Application of gas chromatography–mass spectrometry and gas chromatography–tandem mass spectrometry to assess in vivo synthesis of prostaglandins, thromboxane, leukotrienes, isoprostanes and related compounds in humans

Work on the structure of prostaglandin E1 (PGE1), isolated from natural sources, was completed 25 years ago (1). Shortly after, methods for the chemical synthesis of PG with their natural configuration were developed in the laboratories of the UpJohn Company (2) and of E. J. Corey (3) and, by the late sixties, PGE1 became widely available. The information since accumulated about its biological and clinical effects is more substantial than for any other PG. This review will draw together some of this information, focusing on recent studies of its mechanisms of action.

Prostaglandin E1: A review

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 parathyroidhormone,prostaglandinE2(PGE2)andPGE1,bradykinin-inducedproductionofPGE2).Itwasfoundthat 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 bindingof 3 H-bradykinintoitsreceptorandalsocorrelatedwellwiththebradykinin-inducedsignaltransduction 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-likecelllineMC3T3-E1issynonymousformultiplesublines,representingdifferentstagesofosteoblast 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. (J Bone Miner Res 1997; 12:541‐551)

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

An improved method for the quantitative measurement of the angiotensin-converting enzyme inhibitor lisinopril in human plasma is presented. The assay is based on gas chromatography/negative ion chemical ionization mass spectrometry. The method involves solid phase extraction on C18 sorbent and derivatization to the pentafluorobenzyl diester trifluoroacetamide derivatives.

An improved method for the measurement of the angiotensin-converting enzyme inhibitor lisinopril in human plasma by stable isotope dilution gas chromatography/negative ion chemical ionization mass spectrometry.

Extracellular Ca2+ sensing by the osteoblast-like cell line, MC3T3-E1

A method for measurement of PGF2 alpha, PGE1, PGE2, 6-keto-PGF1 alpha, TXB2, 2,3-dinor-TXB2 as well as 5-, 8-, 9-, 11-, 12-, 15-HETE and HHT, utilizing negative ion chemical ionization GC/MS is presented. A highly efficient separation and purification procedure prior to the derivatization sequence allows quantification of the arachidonic acid metabolites described in two GC/MS runs. The detection limit was in the femtomole range. Application of the method to the quantitative profiling of arachidonic acid metabolites in various tissues and incubation media is demonstrated.

Measurement of prostaglandins, thromboxanes and hydroxy fatty acids by stable isotope dilution gas chromatography/mass spectrometry.

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Measurement of prostaglandins, thromboxanes and hydroxy fatty acids by stable isotope dilution gas chromatography/mass spectrometry.