iPF2α-III-17,18,19,20-d4: Total synthesis and metabolism

Article abstract of DOI:10.1016/j.bmcl.2005.01.062

The first total synthesis of 17,18,19,20-d₄-iPF _2α-III 32, a deuterated analog of iPF_2α-III, is described. We have used this analog in some β-oxidation studies with rat liver homogenates and have shown that 32 was metabolized to 17,18,19,20-tetradeutero-2,3-dinor-iPF_2α-III 36 and 17,18,19,20-tetradeutero-2,3-dinor-5,6-dihydro-iPF_2α-III 37.

Full text of DOI:10.1016/j.bmcl.2005.01.062

Bioorganic & Medicinal Chemistry Letters 15 (2005) 1613–1617
iPF -III-17,18,19,20-d : Total synthesis and metabolism
2
a
4
a
Seongjin Kim, William S. Powell, John A. Lawson, Sheila H. Jacobo,
b
c
a
c
c
d
Domenico Pratico, Garret A. FitzGerald, Kirk Maxey and Joshua Rokach
a,
*
a
Claude Pepper Institute and Department of Chemistry, Florida Institute of Technology, 150 West University Boulevard,
Melbourne, FL 32901, USA
b
Meakins–Christie Laboratories, Department of Medicine, McGill University, Montreal, Quebec, Canada H2X 2P2
c
The Center for Experimental Therapeutics, The University of Pennsylvania, Philadelphia, PA 19104, USA
d
Cayman Chemical, 1180 E. Ellsworth Road, Ann Arbor, MI 48108, USA
Received 23 November 2004; revised 26 January 2005; accepted 26 January 2005
4
Abstract—The first total synthesis of 17,18,19,20-d -iPF2a-III 32, a deuterated analog of iPF2a-III, is described. We have used this
analog in some b-oxidation studies with rat liver homogenates and have shown that 32 was metabolized to 17,18,19,20-tetradeutero-
2
,3-dinor-iPF -III 36 and 17,18,19,20-tetradeutero-2,3-dinor-5,6-dihydro-iPF -III 37.
2a 2a
ꢀ
2005 Elsevier Ltd. All rights reserved.
Recently a new class of naturally occurring lipids, the
1
culate the number of iP groups for any given PUFAs as
a function of the number of skipped double bonds:
N = (n À 2) · 2, where N is the number of iP groups
–3
isoprostanes (iPs), has been discovered.
These mole-
cules are isomeric with prostaglandins. Whereas prosta-
glandins are formed by the action of cyclooxygenase on
arachidonic acid (AA), iPs are produced nonenzymati-
cally by a free-radical peroxidation of polyunsaturated
fatty acids such as AA. These iPs, formed in situ on
phospholipids, are subsequently released in the free
form by the action of phospholipases. Free radical per-
oxidation of AA affords four groups of iPs (III, IV, V,
5
formed, and n the number of skipped double bonds.
For example, the number of iP groups derived from
eicosapentanoic acid (EPA) is N = (5 À 2) · 2 = 6. These
iPs, in particular the more abundant Group VI iPs, have
been used as a marker of oxidative stress in disease
6
7
states such as atherosclerosis and AlzheimerÕs. Some
iPs, in particular Group III, have potent biological ef-
VI) and each group contains 16 stereoisomers (Scheme
4
fects, mainly due to their interaction with the receptors
8–10
1
). We have introduced a formula by which we can cal-
of the enzymatically generated prostanoids.
We have
OH
CO
2
H
2
CO H
COX
OH
AA
OH
1
2
PGF2α
ROS
OH
OH
OH
OH
OH
OH
OH
OH
OH
CO
2
H
CO H
2
CO
2
H
2
CO H
O
H
3
OH Group III
4
Group IV
5
OH
Group V
6
Group VI
Scheme 1. Groups of isoprostanes derived from AA.
Keywords: iPF2a-III; d
*
4
-iPF2a-III; Dinor-iPF2a-III; Dinor-dihydro-iPF2a-III; Arachidonic acid, Metabolism.
Corresponding author. Tel.: +1 321 674 7329; fax: +1 321 674 7743; e-mail: jrokach@fit.edu
0
960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.01.062

1614
S. Kim et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1613–1617
proposed a nomenclature of iPs which is used in the
4
nary in vitro study of one cycle of b-oxidation; and
lastly, (d) to prepare enzymatically the tetradeuterated
analogs of b-oxidation metabolites 8 and 10 from the
present article. Another nomenclature of iPs has also
1
been reported.
