ELSEVIER

URINARY

NITRITE:

MORE THAN A MARKER

J. 0. N. LUNDBERG,
S. CARLSSON,
N. P. WIKLUND,
AND

L. ENGSTRAND,
E. WEITZBERG

OF INFECTION

E. MORCOS,

ABSTRACT

The bacteriostatic gas nitric oxide (NO) is formed when nitrite is acidified. Infected urine may
contain considerable amounts of nitrite as a result of bacterial nitrate reductase activity, and detection of
nitrite in urine is routinely used in the diagnosis of bacterial cystitis. We sought to determine whether NO
was generated from acidified nitrite-containing urine. Furthermore, we also studied the growth of the urinary
pathogen Escherichiu
co/i in acidified nitrite-containing urine.
Methods.
Urine, collected from healthy control subjects or from patients with infected nitrite-containing
urine, was acidified and incubated in a closed syringe with varying amounts of nitrite added. After 30 minutes,
the headspace gas was removed and immediately injected into a chemiluminescence NO analyzer. In addition, NO was measured in urine collected from healthy control subjects after ingestion of vitamin C. Bacterial
growth was measured continuously in control urine for 10 hours after incubation for 2 hours in acidic urine
with varying concentrations of nitrite added.
Results.
Large amounts of NO were released from infected nitrite-containing urine after mild acidification.
NO was also released from acidified control urine if nitrite was added, and this release was greatly potentiated in the presence of vitamin C. Furthermore, the growth of E. co/i was markedly reduced by the addition
of nitrite to acidified urine.
Conclusions.
We propose that nitrite-producing bacteria induce their own death in acidic urine by supplying
substrate for generation of bacteriostatic compounds such as NO. This mechanism might explain why urinary
acidification and vitamin C may be effective in the treatment of bacteriuria. UROLOGY
50: 189- 191, 1997.
0 1997, Elsevier Science Inc. All rights reserved.

Objectives.

cidification of urine (eg, by intake of vitamin C) has long been used in medical praxis
to protect against lower urinary tract infection. Despite this long history, the mechanism of action is
not understo0d.l It was recently shown that the
gas nitric oxide (NO), which may act as a bacteriostatic, is produced in the acidic stomach from
nitrite in swallowed saliva.2*3 Nitrite is formed
when nitrate in saliva is reduced by bacteria in the
oral cavity. Similarly, during infectious cystitis,

From the Department

of Physiology
and Pharmacology,
Karolinska Institute,
Stockholm,
Sweden; Department
of Urology and
Department
of Anaesthesiology
and Intensive
Care, Karolinska
Hospital,
Stockholm,
Sweden; and Departments
of Clinical
Microbiology
and Cancer Epidemiology,
University
Hospital,
Uppsala, Sweden
Reprint
requests:J.
0. N. Lundberg,
M.D., Ph.D., Department
of Physiology
and Pharmacology,
Karolinska
Institute,
S-l 71 77
Stockholm,
Sweden
Submitted
(Rapid Communication):
March
3, 1997, accepted
(with revisions):
April 4, 1997
0 1997,
ELSEVIER
SCIENCE
ALL RIGHTS RESERVED

bacteria may convert urinary nitrate to nitrite,

which can be detected with a conventional urinary
strip reagent. We studied NO release and bacterial
growth in nitrite-containing
urine after mild acidification.
MATERIAL

AND METHODS

Urine was collected

from 10 healthy
control
subjects
(26
to 42 years old) and 8 patients
(41 to 70 years old) with
bacteriuria
as confirmed
by urinary
cultures.
Nitrite
concentration
in infected
urine
was measured
with
capillary
electrophoresis,
and NO release was studied
after adjusting
the pH to 4.5 or 5.0 with
1 M HCl. In control
urine,
NO
formation
was measured
at different
pH values (4 to 8) and
with varying
amounts
of nitrite
(10 to 500 PM).
We also
studied
whether
NO release from acidified
nitrite-containing urine would
be influenced
by the addition
of ascorbate
at a concentration
(10 mM) resembling
that found in urine4
after daily ingestion
of 1 to 2 g. Similar
measurements
were
also performed
in urine
from
5 healthy
control
subjects
before
and after ingestion
of vitamin
C (2 g/day)
for 2 days.
In all experiments,
urinary
samples
(10 mL) were incu0090-4295/97/$17.00

