Excretion Of Sulfate Research Articles

Effect of Feeding Wet Distillers Grains With Solubles to Beef Cattle on Air and Manure Quality

Air quality is becoming a pressing issue for beef feedlot producers. Feeding practices influence the excretion of starch, fiber, nitrogen (N), and sulfur (S) in manure, thereby affecting nutrient content and the production of ammonia (NH3) and odorous compounds. Wet distillers grains with solubles (WDGS) are a common and economical ingredient in feedlot diets. WDGS are high in protein, fiber, phosphorus (P), and S but low in starch. The objective of this study was to compare NH3 concentration in the air and nutrients and volatile organic compounds (VOC) concentration in manure between two dietary treatments fed to feedlot cattle. Five pens of feedlot cattle were fed diets containing 14% to 35% WDGS and five pens were fed a corn-based diet with no ethanol byproducts (Control). Each pen had twelve sampling locations (N = 120) where air and manure samples were collected from the feedlot surface. Air samples were analyzed for NH3 concentration. Manure samples were analyzed for dry matter, pH, volatile solids, VOC, and nutrient composition (N, P, and S). Concentrations of P and S in manure and NH3 in the air were higher in pens fed WDGS compared to pens fed the control diet. Concentrations of VOC were similar across both treatments.

Interdependency of Cystathione -Lyase and Cystathione -Synthase in Hydrogen Sulfide-Induced Blood Pressure Regulation in Rats

BackgroundHydrogen sulfide (H2S), an endogenous vasoactive agent, is produced by cystathionine γ-lyase (CGL) and cystathionine β-synthase (CBS) enzymes. This study was conducted to evaluate the relative contribution of these enzymes in regulating systemic arterial pressure.MethodsSprague–Dawley rats were chronically treated with CGL inhibitor, -propargylglycine (PAG, 37.5 mg/kg/day; intraperitoneally, i.p.) or CBS inhibitor, aminooxyacetic acid (AOA, 8.75 mg/kg/day; i.p.) or in combination for 4 weeks and the effects on arterial pressure (tail-cuff plethysmography) and renal excretory function (24 h urine collections using metabolic cages) were assessed once in a week. Changes in renal blood flow (RBF; Ultrasonic flowmetry) and glomerular filtration rate (GFR; Inulin clearance) were assessed in acute experiments in anesthetized rats at the end of treatment period.ResultsCompared to vehicle treated control group, only the rats with combination therapy showed a decrease in urinary sulfate excretion rate (248 ± 47 vs. 591 ± 70 nmol/24 h; marker for endogenous H2S level) which was associated with an increase in mean arterial pressure (MAP; 130 ± 2 vs. 99 ± 2 mm Hg). Urine flow and sodium excretion were also increased in combination group as consequent to the increase in MAP. GFR did not alter due to these treatments but RBF was lowered (4 ± 0.3 vs. 7 ± 0.4 ml/min/g) only in the combination group compared to the control group.ConclusionThese findings indicate that a deficiency in one enzyme's activity could be compensated by the activity of the other to maintain the endogenous H2S level, the deficiency of which modulates systemic and renal vascular resistances leading to the development of hypertension.

Quantitation of norantipyrine sulphate and norantipyrine glucuronide as major metabolites of antipyrine in man and rat.

Two assay procedures are described for the quantitation of norantipyrine sulphate and norantipyrine glucuronide present in biological material. One, a selective acid hydrolytic assay procedure that affords the discriminative determination of both conjugates without prior separation, measures free norantipyrine by tlc-uv. The other is a tlc separation procedure for intact norantipyrine conjugates, which, in conjunction with radiolabelled material, derived from [3-14C]antipyrine, enables the direct quantitation of both conjugates independently. In man, about 25% of dose of antipyrine (1200 mg) was excreted as norantipyrine glucuronide within 48 h. The amount of norantipyrine sulphate was small. In the rat, norantipyrine sulphate represented about 15-20% of the dose of antipyrine (40-50 mg kg-1); no norantipyrine glucuronide was formed. Free norantipyrine has not been detected in urine of either species after antipyrine dosage. Pretreatment with 3-methylcholanthrene in the rat substantially enhanced excretion of norantipyrine sulphate, whereas induction with phenobarbitone was without effect on the microsomal N-demethylation of antipyrine. The lability of free norantipyrine was examined under different conditions and contrasted with the relative stability of norantipyrine conjugates. Two of the main degradation products of norantipyrine were identified as 4-phenylazo-norantipyrine and a 4,4'-bipyrazole-derivative.

