Note

Combined capillary column gas chromatography—mass spectrometric method for the quantitative analysis of urinary prostaglandins

In an isotope dilution assay, prostaglandin (PG) E₂, 6-keto-PGF_1α, thromboxane (Tx) B₂ and their metabolites PGE-M (11α-hydroxy-9,15-dioxo-2,3,4,5,20-pentanor-19-carboxyprostanoic acid), 2,3-dinor-6-keto-PGF_1α, 2,3-dinor-TxB₂ and 11-dehydro-TxB₂ were determined in urine by gas chromatography—triple stage quadrupole mass spectrometry (GC—MS—MS). After addition of deuterated internal standards, the prostaglandins were derivatized to their methoximes and extracted with ethyl acetate—hexane. The sample was further derivatized to the pentafluorobenzylesters and purified by thin-layer chromatography (TLC). Three zones were scraped from the TLC plate. The prostanoid derivatives were converted to their trimethylsilyl ethers and the products were quantified by GC—MS—MS. In each run, two or three prostanoids were determined.

Simultaneous determination of urinary excretion rates of primary unmetabolized prostanoids and their enzymatic metabolites were performed by gas chromatography-mass spectrometry (GC/MS) or tandem mass spectrometry (GC/MS/MS). Changes in kidney function were induced by acute (4 h) volume expansion. Despite marked changes in urine flow, GFR, urinary pH, osmolality, sodium and potassium excretion, only a insignificant or transient rise in the enzymatic prostanoid metabolites (2,3-dinor-6-keto-PGF_1α, PGE-M, 2,3-dinor-TxB₂ and 11-dehydro-TxB₂) was observed. The excretion rates of the primary prostanoids were elevated in parallel with the rise in urine flow: PGE₂ rose (p < 0.05) from 14.2 ± 4.0 to 86.2 ± 20.7, PGF_2α from 60.0 ± 4.9 to 119.8 ± 24.0, 6-keto-PGF_2α from 7.2 ± 1.3 to 51.5 ± 17.0, and txB₂ from 11.2 ± 3.3 to 13.6 ± 3.6 ng/h/1.73 m² ($\text{x}\text{̄}\text{±}\text{SEM}$) at the maximal urine flow. Except for 6-keto-PGF_1α and TxB₂, this rise in urinary prostanoid levels was only transient despite a sustained fourfold elevated urine flow. We conclude that urine flow rate acutely affect urine prostanoid excretion rates, however, over a prolonged peroid of time these effects are not maintained. The present data support the concept that urinary levels of primary prostanoids mainly reflect renal concentrations whereas those of enzymatic metabolites reflect systemic prostanoid activity. From the excretion pattern of TxB₂ one can assume that this prostanoid represents renal as well as systemic TxA₂ activity.

The role of prostaglandins in the development of aminoglycosideinduced acute renal failure was studied in CD-COBS rats (200 to 250 g). The animals were treated with gentamicin (80 mg/kg), acetylsalicylic acid (ASA, 100 or 200 mg/kg), or both drugs or saline for 5 or 10 days. Renal function was studied measuring creatinine clearance, blood urea nitrogen (BUN), and serum electrolytes, urine osmolality, and maximal urinary concentrating capacity after water deprivation and vasopressin administration. Gentamicin toxicity on the proximal tubule was evaluated by measuring urinary excretion of the lysosomal enzyme N-acetylglucosaminidase (NAG). Renal prostaglandin (PG) production was evaluated measuring the concentration of PGE₂, PGD₂, PGF_2α, 6-keto-PGF_1α, and thromboxane B₂ (TXB₂) in whole renal homogenate after a 15-min incubation at 37°C using gas chromatographymass spectrometry. Gentamicin alone reduced the glomerular filtration rate (GFR) 20 to 30% after 5 and 10 days of treatment. Combination with ASA protentiated the toxic effect of the aminoglycoside after 10 but not after 5 days of treatment. Similarly, gentamicin reduced the urinary concentrating capacity and addition of ASA worsened the effects. Gentamicin markedly increased NAG excretion but this effect was reduced by ASA, probably as a result of lysosomal stabilization. ASA alone inhibited the production of prostaglandins in renal tissue by 70 to 90% after single or multiple doses. The animals treated with gentamicin alone presented a significant, specific increase in PGE₂ production after 10 days of treatment but this increase did not occur when the two compounds were given together. Since PGE₂ has a vasodilatory effect in the kidney these results suggest that it may play a specific role in maintaining normal renal blood flow and GFR during the development of aminoglycoside nephrotoxicity. The inhibition of prostaglandin production by nonsteroid anti-inflammatory drugs prevents this compensatory mechanism and worsens the renal damage.

Radioimmunoassay (RIA) and high-resolution gas chromatography-mass spectrometry (HRGC-MS) were compared for the determination of serum 6-keto-PGF-_1α and TXB₂ in a situation of drug-altered arachidonate metabolism. Results were comparable for TXB₂ in both conditions. 6-keto-PGF_1α RIA determinations (with two different antisera) revealed increased levels in dazoxiben-treated samples, which were not confirmed by HRGC-MS. The assay were repeatedly checked in controlled conditions to investigate these discrepancies. Interference was found with both antisera, due to the drug-induced change in metabolism.

This chapter discusses the role of contemporary gas chromatography (GC) in biochemistry. The ionization detectors used in GC possess unparalleled measurement sensitivity, and are the major reason for using GC in biochemical and biomedical investigations. The flame ionization detector has been extremely useful as a sensitive, universal detector, and there are numerous analytical problems where detection selectivity is required. A mass spectrometer employed in a mass-fragmentographic mode provides almost the ultimate in both selectivity and high sensitivity. The use of other selective devices, such as the electron capture, thermionic or flame-photometric detectors, has been steadily on the increase. Some of these detectors were primarily developed to overcome the problems of sample complexity, and an increasing use of capillary GC does not appear to diminish their importance. On the contrary, the columns of greater resolving power and higher degree of inertness further expand the utility of such detectors. The most frequent use of selective detectors in biochemical analysis is associated with the formation of derivatives that are particularly suitable for such detection.