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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)