Preparation of tetrahydroagathic acid: a serum metabolite of isocupressic acid, a cattle abortifacient in ponderosa pine.

Article abstract of DOI:10.1021/jf011501m

Isocupressic acid (1) was used to synthetically prepare a mixture of (8S,13R,S)-labda-15,19-dioic acid (tetrahydroagathic acid) (5) via a two-step oxidation procedure followed by hydrogenation of the double bonds at C13 and C8. Reduction of the C8,17 double bond was stereospecific producing only the 8S isomer and confirmed by the nOe interaction between the resulting C17 and C20 methyl groups. The 13R and 13S isomers of 5 were separated and analyzed by HPLC/MS, and (13S)-tetrahydroagathic acid was isolated and identified by comparison to a standard prepared by hydrogenation of naturally occurring (13S)-dihydroagathic acid (4). (13R,S)-dihydroagathic acid was prepared by selective sodium metal-catalyzed hydrogenation of the C13,14 allylic double bond of agathic acid (3). The prepared compounds were then used as standards to confirm the presence of 4 and 5 and their respective 13R and 13S isomers in bovine serum samples. Tetrahydroagathic acid was shown to be the only metabolite detected in serum samples taken from a suspected cattle abortion case submitted for diagnosis; and, thus, 5 could be a valuable diagnostic marker for pine needle-induced abortions.

Full text of DOI:10.1021/jf011501m

J. Agric. Food Chem. 2002, 50, 2235−2240 2235
Preparation of Tetrahydroagathic Acid: A Serum Metabolite of
Isocupressic Acid, a Cattle Abortifacient in Ponderosa Pine
MASSOUD GARROSSIAN, DALE R. GARDNER,* KIP E. PANTER, AND
LYNN F. JAMES
Poisonous Plant Research Laboratory, Agricultural Research Service, U. S. Department of Agriculture,
1150 East 1400 North, Logan, Utah 84341
Isocupressic acid (1) was used to synthetically prepare a mixture of (8S,13R,S)-labda-15,19-dioic
acid (tetrahydroagathic acid) (5) via a two-step oxidation procedure followed by hydrogenation of the
double bonds at C13 and C8. Reduction of the C8,17 double bond was stereospecific producing
only the 8S isomer and confirmed by the nOe interaction between the resulting C17 and C20 methyl
groups. The 13R and 13S isomers of 5 were separated and analyzed by HPLC/MS, and (13S)-
tetrahydroagathic acid was isolated and identified by comparison to a standard prepared by
hydrogenation of naturally occurring (13S)-dihydroagathic acid (4). (13R,S)-dihydroagathic acid was
prepared by selective sodium metal-catalyzed hydrogenation of the C13,14 allylic double bond of
agathic acid (3). The prepared compounds were then used as standards to confirm the presence of
4 and 5 and their respective 13R and 13S isomers in bovine serum samples. Tetrahydroagathic acid
was shown to be the only metabolite detected in serum samples taken from a suspected cattle abortion
case submitted for diagnosis; and, thus, 5 could be a valuable diagnostic marker for pine needle-
induced abortions.
KEYWORDS: Isocupressic acid; Pinus ponderosa; cattle abortion; diterpene acids; tetrahydroagathic
acid
INTRODUCTION
of rumen and serum samples from both in vitro and in vivo
studies documented the rapid metabolism of isocupressic acid
to at least four compounds via a series of reduction and oxidation
steps (9, 10). The identified metabolites (Figure 1) include
imbricatoloic acid (2), agathic acid (3), dihydroagathic acid (4),
and a fourth compound tentatively identified as tetrahydroagathic
acid (5). The pine needle toxin is thought to increase uterine
vascular tone resulting in decreased placental perfusion and
reduced blood flow to the fetus, subsequently initiating the
parturition process (11-14). Apparently isocupressic acid does
not appear to act directly at the site of the uterine artery muscle
(15), and the specific abortion mechanism caused by isocupressic
acid or its metabolites remains unknown at this time.
