Analysis of Prostacyclin and Thromboxane
Biosynthesis in Cardiovascular Disease
GARRET A. FITZGERALD, M.D., ANDERS K. PEDERSEN, M.D., AND CARLO PATRONO, M.D.
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THE APPRECIATION that two oxygenated metabolites of arachidonic acid, prostacyclin (PGI2) and
thromboxane A2 (TxA2), have potent and contrasting
effects on vascular tone and platelet function in vitro
has prompted attempts to define their importance in
human vascular disease. Both compounds are formed
from arachidonic acid by the enzyme cyclooxygenase
via cyclic endoperoxide intermediates, PGG2 and
PGH2. TxA2, the major cyclooxygenase product in the
platelet, is a vasoconstrictor and potent stimulus to
platelet aggregation in vitro. I PGJ2, a vasodilator and
inhibitor of platelet aggregation, is the predominant
product of the same enzyme in vascular endothelial
cells.2 The development of techniques to study platelet
function in vivo3 4 and ex vivo5 preceded the discovery
of these compounds, and the application of these tests
suggests that increased platelet activation may well
accompany vascular occlusive events in man.4 6 Thus,
it seems reasonable that PGI2 and TxA2 might play an
important role in this and other syndromes of disordered platelet-vascular homeostasis in man.7
Support for this concept relies upon the development of methods to measure PGI2 and TxA2 in human
biological fluids. However, both PGI2 and TxA2 have
extremely short half-lives' 2 and are rapidly cleared
from the bloodstream. Furthermore, arachidonic acid
release from biological membranes (and thus synthesis
of PGI2 and TxA2) is likely to be phasic rather than
continuous. This phenomenon also renders measurement of these products highly liable to artifact when
performed in media obtained by invasive sampling
techniques, such as plasma. Finally, there are the technical difficulties involved in devising assays of requisite sensitivity and specificity.
Three approaches have been used: bioassay, radioimmunoassay (RIA) and stable isotope dilution assays involving gas chromatography-mass spectrometry (GC/MS). Bioassays have the advantage of
actually measuring biological activity and have been of
critical qualitative importance in the discovery of prostaglandins,2, 8 Because of the evanescence of both PGI2
and TxA2 in the circulation, RIAs and GC/MS methods
are directed toward metabolites. These methods are
more sensitive and specific than bioassay and more
applicable to quantitative study of prostaglandin turnover in man. Initially, the various approaches appeared
to give similar results. PGI2, unlike other prostaglandins, is not extensively metabolized by the lung, and
experiments using bioassay suggested that production
by the lung and perhaps other sources permitted PGI2
to function as a circulating platelet-inhibitory hormone
in rabbits and cats.9 '0 Apparent confirmation of this
observation in man was soon provided by transpulmonary measurements of 6-keto-PGFia by GC/MS
in patients undergoing cardiac catheterization."I The
inactive hydration product of PGJ2, 6-keto-PGFI, , is
formed nonenzymatically and is more stable than the
parent compound. This study'1 suggested that the pulmonary production rate of PGI2 was in the order of 5
ng/kg/min in man, higher than the threshold dose required to inhibit platelet aggregation ex vivo when
synthetic PGI2 was infused into human volunteers.'2
Radioimmunoassays for TxB2, the inactive hydration product of TxA2, were developed and applied to
measurements in human plasma, and many reports
characterized a supposed "imbalance of the PGI2/
TxA2 ratio" in a variety of human diseases. However,
in the past 2 years, evidence has appeared that the
secretion rate of endogenous PGI2 into the circulation
of human volunteers is extremely low (- 0.09 ng/kg/
min),'3 that 6-keto-PGF,, in human plasma obtained
under resting conditions is in the low picogram range
(< 5 pg),'1'8 and that pulmonary release of PGI2 is
undetectable in healthy volunteers.'9 Thus, although
PGI2 is probably important in local platelet-vascular
interactions, it does not function as a circulating hormone under physiologic conditions in man. Similarly,
we have found that immunoreactive TxB2 is undetectable in human plasma obtained from healthy volunteers
when care was taken to avoid artifacts induced by
sampling and technically acceptable assay methods
with limits of sensitivity in the low picogram range
were combined with highly specific antibodies.
The importance of these observations is that they
invalidate the majority of studies pertaining to endogenous biosynthesis of PGI2 and TxA2 in human disease. Studies of PGI2 and TxA2 production in the human coronary circulation illustrate this point. Samples
are usually obtained after insertion of catheters, a procedure that is likely to traumatically stimulate vascular
PGI2 synthesis. Other aspects of the procedure, such as
the use of heparin and radiocontrast media, may also
alter synthesis of either compound. Platelet activation
at the catheter tip and ex vivo prostanoid formation
represent other sources of potential artifact. Finally, it
may not be apparent that levels of these compounds are
artifactually elevated, because the data are often presented as 6-keto-PGF,JTxB2 ratios or coronary sinus/
aortic blood ratios of either compound.
It has been proposed that substantial quantitative
differences between bioassay estimates of prostacyclin-like activity (2-6 ng/ml)20 and measurement of 6keto-PGFia in plasma by negative ion chemical ioniza-
From the Division of Clinical Pharmacology, Vanderbilt University
School of Medicine, Nashville, Tennessee, and the Department of Pharmacology, Catholic University, Rome, Italy.
Supported in part by USPHS grants HL 30400-01, HD 17413-01,
GM 15432 and RR-05424.
Dr. FitzGerald is the recipient of a career development award from
the Pharmaceutical Manufacturer's Association Foundation.
Address for correspondence: G.A. FitzGerald, M.D., Division of
Clinical Pharmacology, Vanderbilt University School of Medicine,
Nashville, Tennessee 37232.
Received February 7, 1982; accepted March 14, 1983.
Circulation 67, No. 6, 1983.
1174
PROSTACYCLIN AND THROMBOXANE BIOSYNTHESIS/FitzGerald
tion GC/MS (0.5-2 pg/ml)'8
may
result from
predominant conversion of PGI2 to 6-keto-PGE,,20 a
biologically active compound21 22 that is a putative me-
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tabolite of PGI2 in vivo.23 However, this seems highly
unlikely because studies of the metabolic disposition
of PGI2 in man have failed to detect metabolites with
the 6-keto-PGE structure24 and because infusion of
synthetic PGI2 in volunteers does not result in accumulation of 6-keto-PGE, in plasma.25
One method of circumventing problems of sampling-induced artifact and ex vivo prostaglandin formation is by measurement of metabolites that have an
extended half-life. Although the 13,14-dihydro-6,15diketo-PGF,, metabolite of PGI2 has a longer half-life
than 6-keto-PGF1a and has been identified in human
plasma after PGI2 infusions,26 experience with other
prostaglandins has shown that the metabolites initially
formed from the hydration products are rapidly replaced by more polar products as the major compounds
in blood. Measurement of tetranor metabolites has
been recently27 utilized to detect phasic release of
PGF2 in patients with primary dysmenorrhea who suffer episodic pain. Quantitative assays for stable oxidative metabolites of PGI2 and TxA2 in plasma are currently being developed.
An alternative approach has been the measurement
of prostaglandin breakdown products in urine. When
this is performed by GC/MS, it represents a highly
specific, noninvasive index of endogenous prostaglandin biosynthesis. Measurement of urinary metabolites
by RIA is even more liable to problems of sensitivity
and specificity than in plasma. Performance of RIAs
on unextracted urine and the use of antibodies with
et
al.
