Department of Medical Chemistry and Biochemistry,
University of Innsbruck,
Innsbruck, Austria
To the Editor:
In their recently published article on nitric oxide (NO) synthesis in
patients with peripheral arterial occlusive
disease Böger et al1 based their
conclusions among others on urinary excretion of cyclic guanosine
monophosphate (cGMP). The increasing use of urinary and plasma cGMP as
a marker of NO production prompts us to critically comment on
the basis of doing this. NO stimulates soluble guanylate
cyclase and elevates intracellular cGMP.2 The
other isoenzyme of guanylate cyclase, particulate
guanylate cyclase, is stimulated by natriuretic
peptides, which also leads to an increase in intracellular
cGMP.3 The induction of cGMP either by
natriuretic peptides, NO, or nitrates in target tissues may
cause an egression of cGMP into the
supernatant.4 5 We observed a release of cGMP
into the medium after stimulation of human internal mammary artery
grafts with either atrial natriuretic peptide (ANP) or
SIN-1 (unpublished results). However, much higher concentrations of
SIN-1 were necessary to achieve comparably high cGMP concentrations in
the medium.
In humans, ANP injections cause a rapid and pronounced increase in
plasma and urinary cGMP,6 7 whereas nitroglycerol
infusions or molsidomine injections lead to a nonsignificant increase
or no increase in peripheral venous plasma cGMP
concentrations.6 8 cGMP is only partly eliminated
from plasma by glomerular filtration, and most of plasma
cGMP is eliminated by extrarenal clearance. Urinary cGMP is primarily
of renal cellular origin and correlated with the natriuresis induced by
ANP.8 Therefore urinary cGMP has been proposed as
a biologic marker for the renal activities of natriuretic
peptides in vivo.8 We found no correlation
between plasma and urinary cGMP concentrations in
humans.9 Urinary and plasma cGMP concentrations
are influenced by renal function. Urinary cGMP per gram of
creatinine was significantly lower and plasma cGMP of
patients with renal diseases was significantly higher than that of
control subjects.6 9 Therefore urinary cGMP is
mainly influenced by the biologic activities of natriuretic
peptides and also by renal function. As a consequence, urinary cGMP
does not appear to be a reliable marker for the in vivo NO
production in humans.
However, Tsutamoto et al10 could demonstrate that
the arteriovenous cGMP difference may be useful for assessing the local
stimulation of the soluble guanylate cyclase in humans.
Although venous cGMP concentrations did not change, the decrease in
arterial cGMP during nitroglycerol infusion indicated a
local cGMP production by stimulation of the soluble
guanylate cyclase. A decrease in ANP plasma concentrations
during nitroglycerol infusion excluded natriuretic peptides
as a cause of the observed cGMP production. cGMP
production correlated with hemodynamics and did
not occur after captopril administration.
In conclusion, most of the plasma cGMP is derived from the
endogenous natriuretic peptides and only a
minor part from other pathways, such as soluble guanylate
cyclase. The change of plasma cGMP concentrations by nitrates is
much smaller than that by ANP, with the same
hemodynamic effect.10 In contrast
to urinary cGMP, the arteriovenous cGMP production may allow
assessment of the activation of soluble guanylatcyclase in vivo.
However, it is mandatory to measure natriuretic peptide
concentrations simultaneously to exclude changes in
natriuretic peptides as the underlying cause of cGMP
production.
References
1.
Böger RH, Bode-Böger SM, Thiele W,
Junker W, Alexander K, Frölich JC. Biochemical evidence for
impaired nitric oxide synthesis in patients with peripheral
arterial occlusive disease. Circulation.. 1997;95:2068–2074.
2.
Murad F. Signal transduction using nitric oxide and cyclic
guanosine monophosphate. JAMA.. 1996;276:1189–1192.
3.
Wilkins MR, Redondo J, Brown LA. The
natriuretic-peptide family. Lancet.. 1997;349:1307–1310.[Medline]
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4.
Hamet P, Pang SC, Tremblay J. Atrial natriuretic
factor-induced egression of cyclic guanosine monophosphate in cultured
vascular smooth muscle and endothelial cells.
J Biol Chem.. 1989;264:12364–12369.
5.
Billiar TR, Curran RD, Harbrecht BG, Stadler J, Williams DL,
Ochoa JB, Di Silvio M, Simmons RL, Murray SA. Association between
synthesis and release of cGMP and nitric oxide biosynthesis by
hepatocytes. Am J Physiol.. 1992;262:C1077–C1082.
6.
Vorderwinkler KP, Artner-Dworzak E, Jakob G, Mair J, Dienstl
F, Pichler M, Puschendorf B. Release of cyclic guanosine monophosphate
evaluated as a diagnostic tool in cardiac diseases.
Clin Chem.. 1991;37:186–190.
7.
