j. Soc. Cosmet. Chem., 41, 173-185 (May/June 1990) Axillary malodor production' A new mechanism C. FROEBE, A. SIMONE, A. CHARIG, and E. EIGEN, Colgate-Palmolive Technical Center, 909 River Road, Piscataway, NJ 08854. Received May 15, 1990. Synopsis The mechanism proposed by Eigen (1) for the generation of axillary malodor from steroids has been ex- plored. Previous work indicated that axillary odor is largely due to the steroids 16,5ot-androstene-313-ol and 16,5ot-androsten-3-one. Following Eigen, we theorize that sterile and odorless apocrine secretions from the axilla contain these steroids as their water-soluble sulfates and glucoronides and that odor is produced only after the volatile free steroids are liberated from these esters by bacterial hydrolyric enzymes such as aryl sulfatase and beta-glucuronidase. Support for this hypothesis was obtained by producing odor from sterile, odorless, apocrine sweat by the addition of either of the two enzymes, or of the corynebacterial strains that produce them. Finally, steroid realodor production can be prevented by inhibitors of beta-glu- curonidase and of aryl sulftase. INTRODUCTION The human axilla is populated with two classes of sweat gland: The eccrine glands produce a watery secretion in response to heat. Apocrine glands produce microdroplets of a viscous secretion in response to emotional stress. Apocrine sweat is a complex mixture containing cholesterol, steroids, and other lipids, as well as 10% protein. It was recognized as early as 1956 that axillary odor is generated from apocrine secretion. Although the sterile fluid is odorless, bacterial action on certain components present in sweat produces the characteristic underarm odor (2). Although the initial view of Strauss and Kligman was that any or all of the axillary population could cause odor, within a few years Shehadeh and Kligman found that it was caused chiefly by gram-pos- itive types (3). Recent work suggests two classes of odorants in the axilla: short-chain fatty acids, e.g. isovaleric acid, and the androgen steroids, especially 5-ot-androstenol and 5-ot-andros- tenone (see Figure 1) (4). It must be mentioned that, while these species have been chemically identified in the axilla through gas chromatographic methods, the identifi- cation of them as axillary odorants has relied upon organoleptic means. Therefore, the positive identification of these three species as the principal axillary odorants has not yet been made. Indeed, other related compounds may contribute to the complex odor. A correlation has been found between the composition of the underarm flora and the 173

174 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS nature of the odor produced: Where the axillary microbial population is dominated by coryneform bacteria (lipophilic diphtheroids), the acrid odor of delta-16 steroids is ap- parent, whereas, if the axillary population is dominated by such micrococci as Staphylo- coccus epidermidis, the odor of isovaleric acid prevails. Pronounced axillary odor is corre- lated with the occurrence of the coryneform flora (5) it is on the steroid compounds associated with these strains that this research focuses. Many studies have identified axillary steroids and have linked their presence with indigenous bacteria (4- 13) how- ever, the mechanism of bacterial action has remained uncertain (9,12). Since they are not typically water-soluble, steroids are normally transported in body fluids as their water-soluble conjugates with sulfate or glucuronic acid (14). When we began to speculate on the origin of volatile free steroids in the axilla, we hypothesized that apocrine secretion contained the steroids as water-soluble conjugates (1). Conver- sion in vivo to the free steroid generally requires the action of hydrolytic enzymes. It seemed likely that it was the production of these enzymes that represented the contri- bution of bacteria to the generation of underarm oder (Figure 2). We theorized that sterile apocrine sweat would deposit the water-soluble, odorless conjugates onto hair and skin in the axilla, where enzymes secreted by local bacteria would release the vola- tile, odorous, free steroids. The enzymes expected to hydrolyze the steroid esters might be any of several bacterial exoesterases -- for example, beta-glucuronidase (beta-G) and aryl sulfatase (AS). These enzymes can be detected with the synthetic substrates 4-methylumbelliferyl glucuro- nide (4-MUG) and 4-methylumbelliferyl sulfate (4-MUS), respectively, both of which release fluorescent 4-methylumbelliferone (4-MU) upon hydrolysis (Figure 3) (14,15). isovaleric acid CH3 %CH --CH= •COOH 5 ,c•-androst- 16-en-3,[5-ol 5,ct-androst- 16-½n-3-on½ Figure 1. Proposed sources of axillary odor.