Genetic analysis of impaired trimethylamine metabolism using whole exome sequencing.

BACKGROUND: Trimethylaminuria (TMAU) is a genetic disorder whereby people cannot convert trimethylamine (TMA) to its oxidized form (TMAO), a process that requires the liver enzyme FMO3. Loss-of-function variants in the FMO3 gene are a known cause of TMAU. In addition to the inability to metabolize TMA precursors like choline, patients often emit a characteristic odor because while TMAO is odorless, TMA has a fishy smell. The Monell Chemical Senses Center is a research institute with a program to evaluate people with odor complaints for TMAU. METHODS: Here we evaluated ten subjects by (1) odor evaluation by a trained sensory panel, (2) analysis of their urine concentration of TMA relative to TMAO before and after choline ingestion, and (3) whole exome sequencing as well as subsequent variant analysis of all ten samples to investigate the genetics of TMAU. RESULTS: While all subjects reported they often emitted a fish-like odor, none had this malodor during sensory evaluation. However, all were impaired in their ability to produce >90% TMAO/TMA in their urine and thus met the criteria for TMAU. To probe for genetic causes, the exome of each subject was sequenced, and variants were filtered by genes with a known (FMO3) or expected effect on TMA metabolism function (other oxidoreductases). We filtered the remaining variants by allele frequency and predicated functional effects. We identified one subject that had a rare loss-of-function FMO3 variant and six with more common decreased-function variants. In other oxidoreductases genes, five subjects had four novel rare single-nucleotide polymorphisms as well as one rare insertion/deletion. Novel in this context means no investigators have previously linked these variants to TMAU although they are in dbSNP. CONCLUSIONS: Thus, variants in genes other than FMO3 may cause TMAU and the genetic variants identified here serve as a starting point for future studies of impaired TMA metabolism.

The Effect of Ethnicity on Human Axillary Odorant Production.

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.

Ethnic/racial and genetic influences on cerumen odorant profiles.

This report describes the volatile organic compounds (VOCs) associated with human cerumen (earwax) and the effects of ethnicity/race and variation on the ATP-binding cassette, sub-family C, member 11 gene (ABCC11). A single nucleotide polymorphism (SNP) in ABCC11 affects the cerumen VOC profiles of individuals from African, Caucasian, and Asian descent. Employing gas chromatography/mass spectrometry (GC/MS) we have identified the nature and relative abundance of cerumen VOCs from 32 male donors. Our results show that cerumen contains a complex mixture of VOCs and that the amounts of these compounds vary across individuals as well as across ethnic/racial groups. In six of the seven compounds whose detected concentrations were found to be statistically different across groups, individuals of African descent (AfD) > Caucasian descent (CaD) > Asians descent (AsD). Our findings also reveal that ABCC11 genotype alone does not predict the type and relative levels of volatiles found in human cerumen, and suggest that other biochemical pathways must be involved. Examination of the composition and diversity of external auditory canal microbiota in a small subset of our subject population revealed that the ear microbiota may not be directly correlated with either ethnic group membership or ABCC11 genotype.

Butylated hydroxytoluene is a ligand of urinary proteins derived from female mice.

Mice secrete substantial amounts of protein, particularly proteins called the major urinary proteins (MUPs), in urine. One function of MUPs is to sequester volatile pheromone ligands, thereby delaying their release and providing a stable long-lasting signal. Previously, only MUPs isolated from male mice have been used to identify ligands. Here, we tested the hypothesis that MUPs derived from females may also sequester volatile organic compounds. We identified butylated hydroxytoluene (BHT), a synthetic antioxidant present in the laboratory rodent diet, as a major ligand bound to urinary proteins derived from C57BL/6J female urine. BHT was also bound to the male-derived proteins, but the binding was less prominent than that in female urine, even though males express approximately 4 times more proteins than females. We confirmed that the majority of BHT in female urine was associated with the high molecular weight fraction (>10 kDa) and the majority of the proteins that sequestered BHT were MUPs as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The sequestration of BHT by MUPs was further confirmed by employing the recombinant MUP8 whose natural analogue has been reported in both sexes. Therefore, our data indicate that MUPs expressed in both sexes can bind, transport, and excrete xenobiotics into urine and raise the possibility that in addition to the known role in chemical communication, MUPs function as a defense mechanism against exogenous toxins.