Consumption of ponderosa pine (Pinus ponderosa Laws)
needles causes late-term abortions in cattle and is a serious
poisonous plant problem in foothill and mountain rangelands
of the western U.S. (1-4). Pine needle abortion often occurs
during the late winter or early spring when cattle are in their
third trimester of pregnancy. Late winter storms often force
animals into pine tree stands for shelter, increasing their
consumption of pine needles and exacerbating the abortion
problem (3-5). Cows may abort as soon as 24 h after
consumption of the pine needles, but typically abortions occur
in 3-4 days, and up to two weeks after exposure to the pine
needles. The abortion often results in the birth of small weak
calves and retained placentas in cows that have aborted. Stillborn
calves may occur if pine needles are ingested during the second
trimester or early third trimester. The incidence of pine needle-
induced abortion increases the closer cows are to their expected
date of parturition. Cow deaths are not uncommon, often
resulting from endometritis, septicemia, or other complications
of premature parturition and retained placenta. The direct
economic losses from pine needle abortions have been estimated
to be as high as $20 million per year (6, 7).
The detected metabolites are present in the serum at parts
per million levels, and thus the proposed compound (5) has not
been isolated in sufficient quantities for complete structural
characterization. Based on the further need for sufficient
quantities of isocupressic acid metabolites for future experiments
we sought to develop methods for their preparation from
isocupressic acid, which can be isolated in large quantities from
ponderosa pine needles or bark. We present here the charac-
terization of (8S,13S)-tetrahydroagathic acid (5) prepared from
naturally occurring isocupressic acid and its confirmation as a
metabolite, along with its corresponding 13R isomer, in serum
of cattle after consumption of plant material containing iso-
cupressic acid.
Isocupressic acid (1) was recently identified as the abortifa-
cient agent in ponderosa pine needles (8). However, analysis
* To whom correspondence should be addressed [telephone (435) 752-
2941; fax (435) 753-5681; e-mail gardnerd@cc.usu.edu].
10.1021/jf011501m This article not subject to U.S. Copyright. Published 2002 by the American Chemical Society
Published on Web 03/16/2002

2236 J. Agric. Food Chem., Vol. 50, No. 8, 2002
Garrossian et al.
Figure 1. Structure of isocupressic acid and identified and proposed serum metabolites.
MATERIALS AND METHODS
(549.7 mg, 4.7 mmol), and tetrapropylammonium perruthenate (55 mg,
0.16 mmol) were added, and after 1 h the solution was filtered through
a plug of silica gel (slurry packed in EtOAc) and concentrated under
reduced pressure to give 0.88 g (89% yield) of the corresponding
aldehyde (1a, Figure 2 - scheme 1) (16).
General Methods. All reaction solvents were dried and distilled by
standard methods prior to use. Reaction materials were purchased from
Aldrich Chemical Co. (Milwaukee, WI) and were generally used
without further purification. Triphenylphosphine was recrystallized
before use by dissolving 20 g in boiling benzene (50 mL). Absolute
ethanol was added slowly until the solution turned slightly cloudy. After
the solution cooled, the triphenylphospine was filtered and dried under
vacuum at 76 °C. Isocupressic acid (1) was isolated from the bark of
Pinus ponderosa using previously described methods (8).
The crude aldehyde (1a) (650 mg, 2.0 mmol) was added to a mixture
of tert-butyl alcohol (25.0 mL) and 2-methyl-2-butene in a 100-mL
round-bottomed flask equipped with a condenser and magnetic stirrer,
and it was cooled to -5 °C (17). Within a 15-min time period, a 1.25
M aqueous solution of KH2PO4 (15 mL, 13.8 mmol) was added. Then
10 mL of sodium chlorite solution (1.65 g, 13.8 mM) was added within
5 min. The reaction mixture was stirred for 30 min at -5 °C, and then
for 15 h at room temperature. The reaction was quenched by slowly
adding dilute HCl (0.7 M) until pH 3 was attained. The solution was
extracted with methylene chloride (3 × 25 mL), and the organic layers
were combined and dried over MgSO4. Removal of the solvent by rotary
evaporation gave 0.68 g (100% yield) agathic acid (3), which was
Gas chromatography/mass spectrometry analyses of the isolated or
prepared diterpene acids were conducted using a Finnigan MAT GCQ
(San Jose, CA) system consisting of a 30 m × 0.25 mm i.d. DB-5ms
capillary column (J&W Scientific, Folsom, CA). Samples (50-100 µg)
were derivatized to their corresponding TMS esters after they were
dissolved in pyridine (200 µL), adding N,O-bis(trimethylsilyl)trifluoro-
actamide (50 µL) (BSTFA, Pierce Chemical, Rockford, IL) and heating
at 70 °C for 30 min. Samples (1-2 µL) were injected splitless with an
injector temperature of 225 °C. The initial column temperature was
100 °C for 1 min, then raised from 100 to 200 °C at 40 °C/min and
from 200 to 300 °C at 5 °C/min, and then held at 300 °C for 1.5 min.
