Quantification of phenolic acid metabolites in humans by LC-MS: a structural and targeted metabolomics approach.

AIM: Co-metabolism between a human host and the gastrointestinal microbiota generates many small phenolic molecules such as 3-hydroxy-3-(3-hydroxyphenyl)propanoic acid (3,3-HPHPA), which are reported to be elevated in schizophrenia and autism. Characterization of these chemicals, however, has been limited by analytic challenges. METHODOLOGY/RESULTS: We applied HPLC to separate and quantify over 50 analytes, including multiple structural isomers of 3,3-HPHPA in human cerebrospinal fluid, serum and urine. Confirmation of identity was provided by NMR, by MS and other detection methods. The highly selective methods support rapid quantification of multiple metabolites and exhibit superior chromatographic behavior. CONCLUSION: An improved ultra-HPLC-MS/MS and structural approaches can accurately quantify 3,3-HPHPA and related analytes in human biological matrices.

Detection of salicylate and its hydroxylated adducts 2,3- and 2,5-dihydroxybenzoic acids as possible indices for in vivo hydroxyl radical formation in combination with catechol- and indoleamines and their metabolites in cerebrospinal fluid and brain tissue.

It has been suggested that salicylate (SA) hydroxylation can be used to detect hydroxyl radical formation in vivo. Here we describe a rapid and sensitive HPLC method using ultraviolet absorbance (UV) and electrochemical detection (EC) to detect SA (UV), its hydroxylated adducts 2,3- and 2,5-dihydroxybenzoic acids (DHBA) and catechol in combination with catechol- and indoleamines and related metabolites (EC) in one isocratic run. These compounds were measured in acidified cerebrospinal fluid (CSF) and perchlorate extracts of striatal tissues of untreated and SA-loaded rats (300 mg/kg SA, i.p.). Peaks were identified by comparing retention times of samples and standards, by adding standards to biological samples, by voltamograms, and by comparing chromatograms of manganese (Mn2+)-injected striata of SA-loaded rats with several control conditions. Six hours after unilateral injection of 0.4 mumol Mn2+ into striatum, 2,3-DHBA and 2,5-DHBA levels in striatum were respectively 4- and 7-fold increased as compared to non-injected (contralateral) striata, suggesting in vivo hydroxyl radical formation. In addition, dopamine and serotonin levels were depleted in Mn(2+)-injected striata by 46% and 64%, respectively. In CSF of Mn(2+)-injected rats, DHBA/SA ratios were not significantly changed as compared to those of control rats. In conclusion, the described technique can be applied to study in vivo hydroxyl radical formation in direct relation with dopaminergic and serotonergic neurotransmitter changes during neurotoxic processes.

On the occurence of vanillic acid in human brain and cerebrospinal fluid.

3-methoxy-4-hydroxyphenylbenzoic (vanillic) acid was previously shown to be one of the endogenous metabolites of adrenaline and noradrenaline. Using thin-layer chromatographic methods for identification and quantification of phenolic acids and phenolic alcohols, the authors identified vanillic acid in different regions of the human brain. The concentration of vanillic acid in the cerebrospinal fluid was also determined and compared to the concentration of 3-methoxy-4-hydroxyphenylethylene glycol. The identification of VA in the human brain suggests that the vanillic acid of the cerebrospinal fluid originates, at least in part, from the catecholamines in the brain. The authors discuss other possible origins of vanillic acid besides the noradrenaline catabolism of dopamine. As the concentration of vanillic acid in the cerebrospinal fluid was found to be greater than the concentration of 3-methoxy-4-hydroxyphenylethylene glycol, it might be important for clinical biological studies to measure vanillic acid in the cerebrospinal fluid as well as the other alcoholic and acid catabolites of the catecholamines.

U74389G prevents vasospasm after subarachnoid hemorrhage in dogs.

OBJECTIVE: Oxygen-derived free radicals may contribute to vasospasm after the rupture of an intracranial aneurysm through direct vasoconstricting effects occurring within the arterial wall or, secondarily, by causing lipid peroxidation in the subarachnoid erythrocytes with secondary induction of vasoconstriction. U74389G is a potent inhibitor of lipid peroxidation and a scavenger of oxygen-derived free radicals. This study determined the relative contributions of oxygen-derived free radicals and lipid peroxidation to vasospasm in the double-hemorrhage dog model. METHODS: Sixteen dogs underwent baseline (Day 0) cerebral angiography and induction of subarachnoid hemorrhage by two injections of blood into the cisterna magna 2 days apart. They were randomized to receive drug vehicle (n=8) or U74389G (n=8, 3 mg/kg of body weight/d) intravenously. Drug administration and end point analysis were blinded. The end points were angiographic vasospasm, as assessed by comparison of angiograms obtained before and 7 days after subarachnoid hemorrhage, and the levels of malondialdehyde and salicylate hydroxylation products (dihydroxybenzoic acids) in cerebrospinal fluid and of malondialdehyde in subarachnoid blood clots and basilar arteries 7 days after hemorrhage. RESULTS: Comparisons within groups of Day 0 and Day 7 angiograms and between groups of angiograms obtained at Day 7, showed significant vasospasm in animals in the vehicle group (mean+/-standard error, 51%+/-4) but not in the U74389G group (25%+/-11, P < 0.05, unpaired t test). High-pressure liquid chromatographic assays of malondialdehyde and dihydroxybenzoic acids in cerebrospinal fluid, subarachnoid blood clots, and basilar arteries showed no significant differences between groups. CONCLUSION: The significant prevention of vasospasm by U74389G without change in levels of indicators of free radical reactions suggests that the effect of the drug is related to other processes occurring in the arterial wall and that cerebrospinal fluid levels of oxygen radicals and lipid peroxides are not useful markers of vasospasm.