The effect of menthol vapor on nasal sensitivity to chemical irritation.

INTRODUCTION: Among other effects, menthol added to cigarettes may modulate sensory response to cigarette smoke either by masking "harshness" or contributing to a desirable "impact." However, harshness and impact have been imprecisely defined and assessed using subjective measures. Thus, the current experiments used an objective measure of sensitivity to chemical irritation in the nose to test the hypothesis that menthol vapor modulates sensitivity to chemical irritation in the airways. METHODS: Nasal irritation thresholds were measured for 2 model compounds (acetic acid and allyl isothiocyanate) using nasal lateralization. In this technique, participants simultaneously sniff clean air in one nostril and chemical vapor in the other and attempt to identify the stimulated nostril. People cannot lateralize based on smell alone but can do so when chemicals are strong enough to feel. In one condition, participants were pretreated by sniffing menthol vapor. In a control condition, participants were pretreated by sniffing an odorless blank (within-subjects design). RESULTS: Pretreatment with menthol vapor decreased sensitivity to nasal irritation from acetic acid (participants required higher concentrations to lateralize) but increased sensitivity to allyl isothiocyanate (lower concentrations were required). CONCLUSIONS: The current experiments provide objective evidence that menthol vapor can modulate sensitivity to chemical irritation in the upper airways in humans. Cigarette smoke is a complex mixture of chemicals and particulates, and further work will be needed to determine exactly how menthol modulates smoking sensation. A better understanding could lead to treatments tailored to help menthol smokers quit by replacing the sensation of mentholated cigarettes.

In search of the chemical basis for MHC odourtypes.

Mice can discriminate between chemosignals of individuals based solely on genetic differences confined to the major histocompatibility complex (MHC). Two different sets of compounds have been suggested: volatile compounds and non-volatile peptides. Here, we focus on volatiles and review a number of publications that have identified MHC-regulated compounds in inbred laboratory mice. Surprisingly, there is little agreement among different studies as to the identity of these compounds. One recent approach to specifying MHC-regulated compounds is to study volatile urinary profiles in mouse strains with varying MHC types, genetic backgrounds and different diets. An unexpected finding from these studies is that the concentrations of numerous compounds are influenced by interactions among these variables. As a result, only a few compounds can be identified that are consistently regulated by MHC variation alone. Nevertheless, since trained animals are readily able to discriminate the MHC differences, it is apparent that chemical studies are somehow missing important information underlying mouse recognition of MHC odourtypes. To make progress in this area, we propose a focus on the search for behaviourally relevant odourants rather than a random search for volatiles that are regulated by MHC variation. Furthermore, there is a need to consider a 'combinatorial odour recognition' code whereby patterns of volatile metabolites (the basis for odours) specify MHC odourtypes.

Psychometric functions for ternary odor mixtures and their unmixed components.

People are often able to reliably detect a mixture of 2 or more odorants, even if they cannot reliably detect the individual mixture components when presented individually. This phenomenon has been called mixture agonism. However, for some mixtures, agonism among mixture components is greater in barely detectable mixtures than in more easily detectable mixtures (level dependence). Most studies that have used rigorous methods have focused on simple, 2-component (binary) mixtures. The current work takes the next logical step to study detection of 3-component (ternary) mixtures. Psychometric functions were measured for 5 unmixed compounds and for 3 ternary mixtures of these compounds (2 of 5, forced-choice method). Experimenters used air dilution olfactometry to precisely control the duration and concentration of stimuli and used gas chromatography/mass spectrometry to verify vapor-phase concentrations. For 2 of the 3 mixtures, agonism was approximately additive in general agreement with similar work on binary mixtures. A third mixture was no more detectable than the most detectable component, demonstrating a lack of agonism. None of the 3 mixtures showed evidence of level dependence. Agonism may be common in ternary mixtures, but general rules of mixture interaction have yet to emerge. For now, detection of any mixture must be measured empirically.

