Method development for fecal lipidomics profiling - PubMed (original) (raw)

. 2013 Jan 15;85(2):1114-23.

doi: 10.1021/ac303011k. Epub 2012 Dec 26.

Affiliations

Method development for fecal lipidomics profiling

Katherine E Gregory et al. Anal Chem. 2013.

Abstract

Robust methodologies for the analysis of fecal material will facilitate the understanding of gut (patho)physiology and its role in health and disease and will help improve care for individual patients, especially high-risk populations, such as premature infants. Because lipidomics offers a biologically and analytically attractive approach, we developed a simple, sensitive, and quantitatively precise method for profiling intact lipids in fecal material. The method utilizes two separate, complementary extraction chemistries, dichloromethane (DCM) and a methyl tert-butyl ether/hexafluoroisopropanol (MTBE) mixture, alone or with high pressure cycling. Extracts were assessed by liquid chromatography-high-resolution mass spectrometry-based profiling with all ion higher energy collisional dissociation fragmentation in both positive and negative ionization modes. This approach provides both class-specific and lipid-specific fragments, enhancing lipid characterization. Solvents preferentially extracted lipids based on hydrophobicity. More polar species preferred MTBE; more hydrophobic compounds preferred DCM. Pressure cycling differentially increased the yield of some lipids. The platform enabled analysis of >500 intact lipophilic species with over 300 lipids spanning 6 LIPID MAPS categories identified in the fecal matter from premature infants. No previous report exists that provides these data; thus, this study represents a new paradigm for assessing nutritional health, inflammation, and infectious disease in vulnerable populations.

PubMed Disclaimer

Figures

Figure 1

Figure 1. Experimental Design

The pooled fecal slurry was made by starting with well-mixed, pooled sample. 150 mg solid fecal sample per 3 ml water was combined and vortexed well to form a slurry. The slurry was diluted 1:3 and 1:10 in water. 180 µl of I.S. was added to 1020 µl of stock and each dilution. This yielded 2 mg, 0.6 mg, and 0.2 mg fecal material and 7.5 µl I.S. per 50 µl sample aliquot. Lipids were extracted using either a modified Bligh and Dyer method with DCM in place of CF, or an MTBE/HFIP mixture. The use of pressure at 35,000 psi for 30 cycles was applied to half of the samples, whereas the other half were vortexed extensively for approximately 10 minutes without the use of PCT.

Figure 2

Figure 2. Lipid Abundance and Precision Comparisons: Pressure vs No Pressure, Positive ion Mode

Each panel presents the following data:

  1. Top left set of panels: The linear scale of lipid abundance as defined by MS relative abundance. This panel emphasizes the most abundant lipids in the sample.
  2. Top right set of panels: The log scale of abundance as defined by MS relative abundance. This panel emphasizes the overall distribution of lipids in the sample. Within these panels, the lower left quadrant is populated by lipids whose overall signal has a relative abundance less than 1000 for either the X or Y condition. We expect that these lipids would be quantitatively unreliable for the LC-MS platform used. In contrast, lipids in the upper right would be expected to be quantitatively reliable, regardless of extraction approach. In contrast, lipids identified in the upper left or lower right panels would be expected to be much more analytically reliable under the "Y" or "X" extraction protocol, respectively.
  3. Bottom left set of panels: The overall CV scale of SIEVE data series. This panel shows 10,000 frames for positive ion mode and 7,000 frames for negative ion mode. These panels emphasize the total lipid yield based on the specific approach.
  4. Bottom right set of panels: The CV scale truncated at 40% CV. This panel emphasizes the "useful peaks" of lipids measured.

Figure 3

Figure 3. Lipid abundance and Precision Comparisons: Pressure vs No Pressure, Negative ion Mode

Details as in Figure 2

Figure 4

Figure 4. Lipid abundance and Precision Comparisons: DCM vs MTBE, Positive ion Mode

Details as in Figure 2

Figure 5

Figure 5. Schematic Showing Relationship between Fecal Lipid Extraction and Elution Characteristics

Panels A and B show two base peak chromatograms from the fecal lipid POOL sample, indicating the difference in signal based on ionization polarity and the need to study these samples in both modes, positive (A) and negative (B). Panel A shows 7 species and Panel B shows 5 species and their individual bar graphs representing their mean signal (±sem) across the 5 samples analyzed across all 3 dilutions for DCMP (DCM, pressure) and DCMS (DCM, no pressure), MTBEP (MTBE, pressure) and MTBES (MTBE, no pressure). The boxes around each graph represent the individual species’ extraction preference, and indicate the need for both extraction solvents and ionization modes to be used to achieve comprehensive lipidome analysis. Equivalent graphs of all 304 identified lipids are in the supplement.

Similar articles

Cited by

References

    1. McGarr SE, Ridlon JM, Hylemon PB. J Clin Gastroenterol. 2005;39:98–109. - PubMed
    1. Zella GC, Israel EJ. Pediatr Rev. 2012;33:207–217. quiz 217–208. - PubMed
    1. Mathai J, Raju B, Bavdekar A. Indian Pediatr. 2011;48:37–42. - PubMed
    1. Hume G, Radford-Smith GL. Pathology. 2002;34:561–567. - PubMed
    1. Dharmani P, Chadee K. Curr Mol Pharmacol. 2008;1:195–212. - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources