In Vitro Biotransformations of Isocupressic Acid by Cow Rumen Preparations:  Formation of Agathic and Dihydroagathic Acids

Abstract

Isocupressic acid [15-hydroxylabda-8(17), 13E-dien-19-oic acid] (1) was incubated under anaerobic conditions for 48 h in an in vitro ruminal fluid mixture and was transformed into two metabolites. The two metabolites were identified by GC/MS as agathic acid [labda-8(17),13(E)-diene-15,19-dioic acid] (4E) and dihydroagathic acid [labda-8(17)-ene-15,19-dioic acid] (6). Metabolite identities were confirmed by chemical conversions of isocupressic acid (1) and imbricataloic acid (5) into 4E and 6, respectively. Structures of synthetic metabolites were confirmed by 1 H and 13 C NMR, specific rotation, GC/MS, and high-resolution mass spectrometry. Plasma obtained from cows that were fed Ponderosa pine needles contained (13R,S)-dihydroagathic acid (6) but not isocupressic acid (1) or 4E. The results suggest that isocupressic acid (1) is metabolically oxidized to agathic acid (4E), subsequently reduced to (13R,S)-dihydroagathic acid (6) in the rumen, and then absorbed into the bloodstream of cattle.Premature parturition in cattle due to the ingestion of Ponderosa pine [Pinus ponderosa Dougl. ex Laws (Pinaceae)] needles is a major economic problem in the Western United States and Canada. 1 Previous investigations have attempted to isolate and characterize the compounds in P. ponderosa responsible for pine needle abortion. A new class of vasoactive lipids 2 and isocupressic acid 3 [15-hydroxylabda-8(17),13(E)-dien-19-oic acid] (1) were identified as possible causative agents in pine needle abortion in pregnant cows. However, the effects of pine needle ingestion on domestic ruminants such as cattle, Bison, sheep, and goats are variable with regard to the parturition response and/or the presence of vasoconstrictive principles present in plasma samples. [4][5][6] The species differences observed in the pine needle abortion phenomenon may result from variability in the differences in digestive systems among species, from differences of possible metabolic transformations of ingested precursor abortifacient compounds, or from a combination of these factors.Ruminants, including cattle, possess a four-compartment stomach. The largest compartment, the rumen, contains a mixed and variable population of symbiotic microorganisms effective in fermenting fibrous feedstuffs into nutrients usable by the host animal. The environment in the rumen is generally anaerobic, neutral to slightly acidic (between pH 5.5-7.0), and at a temperature between 38 and 41°C. During digestion, the rumen microflora cooperatively catalyze enzymatic transformations of nutrients, fiber, and other ingested substances. Digested materials ultimately pass into the bloodstream of the ruminant animal where they are broadly distributed, bound, further metabolized, and ultimately excreted.The mechanism whereby pine-needle ingestion induces abortion in cattle has been shown to involve a reduction in blood flow to the gravid uterus, resulting in a marked and progressive decrease in nutrients and oxygen available to the fetal calf. 4,7 This is consistent with the observa...

Microbial Transformations of Isocupressic Acid

“…Signals for protons and carbons of isocupressic acid (1) were assigned initially by HMBC and HMQC NMR spectral analyses. 12,20,21 N. aurantia gave one major metabolite, which was identified as 2. The preparative-scale incubation with 200 mg of isocupressic acid (1) was terminated after 144 h. Solvent extraction and column chromatography afforded 36 mg (18%) of 2 for spectral analysis.…”

“…Interestingly, the side chain of isocupressic acid was also the target of metabolic transformation by a consortium of bacterial species in cow ruminal fluid incubated under anaerobic conditions. 12 Both of the fungi examined here yielded hydroxylated metabolites of isocupressic acid (1). Metabolites 3 and 5 are simple hydroxylated metabolites likely formed by activation and insertion of molecular oxygen into the respective 7 -and 2R-positions of 1.…”

“…Isocupressic acid (1) was obtained as previously reported. 12 Microorganisms, Media, and Incubation Conditions. Microbial studies were conducted by selecting 31 microorganisms from the culture collection of the College of Pharmacy, University of Iowa.…”

“…However, microbial transformations have been reported for labdane-diterpenes, including sclareol [labd-14-ene-8R,13 -diol] 2-9 and its analogues, 10,11 and with ruminal metabolism of isocupressic acid (1). 12 This investigation utilized microorganisms as in vitro models to prospectively mimic and predict oxidative mammalian biotransformations of isocupressic acid (1) and to produce novel metabolites of the terpene for use as analytical standards for mammalian metabolic studies. The use of selected organisms as "models for xenobiotic metabolism" 13,14 has been successfully exploited in mammalian metabolism studies of many classes of biologically active natural and synthetic compounds.…”

Implication of agathic acid from Utah juniper bark as an abortifacient compound in cattle

“…Isocupressic acid was found to be metabolized in both the rumen and the liver through oxidative and reductive processes and the major metabolites identifi ed included imbricatoloic acid (2), agathic acid (3), dihydroagathic acid (4) and tetrahydroagathic acid (5) (Lin et al, 1998;. It is not known if these metabolites are biologically active as abortifacients in late-term pregnant cattle.…”