Major histocompatibility complex-regulated odortypes: peptide-free urinary volatile signals.

Major histocompatibility complex (MHC) genes influence urinary odors (odortypes) of mice. That volatile odorants are involved is supported by the observation that odortype identity can be detected from a distance. Furthermore, chemical analyses of urines have revealed numerous volatile odorants that differ in relative abundance between mice that differ only in MHC genotypes. In addition, urines from MHC-different mice evoke distinct odor-induced activity maps in the main olfactory bulbs. However, recent studies report that non-volatile MHC class I peptides may directly act as MHC-associated signals and may thereby be seen to call into question the evidence for a volatile MHC signal. To evaluate this question, we designed a procedure to collect peptide-free urinary volatiles and tested these volatiles for their ability to mediate chemosensory discrimination of MHC-congenic mice differing in their MHC genotype. The headspace volatiles from urines of C57BL/6 congenic mice (haplotypes H2(b) and H2(k)) were collected by solid phase microextraction (SPME). These volatiles were then desorbed into a gas chromatograph (GC) and the entire chromatographic eluate was collected into a buffer solution. Our results conclusively demonstrate that mice trained to discriminate between unadulterated urinary signals of the congenic mice generalize the discrimination, without reward or training, to the buffer solution containing the peptide-free urinary volatiles (p<0.001, binomial test). Thus volatile signals, perhaps along with non-volatile ones, are capable of mediating behavioral discriminations of mice of different MHC genotypes.

Synergistic mixture interactions in detection of perithreshold odors by humans.

Laboratory demonstrations of synergistic mixture interactions in human odor perception have been rare. The current study examined perithreshold mixture interactions between maple lactone (ML) and selected carboxylic acids. An air-dilution olfactometer allowed precise stimulus control. Experimenters measured stimulus concentrations in vapor phase using a combination of solid-phase microextraction and gas chromatography/mass spectrometry. A probability of detection versus concentration, or a psychometric, functions was measured for pure ML. Psychometric functions were also measured for ML with the addition of fixed, subthreshold concentrations of carboxylic acids. Relative to statistical independence in detection, clear synergy occurred over a range of ML concentrations. To the best of our knowledge, the current results constitute the first clear demonstration of synergy in odor detection by humans from an experiment that combined precise stimulus control, vapor-phase calibration of stimuli, and a clear statistical definition of synergy.

Human breath odors and their use in diagnosis.

Humans emit a complex array of volatile and nonvolatile molecules that are influenced by an individual's genetics, health, diet, and stress. Olfaction is the most ancient of our distal senses and may be used to evaluate food and environmental toxins as well as recognize kin and potential predators. Many body odors evolved to be olfactory messengers, which convey information between individuals. Consequently, those practicing the healing arts have used olfaction to aid in their diagnosis of disease since the dawn of medical practice. Studies using modern instrumental analyses have focused upon analysis of breath volatiles for biomarkers of internal diseases. In these studies, a subject's oral health status appears to seldom be considered. However, saliva and properly collected alveolar air samples must pass over or come in contact with the posterior dorsal surface of the tongue, a site of bacterial plaque development and source of halitosis-related volatiles. Because of our basic research into the nature of human body odors, our lab has received referrals of people with idiopathic malodor production, from either the oral cavity or body. We developed a protocol to help differentiate individuals with chronic halitosis from those with the genetic, odor-producing metabolic disorder trimethylaminuria (TMAU). In our referred population, TMAU is the largest cause of undiagnosed body odor. Many TMAU-positive individuals present with oral symptoms of dysguesia and halitosis as well as body odor. We present data regarding the presentation of our referred subjects as well as the analytical results from a small number of these subjects regarding their oral levels of halitosis-related malodorants and trimethylamine.

Food odors trigger Drosophila males to deposit a pheromone that guides aggregation and female oviposition decisions.