Effects of imatinib mesylate on the pharmacokinetics of paracetamol (acetaminophen) in Korean patients with chronic myelogenous leukaemia

The major objective of the present study was to investigate the effect of imatinib on the pharmacokinetics of paracetamol in patients with chronic myelogenous leukaemia (CML). Patients (n = 12) received a single oral dose of acetaminophen 1000 mg on day 1 (control). On days 2-8, imatinib 400 mg was administered daily. On day 8 (treatment), another 1000 mg dose of paracetamol was administered 1 h after the morning dose of imatinib 400 mg. Blood and urine samples were collected for bioanalytical analyses. The area under the plasma concentration-time curve (AUC) for paracetamol, paracetamol glucuronide and paracetamol sulphate under control conditions was similar to that after treatment with imatinib; the 90% confidence interval of the log AUC ratio was within 0.8 to 1.25. Urinary excretion of paracetamol, paracetamol glucuronide and paracetamol sulphate was also unaffected by imatinib. The pharmacokinetics of paracetamol and imatinib in Korean patients with CML were similar to previous pharmacokinetic results in white patients with CML. Co-administration of a single dose of paracetamol and multiple doses of imatinib was well tolerated and safety profiles were similar to those of either drug alone. The pharmacokinetics of paracetamol and its major metabolites in the presence of imatinib were similar to those of the control conditions and the combination was well tolerated. These findings suggest that imatinib can be safely administered with paracetamol without dose adjustment of either drug.

The urinary excretion of corticosteroid sulfates by cancer patients

Urinary excretions of free cortisol and corticosteroid sulfates were determined in 31 female controls, 77 breast cancer patients, 14 cases of colonic cancer, and 7 patients with bronchial carcinoma. Elevated corticosteroid sulfate excretion was present in38% of patients with locally recurrent breast cancer and 30% of those with distant metastases, but in only 13% of the “early” breast cancer cases. A similar abnormality was seen in colonic cancer. Urinary free cortisol was usually normal. ACTH stimulation in a normal subject produced marked increases of both urinary free cortisol and corticosteroid sulfates. It is concluded that elevated corticosteroid sulfate excretion in cancer patients arises from an increased cortisol production rate combined with increased sulfurylation of the steroid. In bronchial carcinoma patients, changes similar to those occurring in the ACTH–treated normal subject may have resulted from ectopic ACTH production in the tumor.

Breast Cancer Resistance Protein (BCRP) and Sulfotransferases Contribute Significantly to the Disposition of Genistein in Mouse Intestine

The low bioavailability of genistein has impeded its development into a therapeutic agent. Our earlier studies indicate that glucuronidation is one of the major barriers to genistein oral bioavailability. This study will determine how sulfotransferases and efflux transporters affect its intestinal disposition. A rodent intestinal perfusion model and S9 fractions were used. Sulfate excretion rates were comparable to glucuronide excretion in mouse small intestine but significantly higher than glucuronide excretion in mouse colon, which is different from rat intestinal disposition but similar to disposition in Caco-2 cells. To define efflux transporter(s) involved in sulfate excretion, two organic anion inhibitors (estrone sulfate and dihydroepiandrosterone sulfate) or a multidrug resistance protein inhibitor (MK-571) were used but neither was able to decrease the excretion of genistein sulfates. In contrast, the excretion of genistein sulfate decreased substantially (>90%) in small intestine of breast cancer resistance protein (BCRP) knockout mice and became undetectable in colon of the knockout mice. The excretion rates of genistein glucuronide in the small intestine of BCRP knockout mice were also significant decreased (78%). This study shows clearly that BCRP facilitates the cellular genistein sulfate excretion by removing sulfates to prevent their backward hydrolysis and to limit substrate inhibition, indicating that BCRP plays a dominant role in genistein sulfate excretion and a significant role in genistein glucuronide excretion in the mouse intestine.