1
synthetic tetradeutero-iPF -III 32. These could be used
2
a
Because of the large number of isomeric iPs, a method
of choice for their analysis in biological fluids has been
mass spectrometry. The stereospecific chemical synthesis
of these compounds has been the only way to obtain
pure standards of known stereochemistry that can be
used for mass spectrometric analysis and in metabolic
studies, as well as for biological evaluation. We have
previously reported the total syntheses of several of iPs
and have shown that identical products are formed in
in further metabolic studies and hence save considerable
time for a separate total synthesis of these deuterated
metabolites at this particular juncture.
1. Synthesis of 17,18,19,20-d -iPF -III
4 2a
We have designed the synthesis of tetradeuterated ana-
log of iPF -III in such a way that the deuterium atoms
2
a
3
,12,13
vivo.
We have also prepared the corresponding
were introduced at positions 17,18,19, and 20. Such a
molecule (17,18,19,20-d -iPF -III 32) would be more
resistant to loss of the deuterium atoms as a result of
deuterium-labeled analogs of some of these compounds
to serve as markers for identification and quantitation
4
2a
1
4,15
by mass spectrometry.
metabolized before they are excreted in urine. iPF -
iPs, formed in vivo, are
b-oxidation. Scheme 4 shows the convergent synthesis
of 17,18,19,20-d -iPF -III 32, making use of 21 and
2
a
4
2a
III is the only iP for which some metabolic studies have
been reported. It has been shown that this substance is
metabolized by b-oxidation to 2,3-dinor (8), 2,3-dinor-
24 as key synthons. b-keto phosphonate 21 was synthe-
sized from 1,2,4-butanetriol 12 as shown in Scheme 3.
Aldehyde 13 was prepared from 1,2,4-butanetriol 12 in
two steps. The Wittig reaction of 13 with commercially
available allyltriphenylphosphonium bromide in THF
with lithium hexamethyldisilazane as base afforded cis
and trans mixtures of 14 in 2:3 ratio. Using the Wilkin-
son catalyst, the deuteration of 14 in benzene afforded
5
,6-dihydro (10), and tetranor (11) metabolites as shown
1
6–19
in Scheme 2.
shown in Scheme 2 is based on work that we and oth-
ers
examination of the mechanism of the metabolism of
unsaturated fatty acids. The objectives of the present
study were the following: (a) to synthesize a deuterium
analog of iPF -III that can be used as a quantitative
The proposed metabolic pathway
2
0
2
1,22
did on the metabolism of LTE and on the re-
4
2
3
15 with a very high incorporation of D (greater than
2
99%). After removal of the isopropylidene group in 15
with 4% aqueous H SO , the resulting 6,7-dihydroxy
2 4
2
a
marker for the mass spectrometric analysis of this iP
in biological fluids; (b) to select a deuterium-labeled ana-
log of iPF -III that would be resistant to loss of the
deuterium atoms as a result of b-oxidation and therefore
useful for metabolic studies; (c) to carry out a prelimi-
compound 16 was treated with thiocarbonyl bis (imida-
zole) in acetonitrile to afford 17 in 94% yield. The con-
version of thionocarbonate 17 to iodohydrin 19 was
performed following a methodology described by us
2
a
2
4
recently. Treatment of 17 with methyl iodide in
HO
2
CO H
tas
e
uc
HO
HO
HO
HO
HO
HO
HO
-red
,4
OH 10
1
CO
2
H
1cycle
β-oxidation
CO
2
H
enoyl-CoA
isomerase
CO
2
H
β-
ox
id
ati
on
HO
HO
OH iPF2α-III
OH
OH
7
8
CO
OH
2
H
9
11
Scheme 2. Proposed metabolism pathway for the formation of 8, 10, and 11.
HO
D
D
HO
D
D
D
e
f
O
O
d
i
O
c
a, b
HO
HO
S
CHO
O
O
O
OH
1
3
14
15
16
D
D
1
2
1
D
D
D
D
O
SMe
D
O
O
D
D
O
D
HO
D
D
O
D
j
O
D
D
g
h
MeO
P
I
I
I
O
2
0
MeO
21
7
18
D
D
19
D
D
D
D
D
D
Scheme 3. Reagents and conditions: (a) 2,2-dimethoxypropane, TsOH, CH
C@CHCH P(C
Br, LiHMDS, À78 ꢁC to rt, 2 h, 89%; (d) WilkinsonÕs catalyst, 99.98% D
f) Im CS, CH CN, 94%; (g) CH I, ClCH CH Cl
Cl, 70 ꢁC, 10 h, 86%; (h) DIBAL-H, CH
P(OMe)
, THF, 45 ꢁC, 10 h, 69% (two steps).
