The Volatolome of Chromhidrosis

Abstract

This study investigates the volatolome in an individual with chromhidrosis, utilizing solid-phase microextraction (SPME) and pentane extraction, followed by gas chromatography–mass spectrometry (GC-MS), to identify key volatile organic compounds (VOCs). A total of 31 compounds were identified, including aldehydes, fatty acid esters, and benzoic acid derivatives. SPME was more effective in capturing highly volatile compounds, while pentane extraction primarily isolated lipophilic substances such as squalene and cholesterol. The findings suggest that lipid peroxidation and metabolic dysregulation contribute to the formation of lipofuscin, the pigment responsible for colored sweat. Additionally, the detection of 9-octadecenamide and benzoic acid derivatives highlights the role of oxidative processes in chromhidrosis.

1. Introduction

Chromhidrosis is a rare dermatological condition characterized by the secretion of colored sweat, predominantly from apocrine glands located in areas such as the axillae, malar cheeks, and areolae. This condition is associated with the accumulation of lipofuscin, a pigment produced through oxidative stress and aging processes. Depending on its oxidation state, lipofuscin imparts various colors to sweat, including yellow, blue, green, brown, and black [1].

Lipofuscin is a complex mixture of oxidized proteins, lipids, and smaller amounts of carbohydrates and metals. It forms as a result of oxidative stress and incomplete lysosomal degradation of damaged mitochondria, linking its accumulation to cellular damage. This may explain the abnormal pigmentation in chromhidrosis. Furthermore, lipofuscin can interfere with other cellular systems, such as the ubiquitin–proteasome pathway, potentially contributing to cellular dysfunction and abnormal gland activity. As a biomarker for cellular aging, lipofuscin buildup in apocrine glands may also account for the higher prevalence of chromhidrosis in adults [2].

This study aims to analyze the chemical composition of sweat in a chromhidrosis patient using gas chromatography–mass spectrometry (GC-MS) to identify the volatile organic compounds (VOCs) contributing to the condition’s unique characteristics. The volatolome, which refers to the collection of VOCs emitted from the body, has recently gained attention as a potential source of biomarkers for skin disorders. Previous research has identified 822 unique VOCs from human skin, with variations in detection depending on the methods used. Across 22 studies of disease-free skin, 689 compounds were identified from 32 chemical classes, with the most common being aldehydes (18%), carboxylic acids (12%), alkanes (12%), fatty alcohols (9%), ketones (7%), and benzenes and derivatives (6%) [3].

In this study, we used solid-phase microextraction (SPME), a solvent-free and sensitive technique, to efficiently capture and analyze VOCs from the sweat of a single chromhidrosis patient due to the rarity of the condition. SPME is widely used in analytical chemistry, particularly for volatolome studies, as it allows for the untargeted capture of VOCs emitted from the skin without extensive sample preparation [4].

2. Materials and Methods

2.1. VOCs Extractions

In this study, SPME and pentane (n-pentane, 99.0% purity, VWR, AnalaR NORMAPUR analytical reagent, Bois, France) extraction were used to isolate VOCs from sweat samples collected on cotton pads, followed by their identification using GC-MS. For the SPME method, the cotton pads containing sweat were placed directly into clean 20 mL headspace vials without any additional treatment. The vials were incubated at 60 °C for 30 min to allow VOCs to accumulate in the headspace. After incubation, VOCs were extracted from the headspace using a Supelco SPME fiber (Sigma-Aldrich, Darmstadt, Germany, 57328-U) with a 50/30 μm thickness, coated with divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS), for 20 min in static mode (HS-SPME) at 60 °C. In parallel, another set of cotton pads was prepared using pentane extraction. The sweat-laden cotton pads were transferred from Falcon tubes into a 20 mL glass vial with PTFE-coated silicone septa and metal caps, then submerged in 5 mL of pentane and allowed to soak for 30 min at room temperature to extract lipophilic compounds. Immediately after soaking, the pentane extract was analyzed.

2.2. GC–MS Analysis

Following extractions, the SPME fiber or pentane extract was injected into the GC for analysis. VOCs desorbed from the SPME fiber at 220 °C for 5 min, while the pentane extract was injected directly into the GC. The volatile organic compounds were analyzed using an Agilent 8890 gas chromatograph coupled to a 7000D triple quadrupole mass spectrometer (GC/TQ, Agilent Technologies, Santa Clara, CA, USA). The GC was equipped with an HP-5 MS capillary column (30 m length × 0.25 mm × 0.25 μm). Helium was used as the carrier gas at a flow rate of 1 mL/min. The injector temperature was set to 250 °C, and the injection volume was 1 μL, with a split ratio of 5:1. The transfer line temperature was set to 280 °C, and the ion source temperature was 230 °C.

