Jae Kwak

Individual odortypes: interaction of MHC and background genes

Genes of the major histocompatibility complex (MHC) influence the urinary odors of mice. Behavioral studies have shown (1) that mice differing only at MHC have distinct urinary odors, suggesting an MHC odor phenotype or odortype; (2) that the MHC odortype can be recognized across different background strains; and (3) that the MHC odortype is not an additive trait. Very little is known about the odorants underlying this behavioral phenotype. We compared urinary volatile profiles of two MHC haplotypes (H2b and H2k) and their heterozygous cross (H2b×H2k) for two different background strains (C57BL/6J and BALB/c) using solid phase micro-extraction (SPME) headspace analysis and gas chromatography/mass spectrometry (GC/MS). Both MHC and background genes substantially influence the volatile profile. Of 148 compounds screened, 108 of them significantly differ between the six genotypes. Surprisingly, for numerous compounds, their MHC associations are moderated by background genes (i.e., there is a significant MHC × background interaction effect in the statistical model relating genotype to relative compound concentration). These interactions account for nearly 30% of the total genetic effect on the volatile profile. MHC heterozygosity further extends the odortype diversity. For many compounds, the volatile expression for the heterozygote is more extreme than the expression for either homozygote, suggesting a heterozygous-specific odortype. The remarkable breadth of effects of MHC variation on concentrations of metabolites and the interaction between MHC and other genetic variation implies the existence of as yet unknown processes by which variation in MHC genes gives rise to variation in volatile molecules in body fluids.

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.

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.

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.

Differential binding between volatile ligands and major urinary proteins due to genetic variation in mice

Two different structural classes of chemical signals in mouse urine, i.e., volatile organic compounds (VOCs) and the major urinary proteins (MUPs), interact closely because MUPs sequester VOCs. Although qualitative and/or quantitative differences in each chemical class have been reported, previous studies have examined only one of the classes at a time. No study has analyzed these two sets simultaneously, and consequently binding interactions between volatile ligands and proteins in urines of different strains have not been compared. Here, we compared the release of VOCs in male urines of three different inbred strains (C57BL/6J, BALB/b and AKR) before and after denaturation of urinary proteins, mainly MUPs. Both MUP and VOC profiles were distinctive in the intact urine of each strain. Upon denaturation, each of the VOC profiles changed due to the release of ligands previously bound to MUPs. The results indicate that large amounts of numerous ligands are bound to MUPs and that these ligands represent a variety of different structural classes of VOCs. Furthermore, the degree of release in each ligand was different in each strain, indicating that different ligands are differentially bound to proteins in the urines of different strains. Therefore, these data suggest that binding interactions in ligands and MUPs differ between strains, adding yet another layer of complexity to chemical communication in mice.

Volatile Disease Biomarkers in Breath: A Critique

Hundreds of volatile organic compounds (VOCs) are released from human body fluids. Some of them are produced by endogenous metabolic processes in and on the body, and others are derived from the environment. Expressions of some endogenous VOCs can be affected by pathophysiological changes, and several disease-specific volatile biomarkers have been identified and used as diagnostic aids. Monitoring volatile disease markers is attractive since the procedure can be performed in a noninvasive manner with little or no exposure to biohazardous body fluids. Although many VOCs have been claimed as potential biomarkers, only a few compounds have been consistently demonstrated and approved for clinical applications. This is mainly because (1) many of the putative markers are present in the environment as well as in the body and their levels in the environment are often higher than those in the body, (2) there are a large individual variation in the concentrations of biomarkers within diseased and/or healthy subjects, and (3) the origin and biosynthetic pathway of the claimed biomarkers have been frequently neglected. Unfortunately, these aspects have often been ignored in many studies. Here, we review a number of publications that have identified volatile disease biomarkers in breath, argue that many of these have not demonstrated to actually underlie the differences in volatile profiles between diseased patients and healthy subjects, speculate on the reasons for this lack of success, and suggest potential approaches that may provide a better chance of identifying disease biomarkers.

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.

Comparison of Sampling Probe and Thermal Desorber in HAPSITE ER for Analysis of TO-15 Compounds

The Hazardous Air Pollutants on Site (HAPSITE), a portable Gas Chromatograph-Mass Spectrometer (GCMS), has been used to detect, identify, and quantify Volatile Organic Compounds (VOCs) from environmental samples, providing on-site analysis to aid in operational risk management. HAPSITE is equipped with a hand-held sampling probe in which an air sample is delivered into a concentrator, and the VOCs collected in the concentrator are transferred, separated, and identified in the GC-MS. An upgraded version, HAPSITE ER, has recently been introduced with additional sampling capability for solid phase micro extraction and Thermal Desorption (TD). To our knowledge, however, no study has yet evaluated the performance of the thermal desorber accommodated in HAPSITE ER. In this study, therefore, we analyzed EPA Method TO-15 compounds with two different sampling methods (probe and thermal desorber for TD tubes) in a HAPSITE ER, and compared their results against each other. A major finding was that the peak intensities of the TO-15 compounds, particularly those with high Boiling Point (BP), were substantially higher in the results obtained with the thermal desorber than in those with the sampling probe. The lower peak intensities of the compounds observed in the probe analysis are likely due to the condensation of the VOCs in the probe (transfer) line that is 6 feet long and maintained at 40°C as they are delivered from the probe to the concentrator, whereas the thermal desorber is directly connected to the HAPSITE (no transfer line is used), thereby eliminating the condensation of VOCs. In conclusion, our study suggests that for the analysis of VOCs with high up to 220°C, the use of TD tubes followed by desorption in the thermal desorber offered by the newer version of HAPSITE is recommended.

Volatile organic compounds released by enzymatic reactions in raw nonpareil almond kernel

Abstract Benzaldehyde is well recognized as the predominant aroma in bitter almond (Prunus dulcis var amara) and is released from amygdalin upon enzymatic hydrolysis followed by a loss of hydrogen cyanide. Sweet almond (Prunus dulcis Mill. D.A. Webb) has a sweeter, nuttier aroma than the bitter variety. While benzaldehyde is detected in raw sweet almond, it is not the predominant compound contributing to aroma due to the lower level of amygdalin. Although a variety of volatile organic compounds (VOCs) in sweet almond have been identified, the identity of VOCs due to enzymatic reactions in sweet almond has not been well documented. In this study, we investigated the VOCs released by enzymatic reactions in raw nonpareil (sweet) almond kernel samples and identified several alcohols such as isobutanol, 2-pentanol, 3-methylbutanol, 3-methyl-3-buten-1-ol, and 3-methyl-2-buten-1-ol as the major enzyme-released VOCs. Their released amounts were greater in the sweet almond kernels than in the bitter ones analyzed, suggesting that these alcohols may contribute to the characteristic aroma in the raw sweet almond.