Metabolomic Study of a Diagnostic Model for the Metabolites of Stool Fat

Choong Hwan Lee; Jiyoung Kim; Su-Young Ahn; Sun-Young Lee

doi:10.4166/kjg.2013.61.1.9

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Lee, Kim, Ahn, and Lee: Metabolomic Study of a Diagnostic Model for the Metabolites of Stool Fat

Original Article

Korean J Gastroenterol 2013;61(1):9-16.

Published online: 4 October 2013

DOI: https://doi.org/10.4166/kjg.2013.61.1.9

Metabolomic Study of a Diagnostic Model for the Metabolites of Stool Fat

Choong Hwan Lee, Jiyoung Kim, Su-Young Ahn¹, Sun-Young Lee¹

Department of Bioscience and Biotechnology, Bio/Molecular Informatics Center, Konkuk University, Seoul, Korea

¹Department of Internal Medicine, Konkuk University School of Medicine, Seoul, Korea

Correspondence to: Sun-Young Lee, Department of Internal Medicine, Konkuk University School of Medicine, 120-1 Neungdong-ro, Gwangjin-gu, Seoul 143-729, Korea. Tel: +82-2-2030-7747, Fax: +82-2-2030-7748, E-mail: sunyoung@kuh.ac.kr

Received 11 May 201224 September 2012 Accepted 27 September 2012

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background/Aims

Metabolomics is a powerful tool for measuring low-molecular-weight metabolites in an organism at a specified time under specific environmental conditions. The aim of this study was to determine the usefulness of metabolomics in identifying the metabolites in stool-fat-positive specimens, and to establish whether the results could be used to predict the long-term prognosis.

Methods

Fecal specimens were collected from 52 subjects with bowel habit change. The subjects were accessed using Rome III questionnaires and Bristol stool scale form, and followed after three years. The feces samples were centrifuged and the resulting extracts reconstituted for liquid chromatography/mass spectrometry analysis. The datasets were autoscaled, log-trans-formed, and mean-centered in a column-wise fashion prior to principal-components analysis and partial least-squares-discrim-ination analysis modeling.

Results

Fecal samples from 10 of the 52 patients gave a positive stool-fat result of 30–100 μ m; those of the remaining 42 contained neither fatty acids nor neutral fats. The peak intensities of lithocholic acid (p=0.001), lysophosphatidyl ethanolamine (lysoPE) 16:0 (p=0.015), and lysoPE 18:1/0:0 (p=0.014) were correlated with the size of the fatty acid. Subjects with positive stool-fat result showed higher score in Bristol stool scale form than those with negative stool-fat result at initial (p=0.040) and after three years (p=0.012).

Conclusions

The metabolomic assay of stool fatty acid revealed mainly lysoPEs and lithocholic acid. The size of the fatty acid was correlated with higher concentrations of lysoPEs and lithocholic acid in stool-fat-test-positive specimens and related to loose stool even after three years of follow-up period.

Keywords: Bristol stool scale form, Fatty acid, Metabolomics, Stool fat

References

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Fig. 1.

Partial least-squares discrimination analysis of liquid chromatography/ mass spectrometry-based metabolic profiles for the stool-fat-positive (FAT) group (n=10) and the stool-fat-negative (CON) group (n=42). (A) Score plot between FAT (square) and CON (tri-angle). (B) Loading plot for 66 variables selected by VIP＞1. t[1], variables accounted for 31.6% of the measured variance; P (corr), positive correlation.

Fig. 2.

Box and whiskers plot of metabolite changes in the stool-fat-positive (FAT) group and the stool-fat-negative (CON) group. The findings for eight metabolites differed significantly between the two groups (p＜0.05). m/z, mass-to-charge ration (m/z) data; LysoPE, lysophosphatidyl ethanolamine.

Table 1.

Masses Defined through Analysis Using High-resolution Mass Spectrometry

Raw mass (m/z)	Retention time	Fold	p-value	TOF mass (m/z)	Compound name	HMDB formula	Error (mDa)	Matching MW
727	17.4	4.3	0.036	−	−	−	−	−
452	27.3	2.5	0.014	452.2750	LysoPE 16:0	C21H44NO7P	−2.7	453.26
375	28.7	2.2	0.015	375.2931	Lithocholic acid	C24H40O3	3.2	376.30
153	4.5	2.2	0.042	152.9848	−	−	−	−
282	11.4	2.1	0.027	−	−	−	−	−
478	28.2	2.0	0.030	478.2931	LysoPE 18:1(9z)/0:0	C23H46NO7P	−0.4	479.30
438	25.3	1.6	0.048	438.2627	LysoPE 15:0/0:0	C20H42NO7P	0.6	439.27
391	25.5	1.7	0.048	391.2802	−	−	−	−

m/z, mass-to-charge ration (m/z) data; TOF, time-of-flight; HMDB, human metabolome database; MW, molecular weight.