3
CN, 1 h, 65%; (b) PCC, camphorsulfonic acid, CH
2
Cl , 3 h, 74%; (c)
, THF, 8 h, 99%;
2
H
2
2
6
5
H )
3
2
, benzene, 8 h, 87%; (e) 4% H SO
2
4
(
2
3
3
2
2
2
2
, À78 ꢁC, 40 min, 96%; (i) Jones reagent, acetone, 0 ꢁC; (j)
3

S. Kim et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1613–1617
1615
CH
HO
2
OH
S
TBDMSO
TBDMSO
(a)
TBDMSO
8
steps
O
O
a
O
MeO
2
C
+
O
O
O
HO(H)
OH
TBDMSO OTBDMS
OH
L-glucose
TBDMSO
2
2
23
24
25
(
b) TBDMSO
-
+
COOH
TBDMSO
TBDMSO
TBDMSO
TBDMSO
O
b
OH BrPh
3
P
2
7
d
COOH
O
O
c
OH
TBDMSO
TBDMSO
28
2
4
26
TBDMSO
TBDMSO
TBDMSO
COOMe
D
COOMe
D
COOMe
21
f
D
D
D
e
CHO
HO
2
9
TBDMSO
TBDMSO
D
D
D
3
0
O
31
OH
COOH
D
g, h
D
HO
32
D
7, 18, 19, 20-d -iPF2α-III
4
D
OH
1
Scheme 4. Reagents and conditions: (a) n-Bu
THF, À78 ꢁC to rt, 1 h, 68%; (d) periodinane, CH
g) THF:formic acid:H
O—6:3:1, rt, 8 h; (h) 5% KOH, THF, 0 ꢁC to rt, 3 h, 76% (two steps).
3
SnH, AIBN, benzene, reflux, 5 h, 52%; (b) DIBAL-H, CH
2
Cl
2
, À78 ꢁC, 87%; (c) t-BuOK, HMPA,
2
Cl
2
, rt, 8 h; (e) NaHMDS, THF, À78 ꢁC to rt, 72%; (f) (S)-BINAL-H, THF, À100 ꢁC, 4 h, 99%;
(
2
1,2-dichloroethane at reflux gave 18 in 86% yield. The
reduction of 18 with DIBAL-H, followed by an acidic
3. Results and discussion
work up furnished 19 in 97% yield. The b-keto iodide
iPF -III (i.e., 8-iso-PGF ) 7, a Group III iP, was the
2
a
first iP identified in urine and we have previously per-
2a
1
2
0 derived from the Jones oxidation of 19 was treated
1
2
with trimethylphosphite in THF at room temperature
affording b-keto phosphonate 21 in 62% yield (two
steps).
formed the first total synthesis of this iP. For quantita-
tive analysis of iPF -III, we have used its [ O ]
1
8
2
a
2
2
8
analog. We have described here the first total synthesis
of an isotopically labeled analog of iPF -III (32) in
2
a
Intermediate 24, which was prepared from L-glucose in
1
which the x-side chain has been labeled with four atoms
of deuterium.
2
9
steps, was reduced by DIBAL-H in 87% yield. The
resulting lactol 26 was coupled with phosphorane de-
rived from the phosphonium salt 27 by a Wittig reaction
to afford 28 in 68% yield. Dess–Martin oxidation of 28
gave 29 which was then used in a Horner–Emmons reac-
tion at À78 ꢁC to introduce the lower side chain using
the anion of b-ketophosphonate 21 (prepared following
Scheme 3) afforded 30 in 72% yield. The enantioselective
reduction of the C-15 keto group in 30 with chiral reduc-
ing agent (S)-BINAL-H proceeded well and afforded the
desired pure 15(S) derivative 31 in 99% yield (ee ꢀ 98%).
