Of blood and breath: metabolite-based diagnosis of ovarian cancer

Physicians always knew that breath contains clues to diseases. Chemicals in breath often correlate with chemicals in saliva and blood - be it alcohol, anaesthetics or other metabolites (see, for example, this study by Dr Andreas Hengstenberg).

As one of my interests is breath-based detection of ovarian cancer, I took note of the recent paper claiming 99% to 100% accuracy of detecting ovarian cancer by metabolites in blood.
The authors used customized functional support vector machine-based machine-learning algorithms to classify thousands of metabolites measured by mass spectrometry (JEOL AccuTOF™ DART® that allowed to forego conventional liquid chromatography as sufficient resolution was achieved without separation) in peripheral blood. 

100% sensitivity and 100% specificity was achieved with 64-30 split validation technique, while 100% sensitivity and 98% specificity was the accuracy of leave-one-out-cross-validation. Very large number of metabolites, from 2,000 to 3,000 features, contributed to such discriminatory power (see the list of 14,000+ in supplemental material) 
 

Set of 25 canonical metabolic pathways relevant to the uploaded elemental
formulae ranked according to their p-values (hypergeometric distribution).

Histamine, amino acid, fructose and glucose metabolism were among the most prominent processes discriminating cancer and healthy blood.
It's that simple: sugar feeds cancer. Scientists have long found that cancer cells slurp fructose, and that fructose intake can be linked to some cancers. Histamine/polyamine interplay in cancers is also known. Histamine may be involved in inhibition of the local immune response against cancer. Is amino acid metabolism also linked to cancer? Well, what is not.   




Metabolomic biomarkers were always known to have diagnostic potential - cholesterol and glucose are among the oldest and most widely performed diagnostic tests. Yet, most bleeding edge cancer detection platforms are genomic or proteomic in nature.  Of the thousands of known biomarkers, only a handful have made it into the clinic. Existing ovarian cancer tests mostly rely on detecting a protein -  carbohydrate antigen 125. Vermillion's OVA1 and HealthLinx OvPlex tests use five proteins. This may be extended to 7.

Metabolites represent the end products of the genome and proteome, thus metabolomics-based diagnostics  holds the promise of providing powerful diagnostics,  allowing for differentiation of increased and decreased levels of chemicals with low process coefficient of variation.


Metabolomic tests were used for medical diagnostics starting with Hippocrates and Lavoisier. They continue to be explored by modern scientists. Dr Michael Phillips, for example, developed HeartsBreath Test, approved by the US Food and Drug Agency for early diagnosis of heart transplant rejection. Research proved the potential of inexpensive breath tests in discriminating lung, breast, colon and prostate cancers. Let's hope the new article  - along with others - will lead to novel consumer products, not only more academic research and peer-reviewed publications.



Zhou M, Guan W, Walker LD, Mezencev R, Benigno BB, Gray A, Fernández FM, & McDonald JF (2010). Rapid Mass Spectrometric Metabolic Profiling of Blood Sera Detects Ovarian Cancer with High Accuracy. Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology PMID: 20699376

On cancers and petroleum spills

Researchers have known for years that smell of cancer patients is chemically different from healthy individuals. One more study featured in British Journal of Cancer brings us a bit closer to an inexpensive, easy-to-use, portable device for home diagnostics. 

Exhaled breath collected from 177 volunteers (patients with lung, colon, breast, and prostate cancers and healthy controls) was examined by gold nanoparticle nanosensor arrays (GNPs) and gas chromatography linked to the mass spectrometry technique (GC-MS). 

GNP sensor resistance responses showed remarkable separation between cancer and healthy controls (Principal Component Analysis results are shown in the Figure: LC, lung cancer;  CC, colon cancer; BC, breast cancer; PC, prostate cancer).

Most of the VOCs reported in this study appear for the first time in the literature, adding to the wide spectrum of chemicals previously proposed as cancer biomarkers. Some of the chemicals -  predictive of  lung and prostate cancers -  are frequently released to the environment through petroleum spills. 

1-methyl-4-(1-methylethyl)-benzene - known as p-cymene  or p-isopropyltoluene -   is utilized by various species for chemical communication. It can be derived from the essential oils of herbs and spices and has biocidal properties against foodborne pathogens such as spoilage yeasts and E. coli O157:H7. p-cymene is the biological precursor of carvacrol that is also an antimicrobial agent (Kiskó and Roller, 2005).  It's decreased with cancer, and is present at higher concentrations in healthy individuals.


Toluene, dodecane and other aromatic components of petroleum are among chemicals found in human breath.

