H. Gleispach

Characterization of lipoxygenase metabolites of arachidonic acid in cultured human skin fibroblasts

Cultured human skin fibroblasts were incubated in the presence of [14C]arachidonic acid (50 microM; 1 m Ci/mmol) and the divalent cation ionophore A23187 at 37 degrees C for 60 min. The metabolites formed were extracted from the cell-free medium in diethyl ether, separated by thin layer chromatography and identified unequivocally by GC-MS. The distribution of the arachidonic acid metabolites as estimated from the recovered radioactivity showed as major product prostaglandin E2 (26%). Minor amounts of other prostaglandins, i.e., 6-oxo-prostaglandin F1 alpha (1%), prostaglandin F2 alpha (1%), prostaglandin D2 (0.5%) and prostaglandin A2 (1%) were also present. In addition to the prostaglandins, monohydroxy fatty acids (4.5%) were also detected. This fraction contained 33% 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT), 22% 11-hydroxy-5,8,11,14-eicosatetraenoic acid (11-HETE) and 31% 15-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE). Lipid extracts of the cells did not show any detectable amount of the monohydroxy fatty acids, indicating that they are not incorporated metabolically in the cellular lipids. The monohydroxy fatty acids originate mainly from the exogenously added arachidonic acid as evidenced by the 2H/H ratio (30:1) from experiments with octadeuterated arachidonic acid [( 2H8]arachidonic acid). Indomethacin inhibited the formation of all prostaglandins, HHT and 11-HETE; moreover, eicosatetraynoic acid (also blocked the formation of 15-HETE. From these results, it can be concluded that in human skin fibroblasts prostaglandin E2 is the major product of the cycloxygenase pathway, while 15-HETE is the main lipoxygenase product.

Gamma interferon inhibits basal and interleukin 1-induced prostaglandin production and bone resorption in neonatal mouse calvaria

Production of the osteolytic arachidonic acid metabolites, prostaglandin (PG) E2, PGI2 and PGF2 alpha, by neonatal mouse calvariae was quantitated by gas chromatography/mass spectrometry. Mouse recombinant interleukin 1 (rIL-1) raised medium levels of PGE2 and PGI2 (measured as 6-keto-PGF1 alpha) in the dose range tested (1.0-10.0 U/ml culture medium), while an effect on PGF2 was only observed at 10 U/ml. Bone resorption in response to rIL-1 reached a plateau at 3.0 U/ml. Mouse recombinant gamma-interferon (rIFN-gamma) between 100-500 U/ml suppressed basal PG synthesis and spontaneous resorption of cultured bone. In addition, IFN-gamma at 100 U/ml prevented stimulation of PG synthesis by 3.0 U/ml rIL-1 and thereby reduced the bone resorbing activity of the cytokine by at least 60%. 5 X 10(-7) M indomethacin was equally effective in suppression of PG synthesis and bone resorption. The present study provides evidence that IFN-gamma inhibits PG synthesis and consequently resorption of cultured bone.

Possible inhibitory function of endogenous 15-hydroperoxyeicosatetraenoic acid on prostacyclin formation in bovine aortic endothelial cells

Arachidonic acid is metabolized via the cyclooxygenase pathway to several potent compounds that regulate important physiological functions in the cardiovascular system. The proaggregatory and vasoconstrictive thromboxane A2 produced by platelets is opposed in vivo by the antiaggregatory and vasodilating activity of prostacyclin (prostaglandin I2) synthesized by blood vessels. Furthermore, arachidonic acid is metabolized by lipoxygenase enzymes to different isomeric hydroxyeicosatetraenoic acids (HETEs). This metabolic pathway of arachidonic acid was studied in detail in endothelial cells obtained from bovine aortae. It was found that this tissue produced 6-ketoprostaglandin F1 alpha as a major cyclooxygenase metabolite of arachidonic acid, whereas prostaglandins F2 alpha and E2 were synthesized only in small amounts. The monohydroxy fatty acids formed were identified as 15-HETE, 5-HETE, 11-HETE and 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT). The latter two compounds were produced by cyclooxygenase activity. Nordihydroguaiaretic acid (NDGA), a rather selective lipoxygenase inhibitor and antioxidant blocked the synthesis of 15- and 5-HETE. It also strongly stimulated the cyclooxygenase pathway, and particularly the formation of prostacyclin. This could indicate that NDGA might exert its effect on prostacyclin levels by preventing the synthesis of 15-hydroperoxyeicosatetraenoic acid (15-HPETE), a potent inhibitor of prostacyclin synthetase. 15-HPETE could therefore act as an endogenous inhibitor of prostacyclin production in the vessel wall.

Determination of homovanillic acid and vanillylmandelic acid in neuroblastoma screening by stable isotope dilution GC-MS

A method for the quantitative determination of homovanillic acid (HVA) and vanillylmandelic acid (VMA), two metabolites of catecholamines, is presented. The assay is based on gas chromatography/electron impact mass spectrometry. The preparation of 13C-labeled VMA from [13C6]vanillin is described. Together with purchased deuterated HVA the 13C-labeled VMA is used as an internal standard in stable isotope dilution GC/MS. The method involves elution from soaked filter-papers, determination of creatinine content, extraction of HVA and VMA from eluted and urinary samples and derivatization to the di-and tri(trimethylsilyl) derivatives, respectively. The detection limits were found to be 4.0 pg for HVA and 0.8 pg for VMA. The method was applied to the routine determination of urinary HVA and VMA in a range from 5 to 100 ng HVA and VMA per microgram creatinine. The lower limits of pathological concentrations are set at 35 ng micrograms-1 creatinine for HVA and to 20 ng micrograms-1 creatinine for VMA, which are in close correlation with the values from other methods, but with the main advantage of reducing the amount of questionable or elevated results from 6.7% (high-performance liquid chromatography (HPLC) alone) to 0.9% (HPLC and GC/MS).

Phenotypic Heterogeneity of Osteoblast-like MC3T3-E1 Cells: Changes of Bradykinin-Induced Prostaglandin E2 Production During Osteoblast Maturation

We have examined clonal murine calvarial MC3T3‐E1 cells obtained from different sources to compare their osteoblastic features (alkaline phosphatase [ALP], cyclic adenosine monophosphate [cAMP] response to parathyroid hormone, prostaglandin E2 (PGE2) and PGE1, bradykinin‐induced production of PGE2). It was found that the sublines investigated showed large variation of the above‐mentioned parameters, which may be attributed to distinct differentiated stages of osteoblast development. Increase of ALP activity was paralleled by an increase in cAMP accumulation in response to the above‐mentioned agents. The most striking difference was observed with bradykinin‐induced production of PGE2. Early stage cells (low ALP) produced high levels of PGE2, whereas cells with high ALP activity showed no bradykinin stimulation at all. This was consistent with the results of specific binding of3H‐bradykinin to its receptor and also correlated well with the bradykinin‐induced signal transduction sequence (inositol triphosphate liberation and elevation of intracellular calcium levels). This was confirmed by Northern blot analysis of bradykinin receptor mRNA expression. These results indicate that the widely used osteoblast‐like cell line MC3T3‐E1 is synonymous for multiple sublines, representing different stages of osteoblast development. These sublines were most likely emerging from the early stage cell line due to the applied culture conditions. Moreover, distinct biochemical features are displayed in correlation to the differentiation stage, thus providing a useful model to study the molecular mechanism of osteoblast maturation.

Qualitative and quantitative measurement of hydroxy fatty acids, thromboxanes and prostaglandins using stable isotope dilutions and detection by gas chromatography—mass spectrometry

Methods for measurement of the metabolites of arachidonic acid (AA), namely prostaglandins (PGs), thromboxanes (TXs) and hydroxy fatty acids, using stable isotope dilution gas chromatography--mass spectrometry are described. With a few exceptions, labelled species of the various AA metabolites are not commercially available and were therefore synthesized in our laboratory. [2H8]AA, produced by deuteration of eicosatetraynoic acid, was used for comparing the metabolism of exogenously added and endogenously present AA in fibroblast cultures. After derivatization and catalytic hydrogenation, structure elucidation and quantification of the different hydroxy fatty acids was carried out by determination of the fragment ions resulting from alpha-cleavage at the site of the hydroxy function. During catalytic hydrogenation a significant hydrogen--deuterium exchange was observed. To eliminate this problem, 18O-labelled standards were prepared by exchanging the oxygen of the carboxylic acid group. The preparation and the use of hydroxy fatty acids, PGs and TXs labelled with 18O is described.