Metabolomic Characterization of Nipple Aspirate Fluid by¹H NMR Spectroscopy and GC-MS

Abstract

Nipple aspirate fluid (NAF) is a noninvasively obtained biofluid from the duct openings of the breast. NAF components are constantly secreted, metabolized, and reabsorbed by the epithelial lining of the lactiferous ducts of the breast. NAF has been studied as a potential breast tissue surrogate for the discovery of novel breast cancer risk, early detection, and treatment response biomarkers. We report the first unsupervised metabolite characterization of nipple aspirate fluid using NMR and GC-MS using convenience samples previously collected from four premenopausal and four postmenopausal women. A total of 38 metabolites were identified using the two analytical techniques, including amino acids, organic acids, fatty acids, and carbohydrates. Analytical reproducibility of metabolites in NAF by GC-MS was high across different extraction and analysis days. Overall, 31 metabolites had a coefficient of variation below 20%. By GC-MS, there were eight metabolites unique to NAF, 19 unique to plasma, and 24 shared metabolites. Correlative analysis of shared metabolites between matched NAF and plasma samples from pre- and postmenopausal women shows almost no correlations, with the exception being lactic acid, which was significantly negatively correlated (R2 = 0.57; P = 0.03). These results suggest that NAF is metabolically distinct from plasma and that the application of metabolomic strategies may be useful for future studies investigating breast cancer risk and intervention response biomarkers.

Protein identification from dried nipple aspirate fluid on Guthrie cards using mass spectrometry

“…In ductal carcinoma in situ , overexpression of HER has been observed by immunohistochemistry and is correlated with higher proliferative activity ( 19 ). HER2 has previously been detected in NAF, in addition to breast related hormones, metabolites and growth factors ( 18 – 24 ). Thus, it was suggested that breast cancer biomarkers can be detected using dried NAF spots in Guthrie cards, followed by mass spectrometric analysis.…”

Non-Invasive Biomarkers for Early Detection of Breast Cancer

“…NAF contains high concentrations of proteins, carbohydrates and metabolites, such as amino acids, organic acids, and fatty acids. Several studies have suggested that the levels of drug, protein, and hormone levels in NAF more closely reflect the products of breast tissue metabolism and exposures than those in plasma or serum [ 171 ]. A study analyzed NAFs collected from healthy women using both nuclear magnetic resonance and gas chromatography/mass spectrometry [ 171 ].…”

“…Several studies have suggested that the levels of drug, protein, and hormone levels in NAF more closely reflect the products of breast tissue metabolism and exposures than those in plasma or serum [ 171 ]. A study analyzed NAFs collected from healthy women using both nuclear magnetic resonance and gas chromatography/mass spectrometry [ 171 ]. In that study, a total of 38 metabolites, including amino acids, organic acids, fatty acids, and carbohydrates, were identified by the two analytical techniques.…”

“…In that study, a total of 38 metabolites, including amino acids, organic acids, fatty acids, and carbohydrates, were identified by the two analytical techniques. Eight metabolites are unique to NAF, 19 unique to plasma, and 24 are shared metabolites, indicating that the metabolic profile of NAF is distinct from that of matched plasma samples [ 171 ]. In addition, NAF also contains exfoliated breast epithelial cells, from which breast cancer originates [ 172 ].…”

Metabolomic profiling of breast tumors using ductal fluid

“…However, because they contain proteins and metabolites of breast tissue metabolism in addition to ductal epithelial cells, they are very useful for metabolomic studies, thus providing a unique opportunity to evaluate more directly metabolomic changes in the breast tumor microenvironment itself and avoiding questions of tissue specificity, which arise when evaluating blood and urine. The feasibility of performing metabolomic analysis in NAF was recently demonstrated in a small study of eight subjects (18). The study was conducted on samples obtained from healthy pre- and post-menopausal individuals and compared the findings in NAF with matching plasma samples from the same patients.…”