The oven temperature program started at 70 °C, was held for 1 min, followed by an increase to 120 °C at a rate of 50 °C/min. The temperature was then ramped to 280 °C at a rate of 5 °C/min and maintained at 280 °C for 15 min. The mass spectrometer was operated in positive electron ionization mode (EI+), scanning a mass range of 10–800 m/z.

Data analysis and identification of VOCs were performed using MassHunter Qualitative Analysis software v. 10.0 (Agilent Technologies, Santa Clara, CA, USA), which facilitated the comparison of mass spectra with the NIST database for compound identification. For more reliable compound identification, Kovats retention indices were calculated and compared with reference values in the literature to support the results obtained from the mass spectral comparison. Identified VOCs were cross-referenced with the Human Metabolome Database (HMDB) for accurate compound identification and to elucidate their potential biological origins. Compounds detected in the sweat samples were screened for their potential origin. Those identified as originating from cosmetic products or medications, based on mass spectral data comparison and a review of the relevant literature, were excluded from the final analysis. This step ensured that the reported compounds were more likely to be endogenous in origin.

3. Results

The chemical analysis of sweat samples was performed using two complementary extraction techniques: SPME and pentane extraction. These methods, followed by GC-MS analysis, were applied to comprehensively identify VOCs with a range of polarities and volatilities.

Table 1. VOCs identified in the sweat of a chromhidrosis patient using pentane extraction and SPME.

No	Compound Name	CAS Number	RI	Pentane Extraction	SPME Extraction	HMDB
1	Nonanal	124-19-6	906.5		+	blood, breath, feces, saliva
2	Decanal	112-31-2	1095.7		+	blood, breath, feces, saliva, urine
3	Methyl-10,11-tetradecadienoate	25656-34-2	1306		+	urine
4	Tetradecanal	1240-09-3	1412.8		+	saliva, feces, urine
5	1-Dodecene	112-41-4	1476.3		+	breath, saliva
6	2,4-bis-(1,1-dimethylethyl) Phenol	96-76-4	1516.9	+	+	feces, urine
7	Amyl salicylate	2050-08-0	1588		+	saliva
8	2-Methyldecane	6975-98-0	1600.3		+	breath
9	Diethyl phthalate	84-66-2	1605.2	+	+	feces, urine
10	Methyl dihydrojasmonate	24851-98-7	1664.1	+	+	blood, saliva, urine
11	1-(4-isopropylphenyl)-2-methylpropyl acetate	4728-47-8	1685.8	+	+	not found
12	n-Hexyl salicylate	6259-76-3	1692.9	+		saliva
13	(cis)-2-Nonadecane	6465-99-8	1729.7	+		saliva, feces
14	5-Phenyldodecane	28900-12-7	1741.3		+	not found
15	cis-3-Hexenyl salicylate	65405-77-8	1756.6		+	saliva
16	2-(Phenylmethylene)-octanal	101-86-0	1762.3	+	+	saliva
17	Benzyl benzoate	120-51-4	1783.7	+		blood, urine
18	Nonadecane	629-92-5	1799.3		+	saliva, feces
19	7-Hexadecenal	56219-04-6	1810.5		+	not found
20	2-Ethylhexyl salicylate	118-60-5	1818.6		+	saliva
21	5-α-Cholestanol	80-97-7	1854.5		+	blood, feces
22	1,2-Benzenedicarboxylic acid dibutyl ester	84-74-2	1972.4	+	+	blood
23	Z-10-Methyl-11-tetradecen-1-ol propionate	51597-13-6	2008.2	+	+	feces
24	Isopropyl-hexadecanoate	110-27-0	2025.7	+		urine, saliva
25	11,13-Dimethyl-12-tetradecen-1-ol acetate	179911-07-4	2283.4	+		not found
26	2-Ethylhexyl trans-4-methoxycinnamate	5466-77-3	2332.4	+		saliva
27	(Z)-9-Octadecenamide	301-02-0	2378	+		blood
28	Butyl- 9-tetradecenoate	2425-79-8	2401.8	+		urine, blood, saliva, feces
29	Monoethylhexyl phthalate	4376-20-9	2557.3	+		sweat, blood, urine
30	Squalene	111-02-4	2835.2	+		Sweat, blood, feces
31	Cholesterol	57-88-5	3142.1	+		bile, blood, feces, urine, saliva, cerebrospinal fluid

RI—Kovats retention index; (+)—the compound detected above the detection threshold; HMDB—Human Metabolome Database.

presents the 31 compounds identified from the sweat samples using both extractions. The compounds were categorized into chemical classes, including aldehydes, fatty acid esters, benzoic acid derivatives, hydrocarbons, triterpenoids, and steroids.