Animals use olfactory cues for navigating complex environments. Food odors in particular provide crucial information regarding potential foraging sites. Many behaviors occur at food sites, yet how food odors regulate such behaviors at these sites is unclear. Using Drosophila melanogaster as an animal model, we found that males deposit the pheromone 9-tricosene upon stimulation with the food-odor apple cider vinegar. This pheromone acts as a potent aggregation pheromone and as an oviposition guidance cue for females. We use genetic, molecular, electrophysiological, and behavioral approaches to show that 9-tricosene activates antennal basiconic Or7a receptors, a receptor activated by many alcohols and aldehydes such as the green leaf volatile E2-hexenal. We demonstrate that loss of Or7a positive neurons or the Or7a receptor abolishes aggregation behavior and oviposition site-selection towards 9-tricosene and E2-hexenal. 9-Tricosene thus functions via Or7a to link food-odor perception with aggregation and egg-laying decisions.

The identification of hypoxia biomarkers from exhaled breath under normobaric conditions.

Pilots have reported experiencing in-flight hypoxic-like symptoms since the inception of high-altitude aviation. As a result, the need to monitor pilots, in-flight, for the onset of hypoxic conditions is of great interest to the aviation community. We propose that exhaled breath is an appropriate non-invasive medium for monitoring pilot hypoxic risk through volatile organic compound (VOC) analysis. To identify changes in the exhaled breath VOCs produced during periods of reduced O2 levels, volunteers were exposed to simulated flight profiles, i.e. sea level for 5 min, O2 levels found at elevated altitudes for 5 min or placebo and 5 min at 100% O2 recovery gas, using a modified flight mask interfaced with a reduced O2 breathing device. During the course of these test events, time series breath samples from the flight mask and pre/post bag samples were collected and analyzed by gas chromatography/mass spectrometry (GC/MS). Seven compounds (pentanal, 4-butyrolactone, 2-pentanone, 2-hexanone, 2-cyclopenten-1-one, 3-methylheptane and 2-heptanone) were found to significantly change in response to hypoxic conditions. Additionally, the isoprene, 2-methyl-1,3-butadiene, was found to increase following the overall exposure profile. This study establishes an experimental means for monitoring changes in VOCs in response to hypoxic conditions, a computational workflow for compound analysis via the Metabolite Differentiation and Discovery Lab and MatLab(©) software and identifies potential volatile organic compound biomarkers of hypoxia exposure.

Facts, fallacies, fears, and frustrations with human pheromones.

Among primates in general, pheromones are of variable importance to social communication. Data on humans have generated the greatest controversy regarding the existence of pheromonal communication. In this review, the likelihood of pheromonal communication in humans is assessed with a discussion of chemical compounds produced by the axilla that may function as pheromones; the likelihood that the vomeronasal organ (VNO), a putative pheromone receptor organ in many other mammals, is functional in humans; and the possible ways pheromones operate in humans. In the human axilla, the interactions between the cutaneous microflora and axillary secretions render this region analogous to scent glands found in other primates. Both the chemistry of axillary secretions and their effects on conspecifics in humans appear to be analogous to other mammalian pheromone systems. Whichever chemical compounds serve a pheromonal function in humans, another unknown is the receptor. Although the VNO has been implicated in the reception of pheromones in many vertebrates, it is not the only pathway through which such information has access to the central nervous system; there is ample evidence to support the view that the olfactory epithelium can respond to pheromones. Furthermore, if a chemical activates receptors within the VNO, this does not necessarily mean that the compound is a pheromone. An important caveat for humans is that critical components typically found within the functioning VNO of other, nonprimate, mammals are lacking, suggesting that the human VNO does not function in the way that has been described for other mammals. In a broader perspective, pheromones can be classified as primers, signalers, modulators, and releasers. There is good evidence to support the presence of the former three in humans. Examples include affects on the menstrual cycle (primer effects); olfactory recognition of newborn by its mother (signaler); individuals may exude different odors based on mood (suggestive of modulator effects). However, there is no good evidence for releaser effects in adult humans. It is emphasized that no bioassay-guided study has led to the isolation of true human pheromones, a step that will elucidate specific functions to human chemical signals.

Volatile metabolic monitoring of glycemic status in diabetes using electronic olfaction.

