Jones GRD
More on the inhibition of the urease reaction by boric
acid
Ann Clin Biochem
(Jul) 34:430-432 1997

Boric acid is commonly used as a preservative for urinary
specimens to inhibit bacterial growth during the collection
period. Concentrations of 5-15 g/L boric acid were used in the
past to inhibit bacterial growth. Other urine preservatives
include thiomersal and sodium azide, not popular because of their
toxicity, or hydrochloric acid, which apparently needs to reduce
urine pH to below 2.0 to prevent bacterial growth.
There are four techniques in current use to measure urea
concentrations. In one method, urea reacts directly with
diacetyl monoxime to form a colored product (Fearon reaction).
In the other three techniques, urea is first converted to ammonia
by reacting it with urease. Ammonia is a charged ion and will
increase the conductivity of the reaction mixture. In one urease
method the amount of generated ammonia is assessed directly by
conductivity measurements. In a second technique, the ammonium
reacts with phenol or salicylate to form a blue or green dye
(Berthelot reaction). In a third method, widely used in
autoanalyzers, the generated ammonia is used to drive the
enzymatic conversion of alpha-ketoglutarate to glutamate in a
reaction that involves oxidation of NADH to NAD. The
concentration of NAD that is generated is assessed by UV
spectroscopy, using either an endpoint or a kinetic method (for a
review of all of these methods, see Taylor AJ and Vadgama P.
Analytical reviews in clinical biochemistry: the estimation of
urea. (Ann Clin Biochem 1992;29:245-264).
In an earlier, 1993 paper, Mazurkiewicz and colleagues (Ann
Clin Biochem 1993; 30:215-216) found that boric acid
inhibited urease-based methods of urea analysis. The inhibition
was not present with an older, colorimetric diacetyl monoxime
method of urea analysis. The inhibition was about 10% at 10g/L
boric acid concentration with an automated paired enzyme method,
but only 5% using a urease method in which ammonia is detected by
a non-enzymatic technique (Berthelot reaction). From this result
it was hypothesized
that perhaps boric acid was inhibiting BOTH urease and the
glutamate dehydrogenase reaction, although this hypothesis was
not tested further. The conclusion of the Mazurkiewicz paper
was, that the boric acid concentration should not exceed 2g/L.
In the present paper, Jones studied the effects of boric acid on
urine urea samples using the urease-conductivity method (Beckman)
and the urease-glutamate dehydrogenase paired enzyme method
(Hitachi 747 autoanalyzer). They found that the inhibitory
effect of boric acid was much stronger than that reported
previously by Mazurkiewicz et al; at a 10 g/L boric acid
concentration urea concentration was reduced by 80% using the
Beckman analyzer, and by about 70% using the Hitachi paired
enzyme analyzer. Even at 2 g/L, where Mazurkiewicz found only a
minimal effect, the inhibition was 60% by the Beckman method and
40% by the paired enzyme method. Whereas Mazurkiewicz found that
the paired enzyme method was MORE sensitive to boric acid
inhibition than the Berthelot reaction, in the present paper, the
sensitivity pattern was reversed; the urease-conductivity method
was more sensitive the paired enzyme method. This reverse
sensitivity pattern did not support the hypothesis of
Mazurkiewicz, that boric acid was inhibiting not only urease, but
also the glutamate dehydrogenase reaction.
Comment: The clinical message is, urine for residual
urine function measurements (Kru) should not contain boric acid.
Refrigeration should be used as the primary method of inhibiting
bacterial growth. The sensitivity of the paired enzyme method to
boric acid makes one wonder whether or not uremic inhibitors in
the plasma may cause underestimation of predialysis BUN
measurements.
(John T. Daugirdas, M.D., Univ of IL at Chicago)