Reversed-phase liquid chromatography/mass spectrometry (HPLC/
MS) was preformed using a system consisting of an HP 1100 (Agilent
Technologies, Wilmington, DE), binary solvent pump and autosampler,
a 100 × 2 mm i.d. Betasil C18 reversed-phase HPLC column (Keystone
Scientific, Bellefonte, PA), and a Finnigan LCQ mass spectrometer.
The mobile phase was a mixture of methanol (A) and 20 mM
ammonium acetate (B) at a flow rate of 0.5 mL/min starting with 50%
A for 1 min, followed by a linear gradient increasing to 100% A in 20
min. Ionization was achieved using an atmospheric pressure chemical
ionization (APCI) source with a vaporizer temperature of 450 °C and
corona discharge current of 5 µA. The capillary inlet temperature and
voltage were 150 °C and -30V, respectively. Operational parameters
of the mass spectrometer were optimized using the standard “autotune”
procedure to optimize the signal [m/z 319, (M - H)-] for a standard
solution of isocupressic acid infused into a flow (0.5 mL/min) of 60:
40 methanol/20 mM ammonium acetate. The mass spectrometer was
set to scan negative ions in a mass range of 150-1000 amu. Maximum
ion trap inject time was 200 ms and two micro scans were averaged
for each data point. Under these conditions the two most prominent
negative ions observed for isolated or prepared diterpene acids were
(M - H)- and (M + OAc)-.
1
identical with authentic compound by GC/MS and H NMR.
Preparation of Tris (Triphenylphosphine) Chlororhodium Cata-
lyst. To a solution of freshly recrystallized triphenylphosphine (6.0 g,
0.6 M excess) in hot absolute ethanol (175 mL) was added a solution
of rhodium trichloride trihydrate (1.0 g) in hot absolute ethanol (35
mL), and the mixture was refluxed for 45 min. The hot solution was
filtered, and the burgundy-red crystals were washed with degassed
diethyl ether (50 mL) and dried under vacuum at room temperature to
yield 3.1 g (84% yield); mp 156-158 °C (lit. mp 157-158 °C) (18).
Conversion of Agathic Acid to (13R,S)-Dihydroagathic Acid (4).
Agathic acid (3) (140 mg) was added to a 50-mL round-bottomed flask
(equipped with a condenser and magnetic stirrer) containing absolute
ethanol (14 mL) and sodium metal (800 mg). The reaction mixture
was refluxed for 3 h. After the mixture cooled, deionized water (25
mL) was added, and the reaction mixture was extracted twice with
methylene chloride (40 mL). The organic portions were combined and
dried over Na2SO4. The solvent was removed under reduced pressure
to give 129 mg (92% yield) of (13R,S)-dihydroagathic acid. The 13R,S
isomers coeluted on GC/MS analysis with retention time and mass
spectrum identical to that of the naturally occurring (13S)-dihydro-
agathic acid (4) isolated from P. ponderosa needles. Analysis by LC/
MS resolved the two compounds with (13R)-dihydroagathic acid (Rt
4.19 min) and (13S)-dihydroagathic acid (Rt 5.26 min) being identified
by comparison of retention time with that of naturally occurring (13S)-
dihydroagathic acid.
Preparation of (8S,13R,S)-Tetrahydroagathic Acid. To a high-
pressure hydrogenation reactor, agathic acid (3) (211 mg, 0.65 mmol)
and dry benzene (30 mL) were added, and the solution was degassed
for 10 min using dry, ultrapure argon. While under argon the
tris(triphenylphosphine) chlororhodium catalyst (10 mg; 1.08 × 10-2
mmol) was added (19), and dry hydrogen gas was introduced for 3
Synthesis of Agathic Acid (3). A solution of isocupressic acid (1.00
g, 3.1 mmol) in methylene chloride (45 mL) was placed into a 100-
mL round-bottomed flask containing oven-dried 4 Å molecular sieves
(2.0 g) and equipped with a rubber septum and a magnetic stir bar.