The increased incidence of Type I and Type II diabetes among adults and adolescents is a growing public health concern worldwide. The primary objective of diabetes mellitus management involves keeping glycemia levels within the euglycemic range to prevent a variety of serious health complications. Unfortunately, daily self-monitoring is both a requirement and a problem for many patients with diabetes, particularly children and adolescents. Studies have shown that as many as 43% of adolescents and 30% of children (<14 years old) regularly forget to use glycemic tests and are significantly poorer at recognizing and reporting symptoms and signs of hypoglycemia/hyperglycemia. For this reason, methods for noninvasive, continuous monitoring that can signal glycemic status to a parent, teacher, or other caregiver would improve the care and management of symptoms of diabetes among these individuals. The goal of this review is to describe and evaluate electronic olfaction technology ("electronic nose") for monitoring the presence and levels of volatile chemicals from human body and breath that can be used to evaluate status of diabetes. The review is organized in four sections. The first section reviews the chemistry of the volatile signals that are produced by the body that are indicative of metabolic status. The second section provides an overview of novel sensor technology, e.g., "electronic olfaction," that mimics the biological olfactory system and can be used to monitor and identify complex plumes of volatiles that are signatures of metabolic states. The third section reviews studies that have employed electronic "nose" technology for diagnosis and monitoring of diabetes via urine and breath, and the final section discusses needed future directions for the development of olfactory-based metabolic monitoring, particularly among noncompliant populations.

Effects of the dietary supplements, activated charcoal and copper chlorophyllin, on urinary excretion of trimethylamine in Japanese trimethylaminuria patients.

Trimethylaminuria (TMAU) is a metabolic disorder characterized by the inability to oxidize and convert dietary-derived trimethylamine (TMA) to trimethylamine N-oxide (TMAO). This disorder has been relatively well-documented in European and North American populations, but no reports have appeared regarding patients in Japan. We identified seven Japanese individuals that showed a low metabolic capacity to convert TMA to its odorless metabolite, TMAO. The metabolic capacity, as defined by the concentration of TMAO excreted in the urine divided by TMA concentration plus TMAO concentration, in these seven individuals ranged from 70 to 90%. In contrast, there were no healthy controls examined with less than 95% of the metabolic capacity to convert TMA to TMAO. The intake of dietary charcoal (total 1.5 g charcoal per day for 10 days) reduced the urinary free TMA concentration and increased the concentration of TMAO to normal values during charcoal administration. Copper chlorophyllin (total 180 mg per day for 3 weeks) was also effective at reducing free urinary TMA concentration and increasing TMAO to those of concentrations present in normal individuals. In the TMAU subjects examined, the effects of copper chlorophyllin appeared to last longer (i.e., several weeks) than those observed for activated charcoal. The results suggest that the daily intake of charcoal and/or copper chlorophyllin may be of significant use in improving the quality of life of individuals suffering from TMAU.

Identification of volatile organic compounds in human cerumen.

We report here the initial examination of volatile organic compounds (VOCs) emanating from human earwax (cerumen). Recent studies link a single nucleotide polymorphism (SNP) in the adenosine triphosphate (ATP) binding cassette, sub-family C, member 11 gene (ABCC11) to the production of different types of axillary odorants and cerumen. ABCC11 encodes an ATP-driven efflux pump protein that plays an important function in ceruminous apocrine glands of the auditory canal and the secretion of axillary odor precursors. The type of cerumen and underarm odor produced by East Asians differ markedly from that produced by non-Asians. In this initial report we find that both groups emit many of the same VOCs but differ significantly in the amounts produced. The principal odorants are volatile organic C2-to-C6 acids. The physical appearance of cerumen from the two groups also matches previously reported ethnic differences, viz., cerumen from East Asians appears dry and white while that from non-Asians is typically wet and yellowish-brown.

Differentiation of complex vapor mixtures using versatile DNA-carbon nanotube chemical sensor arrays.