INC.
PII

soogo-4295(97>ooz57-4

189

bated in a closed syringe at 37C with a head space of 50

mL. After 30 minutes, the head space gas was removed and
immediately
injected into a chemiluminescence
NO analyzer (Eco Physics, Switzerland).
Ambient NO levels were
below 4 ppb in all experiments.
A reference strain, Escherichia cob (ATCC 25922) was
grown in Mueller-Hinton
broth for 6 hours at 37C resulting in 3 X lo8 colony-forming
units (CFU)/mL.
The
strain was diluted to a bacterial density of approximately
lo6 CFU/mL in acidified urine with or without the addition
of nitrite and kept for 2 hours at 37C in a closed tube.
Bacterial growth was measured continuously
in control
urine medium for 10 hours by vertical photometry in a
computerized
incubator for bacteria (Bioscreen C, Labsysterns, Helsinki, Finland).

a)
100000
1

RESULTS

Basal NO formation
was low both in control
urine and in infected urine. In contrast, large
amounts of NO were generated from infected
urine (containing
8 to 400 PM nitrite) when the
urine was acidified to pH 4.5 or 5.0 and from acidified noninfected urine if nitrite was added (Fig.
la). Urinary NO release was strongly pH dependent in the presence of nitrite and was greatly enhanced by the addition of ascorbic acid (Fig. lb).
Also, NO formation increased with higher nitrite
concentrations
in urine. At pH 5, SO-ppb NO was
released from 10 PM nitrite, whereas 100,250, and
500 PM nitrite yielded 400-, 1,500-, and 4,000ppb NO, respectively. After ingestion of vitamin
C, urinary NO release (at pH 5.0, 100 PM nitrite)
increased sevenfold compared with control conditions.
The addition
of nitrite to acidified urine (pH
5.0) dose dependently
reduced the growth of E.
coli (Fig. 2a). The inhibition
of bacterial growth
was greater at lower urinary pH when a fixed
concentration
of 100 PM nitrite was used (Fig.
2b).
COMMENT

We show here that the growth of E. coli, the

most common
pathogen in the lower urinary
tract, is markedly
inhibited
in mildly acidified
urine when nitrite is present. The bacteriostatic
effects of acidified nitrite described here and by
otherP
may be related to the release of NO, a
gas known to inhibit the growth of a variety of
microorganisms.
Indeed, large amounts of NO
were released from nitrite-containing
infected
urine when acidified, and the levels seemed to
correspond
well to the bacteriostatic
effects.
When nitrite is acidified, other compounds
are
formed in addition
to N0.8 These include sodium nitrite (NaNO,)
and nitrous acid (HN02),
both of which have been reported to have bacteriostatic properties.8,9 Thus, NO alone may not
account for all antibacterial
properties of acidified urine.
190

5.5-6.5

5.0

Basal

4.5
Acidified

W
s
8
%
.::
'C0
.t
z

1o5
1 o4
1000
100
10
1
4

4.25

4.5

4.75

5.5

6.5

PH
FIGURE 1. (a) Urinary NO release from control urine
(open bars), control urine with 100 PM NaN02 (striped
bars), and infected urine containing
8 to 400 PM nitrite
(hatched bars). (b) NO release from control urine (100
PM nitrite) at different pH values with (solid bars) or
without (striped bars) 10 mM ascorbic acid.