Karrenbrock B, Heim JM, Gerzer G. Effect of molsidomine on ex
vivo platelet aggregation and plasma guanosine cyclic monophosphate
levels in healthy volunteers. Klin Wochenschr.. 1990;68:213–217.[Medline]
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8.
Heim JM, Gottmann K, Weil J, Schiffl H, Lauster F, Loeschke
K, Gerzer R. Effects of a small bolus dose of ANF in healthy volunteers
and in patients with volume retaining disorders. Klin
Wochenschr.. 1990;68:709–717.[Medline]
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9.
Jakob G, Mair J, Vorderwinkler KP, Judmaier G, König P,
Zwierzina H, Pichler M, Puschendorf B, Clinical significance of urinary
cyclic guanosine monophosphate in diagnosis of heart failure.
Clin Chem.. 1994;40:96–100.
10.
Tsutamoto T, Kinoshita M, Ohbayashi Y, Wada A, Maeda Y, Adachi
T. Plasma arteriovenous cGMP difference as a useful indicator of
nitrate tolerance in patients with heart failure.
Circulation.. 1994;90:823–829.
Institute of Clinical Pharmacology,
Medical School,
Hannover, Germany
The letter by Drs Mair and Puschendorf gives us an opportunity
to comment on the use of plasma and urinary nitrate and cGMP as
indicators of in vivo nitric oxide formation.
Nitric oxide is formed in the vascular
endothelium and in other tissues. One of its major
targets is the soluble guanylyl cyclase (sGC), which leads to the
formation of cGMP. NO is rapidly inactivated through
oxidation to nitrite and nitrate; under pathophysiologic conditions,
oxidative inactivation may occur even before the sGC has been
stimulated.1 Both cGMP and nitrite/nitrate can be
found in conditioned endothelial cell media, in plasma,
and in urine. Because the chemical half-life of NO is in the range of
seconds, NO itself can hardly be measured in vivo. Two main strategies
have therefore been followed to assess NO activity in vivo. One is to
determine NO-dependent vasodilation and the other is to measure the
metabolites and/or second messenger of NO, nitrite/nitrate and cGMP, as
biochemical surrogates for NO. However, both of these strategies may be
limited by some constraints: NO-dependent vasodilation allows one to
assess the biologic activity of NO irrespective of whether decreased
elaboration of NO or enhanced oxidative inactivation may underlie this
disorder.1 On the other hand, the
simultaneous quantitation of nitrite/nitrate and cGMP
allows differentiation between impaired NO synthesis (in which case
nitrite/nitrate levels and cGMP levels are expected to be low) and
oxidative inactivation (in which case cGMP levels are expected to be
low, but nitrite/nitrate levels should be normal or elevated). This
approach is curtailed by the potential influence of dietary nitrate
intake2 and by cGMP formation by the particulate
guanylyl cyclase as discussed by Drs Mair and Puschendorf. However,
because dietary nitrate would only affect nitrate levels but not those
of cGMP, and activation of the particulate GC would only affect cGMP
levels but not those of nitrate, we have repeatedly advocated the
parallel use of both indicators to estimate NO
elaboration.3 4 5 6 Furthermore, plasma levels of
these index molecules may only reflect a momentary situation in a
localized area of the circulation, whereas urinary levels reflect
systemic NO production rates but may be affected by renal
excretory function. We have addressed this latter question in an
experimental study and found that correction of urinary nitrate and
cGMP concentrations by urinary creatinine concentration
(ie, urinary excretion rates of these metabolites instead of urinary
concentrations) eliminates the dependency on renal excretory
function.3 Using this approach, we and others
have adopted nitrate and cGMP measurements as reliable indicators of NO
elaboration during physiologic (eg, physical
exercise)4 7 and pharmacologic
stimulation.5 8 9 In our recent study to which
Drs Mair and Puschendorf refer,6 we have
analyzed basal urinary nitrate and cGMP excretion rates in
patients with peripheral arterial occlusive
disease of the legs. Twenty-three of these patients had impaired renal
function as judged by creatinine clearance. We have
reanalyzed urinary nitrate and cGMP excretion rates in the
subgroups of patients with normal or impaired renal function and found
no statistically significant differences in these
parameters despite a 50% reduction of
creatinine clearance in the patients with impaired renal
function (Figure
The in vitro studies cited by Drs Mair and Puschendorf confirm that
cGMP levels are influenced both by ANP and NO donors. However, it is
difficult to us to extrapolate results obtained with pharmacologic
concentrations of ANP and NO donors in isolated arteries in vitro to
physiologic situations in vivo. Indeed, Arnal and
coworkers10 found that the in vivo basal aortic
cGMP levels in rats were mainly dependent on NO synthase:soluble
guanylyl cyclase activity. During chronic NO synthase inhibition,
aortic cGMP levels significantly decreased; in this setting cGMP levels
were correlated with ANP levels. The reduction of aortic cGMP levels
during NO synthase inhibition was reversed by L-arginine.
