ABSTRACT
OBJECTIVE: Histamine plays a role in several (patho) physiological processes that are commonly studied in mouse models. However, a systematic quantification of histamine and its metabolite N-methylhistamine in mouse organs has not been reported so far. METHODS: Balb/c and C57Bl/6 mice were grouped according to their sex and age. Brains, hearts, lungs, livers, kidneys, stomachs, intestines, thymi, spleens, and lymph nodes were excised, weighed, and homogenized. Histamine and N-methylhistamine were quantified simultaneously by a HPLC-mass spectrometry method. RESULTS: In all organs analyzed, histamine and N-methylhistamine were detected; however, with quantitative differences. Histamine was present most abundantly in the stomach, lymph nodes, and thymus. The lowest histamine concentrations were detected in brain, liver, lung, and intestine. In most organs, the histamine concentrations increased age-dependently. Substantial concentrations of N-methylhistamine were detected only in lung, intestine and kidney, while in all other organs it was present only in minor quantities. CONCLUSION: HPLC-mass spectrometry is a useful method for the highly sensitive and simultaneous detection of histamine and N-methylhistamine. Histamine is present in virtually all organs, not only in those traditionally associated with histamine-mediated disease. Highest concentrations are found in stomach, lymph node, and thymus; medium concentrations in heart, spleen, and kidney; and lowest concentrations detected in intestine, brain, liver, and lung.
Subject(s)
Histamine/analysis , Methylhistamines/analysis , Mice, Inbred BALB C , Mice, Inbred C57BL , Animals , Brain Chemistry , Chromatography, High Pressure Liquid , Female , Intestines/chemistry , Kidney/chemistry , Liver/chemistry , Lung/chemistry , Lymph Nodes/chemistry , Male , Mice , Myocardium/chemistry , Spleen/chemistry , Stomach/chemistry , Tandem Mass Spectrometry , Thymus Gland/chemistryABSTRACT
The macrocyclic polyketides FK506, FK520, and rapamycin are potent immunosuppressants that prevent T-cell proliferation through initial binding to the immunophilin FKBP12. Analogs of these molecules are of considerable interest as therapeutics in both metastatic and inflammatory disease. For these polyketides the starter unit for chain assembly is (4R,5R)-4,5-dihydroxycyclohex-1-enecarboxylic acid derived from the shikimate pathway. We show here that the first committed step in its formation is hydrolysis of chorismate to form (4R,5R)-4,5-dihydroxycyclohexa-1,5-dienecarboxylic acid. This chorismatase activity is encoded by fkbO in the FK506 and FK520 biosynthetic gene clusters, and by rapK in the rapamycin gene cluster of Streptomyces hygroscopicus. Purified recombinant FkbO (from FK520) efficiently catalyzed the chorismatase reaction in vitro, as judged by HPLC-MS and NMR analysis. Complementation using fkbO from either the FK506 or the FK520 gene cluster of a strain of S. hygroscopicus specifically deleted in rapK (BIOT-4010) restored rapamycin production, as did supplementation with (4R,5R)-4,5-dihydroxycyclohexa-1,5-dienecarboxylic acid. Although BIOT-4010 produced no rapamycin, it did produce low levels of BC325, a rapamycin analog containing a 3-hydroxybenzoate starter unit. This led us to identify the rapK homolog hyg5 as encoding a chorismatase/3-hydroxybenzoate synthase. Similar enzymes in other bacteria include the product of the bra8 gene from the pathway to the terpenoid natural product brasilicardin. Expression of either hyg5 or bra8 in BIOT-4010 led to increased levels of BC325. Also, purified Hyg5 catalyzed the predicted conversion of chorismate into 3-hydroxybenzoate. FkbO, RapK, Hyg5, and Bra8 are thus founder members of a previously unrecognized family of enzymes acting on chorismate.