While the flask contents were stirring, 4-methylmorpholine N-oxide

Preparation of Tetrahydroagathic Acid
J. Agric. Food Chem., Vol. 50, No. 8, 2002 2237
Figure 2. Reaction schemes for production of tetrahydroagathic acid (5).
min to displace all argon. The pressure of the reaction vessel was
increased to 60 psi hydrogen, and then it was placed into an oil bath
and the temperature was maintained at 50-55 °C. After 14 h GC/MS
analysis (TMS derivative) indicated complete conversion of 3 to 5. To
purify the product from the rhodium catalyst, the solvent was removed
at reduced pressure; the residue was dissolved in chloroform (40 mL)
and extracted with 0.75 N NaOH (2 × 30 mL). The aqueous layers
were combined and adjusted to pH 2 by dropwise addition of 1 N HCl.
The aqueous acid solution was then extracted with chloroform (2 ×
50 mL). The organic layers were combined, dried with anhydrous
Na2SO4, and filtered, and the solvent was removed under reduced
pressure by rotary evaporation to give 135 mg of (8S,13R,S)-
tetrahydroagathic acid (5).
(100 MHz, CDCl3) δ 14.7 (CH3, C-20), 14.8 (CH3, C-17), 18.8 (CH2,
C-6), 19.0 (CH2, C-2), 21.5 (CH3, C-16), 23.7 (CH2, C-11), 29.0 (CH3,
C18), 29.2 (CH, C-8), 31.1 (CH, C-13), 35.0 (CH2, C-7), 36.3 (CH2,
C-12), 37.9 (CH2, C-3), 38.9 (C-10), 39.8 (CH2, C-1), 41.8 (CH2, C-14),
43.9 (C-4), 53.3 (CH, C-9), 57.5 (CH, C-5), 180.0 (CdO, C-15), 184.6
(CdO, C-19). Methylabda-15,19-dionate (tetrahydroagathic acid di-
methyl ester) was prepared by reaction of (5) with idomethane, K2CO3,
and dimethylformamide, room temperature (1 h), followed by extraction
with water and ethyl acetate. The ethyl acetate fraction was dried with
Na2SO4, and the solvent was removed by evaporation under N2 flow
at 70 °C. HRCIMS (DIP, isobutane), obsd m/z 367.28567 (MH+),
C22H39O4 requires 366.28483.
The individual 13R and 13S isomers of 5 were detected by HPLC
and were separated by semipreparatory HPLC. Aliquots (10 mg/mL)
were injected onto a 250 × 10 mm i.d. C18 reversed-phase Beckman
Ultrasphere column and eluted with a mixture of 20 mM ammonium
acetate and methanol starting with 50% methanol for 2 min followed
by a linear gradient to 100% methanol after 30 min. Fractions (4 mL)
were collected throughout the run and then analyzed by APCI negative
ion MS. Repeated runs were combined to give 10 mg of a 9:1 enriched
13S/13R isomer which was identical to that prepared by hydrogenation
of naturally occurring 13S-dihydroagathic acid, (Figure 2 - scheme 2)
isolated from Pinus ponderosa needles by LC/MS analysis.
RESULTS AND DISCUSSION
Preparation of (13R,S)-Tetrahydroagathic Acid (5). Iso-
cupressic acid (1) was the first choice as the starting material
for the preparation of tetrahydroagathic acid (5), as it was readily
available from previous preparatory extractions of ponderosa
pine needles and bark (8). The conversion of 1 to 5 (Figure 2
- scheme 1) proceeded via a two-step oxidation, followed by
reduction of both double bonds to yield (13R,S)-5. Lin et al.
(9) previously used a similar procedure in the preparation of 3
and 4; however, their use of pyridinium chlorochromate in the
initial oxidation of 1 led to an equal mixture of the E and Z
isomers of the corresponding aldehyde (1a). The use of the
tetrapropylammonium perruthenate oxidation (16) followed by
the sodium chlorite oxidation method (17) maintained the C13,
14 cis configuration in the conversion of 1 to 3. From 3 it was
then possible to selectively reduce the C13,14 allylic double
bond to first provide (13R,S)-dihydroagathic acid (4) (step 2a,
scheme 1) which then could be further reduced to 5; or both
(8S,13S)-Labda-15,19-dioic acid (tetrahydroagathic acid) (5).