Vapor sensors based on functionalized carbon nanotubes (NTs) have shown great promise, with high sensitivity conferred by the reduced dimensionality and exceptional electronic properties of the NT. Critical challenges in the development of NT-based sensor arrays for chemical detection include the demonstration of reproducible fabrication methods and functionalization schemes that provide high chemical diversity to the resulting sensors. Here, we outline a scalable approach to fabricating arrays of vapor sensors consisting of NT field effect transistors functionalized with single-stranded DNA (DNA-NT). DNA-NT sensors were highly reproducible, with responses that could be described through equilibrium thermodynamics. Target analytes were detected even in large backgrounds of volatile interferents. DNA-NT sensors were able to discriminate between highly similar molecules, including structural isomers and enantiomers. The sensors were also able to detect subtle variations in complex vapors, including mixtures of structural isomers and mixtures of many volatile organic compounds characteristic of humans.

Changes in volatile compounds of human urine as it ages: their interaction with water.

The urinary odors are commonly perceived as unpleasant. While numerous studies have identified the volatile organic compounds (VOCs) released from urine, the odorants responsible for the urine odor are not well characterized. Furthermore, anecdotal reports suggest that the odor of aged urine is different from that of fresh urine. However, no study has yet to investigate the specific VOCs released from aged urine. In this study, we analyzed and compared the VOCs released from fresh and aged urine samples, investigating the changes in the urinary VOCs as urine aged. We found an overall decrease in concentration of many urinary VOCs, and concluded this was due to the urine evaporating as it aged. On the contrary, some highly water-soluble compounds such as short and branched-chain organic acids and trimethylamine, increased. Their increased release is most likely due to the loss of water and the subsequent release of water-soluble VOCs as urine ages. We suggest that these VOCs may contribute to the odor of the aged urine.

Changes in volatile compounds of mouse urine as it ages: their interactions with water and urinary proteins.

Mice release a variety of chemical signals, particularly through urine, which mediate social interactions and endocrine function. Studies have been conducted to investigate the stability of urinary chemosignals in mice. Neuroendocrine and behavioral responses of mice to urine samples of male and female conspecifics which have aged for different amounts of time have been examined, demonstrating that the quality and intensity of signaling molecules in urine change over time. In this study, we monitored changes in volatile organic compounds (VOCs) released from male and female mouse urine following aging the urine samples. Substantial amounts of some VOCs were lost during the aging process of urine, whereas other VOCs increased. Considerable portions of the VOCs which exhibited the increased release were shown to have previously been dissolved in water and subsequently released as the urine dried. We also demonstrated that some VOCs decreased slightly due to their binding with the major urinary proteins (MUPs) and identified MUP ligands whose headspace concentrations increased as the urine aged. Our results underscore the important role of MUPs and the hydration status in the release of VOCs in urine, which may largely account for the changes in the quality and intensity of urinary signals over time.

Volatile biomarkers from human melanoma cells.

Dogs can identify, by olfaction, melanoma on the skin of patients or melanoma samples hidden on healthy subjects, suggesting that volatile organic compounds (VOCs) from melanoma differ from those of normal skin. Studies employing gas chromatography-mass spectrometry (GC-MS) and gas sensors reported that melanoma-related VOCs differed from VOCs from normal skin sources. However, the identities of the VOCs that discriminate melanoma from normal skin were either unknown or likely derived from exogenous sources. We employed solid-phase micro-extraction, GC-MS and single-stranded DNA-coated nanotube (DNACNT) sensors to examine VOCs from melanoma and normal melanocytes. GC-MS revealed dozens of VOCs, but further analyses focused on compounds most likely of endogenous origin. Several compounds differed between cancer and normal cells, e.g., isoamyl alcohol was higher in melanoma cells than in normal melanocytes but isovaleric acid was lower in melanoma cells. These two compounds share the same precursor, viz., leucine. Melanoma cells produce dimethyldi- and trisulfide, compounds not detected in VOCs from normal melanocytes. Furthermore, analyses of the total volatile metabolome from both melanoma cells and normal melanocytes by DNACNT sensors, coupled with the GC-MS results, demonstrate clear differences between these cell systems. Consequently, monitoring of melanoma VOCs has potential as a useful screening methodology.