Identification of Noninvasive Biomarkers for Alcohol-Induced Liver Disease Using Urinary Metabolomics and the Ppara-null Mouse

Alcohol-induced liver disease (ALD) is a leading cause of nonaccident-related deaths in the United States. Although liver damage caused by ALD is reversible when discovered at the earlier stages, current risk assessment tools are relatively nonspecific. Identification of an early specific signature of ALD would aid in therapeutic intervention and recovery. In this study, the metabolic changes associated with ALD were examined using alcohol-fed male Ppara-null mouse as a model of ALD. Principal components analysis of the mass spectrometry-based urinary metabolic profile showed that alcohol-treated wild-type and Ppara-null mice could be distinguished from control animals without information on history of alcohol consumption. The urinary excretion of ethyl-sulfate, ethyl-beta-d-glucuronide, 4-hydroxyphenylacetic acid, and 4-hydroxyphenylacetic acid sulfate was elevated and that of the 2-hydroxyphenylacetic acid, adipic acid, and pimelic acid was depleted during alcohol treatment in both wild-type and the Ppara-null mice albeit to different extents. However, indole-3-lactic acid was exclusively elevated by alcohol exposure in Ppara-null mice. The elevation of indole-3-lactic acid is mechanistically related to the molecular events associated with development of ALD in alcohol-treated Ppara-null mice. This study demonstrated the ability of a metabolomics approach to identify early, noninvasive biomarkers of ALD pathogenesis in Ppara-null mouse model.

Copper(II)-selective chelation improves function and antioxidant defences in cardiovascular tissues of rats as a model of diabetes: comparisons between triethylenetetramine and three less copper-selective transition-metal-targeted treatments

Treatment with the Cu(II)-selective chelator triethylenetetramine (TETA) improves cardiovascular disease in human patients, and cardiac and vascular/renal disease in rats used as a model of diabetes. Here we tested two hypotheses: first, that TETA elicits greater improvement in organ function than less Cu-selective transition-metal-targeted treatments; second, that the therapeutic actions of TETA are consistent with mediation through suppression of oxidative stress. Rats were made diabetic with streptozotocin (55 mg/kg, i. v.) and treated from 8 weeks after disease induction for the following 8 weeks with effective dosages of oral TETA, or one of three less Cu-selective transition-metal-targeted treatments: D-penicillamine, deferiprone or Zn acetate. Treatment effects were measured in ex vivo cardiac and aortic tissues, plasma and urine. Diabetes damaged both cardiac and renal/vascular function by impairing the ability of cardiac output to respond physiologically to rising afterload, and by significantly elevating the urinary albumin/creatinine ratio. Diabetes also lowered total antioxidant potential and heparan sulphate levels in cardiac and arterial tissues, and serum ferroxidase activity, whereas it elevated urinary heparan sulphate excretion. TETA treatment rectified or partially rectified all these defects, whereas the other three experimental treatments were ineffectual. By contrast, none of the four drug treatments lowered diabetes-mediated elevations of plasma glucose or lipid concentrations. TETA may limit the cardiac and renal/vascular damage inflicted by diabetes through its actions to reinforce antioxidant defence mechanisms, probably acting through selective chelation of 'loosely-bound'/chelatable Cu(II). It may also improve heparan sulphate homeostasis and bolster antioxidant defence by increasing vascular extracellular superoxide dismutase activity. Urinary albumin/creatinine ratio might prove useful for monitoring TETA treatment.

