THE EFFECT OF ETHNICITY ON HUMAN AXILLARY ODORANT PRODUCTION (2024)

Abstract

Previous findings from our laboratory highlighted marked ethnic differences in volatile organic compounds (VOCs) from cerumen among individuals of Caucasian, East Asian, and African-American descent, based, in part, on genetic differences in a gene that codes for a transport protein, which is a member of the ATP-binding cassette transporter, sub-family C, member 11 (ABCC11). In the current work, we hypothesized that axillary odorants produced by East Asians would differ markedly from those obtained from individuals of European or African descent based on the pattern of ethnic diversity that exists in ABCC11. Using gas chromatography/mass spectrometry (GC/MS) we examined differences in axillary odorant VOCs among 30 individuals of African-American, Caucasian, and East Asian descent with respect to their ABCC11 genotype. While no qualitative differences in the type of axillary odorants were observed across ethnic groups, we found that characteristic axillary odorants varied quantitatively with respect to ethnic origin. We propose that ABCC11 is not solely responsible for predicting the relative amounts of volatiles found in axillary secretions and that other biochemical pathways must be involved.

Keywords: Axillary odor, ethnicity/race, volatile organic compounds, genetics, analytical chemistry

INTRODUCTION

The axillae of humans contain a large number of apocrine, eccrine, and sebaceous glands. Volatile organic compounds (VOCs) emanating from this area give rise to the canonical axillary odor (commonly referred to as “body odor”). Although this odor is mainly perceived by modern society as unpleasant, several studies indicate that axillary odorants contain chemical signals that affect the menstrual cycle (Preti et al. 2003) or that may be involved in major histocompatibility complex, allele-dependent, mate selection (Wedekind et al. 1995). These studies point to the importance of understanding the factors that contribute to human odor production. Current evidence suggests that genetically based odor profiles play a role in individual identity, immune and stress responses, and that odor may be useful for monitoring physical, emotional, and disease states in humans (Dormont et al. 2013). For this potential to be fully realized, however, careful analytical studies, in well-characterized human populations, are needed. Indeed, recently NIH has urged ethnic/racial diversity and genetic analyses to be inclusion criteria in clinical/medical research (Green et al. 2011). Our approach, herein, is to study the influence of ethnic and genetic factors contributing to human odorant profiles.

Human body odor is a complex mixture of VOCs. Organoleptic and analytical chemistry methodologies have revealed that axillary odor derives from a complex mixture of C₂–C₁₁ normal, branched, and unsaturated acids, with the main components being (E)-3-methyl-2-hexenoic acid (E-3M2H) (Pierce et al. 1995; Zeng et al. 1991, 1992, 1996a, b) and 3-hydroxy-3-methylhexanoic acid (3H3M) (Natsch et al. 2003), as well as volatile sulfur compounds, particularly (S)-3-methyl-3-sulfanylhexan-1-ol) (Hasegawa et al. 2004; Natsch et al. 2004; Troccaz et al. 2004). The latter sulfur-containing compounds often are present at very low levels but have high odor impact (i.e., low olfactory threshold). Volatile steroids, such as 5α-androst-16-en-3-one (androstenone) and 5α-androst-16-en-3β-ol (androstenol), originally were thought to play a role in axillary odor (Bird and Gower 1981), but later were found to be minor sensory and analytical contributors relative to the organic acids (Zeng et al. 1996a, b).

Apocrine secretions, which contain the precursors to axillary odor, are odorless upon secretion and become odorous after interaction with the axillary microbial communities residing on the skin's surface (Labows et al. 1979; Leyden et al. 1981; Natsch et al. 2003, 2006; Troccaz et al. 2009; Starkenmann et al. 2005; Zeng et al. 1992). In those studies that have collected and used sterile, pure apocrine secretions, the odorless precursors are proteins that contain the odorants within lipid-friendly calyxes (Spielman et al. 1995; Zeng et al. 1992, 1996a). The axillae support a dense bacterial population dominated by two types, Staphylococcus and Corynebacterium. A strong correlation has been found between a dense population of corynebacteria and robust axillary odor production (Barzantny et al. 2012; Leyden et al. 1981).