Colorless resin. IR (drifts, KBr) 3400-3000 broad, 2960, 2842, 2677,
1
1697, 1471, 1411, 1388, 1268, 1182, 946 cm-1. H NMR (400 MHz,
CDCl3) δ 0.72 (3H, s, H-20), 0.85 (1H, m, H-1), 0.89 (3H, d, J ) 7.4
Hz, H-17), 0.98 (3H, d, J ) 6.7 Hz, H-16), 1.0 (2H, m, H-3,9), 1.08
(1H, m, H-5), 1.09 (1H, m, H-12), 1.22 (1H, m, H-11), 1.22 (3H, s,
H-18), 1.32 (1H, m, H-12), 1.4 (2H, m, H-2), 1.42 (1H, m, H-11),
1.43 (1H, m, H-7), 1.68 (1H, m, H-6), 1.70 (1H, m, H-7), 1.78 (1H,
m, H-1), 1.80 (1H, m, H-2), 1.88 (1H, m, H-6), 1.90 (1H, m, H-8),
1.98 (1H, m, H-13), 2.12 (1H, m, H-3), 2.25 (2H, m, H-14). 13C NMR

2238 J. Agric. Food Chem., Vol. 50, No. 8, 2002
Garrossian et al.
Figure 3. Observed nOe difference spectra and dipolar interaction between C17 and C20 confirming the assignment as the 8S isomer.
double bonds of 3 could be reduced in a single step using the
homogeneous catalytic hydrogenation procedure (18, 19) to
produce (13R,S)-5.
Use of the large tris(triphenylphosphine)chlororhodium cata-
lyst for general hydrogenation was nonspecific for the C13,14
double bond but was stereospecific with respect to the reduction
of the C8,17 double bond, producing only the 8S isomer and a
final mixture of (8S,13R,S)-tetrahydroagathic acid. The proposed
8S configuration was confirmed from the nOe interaction
between C-20 and C-17 (Figure 3).
A simpler procedure was to start with naturally occurring
(13S)-dihydroagathic acid, a minor diterpene acid in ponderosa
pine needles, and convert it directly to (8S,13S)-tetrahydro-
agathic acid by reduction of the C8,17 exomethylene group
(scheme 2). Because of a limited supply of (13S)-4 this
procedure was only used to provide a few mg of analytical
standard material that was useful for later identification of the
13R and 13S isomers by HPLC/MS.
GC/MS analysis of the TMS derivatives was useful in
following each reaction step, but it was not suitable for
separating the 13R,S isomers. HPLC/MS analysis of products
from schemes 1 and 2 produced the expected products as shown
in Figure 4, and enabled the assignment of the later eluting
compound as (13S)-5 by comparison with the single product
from scheme 2 and the (13R,S) mixture produced by scheme 1.
Identification of (13R,S)-Tetrahydroagathic Acid in Bo-
vine Serum. Previous isolation and analysis of bovine serum
from animals after consumption of P. ponderosa, Juniperus
communis, or isocupressic acid, identified 3 and 4 as metabolites
by direct comparison of GC/MS data (retention time, mass
spectra, and coelution) with the natural occurring compounds
(9, 10). A third metabolite, tetrahydroagathic acid (5), was
proposed, based on mass spectral data alone, but could not be
confirmed at the time because no naturally occurring compounds
were available (10). After preparation of standards of known
configuration we reexamined the serum metabolites to confirm
their identity.
Figure 4. HPLC/MS ion chromatograms for reaction products: (a) starting
material; (b) agathic acid (3) product prepared from (1); (c) dihydroagathic
acid product prepared from (3); (d) the final product, tetrahydroagathic
acid, prepared from (4); (e) naturally occurring (13S)-dihydroagathic acid
isolated from ponderosa pine needles; and (f) (13S)-5 isomer prepared
from naturally occurring (13S)-4.
A pooled serum sample taken 52-60 h after a cow was dosed
with 1 was extracted using the previous described procedures
(10) and then analyzed by HPLC-MS (Figure 5). Analysis of
selected ion chromatograms for 4 (m/z 394, M + 59) and 5
(m/z 396, M + 59), and comparison to the standard compounds
confirmed their presence in the serum. Both the 13R and the
13S isomers were observed for compounds 4 and 5. Lin et al.
(9) had previously reported the presence of (13R,S)-4 as a serum
metabolite of isocupressic acid in vitro and reported the S/R
ratio was about 2:1. We could directly observe the individual
R and S isomers by HPLC and found S/R to be about 3:1,
demonstrating that the reductive enzyme is stereoselective but
not stereospecific. However, in vivo reduction of C8,17 is
stereospecific as only the (8S)-5 isomer was observed.