The deprotection of bis-silyl groups in 31 was carried
out using formic acid. Finally, hydrolysis with aqueous
iPF -III is the only isoprostane for which some meta-
2
a
bolic studies have been described in any detail. The
major urinary metabolite formed after administration
of tritium-labeled iPF -III to humans was identified by
2a
gas chromatography–mass spectrometry as 2,3-dinor-
1
6
5,6-dihydro-iPF -III. There is also evidence for the
2
a
formation of 2,3-dinor-iPF -III in humans and
2
a
1
7,18
rats.
metabolites identified following administration of tri-
tium-labeled iPF -III was 5,6-dihydro-2,3,4,5-tetra-
In rabbits, the major radiolabeled urinary
2
a
1
9
nor-15-keto-13,14-dihydro-iPF -III. The 17,18,19,20-
2
a
2
5
KOH afforded the desired d -iPF -III 32.
4 2a
d analog of iPF -III reported in the present study
4 2a
would be very useful for studies on the turnover of these
metabolites in vivo, as none of the deuterium atoms
would be lost, in contrast to the tetradeutero 33 where
we expect some loss of deuterium.
2
. Metabolism and MS studies
To determine whether 17,18,19,20-tetradeutero-iPF -
a
2
III 32 would be resistant to the loss of deuterium atoms
as a result of b-oxidation and therefore useful for meta-
bolic studies, 32 was incubated with rat liver homoge-
nates as described by us earlier. The metabolites that
were formed were extracted and analyzed by GC/MS.
We also have decided to include in these metabolic stud-
ies the commercially available deuterated analog of
The experimental results (Table 1) bear this out. Incuba-
tion of 32 with rat liver homogenates, under the condi-
tions briefly described in the footnotes of Table 1,
resulted in the formation of two main compounds, the
2
6
17,18,19,20-tetradeutero-2,3-dinor-iPF -III 36 and
2
a
17,18,19,20-tetradeutero-2,3-dinor-5,6-dihydro-iPF -III
2a
37. Under the same conditions, incubation of 33
yielded exclusively the non-deuterated metabolites,
namely 2,3-dinor-iPF -III 34 and 2,3-dinor-5,6-dihydro-
2
iPF -III, 3,3,4,4-tetradeutero-iPF -III 33, which has
2
a
2a
2
7
the deuterium atoms at positions 3 and 4. The results
of these experiments are summarized in Table 1.
iPF -III 35. It is interesting to note that the enzymatic
2
a

1616
S. Kim et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1613–1617
Table 1. Metabolites formed from deuterated analogs of iPF2a-III
a,b
Substrate
Metabolites
% Conversion
HO
HO
COOH
D
D
3
.6
CO H
2
D
D
3
D
6
HO
D
OH
3
2
HO
D
D
17,18,19,20-tetradeutero-
,3-dinor-iPF2α-III
OH
2
1
7,18,19,20-tetradeutero-iPF2α-III
HO
COOH
D
D
37
2
.2
HO
D
D
OH
1
7,18,19,20-tetradeutero-2,3-
dinor-5,6-dihydro-iPF2α-III
HO
COOH
0
0
.2
D
D
HO
HO
3
4
HO
OH
CO H
2
D
D
2,3-dinor-iPF2α-III
3
3
HO
OH
3
,3,4,4-tetradeutero-iPF2α-III
COOH
.03
3
5
HO
OH
2
,3-dinor-5,6-dihydro-iPF2α-III
a
The substrate (20 lM; either 32 or 33) was incubated with rat liver homogenates in the presence of ATP, CoA, L-carnitine, and NAD+ for 90 min at
37 ꢁC. The incubation was quenched by addition of MeOH and the resulting mixture was centrifuged. The metabolites were isolated from the
supernatant by SPE following a methodology described by us previously.
The metabolites were derivatized and analyzed by GC/MS as described by us previously. Briefly, the metabolites were esterified with pentafluo-
b
robenzyl bromide in the presence of DIPEA using CH
using bis(trimethylsilyl) trifluoroacetamide. The mixture was dried under N
Packard 5973 mass spectrometer interfaced with a 6890 gas chromatograph and a 7683 autosampler was used for analysis.
3
CN as solvent. The resulting esters were purified by TLC and derivatized in pyridine by
2
and was redissolved in dodecane for GC/MS analysis. A Hewlett-
b-oxidation of 33 is much slower than that of 32, a fact
we attribute to the deuterium isotope effect in 33, which
generally slows down the rate of reaction.
activated by carbonyl functions and are the most likely
candidates in which the deuterium could have been ex-
changed with hydrogen. To our knowledge this is the
first time such an observation has been made in the iso-
2
9
The b-oxidation of 3,3,4,4-tetradeutero- iPF -III merits
prostane field.