Recent studies have suggested that human axillary odor is influenced by genetic factors. The human adenosine triphosphate (ATP)-Binding Cassette transporter, sub-family C, member 11 (ABCC11), encodes an ATP-driven efflux transporter that is expressed and localized in human apocrine sweat glands. The ABCC11 protein is known to transport a variety of small molecules (Chen et al. 2005; Kruh et al. 2007) and has been shown to play a role in both the generation of axillary malodor (Baumann et al. 2014; Martin et al. 2010; Preti and Leyden 2010) and earwax (cerumen) phenotype (Toyoda et al. 2009; Yoshiura et al. 2006). A single nucleotide polymorphism (SNP, rs17822931) in ABCC11 (538G→A) leads to a glycine-to-arginine substitution (G180R) in the resulting protein. The G180R variant of ABCC11 results in a dry, white earwax phenotype, which is predominant (80-95%) among East Asian populations (e.g., Japanese, Korean, and Chinese), but is quite rare (0-3%) among individuals of European and African descent. TT hom*ozygous individuals for the G180R variant produce significantly less characteristic axillary odor than both heterozygous (CT) and hom*ozygous (CC) individuals (Harker et al. 2014; Inoue et al. 2010; Martin et al. 2010; Nakano et al. 2009). Previous findings from our laboratory highlighted marked ethnic differences in cerumen VOCs between individuals of Caucasian, East Asian, and African American descent (Prokop-Prigge et al. 2014, 2015). Our current work extends that of previous reports, both ours and that of others, and investigates the influence of ethnicity on the relative amounts of axillary VOCs produced. Based on the pattern of ethnic diversity that exists in ABCC11, we hypothesized that axillary odorants produced by East Asians will differ markedly from those obtained from individuals of European and African descent. Using gas chromatography/mass spectrometry [GC/MS], we examined differences in VOCs found in the axillary secretions of Caucasian, East Asian, and African-American individuals with respect to their ABCC11 genotype.

METHODS AND MATERIALS

RESULTS

The ABCC11 genotype (rs17822931) of each donor was analyzed (Fig. 1). Nine East Asian donors were identified as TT hom*ozygotes, while one was found to be a CT heterozygote. The remaining 20 donors (Caucasian and African-American) were all CC hom*ozygotes. These findings match the expected allele frequencies for these populations (Yoshiura et al. 2006).

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Fig. 1

ABCC11 genotype (rs17822931) of 30 male donors with respect to ethnicity/race.

We examined whether (a) the ABCC11 genotype and (b) ethnicity contributed to variability in axillary sweat samples collected from all genotyped donors. The results revealed that all donor groups, at least qualitatively, produced the same volatile acid profile. However, subtle differences in the amounts of short, C₂-C₆, straight- and branched-chain organic acids were observed (Fig. 2). While these differences were not found to vary significantly across groups, it is interesting to note that relative differences were found for the branched-chain acids specifically. As a group, African-Americans produced relatively higher amounts of all straight-chain acids: acetic, propanoic, butyric, valeric, and hexanoic; however, when branch-chained acids (isobutyric, 2-methylbutyric, and isovaleric acid) were examined, Caucasian donors appeared to produce relatively higher amounts than East Asian and African-American donors. These shifts between Caucasians and other donor groups may have contributed to our non-significant statistical results across all compounds (see below).

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Fig. 2

Comparison of short-chain volatile acids detected in axillary sweat samples collected from East Asian, Caucasian, and African-American donors. Variation across the three donor groups was nearly significant (F_(16,42) = 1.76, P = 0.07).

All 12 compounds measured in all individuals (see Fig. 2-​-3)3) were subjected to a multivariate analysis of variance (MANOVA) with donor group as a between-subjects factor. Granted, many assumptions underlying this parametric analysis were violated, this statistical approach revealed no significant difference across donor group (F_(24,34) = 1.03, P = 0.46), although there were some suggestions of differences that were probed with ANOVA and non-parametric tests (see below).

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Fig. 3

a 3-Methyl-2-hexenoic acid (3M2H) comparison revealing significant differences (P = 0.009, Kruskal-Wallis test) in the relative amounts of the characteristic axillary odorant, E-3M2H, among the three donor groups (also, see text for a significant interaction generated by a repeated measures ANOVA). b Comparison of 7- and 2-octenoic acid detected in axillary sweat samples from East Asian, Caucasian, and African-American donors. For 7-octenoic acid, differences in the distributions across the three groups of donors was suggestive (Jonckheere-Terpstra test for ordered alternatives; P = 0.065; across the three groups, this test has more statistical power than the Kruskal–Wallis test).