A Pharmacokinetics Evaluation of a New, Low‐Volume, Oral Sulfate Colon Cleansing Preparation in Patients With Renal or Hepatic Impairment and Healthy Volunteers

The pharmacokinetics (PK) of an oral sulfate solution (OSS) for bowel cleansing preparation was studied. OSS (30 g of sulfate) was split between 2 doses, 12 hours apart. Safety measures included electrocardiography, vital signs, adverse events, hematology, blood chemistry, and urinalysis. Six adult patients with moderate renal disease (MRD), 6 with mild-moderate hepatic disease (M/MHD), and 6 normal healthy volunteers (NHVs) completed the study. Adverse events were mild to moderate in severity and were mainly limited to headache and expected gastrointestinal symptoms. Serum sulfate levels were highly variable at all times, even after adjusting for baseline. Sulfate was higher in MRD in comparison to the other groups. The C(max) and AUC were higher in the patients, but no statistically significant differences emerged. Sulfate levels returned to predose values within 54 hours after dosing. No electrolyte disturbances occurred. Urinary sulfate excretion was approximately 20% of the dose. OSS was well tolerated. The types and severity of adverse events were similar to those seen in large phase III trials. While patients with MRD had elevated sulfate, the levels were less than those in renal failure and did not alter biochemical parameters that are associated with hypersulfatemia.

Using phenotypic plasticity: focus on “Identification of renal transporters involved in sulfate excretion in marine teleost fish”

for osmoregulating vertebrates, diffusive water loss is an unavoidable consequence of living in seawater. Marine teleost fish drink seawater to offset this diffusive water loss dictated by the steep osmotic gradient between blood and the surrounding seawater, a water loss occurring mainly across the

Identification of renal transporters involved in sulfate excretion in marine teleost fish

Sulfate (SO(4)(2-)) is the second most abundant anion in seawater (SW), and excretion of excess SO(4)(2-) from ingested SW is essential for marine fish to survive. Marine teleosts excrete SO(4)(2-) via the urine produced in the kidney. The SO(4)(2-) transporter that secretes and concentrates SO(4)(2-) in the urine has not previously been identified. Here, we have identified and characterized candidates for the long-sought transporters. Using sequences from the fugu database, we have cloned cDNA fragments of all transporters belonging to the Slc13 and Slc26 families from mefugu (Takifugu obscurus). We compared Slc13 and Slc26 mRNA expression in the kidney between freshwater (FW) and SW mefugu. Among 14 clones examined, the expression of a Slc26a6 paralog (mfSlc26a6A) was the most upregulated (30-fold) in the kidney of SW mefugu. Electrophysiological analyses of Xenopus oocytes expressing mfSlc26a6A, mfSlc26a6B, and mouse Slc26a6 (mSlc26a6) demonstrated that all transporters mediate electrogenic Cl(-)/SO(4)(2-), Cl(-)/oxalate(2-), and Cl(-)/nHCO(3)(-) exchanges and electroneutral Cl(-)/formate(-) exchange. Two-electrode voltage-clamp experiments demonstrated that the SO(4)(2-)-elicited currents of mfSlc26a6A is quite large (approximately 35 microA at +60 mV) and 50- to 200-fold higher than those of mfSlc26a6B and mSlc26a6. Conversely, the currents elicited by oxalate and HCO(3)(-) are almost identical among mfSlc26a6A, mfSlc26a6B, and mSlc26a6. Kinetic analysis revealed that mfSlc26a6A has the highest SO(4)(2-) affinity as well as capacity. Immunohistochemical analyses demonstrated that mfSlc26a6A localizes to the apical (brush-border) region of the proximal tubules. Together, these findings suggest that mfSlc26a6A is the most likely candidate for the major apical SO(4)(2-) transporter that mediates SO(4)(2-) secretion in the kidney of marine teleosts.

Multiple sulfatase deficiency: clinical report and description of two novel mutations in a Brazilian patient