2
a
some comments. A look at the accepted metabolic
sequence for b-oxidation as applied to this 3,3,4,4-tetra-
deutero derivative 33 is shown in Scheme 5.
The availability of synthetic 32 in addition to its use as a
quantitative marker will allow the enzymatic prepara-
tion and use of 36 and 37 in further stepwise metabolic
1
8
Instead of the anticipated dideutero metabolite 44, we
obtained the non-deuterated metabolite, 2,3-dinor-
studies. We have previously prepared [ O ] analog of 10
2
(non-deuterated analog of 37), which we have used as
1
7
18
a marker in our MS studies. While the use of [ O ]
2
iPF -III 34. This was somewhat surprising. A look at
a
2
3
0
the b-oxidation sequence shown in Scheme 5 indicates
that biosynthetic intermediate 41, or possibly 42, are
derivatives is extremely helpful, the preparation is
sometimes tedious and time consuming. Tetradeuterated
D
D
D
D
OH
OH
OH
D
D
OH
OH
COSCoA
COSCoA
COSCoA
OH
D
D
D
D
OH
OH
3
8
OH
D
OH
39
OH
40
D
SR
D
D
O
D
D
O
OH
OH
OH
SR
OH
COSCoA
RSH
+
CH COSCoA
3
O
4
3
OH
OH
OH 41
OH 42
OH 44
Scheme 5.

S. Kim et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1613–1617
1617
metabolite 37, prepared as described in Table 1, would
be a good replacement to the [ O ] analog. 36 and 37
10. Kunapuli, P.; Lawson, J. A.; Rokach, J.; FitzGerald, G.
A. J. Biol. Chem. 1997, 272, 27147.
1
8
2
1
1
1
1. Taber, D. F.; Morrow, J. D.; Roberts, L. J., II. Prosta-
glandins 1997, 53, 63.
2. Hwang, S. W.; Adiyaman, M.; Khanapure, S.; Schio, L.;
Rokach, J. J. Am. Chem. Soc. 1994, 116, 10829.
3. Adiyaman, M.; Lawson, J. A.; FitzGerald, G. A.; Rokach,
J. Tetrahedron Lett. 1998, 39, 7039.
separate very easily by HPLC (retention time: 12.9 min
1
3
(
1
36) and 14.6 min (37). In addition, since metabolite
0 has been found to be more abundant in urine than
its parent compound, its measurement in biological flu-
ids with 37 as marker could be a better index of oxida-
tive stress than iPF -III.
2
a
14. Li, H.; Lawson, J. A.; Reilly, M.; Adiyaman, M.; Hwang,
S. W.; Rokach, J.; FitzGerald, G. A. Proc. Natl. Acad. Sci.
U.S.A. 1999, 96, 13381.
15. Pratico, D.; Barry, O. P.; Lawson, J. A.; Adiyaman, M.;
Hwang, S. W.; Khanapure, S. P.; Iuliano, L.; Rokach, J.;
FitzGerald, G. A. Proc. Natl. Acad. Sci. U.S.A. 1998, 95,
The spread of the deuterium arrangement in 32 could
provide us with additional advantages for the study of
iPF -III metabolism. For example, x-oxidation which
2
a
is known to occur in prostaglandins, will transform
the x-carbon into a hydroxy and carboxy derivative.
The product of such a metabolic step, in the case of
3
449.
1
1
1
6. Roberts, L. J., II; Moore, K. P.; Zackert, W. E.; Oates, J.
A. J. Biol. Chem. 1996, 271, 20617.
3
2, would be a molecule with two or three deuteriums
7. Burke, A.; Lawson, J. A.; Meagher, E. A.; Rokach, J.;
FitzGerald, G. A. J. Biol. Chem. 2000, 275, 2499.
8. Chiabrando, C.; Valagussa, A.; Rivalta, C.; Durand, T.;
Guy, A.; Zuccato, E.; Villa, P.; Rossi, J.-C.; Fanelli, R.
J. Biol. Chem. 1999, 274, 1313.
at 17, 18 and/or 19, which would be enough to identify
and quantitate the initial metabolism at C-20.