While differences were marginal for short-chain volatile acids across donor groups (a MANOVA revealed F_(16,42) = 1.76, P = 0.07; Fig. 2), differences were noted for the longer-chain, characteristic axillary, odorants (e.g., 3-methyl-2-hexenoic acid, 7-octenoic acid, Fig. 3). Both isomers of 3M2H were subjected to repeated measures ANOVA with donor group as a between-subjects factor. The main effect of isomer was significant (F_(1,27) = 8.62, P = 0.007; E > Z), but so was the interaction between isomer and donor group (F_(2,27) = 4.51, P = 0.02; i.e., the pattern of results across donor groups was significantly different for the two isomers). We used the Kruskal-Wallis nonparametric test to explore further variation in the E-and Z-isomers of 3M2H. For the Z-isomer, there was no significant difference across groups (P = 0.238,); however, the E-isomer did vary (P = 0.010) with African-American > Caucasian > East Asian in the relative amounts produced. Mann-Whitney pairwise comparisons, using a Bonferroni-corrected P = 0.017, revealed a significant difference between samples from African-Americans vs. East Asians (U = 10.0, P = 0.002); no other pairwise comparison was significant. A similar trend also was observed for 3-hydroxy-3-methylhexanoic acid (3H3M) with African-American > Caucasian > East Asian in relative amounts observed. While difficult to quantitate using the GC/MS parameters employed, there was a significant difference across donor groups in the number of individuals who produced detectable levels of 3H3M (Fig. 4; Χ² = 10.05, P = 0.007). Post hoc tests using adjusted residuals and a Bonferroni correction revealed a significant effect of African-American vs. East Asian donors.

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Fig. 4

Percentage of donors from whom 3H3M was detected in axillary secretions. Chi Square analysis revealed a significant difference across groups (Χ² = 10.05, P = 0.007).

DISCUSSION

The sources that contribute to human body odor and an individual's odor profile are diverse, and recent studies have suggested that there may be an underlying genetic factor that controls axillary odorant formation. A single gene, viz., ACBCC11 (rs17822931), is believed to be a major component for predicting the level of axillary odorants and earwax phenotype (Baumann et al. 2014; Martin et al. 2010; Preti and Leyden 2010; Toyoda et al. 2009; Yoshiura et al. 2006). Our results, however, suggest that the control of odor production is not so straight forward. Examination of 30 male individuals of three diverse ethnic/racial backgrounds (African-American, Caucasian, and East Asian) revealed that key characteristic axillary odorants vary with respect to ethnic origin, not strictly ABCC11 genotype, as previously reported.

The ABCC11 genotype (rs17822931) of each donor was analyzed, and the findings are in excellent agreement with the expected allele frequencies for the three populations (Yoshiura et al. 2006). Nine East Asian donors were identified as TT hom*ozygotes, while all Caucasian and African-American donors were CC hom*ozygotes. Previous findings suggest that individuals hom*ozygous for the SNP 538G→A produced significantly less characteristic axillary odorant precursors than both heterozygous (C/T) and wild-type (C/C) individuals (Harker et al. 2014; Inoue et al. 2010; Martin et al. 2010; Nakano et al. 2009). Based on these findings, and the marked ethnic diversity of the ABCC11 allele frequencies, one would predict East Asian donors to exhibit lower levels of axillary odorants as compared to donors of Western descent. Our findings are consistent with these predictions. The results, however, also reveal that despite exhibiting the same ABCC11 genotype, there were marked differences in the levels of characteristic axillary odors between African-American and Caucasian donors. For example, a major contributor to axillary odor, E-3M2H, was significantly higher in African-Americans when compared to Caucasians. In addition, there were significant differences across donor groups in the number of individuals who produced detectable levels of 3H3M, with African-American > Caucasian > East Asian. The lack of 3-methyl-3-sulfanylhexan-1-ol in any of our extracts is due most likely to the method of collection. Its oniony-sweaty odor was not evident in the extracts (though present on some T-shirts evaluated organoleptically), suggesting that it was not extracted efficiently under the extraction conditions employed. Previous studies (Hasegawa et al. 2004; Natsch et al. 2004; Troccaz et al. 2004) have isolated it from pooled, liquid sweat collected directly from the axillae of multiple donors.