Multiple Sulfatase Deficiency (MSD) is a rare autosomal recessive disease in which the activities of all sulfatases are reduced; its estimated prevalence is 1:1.4 million births. The disease is caused by mutations in SUMF1, which encodes an enzyme involved in the post-translational modification of sulfatases. The MSD phenotype is a combination of the clinical features found in diseases resulting from a deficiency of the individual sulfatases; i.e., mucopolysaccharidosis II, IIIA, IIID, IVA and VI, metachromatic leukodystrophy, X-linked ichthyosis, and the X-linked recessive form of chondrodysplasia punctata. We describe herein the first case of a Brazilian patient with MSD. The case was initially diagnosed as having mucopolysaccharidosis (MPS), due to skeletal alterations, coarse facial features, and urinary excretion of dermatan sulfate and heparan sulfate. Later, after a detailed biochemical investigation, the diagnosis of MSD was established. The analysis of the SUMF1 showed the patient was a compound heterozygote for two novel mutations (p.R349G and p.F244S). This case illustrates the challenges in the diagnosis of a disease considered rare, such as MSD. We point out that the availability of therapy for certain MPS disorders necessitates correct disease assignment, and the need to exclude the likelihood of MSD.

Nocturnal urinary melatonin levels and urine biochemistry in microwave-irradiated rats

The aim of this study was to explore whether, during the course of a 15 days-lasting experiment, a two hours per day and five days per week, 2.45 GHz microwave whole-body irradiation may substantially and therefore provably affect rats’ nocturnal urinary 6-hydroxy-melatonin sulphate (aMT6s) excretion and urinalysis parameters. The average whole-body specific absorption rate (SAR) equalled to 1.25 (± 0.36 SE) W/kg. To collect nocturnal urine samples, animals were held in individual metabolic cages every experimental night from 7:00 PM till 7:00 AM next day. The concentration of aMT6s in rat urine samples was determined by a direct radioimmunoassay. Bilirubin, ketones, and urine protein content have been determined via multiple-use reagent strips. In comparison to the sham-exposed group, no significant changes in body temperature and food or water intake were observed in the exposed group. A decline in aMT6s, determined in the exposed rats, was observed from day 8 to day 11 of the experiment (P < 0.05). The aMT6s level remained consistently low until the end of the experiment, but not significantly lower than the control values. The results of the urine samples biochemical workup failed to reveal any significant differences between the exposed and the control animal groups. The results of this study suggest that, under the above described experimental conditions, repeated 2.45 GHz irradiation could act as a stressor and therefore influence the melatonine balance in rat.

Bifurcated degradative pathway of 3-sulfolactate in Roseovarius nubinhibens ISM via sulfoacetaldehyde acetyltransferase and (S)-cysteate sulfolyase.

Data from the genome sequence of the aerobic, marine bacterium Roseovarius nubinhibens ISM were interpreted such that 3-sulfolactate would be degraded as a sole source of carbon and energy for growth via a novel bifurcated pathway including two known desulfonative enzymes, sulfoacetaldehyde acetyltransferase (EC 2.3.3.15) (Xsc) and cysteate sulfo-lyase (EC 4.4.1.25) (CuyA). Strain ISM utilized sulfolactate quantitatively with stoichiometric excretion of the sulfonate sulfur as sulfate. A combination of enzyme assays, analytical chemistry, enzyme purification, peptide mass fingerprinting, and reverse transcription-PCR data supported the presence of an inducible, tripartite sulfolactate uptake system (SlcHFG), and a membrane-bound sulfolactate dehydrogenase (SlcD) which generated 3-sulfopyruvate, the point of bifurcation. 3-Sulfopyruvate was in part decarboxylated by 3-sulfopyruvate decarboxylase (EC 4.1.1.79) (ComDE), which was purified. The sulfoacetaldehyde that was formed was desulfonated by Xsc, which was identified, and the acetyl phosphate was converted to acetyl-coenzyme A by phosphate acetyltransferase (Pta). The other portion of the 3-sulfopyruvate was transaminated to (S)-cysteate, which was desulfonated by CuyA, which was identified. The sulfite that was formed was presumably exported by CuyZ (TC 9.B.7.1.1 in the transport classification system), and a periplasmic sulfite dehydrogenase is presumed. Bioinformatic analyses indicated that transporter SlcHFG is rare but that SlcD is involved in three different combinations of pathways, the bifurcated pathway shown here, via CuyA alone, and via Xsc alone. This novel pathway involves ComDE in biodegradation, whereas it was discovered in the biosynthesis of coenzyme M. The different pathways of desulfonation of sulfolactate presumably represent final steps in the biodegradation of sulfoquinovose (and exudates derived from it) in marine and aquatic environments.