Acknowledgements
19. Basu, S. FEBS Lett. 1998, 428, 32.
2
0. Tagari, P.; Foster, A.; Delorme, D.; Girard, Y.; Rokach,
J. Prostaglandins 1989, 37, 629.
Supported by grants from the National Institutes of
Health (DK-44730 (JR), HL-69835 (JR), HL-70128
GAF), AG-11542 (DP); the NSF for an AMX-360
NMR instrument (CHE-90-13145); the Canadian Insti-
tutes of Health Research (MOP-6254) (WSP); the Heart
and Stroke Foundation of Qu e´ bec (WSP).
2
1. Murphy, R. C.; Wheelan, P. In SRS-A to Leukotrienes:
The Dawning of a New Treatment; Holgate, S., Dahlen, S.
E., Eds.; Blackwell Science Ltd: London, 1997; pp 101–
(
120.
2. Keppler, D.; Huber, M.; Hagmann, W.; Ball, H. A.;
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24, 68.
Reference and notes
24. Adiyaman, M.; Khanapure, S. P.; Hwang, S. W.; Rokach,
J. Tetrahedron Lett. 1995, 36, 7367.
1
25. H NMR (360 MHz, CD
1
. Morrow, J. D.; Hill, K. E.; Burk, R. F.; Nammour, T. M.;
Badr, K. F.; Roberts, L. J., II. Proc. Natl. Acad. Sci.
U.S.A. 1990, 87, 9383.
. Morrow, J. D.; Harris, T. M.; Roberts, L. J., II. Anal.
Biochem. 1990, 184, 1.
. Adiyaman, M.; Lawson, J. A.; Hwang, S. W.; Khanapure,
S. P.; FitzGerald, G. A.; Rokach, J. Tetrahedron Lett.
1996, 37, 4849.
. Rokach, J.; Khanapure, S. P.; Hwang, S. W.; Adiyaman,
M.; Lawson, J. A.; FitzGerald, G. A. Prostaglandins 1997,
54, 853.
. Rokach, J.; Kim, S.; Bellone, S.; Lawson, J. A.; Pratico,
D.; Powell, W. S.; FitzGerald, G. A. Chem. Phys. Lipids
2004, 128, 35.
. Pratico, D.; Tangirala, R. K.; Rader, D. J.; Rokach, J.;
FitzGerald, G. A. Nat. Med. 1998, 4, 1189.
. Pratico, D.; Rokach, J.; Lawson, J.; FitzGerald, G. A.
Chem. Phys. Lipids 2004, 128, 165.
. Janssen, L. J. Am. J. Physiol. (Lung Cell Mol. Physiol.)
OD) d 5.51–5.58 (m, 4H), 3.93
3
(m, 2H), 3.81 (q, 1H, J = 6.3 Hz), 3.06 (br, 2H), 2.62 (m,
1H), 2.42 (m, 1H, J = 7.0, 6.8 and 14.1 Hz), 2.23 (t, 2H,
J = 7.3), 2.12–1.91 (m, 4H), 1.64–1.18 (m, 9H), 0.82 (t,
2
+
2H); MS (EI) (m/z) 357.6 [M ], 339 [M À 18], 313
3
[M À 44], 251, 197, 193 (base peak).
26. Powell, W. S. Prostaglandins 1980, 20, 947.
27. Compound 33 is available commercially from Cayman
Chemical Co., Inc.
4
28. Meagher, E. A.; Barry, O. P.; Burke, A.; Lucey, M. R.;
Lawson, J. A.; Rokach, J.; FitzGerald, G. A. J. Clin.
Invest. 1999, 104, 805.
29. We thank the reviewer for bringing to our attention a
report discussing the loss of deuterium in the metabolism
5
6
7
8
9
of 3,3,4,4-d -PGE
4
2
by hepatocytes; Hankin, J. A.;
Wheelan, P.; Murphy, R. C. Arch. Biochem. Biophys.
1997, 340, 317.
30. Pickett, W. C.; Murphy, R. C. Anal. Biochem. 1981, 111,
115.
2001, 280, L1067.
. Kunapuli, P.; Lawson, J. A.; Rokach, J.; Meinkoth, J. L.;
FitzGerald, G. A. J. Biol. Chem. 1998, 273, 22442.
31. HyperClone C18-BDS, 5 lm, 150 · 2 mm column; mobile
phase:
MeOH:CH
H
2
O:CH
3 3
COOH (solvent A) and CH CN:
3
COOH (solvent B).