Previous results suggest that the ABCC11-controlled transporter protein should not be functional in our cohort of East Asian donors with the TT genotype (Martin et al. 2010). In contrast, Harker et al. (2014) noted that their donors with TT genotype had measurable, albeit significantly lower, levels of axillary malodorant precursors (measured as both their N-acyl glutamine conjugates and the corresponding free acids). The findings also demonstrate that odorant molecules, like 3M2H, are still able to reach the skin's surface. Preti and Leyden (2010) noted that research groups examining the influence of ABCC11 had failed to consider the influence of the lipocalin-type proteins, namely apocrine apolipoprotein D (ApoD) (Spielman et al. 1998; Zeng et al. 1996a). Quantitative measures of this protein in both axillary and apocrine secretions demonstrate that it is present in Asian populations, albeit at significantly lower concentrations than in Caucasians or African-Americans (Jacoby et al. 2004). A second protein found in apocrine secretions also was shown by Spielman et al. to carry 3M2H, but its blocked N-terminus failed to provide structural identity by classical means (Spielman et al. 1995, 1998). Spielman and co-workers designated this protein ASOB1 (apocrine secretion odor binding protein 1; molecular weight approx. 45kD). Studies involving antibodies to apocrine ApoD as well as ASOB1 demonstrate that these proteins have wide-spread presence in various body fluids/secretions (Gallagher et al. 2008; Spielman et al. 1995). Consequently, these findings suggest that biochemical pathways involving protein carriers of odorants other than the protein made by the ABCC11 gene are involved in the regulation of transport and/or release of volatile odorants in the axilla. Recent studies and reviews of axillary odor formation (Harker et al., 2014; Natsch 2015) appear to have neglected incorporating the ApoD protein precursor into their narrative, as noted by Preti and Leyden (2010). The isolation procedures used by these investigators (Harker et al., 2014; Natsch 2015) and others did not focus on sterile-collected, apocrine secretions as did the study by Zeng et al. (1996a). Instead, they used non-sterile axillary washings, thus providing ample time for the axillary bacteria to convert protein carriers to the N-acyl glutamine conjugates, as previously indicated (Preti and Leyden, 2010).

Odorous, volatile short-chain fatty acids, albeit more minor, are contributors to human axillary odor (Harker et al. 2014). Our results reveal a marginal effect on the relative amounts of these compounds across the ethnic groups studied. The reduced presence of these short-chain acids, in addition to low levels of E- and Z-3M2H documented here and by others (Akutsu et al. 2006; Harker et al. 2014), most likely contribute to the reduced malodor reported for individuals of Asian descent (typically TT hom*ozygotes). In addition, interesting trends were revealed here for the relative amounts of branched chain vs. straight short-chain volatile acids. Individuals from the cohort of African-American donors produced slightly higher amounts of all straight-chain acids: acetic, propanoic, butyric, valeric, and hexanoic. When branched chain acids (isobutyric, 2-methylbutyric, and isovaleric acid) were examined, Caucasian donors appeared to produce relatively higher amounts than both Asian and African-American donors. Perhaps differences in dietary intake can account for this trend. The amino acids leucine and isoleucine are found in high levels in dairy products (Annigan 2014). The branched-chain volatile acids, isovaleric acid and 2-methylbutyric acid, stem from the metabolism of these two amino acids, respectively. East Asians and African-Americans experience higher levels of lactose intolerance than do Caucasians (ProCon.org 2010); hence we speculate that increased intake of dairy products by individuals of Caucasian descent results in increased levels of the branched chain amino acids in these individuals. There has been considerable speculation concerning the influence, if any, of diet on axillary odor, but little to no experimental data exists to date. However, Havlicek and Lenochova (2006) have shown that red meat consumption increases the negative hedonics of axillary odors (e.g., less attractive, more intense and unpleasant). While the measured differences in branched chain volatile acids in this study did not vary significantly across donor groups; perhaps a larger sample size and a controlled study monitoring dietary intake would provide insight. Further studies on the effect of diet on axillary odorant profiles are warranted.

Another interesting trend was seen in the data from East Asian donors (as shown in Fig. 3a). The ratio of E-to Z-isomers for 3M2H appears different from that seen for African-Americans and Caucasian donors, and this appears to be the reason for the significant interaction between donor group and 3M2H isomer (see Results section). The E-to Z-isomer ratio for East Asian individuals favors the Z-isomer vs. the larger relative amount of the E-isomer seen in the other two donor groups (Zeng et al. 1991, 1996a). Hence, the biogenesis of the 3M2H may differ across the ethnic groups, where conditions in the apocrine glands of the East Asians energetically favor formation and/or retention of the Z-isomer.

Overall, our findings suggest that an individual's ethnicity has a significant impact on human axillary odor production. Ongoing sensory studies will address whether differences in ethnicity can be discriminated by human panelists. Furthermore, as advancements are made in studies of disease-related alterations in metabolism, some of which may affect qualitative/and or quantitative expression of odorous VOCs that emanate from the body or its excretions, e-nose technologies may prove to be an invaluable supplement to diagnostic protocols. When this occurs, differences in baselines across different groups will have to be taken into account.

Acknowledgments

The authors thank Jason Eades for technical support. KAP acknowledges support from NIH-NIDCD Postdoctoral Training Grant 5T32DC0014 as well as Monell Institutional Funds. The genotyping was performed at the Monell Genotyping and DNA/RNA Analysis Core, which is supported, in part, by funding from the NIH-NIDCD Core Grant P30DC01173.

Footnotes

REFERENCES