess the effectiveness of cholestyramine as a binding agent for fumonisins.

Several adsorbent materials were tested at I mg/ml for their in vitro capacity to adsorb fumonisin B1(FB1) from aqueous solutions. Cholestyramine showed the best adsorption capacity (85% from a solution containing 200 microg/ml FB1) followed by activated carbon (62% FB1). Bentonite adsorbed only 12% of the toxin from a solution containing 13 microg/ml FB1, while celite was not effective even at the lowest tested FB1 concentration (3.2 microg/ml). Cholestyramine was tested in vivo to evaluate its capacity to reduce the bioavailability of fumonisins (FBs) in rats fed diet contaminated with toxigenic Fusarium verticillioides culture material. Rats were exposed for one week to FBs-free diet, FBs-contaminated diet containing 6 or 20 microg/g FB1 + FB2 and the same FBs-contaminated diet added of 20 mg/g cholestyramine. The increase of sphinganine/sphingosine (SA/SO) ratio in urine and kidney of treated rats was used as specific and sensitive biomarker of fumonisin exposure. The addition of cholestyramine to the FBs-contaminated diets consistently reduced the effect of FBs by reducing significantly (P < 0.05) both urinary and renal SA/SO ratios.