CORTISONE and the metabolic response to injury.

THE pattern of the metabolic response of man to injury (for example, fracture of a long bone), in particular its effect on protein catabolism, has been under investigation for some time by one of us1 as this phenomenon is of basic importance to our understanding of protein metabolism in general. The reaction is characterized in man by a marked increase in the urinary excretion of nitrogen and sulphur, which reaches a peak from the fourth to the eighth day after the injury and then slowly declines. There is also a loss of phosphorus and potassium. This phenomenon has been confirmed by many workers and in other forms of injury2. Cuthbertson, McGirr and Robertson3 have also found the same thing to occur in the rat, though rather earlier. The losses appear to be more general than local in origin.

Taurine

Taurine, a sulfur-containing amino acid (Figure), has been termed a functional nutrient that could be used to protect against, among others, diabetes mellitus and atherosclerosis.1 Indeed, an increasing body of literature supports the use of taurine supplements. Because taurine has very diverse functions, notably, intracellular osmoregulation and bile acid formation, and is abundantly present in several organs, multiple pathways could be involved. Some of these are discussed in this editorial. Figure. A simplified biosynthesis pathway from methionine to taurine. Methionine is converted to homocysteine in 3 steps. Homocysteine is used as a substrate to form cysteine in 2 steps but can also be converted back to methionine by 2 different mechanisms. Cysteine, precursor for the important antioxidant glutathione and the gaseous transmitter/signaling molecule H2S, can be further converted to cysteine sulfinate. Cysteine sulfinate is converted to taurine in 2 steps via 2 different pathways. Taurine can decrease methionine by decreasing uptake. Whether taurine decreases homocysteine directly or via a reduction of methionine is unclear. Of the 20 canonical amino acids, 18 are composed of carbon, hydrogen, nitrogen, and oxygen only. The remaining 2, methionine and cysteine, also contain 1 atom of sulfur. Because sulfur is not as electronegative as oxygen, the sulfur-containing amino acids play a key role in protein structure and synthesis.2 Methionine and cysteine also play important roles in cell metabolism. For instance, methionine serves as a substrate for S-adenosylmethionine, which is vital for methylation of nucleic acids, proteins, lipids, etc. In proteins, cysteine easily forms double bonds with other cysteine residues, thus determining tertiary structure and binding sites. Moreover, cysteine is substrate for glutathione, an important intracellular antioxidant, and H2S, a gas that can induce endothelial-dependent relaxation.3 Cysteine is considered a nonessential amino acid because it is synthesized …

123. Construction & characterization of mutation-specific mouse models for Types A and B Niemann–Pick disease

due to deficiency of N-acetyl-galactosamine-6-sulphatase (MPS IVA) or beta-galactosidase (MPS IVB). The aim of this study was to describe the Argentinean experience in first assessment and biochemical diagnosis of the disease. Results: Of a total of 36 patients, MPS IVA was diagnosed in 33 (92%) and MPS IV B in 3 (8%) through enzymatic assay. All but one (3%) had abnormal urinary excretion of keratan sulphate in urine. The referring physician speciality was: paediatrician 36%, geneticist 36%, pediatric neurologist 21% and orthopedistt 7%. At the time of diagnosis, disostosis was present in all the patients 100%, liver and spleen enlargement in 16% and corneal clouding in 13%. Median age at the diagnosis was 3.5 years old. Conclusions: At our knowledge, this is one of the largest populations of MPS IV 4s patients. Detailed reports on this pathology would probably allow its earlier recognition and a better therapeutic approach.

