Metabonomic approaches are believed to have the capability of revolutionizing diagnosis of diseases and assessment of patient conditions after medical interventions. In order to ensure comparability of metabonomic 1H NMR data from different studies, we suggest validated sample preparation guidelines for human urine based on a stability study that evaluates effects of storage time and temperature, freeze-drying, and the presence of preservatives. The results indicated that human urine samples should be stored at or below -25 degrees C, as no changes in the 1H NMR fingerprints have been observed during storage at this temperature for 26 weeks. Formation of acetate, presumably due to microbial contamination, was occasionally observed in samples stored at 4 degrees C without addition of a preservative. Addition of a preserving agent is not mandatory provided that the samples are stored at -25 degrees C. Thus, no differences were observed between 1H NMR spectra of nonpreserved urines and urines with added sodium azide and stored at -25 degrees C, whereas the presence of sodium fluoride caused a shift of especially citrate resonances. Freeze-drying of urine and reconstitution in D2O at pH 7.4 resulted in the disappearance of the creatinine CH2 signal at delta 4.06 due to deuteration. A study evaluating the effects of phosphate buffer concentration on signal variability and assessment of the probability of citrate or creatinine resonances crossing bucket border (a boundary between adjacent integrated regions) led to the conclusion that a minimum buffer concentration of 0.3 M is adequate for normal urines used in this study. However, final buffer concentration of 1 M will be required for very concentrated urines.

1. Sixteen naturally occurring flavonoids were investigated as substrates for cytochrome P450 in uninduced and Aroclor 1254-induced rat liver microsomes. Naringenin, hesperetin, chrysin, apigenin, tangeretin, kaempferol, galangin and tamarixetin were all metabolized extensively by induced rat liver microsomes but only to a minor extent by uninduced microsomes. No metabolites were detected from eriodictyol, taxifolin, luteolin, quercetin, myricetin, fisetin, morin or isorhamnetin. 2. The identity of the metabolites was elucidated using lc-ms and 1H-nmr, and was consistent with a general metabolic pathway leading to the corresponding 3',4'-dihydroxylated flavonoids either by hydroxylation or demethylation. Structural requirements for microsomal hydroxylation appeared to be a single or no hydroxy group on the B-ring of the flavan nucleus. The presence of two or more hydroxy groups on the B-ring seemed to prevent further hydroxylation. The results indicate that demethylation only occurs in the B-ring when the methoxy group is positioned at C4', and not at the C3'-position. 3. The CYP1A isozymes were found to be the main enzymes involved in flavonoid hydroxylation, whereas other cytochrome P450 isozymes seem to be involved in flavonoid demethylation.

Bulk electrolysis of the antioxidant flavonoids quercetin and kaempferol in acetonitrile both yield a single oxidation product in two-electron processes. The oxidation products are more polar than their parent compounds, with an increased molecular weight of 16g/mol, and were identified as 2-(3,4-dihydroxybenzoyl)-2,4,6-trihydroxy-3(2H)-benzofuranone and 2-(4-hydroxybenzoyl)-2,4,6-trihydroxy-3(2H)-benzofuranone for quercetin and kaempferol, respectively. Two-electron oxidation of the parent flavonoid is suggested to yield a 3,4-flavandione with unchanged substitution pattern in the A- and B-ring, which may rearrange to form the substituted 3(2H)-benzofuranone through the chalcan-trione ring-chain tautomer. The acidity of the 3-OH group is suggested to determine the fate of the flavonoid phenoxyl radical, originally formed by one-electron oxidation, as no well-defined oxidation product of luteolin (lacking the 3-OH group) could be isolated despite rather similar half-peak potentials: Ep/2 = 0.97V, 0.98 V and 1.17 V vs. NHE for quercetin, kaempferol and luteolin, respectively, as measured by cyclic voltammetry in acetonitrile.

The mitochondrial pH gradient across the inner-membrane is stabilised by buffering of the matrix. A low-molecular mass buffer compound has to be localised in the matrix to maintain its alkaline pH value. Taurine is found ubiquitously in animal cells with concentrations in the millimolar range and its pKa value is determined to 9.0 (25°C) and 8.6 (37°C), respectively. Localisation of such a low-molecular buffer in the mitochondrial matrix, transforms the matrix into a biochemical reaction chamber for the important matrix-localised enzyme systems. Three acyl-CoA dehydrogenase enzymes, which are pivotal for beta-oxidation of fatty acids, are demonstrated to have optimal activity in a taurine buffer. By application of the model presented, taurine depletion caused by hyperglycemia could provide a link between mitochondrial dysfunction and diabetes.

We have determined the absorption, conjugation and excretion of naringenin-7-O-rutinoside (narirutin) compared to the corresponding glucoside in an orange juice matrix in human subjects. Healthy volunteers (eight men and eight women), in a double blind, randomised, crossover study, consumed orange juice with (1) natural content of naringenin-7-O-rutinoside; (2) a-rhamnosidase-treated to yield naringenin-7-O-glucoside. Blood was sampled at twelve time points and three fractions of urine were collected over 24 h. The area under the plasma-time curve of naringenin from (2) a-rhamnosidase-treated orange juice was increased about 4-fold (P,0·0001), peak plasma concentration (C max ) was 5·4-fold higher (P,0·0001) and T max was decreased from 311 to 92 min (P¼ 0·002) compared to untreated orange juice (1), indicating a change in absorption site from the colon to the small intestine. Furthermore, the amount in urine was increased from 7 to 47 % (P, 0·0001) of the dose after consumption of the a-rhamnosidase-treated orange juice (2). All urine samples contained both naringenin-7-and -4 0 -O-glucuronides. In addition, to examine the effect of dose and a-rhamnosidase treatment on hesperetin conjugate profiles, a further treatment where (3) orange juice fortified with three times the original content of hesperetin-7-O-rutinoside was used. Five hesperetin metabolites (3 0 -O-glucuronide; 7-O-glucuronide; 5,7-O-diglucuronide; 3 0 ,7-O-diglucuronide; 3 0 -O-sulphate) were present after all treatments (1-3), with the same profile of the conjugates. The present data show that bioavailability of naringenin is increased by conversion from rutinoside to glucoside, but the profile of the conjugates of flavanones formed and excreted in urine is neither affected by the absorption site nor by a 3-fold change in dose.