high cross-reactivity to structurally related compounds
have been common sources of error. However, the
combination of rigorous sample purification with highly specific antibodies has permitted the measurement
of 6-keto-PGF 16, 28 and TxB229 in urine by RIA. Although 6-keto-PGF,24 and TxB230 are urinary metabolites of infused PGI2 and TxB2 in man, these indexes
are likely to predominantly reflect renal PGI2 and TxA2
generation under physiologic conditions. 16, 31 Should
extrarenal production of either compound be increased
(e.g., during a vascular catastrophe), one would anticipate an increase in the urinary levels of the relevant
hydration product. For example, urinary 6-keto-PGF1a
excretion is increased in preterm infants with persistent
ductus arteriosus and respiratory distress syndrome in
whom an increase in systemic prostaglandin biosynthesis is likely.32 The dinor metabolites of 6-ketoPGF1a and TxB2 are the most abundant products of
systemically administered, radiolabeled PGI224 and
TxB2,30 respectively, in man (table 1). Thus, urinary
levels of 2,3-dinor-6-keto-PGF,a (PGI-M) and 2,3dinor-TxB2 (Tx-M) have been used to quantify the
effects of aspirin and similar drugs upon systemic PGI2
and TxA2 synthesis in man33-35 and to accurately predict plasma levels of endogenous PGI2 in the low picogram range.'3
Although these measurements represent the only
noninvasive method of quantifying endogenous generation of PGI2 and TxA2, they are not specific to the
tissue or origin of these compounds. More specific
indexes have been studied ex vivo, such as TxB2 formation in serum28 36 or in stimulated platelet-rich plasma33 or 6-keto-PGF1a formation by biopsies of vascular
TABLE 1. Indexes of Endogenous Prostacyclin and Thromboxane A2 Biosynthesis in Man
Notes
Assay method
Biological fluid
Prostacyclin:
GC/MS and RIA Levels 2 pg/ml under physiologic condiPlasma
1. 6-keto-PGF1a
tions.
RIA
Plasma
2. 6,15-diketo-13,14-dihydroLonger half-life than 6-keto-PGFI, in plasma.26
PGFIa
3. 6-keto-PGE,
Plasma
4. 6-keto-PGF1a
Urine
5. 2,3-dinor-6-keto-PGFia
Urine
6. 6,15-diketo-13,14-dihydro-
Urine
2,3-dinor-PGF,a
Thromboxane A2:
1. Thromboxane B2
Maximal estimates in the low picogram range.
No conversion to this compound from infused
PG12. 25
GC/MS and RIA Many RIAs unreliable due to problems of urine
purification and/or antibody specificity. May
largely reflect renal production under physiologic conditions.
Reflects mainly systemic (extrarenal) PGI2 bioGCIMS
synthesis'3, 24
Reflects mainly systemic (extrarenal) PG12 synGCIMS
thesis. 13' 24
GC/MS
Levels < 15 pg/ml under physiologic conditions.
Urine
GC/MS and RIA Many RIAs unreliable due to problems of urine
2. Thromboxane B2
purification or antibody specificity. May
largely reflect renal production under physiologic conditions.
Reflects mainly systemic (extrarenal) thromUrine
3. 2,3-dinor-thromboxane B2
GC/MS
boxane synthesis.30. 33
=
= radioimmunoassay.
RIA
Abbreviations: GC/MS gas chromatography-mass spectrometry;
Plasma
RIA
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CIRCULATION
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tissue.37 However, these measurements are related to
capacity rather than to actual production rates in vivo.
For example, although biopsies of atherosclerotic vessels have a lower capacity to release 6-keto-PGF a than
do those of healthy vessels ex vivo,38 endogenous PGI2
biosynthesis is actually increased in patients with diffuse atherosclerosis and platelet activation, perhaps
resulting from traumatic platelet-vascular interactions
in ViVo.39
In conclusion, measurement of urinary metabolites
represents the only noninvasive approach to quantitation of endogenous PGI2 and TxA2 biosynthesis. Methodologic difficulties and sampling artifacts have cast
serious doubt on much of the published data, which are
dependent on plasma measurements of TxB2 and 6keto-PGF . The relationship of tissue-specific, capacity-related indexes to endogenous production rates of
PG12 and TxA2 under conditions of stimulation and
depression is largely unknown. Clarification of the
importance of PGI2 and TxA2 in human cardiovascular
disease awaits the more widespread application of reliable analytical techniques in this area.
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Analysis of prostacyclin and thromboxane biosynthesis in cardiovascular disease.
G A FitzGerald, A K Pedersen and C Patrono
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Circulation. 1983;67:1174-1177
doi: 10.1161/01.CIR.67.6.1174
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