Methionine-Adequate Cysteine-Free Diet Does Not Limit Erythrocyte Glutathione Synthesis in Young Healthy Adult Men

Most methods of determining amino acid (AA) requirements are based on endpoints that determine adequacy for protein synthesis. However, the sulfur AA (SAA) cysteine is believed to be the rate-limiting substrate for synthesis of the most abundant intracellular antioxidant, glutathione (GSH). Our objectives were to determine whether supplementation of cysteine in a diet containing adequate SAA for protein synthesis, as methionine, increased GSH synthesis by measuring the fractional and absolute synthesis rates, and if concentration of GSH changed in response to feeding 5 graded intakes of cysteine (0, 10, 20, 30, and 40 mg·kg−1·d−1) in a random order with a fixed methionine intake of 14 mg·kg−1·d−1 and a protein intake of 1 g·kg−1·d−1. Each subject received a multivitamin and choline supplement during the study. Four healthy adult men each underwent 5 isotope infusion studies of 7-h duration after a 2-d adaptation to the level of cysteine intake being studied on the isotope infusion day. The isotope used was [U-13C2 −15N]glycine. Analyses included erythrocyte GSH synthesis rates and concentration and urinary sulfate excretion. The GSH synthesis rates and concentration, measured at a methionine intake of 14 mg·kg−1·d−1, did not change with increasing intakes of cysteine. Urinary sulfate excretion showed a significant positive relationship with cysteine intake (r = 0.92; P < 0.01). In conclusion, this study provides preliminary evidence that consumption of SAA adequate to meet the requirement for protein synthesis does not limit GSH synthesis in healthy adult men receiving an otherwise adequate diet.

The effects of milk as a food matrix for polyphenols on the excretion profile of cocoa ( − )-epicatechin metabolites in healthy human subjects

The effect of different food matrices on the metabolism and excretion of polyphenols is uncertain. The objective of the study was to evaluate the possible effect of milk on the excretion of (2)-epicatechin metabolites from cocoa powder after its ingestion with and without milk. Twenty-one volunteers received the following three test meals each in a randomised cross-over design with a 1-week interval between meals: (1) 250 ml whole milk as a control; (2) 40 g cocoa powder dissolved in 250 ml whole milk (CC-M); (3) 40 g cocoa powder dissolved in 250 ml water (CC-W). Urine was collected before consumption and during the 0-6, 6-12 and 12-24 h periods after consumption. (2)-Epicatechin metabolite excretion was measured using liquid chromatography-MS. One (2)-epicatechin glucuronide and three (2)-epicatechin sulfates were detected in urine excreted after the intake of the two cocoa beverages (CC-M and CC-W). The results show that milk does not significantly affect the total amount of metabolites excreted in urine. However, differences in metabolite excretion profiles were observed; there were changes in the glucuronide and sulfate excretion rates, and the sulfation position between the period of excretion and the matrix. The matrix in which polyphenols are consumed can affect their metabolism and excretion, and this may affect their biological activity. Thus, more studies are needed to evaluate the effect of these different metabolite profiles on the body.

7008 POSTER Locally generated elastin peptides increase invasive potential of melanoma cells dominantly by galectin-3

GM1-gangliosidosis is a lysosomal storage disorder caused by acid β-galactosidase deficiency. Aside from the lysosomal β-galactosidase enzyme, the β-galactosidase gene also encodes the elastin-binding protein (EBP), deficiency in which impairs elastogenesis. Using expression studies and Western blots of COS-1 cells, we identified and characterized four new and two known β-galactosidase gene mutations detected in GM1-gangliosidosis patients with infantile, juvenile, or adult forms of disease. We then focused on impaired elastogenesis detected in fibroblasts from patients with infantile and juvenile disease. The juvenile patient showed connective-tissue abnormalities, unusual urinary keratan sulfate excretion, and an EBP reduction, despite mutations affecting only β-galactosidase. Because galactosugar-bearing moieties may alter EBP function and impair elastogenesis, we assessed infantile and juvenile patients for the source of altered elastogenesis. We confirmed that the infantile patient's impaired elastogenesis arose from a primary EBP defect, according to molecular analysis. We examined the juvenile's fibroblasts by immunohistochemistry, addition of keratanase, soluble/insoluble elastin assay, and radiolabeling of tropoelastin. These experiments revealed that the juvenile's impaired elastogenesis likely arose from secondary EBP deficiency caused by keratan sulfate accumulation. Thus, impaired elastogenesis in GM1-gangliosidosis can arise from primary or secondary EBP defects in fibroblasts from infantile and juvenile patients, respectively.