Can Fragrance Ingredients Inhibit the Reactions That Produce Urine Odor?

Focusing only on ammonia when considering urine odor may overlook another important pathway. A 2016 study examined how substances originally present in urine in a poorly volatile form are broken down by an enzyme and release odor.

Screening fragrance ingredients revealed compounds that inhibited this breakdown enzyme. In experiments with enzyme-treated urine, substances remained in their nearly odorless form, and the increase in urine odor was also suppressed. The idea is to inhibit the reaction that generates odor, rather than simply adding fragrance on top.

Potential odor sources are present in a poorly volatile form

The study focused on phenolic compounds such as p-cresol. In freshly voided urine, many of these components occur as conjugates, bound to glucuronic acid or sulfate. Binding makes them more water-compatible and less volatile, so they are normally odorless.

When those bonds are broken, however, volatile components reappear. The researchers examined which type of conjugate declined in urine, searched for compounds inhibiting the enzyme responsible for breakdown, and then investigated both the inhibition mechanism and effects on urine odor.

The glucuronic-acid conjugate was the one that declined

Urine was collected from four healthy men in their thirties to fifties and pooled in equal amounts. In the first experiment, filtered urine from which bacteria had been removed and unfiltered urine were stored at room temperature, and the two p-cresol conjugates were tracked by liquid chromatography–mass spectrometry.

In unfiltered urine, the glucuronide conjugate declined substantially over time. It did not decline in filtered urine, and the sulfate conjugate did not decline under either condition. These results led the researchers to focus on microorganism-associated breakdown of glucuronide conjugates.

They next added commercially available Escherichia coli-derived β-glucuronidase to filtered urine and allowed it to react for one day at room temperature. Phenolic compounds and indole were produced, and two expert assessors perceived stronger urine odor. This experiment confirmed that the conjugate-cleaving enzyme reaction was associated with increased urine odor.

Screening 193 fragrance compounds for enzyme inhibition

The researchers tested 193 structurally diverse fragrance compounds, adding each at 100 ppm to an enzyme assay. At this stage they used an artificial substrate that permits color measurement after enzymatic breakdown, rather than urine itself, to compare how far the reaction proceeded.

The top five compounds showed at least 90% inhibition. The top two, including 8-cyclohexadecen-1-one (8Cy16), required low concentrations to halve enzyme activity; both were macrocyclic compounds with large molecular rings.

A closer comparison of 15 related macrocyclic compounds showed a tendency for compounds with ketone structures or double bonds within the ring to display strong inhibition. Under these assay conditions, the concentration of 8Cy16 needed for 50% inhibition was about one-fortieth that of the known inhibitor D-glucaro-1,4-lactone (1,4-GL).

This ratio compares concentrations required in a particular enzyme assay. It does not mean a product has 40 times the deodorizing power; it was a measure for selecting candidates for further experiments.

Temporarily inhibiting the enzyme rather than destroying it

To investigate 8Cy16’s action, the researchers mixed it with the enzyme and then removed it using gel filtration. Enzyme activity returned to its original level. This is consistent with reversible inhibition that disappears when the compound is removed, rather than irreversible destruction of the enzyme.

They also measured reaction rates while varying substrate concentration. The pattern for 8Cy16 was consistent with noncompetitive inhibition, in which the enzyme’s maximum processing rate remains lower even when more substrate is supplied. This differed from the competitive inhibition observed for the comparator, 1,4-GL.

The study did not visualize binding to the enzyme’s three-dimensional structure, but it went beyond observing a slower reaction to characterize the type of inhibition.

In urine, too, conjugate breakdown and the increase in odor were suppressed

Finally, the researchers added the E. coli-derived enzyme to filtered urine and allowed it to react for two hours at 37°C. Without an inhibitor, approximately 40% of the p-cresol glucuronide was broken down, and mean urine-odor intensity rated by two assessors increased from 1 to 3 on a 0–5 scale.

Adding either 0.01% 8Cy16 or 0.1% 1,4-GL suppressed conjugate breakdown and reduced the increase in urine odor. Perceived urine-odor intensity was lower with 8Cy16 than with 1,4-GL added at ten times the concentration.

Because 8Cy16 has an odor of its own, masking that makes urine odor less noticeable is also possible. However, chemical analysis showed that conjugates were preserved, and even odorless 1,4-GL limited the increase in urine odor to no more than 0.5. These observations support the explanation that inhibiting the enzyme reaction itself reduced odor generation. The experiment did not completely separate the contribution of fragrance effects.

This was preliminary work involving two assessors, with enzyme assays also based mainly on averages of duplicate measurements. Microorganisms and their enzymes in actual care settings, and safety for people during use, were not tested. The authors identify determining the microorganisms present in those settings and verifying efficacy against them as future tasks.

Summary

In this study, p-cresol glucuronide in urine declined under unfiltered conditions, and adding its breakdown enzyme increased urine odor. The fragrance compound 8Cy16 reversibly inhibited that enzyme and reduced both conjugate breakdown and the increase in odor in enzyme-treated urine.

In addition to capturing odors already released, urine-odor control can aim to maintain molecules in a form less likely to release odor. Although conditions for practical application remain to be established, the study demonstrates the potential for a fragrance compound to act on an enzyme reaction.

All four authors were listed as affiliated with Kao’s Fragrance Development Research Laboratory at publication. No separate research-funding or competing-interest declaration section was identified.

Reference

Mori I, Yabuki M, Ishida H, Kashiwagi M. (2016). Suppression of Urinary Odor Generation by β-Glucuronidase Inhibitors. Journal of Japan Association on Odor Environment, 47(3), 222–229. doi:10.2171/jao.47.222 (Original full text)