Typical "octane booster" toluene  - present at higher concentrations in lung and prostate cancers - is toxic to living organisms although some bacteria (like P. putida that has toluene operon) are able to grow in its presence (Eaton 1997).

Dodecane, a biogasoline component, is higher in the breath of healthy individuals. It is decreased in lung cancer. One of its derivatives - 2,6,11-trimethyl-dodecane was found in 80% of the males, but in none of the females participating in the study. 

Another aromatic compound, 2-amino-5-isopropyl-8-methyl-1-azulenecarbonitrile, similar to carbonitriles used in manufacturing of fragrance agents, is present at higher concentrations in breast, colon and prostate cancers when compared to healthy controls.

An alcane 3,7-dimethyl-undecane was found to be indicative of allergies. It was previously found to be eliminated from mice odors when they enter reproductive cycle (Achiraman & Archunan, 2006) and proposed to be used in diagnostics of asthma (Dragonieri et al., 2007).

Chemicals in breath can tell not only about cancers, but also relate to other diseases, environmental exposures and dietary behavior. This might decrease discriminative power of  expensive metabolomics technologies and bioinformatics approaches not based on additional knowledge, yet custom-made sensor arrays show great promise.


References


Peng G, Hakim M, Broza YY, Billan S, Abdah-Bortnyak R, Kuten A, Tisch U, & Haick H (2010). Detection of lung, breast, colorectal, and prostate cancers from exhaled breath using a single array of nanosensors. British journal of cancer, 103 (4), 542-51 PMID: 20648015



Gabriella Kiskó, Sibel Roller. (2005) Carvacrol and p-cymene inactivate Escherichia coli O157:H7 in apple juice.  BMC Microbiol. 2005; 5: 36

Eaton RW.  (1997) p-Cymene catabolic pathway in Pseudomonas putida F1: cloning and characterization of DNA encoding conversion of p-cymene to p-cumate. J Bacteriol. 1997 May;179(10):3171-80.

Shanmugam Achiraman, Govindaraju Archunan (2006) 1-Iodo-2methylundecane, a putative estrus-specific urinary chemo-signal of female mouse (Mus musculus) Theriogenology 66, 1913–1920

Dragonieri S, Schot R, Mertens BJ, Le Cessie S, Gauw SA, Spanevello A, Resta O, Willard NP, Vink TJ, Rabe KF, Bel EH, Sterk PJ. (2007) An electronic nose in the discrimination of patients with asthma and controls. J Allergy Clin Immunol. 120(4):856-62. 

Hormonal Manipulation of Olfactory Cues, or How to Lose a Guy in 10 days

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Body odors are important cues used for social and sexual discrimination. As was shown many times, animals can easily smell age-, health- and genetics-related  differences.  Recent study of our large-eyed relatives, ring-tailed lemurs, demonstrate that drugs can alter body scents and change behavior.

Researchers examined changes in endocrine and  semiochemical profiles of sexually mature female lemurs treated with hormonal contraceptives during their breeding season. Genetic diversity and kinship were estimated using 11–14 microsatellite loci and pairwise genetic distances. Gas chromatography-mass spectrometry (GCMS) was used to detect the volatile compounds in odor. A rater blind to the treatments scored lemur male behavior in regards to female odors. 

The conclusion? Contraceptives change chemical ‘signature’, minimizing distinctiveness and genetic fitness cues. No more can the males determine which females are genetically and physically beautiful. All contracepted females lost their individuality and started to smell funny.  

What about hormones and chemicals in our food?  Maybe one day humans will wake up and realize that something is lost? May it will happen  sooner rather than later...

For those interested in helping with our research of human environmental malodor - check our studies or this call for collaboration.   



Jeremy Chase Crawford,, Marylène Boulet,, & Christine M. Drea (2010). Smelling wrong: hormonal contraception in lemurs alters critical female odour cues Proc. R. Soc. B published online before print July 28, 2010

Odor-prints: individual but genetic connections unclear

Odor is like fingerprints or facial features - it's unique.  Yet no single measurement could be easily applied to recognize an individual.

GC/MS measurements can be used to analyze mixtures of acids, alcohols, aldehydes, hydrocarbons, esters, ketones, and nitrogenous molecules in human odor. Complex algorithms mining patterns help to pinpoint the signatures. But could these signatures be easily derived from genetic makeups?

Recent article published in the Journal of Chemical Ecology looked at the usual suspects -  major histocompatibility locus (MHC) and found that these genes do not determine major patterns. 