The great potentiation

of urinary NO release observed here in the presence of ascorbate is in accordance with the knowledge that vitamin C may
enhance NO formation from nitrite within a wide
pH range. Thus, intake of ascorbate may induce
urinary NO formation during infection, not only
by decreasing urinary pH but also by its intrinsic
ability to reduce nitrite to NO. This may also result
in the formation of NO at bacteriostatic
concentrations when urinary pH is somewhat higher.
The ability of urinary pathogens such as E. coli
to generate nitrite may, under acidic conditions,
lead to the production of NO, as shown here. UriUROLOGY

50 (21, 1997

A
.=
l!
43

0.25

5.z
0

0
0

Time

IO

(h)

Thus, nitrate-reducing
bacteria may induce their
own destruction, provided that the urine is made
acidic. Infected urine is often slightly alkaline, and
therefore NO release is low despite a high nitrite
concentration.
However, ingestion of ascorbate or
ammonium
chloride, for example, will result in
acidification
of the urine, with a concomitant
increase in urinary NO output.
The exact mechanism underlying
the effects of
urinary acidification
on bacterial growth needs to
be further elucidated.
Nevertheless,
the results
presented here offer a novel explanation
for the
beneficial effects of urinary acidification
and vitamin C on lower urinary tract infections, expanding
the role of nitrite beyond diagnosis of disease.
ACKNOWLEDGMENT.

To Tord Nystrom for expert technical

assistance.

REFERENCES

5.5

4.5

PH
Growth of Escherichiacob after exposure to
acidified nitrite-containing urine. Experiments were performed using (a) different concentrationsof nitrite (@Vl)at
pH 5.0 and with (b) different pH values using a fixed concentration of nitrite (100 fl, solid bars). Optical density
in (b) was measuredat 4 hours. Asterisk indicatessignificant differencefrom control conditionswithout nitrite (open
bars, P ~0.05, Mann-Whitney U test, mecmof 15 experiments).
FIGURE 2.

nary NO generation seems to be autoregulated

in
vivo because the release of NO is proportional
to
the amount of nitrite produced by the bacteria.

UROLOGY

50 (21, 1997

1. Quinn EL, and Kass EH (Eds): Principles in the Long

Term Management of Chronic Infection of the Urinary Tract.
Boston, Little Brown and Co, 1960, p 663.
2. Lundberg JON, Weitzberg E, Lundberg JM, and Alving
K: Intragastric nitric oxide production in humans: measurements in expelled air. Gut 35: 1543-1546,
1994.
3. Benjamin N, ODriscoll F, Dougall H, Duncan C, Smith
L, Golden M, and McKenzie H: Stomach NO. Nature 368:
502,1994.
4. Brandt R, Guyer KE, and Banks WL Jr: Urinary glucose
and vitamin C. Am J Clin Path01 68: 592-594, 1997.
5. Tarr H: Bacteriostatic actions of nitrates. Nature 147:
417-4181941.
6. Dykhuizen RS, Frazer R, Duncan C, Smith CC, Golden
M, Benjamin N, and Leifert G: Antimicrobial
effect of acidified
nitrite on gut pathogens: importance of dietary nitrate in host
defence. Antimicrob
Agents Chemother 40: 1422-1425,
1996.
7. Moncada S, Palmer RM, and Higgs EA: Nitric oxide:
physiology, pathophysiology
and pharmacology. Pharmacol
Rev 43: 109-141,1991.
8. Kaplan SS, Lancaster JR Jr, Basford RE, and Simmons
RL: Effect of nitric oxide on staphylococcal killing and interactive effect with superoxide. Infect Immun 64: 69-76,1996.
9. Kono Y, Shibata H, Adachi K, and Tanaka K: Lactatedependent killing of Escherichia coli by nitrite plus hydrogen
peroxide: a possible role of nitrogen dioxide. Arch Biochem
Biophys 311: 153-159, 1994.
10. Bartsch H, Ohshima H, and Pignatelli B: Inhibitors of
endogenitric oxide nitrosation. Mechanisms and implications
in human cancer prevention. Mutation Res 202: 307-324,
1988.

191