The physiological role of the serum metabolites of iso-
cupressic acid is not known at this time. The observed

Preparation of Tetrahydroagathic Acid
J. Agric. Food Chem., Vol. 50, No. 8, 2002 2239
Table 1. Detection of Tetrahydroagathic Acid in Serum from Cattle
Reported to Abort after Consumption of Ponderosa Pine Needles
serum sample
THAA (units/g)a
DHAA (units/g)a
4691-00-6G
4691-00-8
4691-00-9
4691-00-11
4691-00-26
controlb
10.4
35.3
60.4
5.5
32.1
8.3
17.0
a THAA ) tetrahydroagathic acid (5); DHAA ) dihydroagathic acid (4); units/g
) (peak area counts/sample weight) × 10-3 b Control serum was taken 48 h
.
post-dosage from test animal treated with 2.2 kg of ground ponderosa pine needles,
a dose sufficient to induce abortion in cattle (5).
animal at 48-h postdosage (dosed 2.2 kg of ponderosa pine
needles) (Table 1). The detection of tetrahydroagathic acid in
the serum of these five animals confirmed the animals’
consumption of isocupressic acid-containing plant material, and
the relative concentrations demonstrate a likelihood that the
animals consumed quantities sufficient to induced the abortions.
ACKNOWLEDGMENT
We thank Rosalind Wong (Western Regional Research Labora-
tory, ARS, USDA, Albany, CA) for assistance in obtaining high-
resolution NMR data and Tom Foderaro (Roche Colorado Corp.,
Boulder, CO) for assistance in obtaining nOe difference spectra.
LITERATURE CITED
(1) Bruce, E. A. Astragalus serotinus and other stock-poisoning
plants of British Columbia; Bulletin 88; Dominion of Canada,
Department of Agriculture: Ottawa, ON, 1927; p 44.
(2) MacDonald, M. A. Pine needle abortion in range beef cattle. J.
Range Manage. 1952, 5, 150-155.
(3) James, L. F.; Call, J. W.; Stevenson, A. H. Experimentally
induced pine needle abortion in range cattle. Cornell Vet. 1977,
67, 294-299.
(4) James, L. F.; Short, R. E.; Panter, K. E.; Molyneux, R. J.; Stuart,
L. D.; Bellows, R. A. Pine needle abortion in cattle: A review
and report of 1973-1984 research. Cornell Vet. 1989, 79, 39-
52.
(5) Stevenson, A. H.; James, L. F.; Call, J. W. Pine needle (Pinus
ponderosa)-induced abortions in range cattle. Cornell Vet. 1972,
62, 519-524.
(6) Miner, J. L.; Bellows, R. A.; Staigmiller, R. B.; Peterson, M.
K.; Short, R. E.; James, L. F. Montana pine needles cause
abortion in beef cattle. Mont. Agric. Res., Mont. Agric. Exp. Sta.
1987, 4, 6-9.
(7) Lacey, J. R.; James, L. F.; Short, R. E. Ponderosa pine:
Economic impact. In The Ecology and Economic Impact of
Poisonous Plants on LiVestock Production; James. L. F., Ralphs,
M. H., Nielson, D. B., Eds.; Westview Press: Boulder, CO, 1988;
pp 95-106.
(8) Gardner, D. R.; Molyneux, R. J.; James, L. F.; Panter, K. E.;
Stegelmeier, B. L. Ponderosa pine needle-induced abortion in
beef cattle: Identification of isocupressic acid as the principal
active compound. J. Agric. Food Chem. 1994, 42, 756-761.
(9) Lin, S. J.; Short, R. E.; Ford, S. P.; Grings, E. E.; Rosazza, J. P.
N. In vitro biotransformations of isocupressic acid by cow rumen
preparations: Formation of agathic and dihydroagathic acids. J.
Nat. Prod. 1998, 61, 51-56.