Volatile carboxylic acids are the most diverse class of known axillary odorants, and the pattern of these acids is genetically determined. These acids  - like vast majority of human odorous compounds - are produced by human microbiome, in this case by skin bacteria. Odors of 12 families, comprising 3 to 6 siblings,were analyzed with comprehensive two-dimensional gas chromatography (GC x GC) and time-of-flight mass spectrometry (ToF MS). the analysis onfirmed the presence of individual signatures. but failed to find odors specific to HLA genes.

Even though paternally inherited HLA-associated odors were proposed to influence women odor preferences, genetic basis of odors may be more complicated than previously thought.

References

Natsch A, Kuhn F, & Tiercy JM (2010). Lack of Evidence for HLA-Linked Patterns of Odorous Carboxylic Acids Released from Glutamine Conjugates Secreted in the Human Axilla. Journal of chemical ecology PMID: 20623248

Thompson EE, Haller G, Pinto JM, Sun Y, Zelano B, Jacob S, McClintock MK, Nicolae DL, Ober C. (2010) Sequence variations at the human leukocyte antigen-linked olfactory receptor cluster do not influence female preferences for male odors. Hum Immunol. 2010 Jan;71(1):100-3. PMID: 19833159 
 
Jacob S, McClintock MK, Zelano B, Ober C (2002) Paternally inherited HLA alleles are associated with women's choice of male odor. Nature Genet 30: 175-179  PMID: 11799397  PDF
 

Foods and Smells

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How many flavors are out there? We often hear only about these five - sweet, sour, salty, bitter and savory (umami), but there are so many more and they are important not only to our tastes but also health.

Remember that fresh grassy smell wafting up from the newly sliced tomato? It may be it's way of saying  "I'm good for you".

Stephen A. Goff and Harry J. Klee's article "Plant Volatile Compounds: Sensory Cues for Health and Nutritional Value?" published in a 2006 issue of Science explains why odors from foods may be nutritional or health signals that the human nose has learned to recognize.

Among the things emmigrants from less developed countries miss in USA is the scent of fresh tomatoes. One of the volatile compounds associated with the “grassy” tomato flavor, cis-3-Hexenal, is also an indicator of fatty acids essential to the human diet. Wild tomato contained more than three times the amount of that chemical than the cultivated version in the developed world. Two other contributors to tomato flavor — 2- and 3-methylbutanal — are indicators of the presence of essential amino acids and are also three times more common in the wild tomato. Same applies to commercial apples, strawberries, bread, cheese, even wine and beer.

Flavorful curcumin in tumeric has anti-inflammatory properties, compounds in ginger have antioxidants, and there are antimicrobial chemicals that contribute to the scent of onions, garlic, rosemary, sage, clove, mustard, chili peppers and thyme.

There are hundreds of volatile compounds in foods and beverages, often a major factor in how taste of foods is perceived.

What smells people enjoy the most?

Joanne Camas from Epicurious.com lists these 5 food smells:

1. Fried onions cooking
2. Banana bread baking (extra points if it has chocolate chips in it)
3. A perfectly ripe tomato as you slice into it, especially on a warm, sunny day
4. Coffee brewing
5. Garlic bread, fresh out of the oven
Most people commenting on this post listed baked breads and coffee as their top favorites too. Other choices include pies, spices and meats.

Here are some of the responses pulled from different blogs. What are your top five?

chefrosey 12:23:21 PM on 02/01/10

Chocolate
Fresh brewed coffee
Fresh baked bread
Fresh picked strawberries or an orange being peeled!
Any baked good coming out of the oven!

chef330 12:14:17 PM on 02/01/10

1. Onions sauteeing in butter
2. Chocolate Chip Cookies coming out of the oven
3. Just-picked peaches
4. Hot Apple Pie
5. European Butter - you can smell the flavor

Janet Tue Feb 2, 2010 2:22pm PST

apple pie baking in the oven
tralala311 Tue Feb 2, 2010 2:25pm PST

mmmm... GUMBO!!!

Habanero♥™ Tue Feb 2, 2010 2:32pm PST

Bacon, Baking Bread, Turkey, Pumpkin Pie, Molasses Cookies, Cinnamon Rolls.

Sherri Tue Feb 2, 2010 2:53pm PST

Coffee brewing, Chocolate Chip Cookies, Cinnammon Rolls, Bread, Pumkin Pie
__A_YAHOO_USER__ Wed Feb 3, 2010 9:21am PST
i think there's something about a roast that's been slow cooking all day that smells delicious, it'd be on my top 5 for sure.