Acidification 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 understood.' 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, 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 HC1. In control urine, NO formation was measured at different pH values (4 to 8) and with varying amounts of nitrite (10 to 500 MM). 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 incubated in a closed syringe at 37°C 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 coli (ATCC 25922) was grown in Mueller-Hinton broth for 6 hours at 37°C, re­sulting in 3 X 10⁸ colony-forming units (CFU)/mL. The strain was diluted to a bacterial density of approximately 10⁶ CFU/mL in acidified urine with or without the addition of nitrite and kept for 2 hours at 37°C 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, Labsys­tems, Helsinki, Finland).

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 .tM 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, 50-ppb NO was released from 10 µM nitrite, whereas 100, 250, and 500 1tM nitrite yielded 400-, 1,500-, and 4,000-ppb NO, respectively. After ingestion of vitamin C, urinary NO release (at pH 5.0, 100 .tM 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 µM 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 others'' may be related to the release of NO, a gas known to inhibit the growth of a variety of microorganisms.⁷ 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 NO.⁸ These include so­dium nitrite (NaNO₂) and nitrous acid (HNO₂), 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.

FIGURE 1. (a) Urinary NO release from control urine (open bars), control urine with 100 µM NaNO₂ (striped bars), and infected urine containing 8 to 400 µM nitrite (hatched bars). (b) NO release from control urine (100 µM 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.¹⁰ 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. Uri-

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.

FIGURE 2. Growth of Escherichia coil after exposure to acidified nitrite-containing urine. Experiments were per­formed using (a) different concentrations of nitrite (.tM) at pH 5.0 and with (b) different pH values using a fixed con­centration of nitrite (100 pM, solid bars). Optical density in (b) was measured at 4 hours. Asterisk indicates signifi­cant difference from control conditions without nitrite (open bars, P <0.05, Mann-Whitney U test, mean of 15 exper­iments).

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

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