Figure 5. HPLC/MS analysis and detection of (13R,S)-dihydroagathic acid
(4) and (13R,S)-tetrahydroagathic acid (5) in bovine serum. Figures (a)
and (b) are the reconstructed ion chromatograms selected to isolate the
(M − H)- ions for tetrahydoagathic acid (m/z 397) and dihydroagathic
acid (m/z 395), respectively, and are compared to the total ion
chromatogram (c) from which these ions were extracted. (*) Peaks in the
total ion chromatogram corresponding to dihydro- and tetrahydroagathic
acid.
metabolites might result from a detoxification process, or one
or more metabolites could be key compounds in inducing
abortions after consumption of ponderosa pine needles by late-
term pregnant cattle. Further research is needed to establish the
exact role of each of the metabolites. The developed synthetic
procedures might be useful in providing sufficient materials for
continued animal studies which must be done in cattle because
isocupressic acid appears to be not abortifacient in other animal
species.
Serum metabolites of isocupressic acid may have diagnostic
value as well, and because tetrahydroagathic acid is observed
for longer periods of time in the serum than the other metabolites
are (10), it may be the most important compound to use as a
diagnostic marker for pine needle-induced abortion. A recent
diagnostic case provides an example. The animals in question
had been exposed to pine trees for 2-3 days prior to abortions,
and serum samples were taken 1-2 days postabortion (personal
communication, Dr. Merl Raisbeck, University of Wyoming,
Wyoming State Veterinary Diagnostic Laboratory). Samples
were submitted, extracted, and analyzed by GC/MS using
methods previously described (10). Of the four isocupressic acid
metabolites previously identified in experiments with cows fed
a controlled diet, only compound 5 was observed in these
diagnostic samples taken from actual cattle abortions in the field.
Four of the five samples contained higher concentrations of 5
when compared to that of a control sample taken from a test
(10) Gardner, D. R.; Panter, K. E.; James, L. F. Pine needle abortion
in cattle: Metabolism of isocupressic acid. J. Agric. Food Chem.
1999, 47, 2891-2897.
(11) Ford, S. P.; Christenson, L. K.; Rosazza, J. P.; Short, R. E. Effect
of ponderosa pine needle ingestion on uterine vascular function
in late-gestation beef cows. J. Anim. Sci. 1992, 70, 1609-1614.

2240 J. Agric. Food Chem., Vol. 50, No. 8, 2002
Garrossian et al.
(12) Ford, S. P.; Rosazza, J. P. N.; Short, R. E. Pinus ponderosa
needle-induced toxicity. In Handbook of Plant and Fungal
Toxicants; Felix D’Mello, J. P., Ed.; CRC Press Inc.: Boca
Raton, FL, 1977; pp 219-229.
(17) Bal, B. S.; Childers, W. E., Jr.; Pinnick, H. W. Oxidation of
R,â-unsaturated aldehydes. Tetrahedron 1981, 37, 2091-2096.
(18) Osborn, J. A.; Jardine, F. H.; Young, J. F.; Wilkinson, G. The
preparation and properties of tris (triphenylphosphine) halo-
genorhodium (I) and some reactions thereof including catalytic
homogeneous hydrogenation of olefins and acetylenes and their
derivatives. J. Chem. Soc. (A) 1966, 1711-1735.
(19) Harmon, R. E.; Parsons, J. L.; Cooke, D. W.; Gupta, S. K.;
Schoolenberg, J. Homogeneous catalytic hydrogenation of
unsaturated organic compounds. J. Org. Chem. 1969, 34, 3684-
3685.
(13) Christenson, L. K.; Short, R. E.; Ford, S. P. Effects of ingestion
of ponderosa pine needles by late-pregnant cows on uterine blood
flow and steroid secretion. J. Anim. Sci. 1992, 70, 531-537.
(14) Christenson, L. K.; Short, R. E.; Farley, D. B.; Ford, S. P. Effects
of ingestion of pine needles (Pinus ponderosa) by late-pregnant
beef cows on potential sensitive Ca2+ channel activity of
caruncular arteries. J. Reprod. Fertil. 1993, 98, 301-306.
(15) Short, R. E.; Ford, S. P.; Rosazza, J. P. N.; Farley, D. B.;
Klavons, J. A.; Hall, J. B. Effects of feeding pine needles and
pine needle components to late pregnant cattle. Proc. - Am.
Soc. Anim. Sci., West. Sect. 1996, 47, 193-196.
Received for review November 13, 2001. Revised manuscript received
February 7, 2002. Accepted February 8, 2002.
(16) Ley, S. V.; Norman, J.; Griffith, W. P.; Marsden, S. P.
Tetrapropylammonium perruthenate, Pr4N+RuO4-, TPAP: A
catalytic oxidant for organic synthesis. Synthesis 1994, 639-
665.
JF011501M