Tolins and coworkers11 reported similar findings
from an in vivo study in which they infused acetylcholine into rats
under euvolemic conditions. Acetylcholine induced hypotension with
concomitant increased urinary cyclic GMP excretion. These effects were
reversed by L-NMMA, they were not paralleled by increased plasma
ANP levels. These authors also showed a correlation between the changes
in urinary cGMP excretion and the acetylcholine-induced decrease in
systemic blood pressure. We have shown that changes in urinary cGMP
excretion rates could not be explained by differences in ANP levels in
healthy humans at baseline4 and even after
intravenous volume loading.12 This
may be different in patients in whom the ANP system is
activated, as in chronic heart failure. The usefulness of
urinary nitrate and cGMP as markers for systemic NO elaboration in vivo
should therefore be evaluated in any patient group separately.
References
1.
Böger RH, Bode-Böger SM, Frölich JC.
The L-arginine–nitric oxide pathway: role in
atherosclerosis and therapeutic implications.
Atherosclerosis. 1996;127:1–11.[Medline]
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2.
Green LC, Ruiz de Luzuriaga K, Wagner DA, Rand W, Istfan N,
Young VR, Tannenbaum SR: nitrate biosynthesis in man. Proc Natl
Acad Sci USA. 1981;78:7764–7768.
3.
Böger RH, Bode-Böger SM, Gerecke U, Gutzki FM,
Tsikas D, Frölich JC. Urinary NO3- excretion as an indicator of
nitric oxide formation in vivo during oral administration of L-arginine
or L-NAME in rats. Clin Exp Pharmacol Physiol. 1996;23:11–15.[Medline]
[Order article via Infotrieve]
4.
Bode-Böger SM, Böger RH, Schröder EP,
Frölich JC. Exercise increases systemic NO production in
men. J Cardiovasc Risk. 1994;1:173–178.[Medline]
[Order article via Infotrieve]
5.
Bode-Böger SM, Böger RH, Alfke H, Heinzel D,
Tsikas D, Creutzig A, Alexander K, Frölich JC.
L-Arginine induces nitric oxide-dependent vasodilation in
patients with critical limb ischemia: a randomized, controlled
study. Circulation. 1996;93:85–90.
6.
Böger RH, Bode-Böger SM, Thiele W, Junker
W, Alexander K, Frölich JC. Biochemical evidence for impaired
nitric oxide synthesis in patients with peripheral
arterial occlusive disease. Circulation. 1997;95:2068–2074.
7.
Jungersten L, Ambring A, Wall B, Wennmalm A. Both physical
fitness and acute exercise regulate nitric oxide formation in healthy
humans. J Appl Physiol. 1997;82:760–764.
8.
Kanno K, Hirata Y, Emori T, Ohta K, Eguchi S, Imai T, Marumo
F. L-arginine infusion induces hypotension and
diuresis/natriuresis with concomitant increased urinary
excretion of nitrite/nitrate and cyclic GMP in humans. Clin Exp
Pharmacol Physiol. 1992;19:619–625.[Medline]
[Order article via Infotrieve]
9.
Suzuki H, Ikenaga H, Hishikawa K, Nakaki T, Kato R, Saruta T.
Increases in NO2-/NO3-
excretion in the urine as an indicator of the release of
endothelium-derived relaxing factor during elevation of
blood pressure. Clin Sci. 192;82:631–634.
10.
Arnal JF, Warin L, Michel JB. Determinants of aortic cyclic
guanosine monophosphate in hypertension induced by chronic inhibition
of NO synthase. J Clin Invest. 1992;90:6647–6652.
11.
Tolins JP, Palmer RMJ, Moncada S, Raij L. Role of
endothelium-derived relaxing factor in regulation of
renal hemodynamic responses. Am J
Physiol. 1990;258:H655–H662.
12.
Bode-Böger SM, Böger RH, Creutzig A, Tsikas D,
Gutzki FM, Alexander K, Frölich JC. L-arginine infusion decreases
peripheral arterial resistance and inhibits
platelet aggregation in healthy subjects. Clin Sci. 1994;87:303–310.[Medline]
[Order article via Infotrieve]
© 1998 American Heart Association, Inc.
Correspondence
Is Measurement of Cyclic Guanosine Monophosphate in Plasma or Urine Suitable for Assessing In Vivo Nitric Oxide Production?
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Figure 1. Urinary nitrate and cGMP excretion rates and
creatinine clearances (CL creatinine) in
patients with peripheral arterial occlusive
disease and normal or moderately impaired renal function. Data are
mean±SEM.
Response
). This demonstrates again that urinary
nitrate and cGMP excretion rates are independent of renal excretory
function.
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