4. Discussion

The analysis of VOCs identified in the sweat of a chromohidrosis patient revealed significant insights into the metabolic processes involved. The presence of aldehydes, including nonanal, decanal, tetradecanal, 2-(phenylmethylene)-octanal and 7-hexadecanal, were predominantly identified through SPME, aligning with their known role as byproducts of lipid peroxidation in cellular membranes [5,6]. These aldehydes are commonly linked with human skin emissions and lipid oxidation pathways, as demonstrated in previous studies [7,8]. 7-Hexadecenal is highlighted in the literature as a potential biomarker for oxidative stress, suggesting its relevance in conditions like chromhidrosis, where oxidative processes are implicated [9]. Their detection in chromhidrosis further supports the hypothesis of oxidative stress as a key factor in lipofuscin accumulation [10].

Figure 1 presents a Venn diagram illustrating the overlap and unique compounds detected through SPME and pentane extraction. The diagram highlights the number of compounds detected by each method and the shared compounds, providing a visual representation of their complementary nature in the analysis of VOCs in chromhidrosis. While both methods detected several common compounds, each extraction method generates a unique profile of compounds, both of which are important for a comprehensive understanding of lipid metabolism in chromhidrosis.

Fatty acid esters such as isopropyl-hexadecanoate and butyl- 9-tetradecenoate, which play a significant role in skin lipid composition, were predominantly detected through pentane extraction. These esters, originating from saturated fatty acids, align with the findings of Duffy and Morrin [11], emphasizing their role in both normal and pathological conditions of the skin. Their presence in chromhidrosis suggests involvement in lipid peroxidation processes, further supporting the hypothesis of oxidative stress as a key factor in lipofuscin accumulation.

Furthermore, several benzoic acid derivatives identified in the samples likely reflect the body’s metabolic response to stress. These compounds are involved in detoxification pathways and may exhibit pro-oxidant activity under certain conditions, especially in the presence of transition metals that catalyze lipid peroxidation. Their presence in sweat could signify the body’s efforts to mitigate oxidative damage or detoxify external contaminants [12].

The detection of hydrocarbons, including methylated hydrocarbons, is of particular note as these compounds have been reported as endogenous products of oxidative stress, [10].

Squalene and cholesterol, hydrophobic compounds integral to sebaceous secretions, were primarily detected in pentane extracts. Squalene, known for its antioxidant and anti-inflammatory properties, plays a crucial role in skin protection by neutralizing reactive oxygen species (ROS) and preventing lipid peroxidation. In the context of chromhidrosis, the oxidative degradation of these compounds may contribute to the formation of lipofuscin, linking their presence to the discoloration of sweat [13,14].

Finally, 9-octadecenamide (oleamide), a fatty acid identified in the sweat sample, marks the first detection of oleamide in human sweat. Oleamide is known for its involvement in lipid signaling and oxidative stress pathways through its interaction with the TRPV1 receptor (transient receptor potential vanilloid) [15,16]. Its presence may indicate a role in the oxidative lipid breakdown seen in chromhidrosis, contributing to the formation of lipofuscin and the characteristic colored sweat.

5. Conclusions

This study provides the first comprehensive analysis of the volatolome in a patient with chromhidrosis, utilizing both SPME and pentane extraction. The results highlight the complementary nature of these methods in capturing a diverse range of VOCs. Notably, the detection of 9-octadecenamide (oleamide) suggests a potential link between lipid metabolism and oxidative stress, contributing to the biochemical mechanisms underlying chromhidrosis.

The unique profiles generated by both extraction methods offer valuable insights into disease-related biochemical processes, indicating that these compounds may play a significant role in elucidating the mechanisms underlying chromhidrosis. This underscores the need for further research to explore these mechanisms and their implications for understanding lipid metabolism in chromhidrosis.