Table 2.

Link between the Size of a Fatty Acid and Its Metabolites

	Size of fatty accid			p-value
	0 μ M	1–50 μ M	51–100 μM	p-value
Lithocholic	4,640	10,556	24,2170	0.001
acid	(301–28,648)	(4,901–22,478)	(21,085–27,348)	0.001
LysoPE	7,355	34,642	37,391	0.015
(16:0)	(730–87,232)	(7,105–82,036)	(14,614–60,168)	0.015
LysoPE	15,144	30,091	52,208	0.014
(18:1/0:0)	(388–90,613)	(15,829–76,560)	(22,037–82,378)	0.014
LysoPE	9,994	19,311	31,674	0.183
(15:0/0:0)	(240–68,382)	(5,438–36,412)	(23,879–39,468)	0.183

Values are expressed as median (range). LysoPE, lysophosphatidyl ethanolamine.

Table 3.

Sizes of Fatty Acids/Neutral Fats and Peak Intensities of Metabolites in Stool-fat-test-positive Patients

Gender/Age (yr)	Size of fatty acid (μM)	Size of neutral fat (μ M)	Peak intensity of lithocholic acid	Peak intensity of lysoPE 16:0	Peak intensity of lysoPE 18:1(9z)/0:0	Peak intensity of lysoPE 15:0/0:0
M/35	30	0	16,966.96	45,049.74	76,560.70	21,759.79
F/47	30	0	22,478.13	82,036.39	56,704.74	16,863.56
F/67	30	0	5,277.719	28,120.99	75,822.84	14,606.92
F/38	40	40	4,901.545	7,105.938	34,684.49	5,438.531
F/48	45	50	18,506.04	41,164.15	24,853.82	36,412.34
M/31	50	70	6,414.613	12,602.15	25,498.29	15,581.43
M/46	50	0	13,973.96	43,429.49	20,507.02	25,836.44
M/62	50	30	7,138.484	9,633.017	15,829.03	35,252.89
F/30	80	0	21,085.16	60,168.72	82,378.52	23,879.18
F/29	100	0	27,348.93	14,614.56	22,037.56	39,468.80

LysoPE, lysophosphatidyl ethanolamine.

Table 4.

Bristol Stool Form Scale in Stool-fat-test-positive and Negative Patients

Bristol stool form and metabolites	Positive stool fat test (n=10)	Negative stool fat test (n=42)	p-value
Bristol stool form at the time of enrollment			0.040
Type 1 – Separate hard lump (like nuts)	0	1 (2.4)
Type 2 – Sausage-shaped but lumpy	0	1 (2.4)
Type 3 – Like a snake with crack	0	14 (33.3)
Type 4 – Like a smooth and soft snake	1 (10)	13 (30.9)
Type 5 – Soft blobs with clear-cut edge	3 (30)	7 (16.7)
Type 6 – Fluffy pieces with ragged edge	5 (50)	5 (11.9)
Type 7 – Watery (no solid pieces)	1 (10)	1 (2.4)
Bristol stool form after 3 years of follow-up			0.012
Type 1 – Separate hard lump (like nuts)	0	0
Type 2 – Sausage-shaped but lumpy	0	2 (4.8)
Type 3 – Like a snake with crack	1 (10)	11 (26.2)
Type 4 – Like a smooth and soft snake	1 (10)	12 (28.6)
Type 5 – Soft blobs with clear-cut edge	1 (10)	9 (21.4)
Type 6 – Fluffy pieces with ragged edge	5 (50)	8 (19.0)
Type 7 – Watery (no solid pieces)	2 (20)	0
Lithocholic acid	15,470 (4,901–27,348)	4,640 (301–28,648)	0.004
LysoPE (16:0)	34642 (7,105–82,036)	7,355 (730–87,232)	0.004
LysoPE (18:1/0:0)	30091 (15,829–82,378)	15,144 (388–90,613)	0.004
LysoPE (15:0/0:0)	22,819 (5,438–39,468)	9,994 (240–68,382)	0.103

Values are expressed as number (%) or median (range). LysoPE, lysophosphatidyl ethanolamine.

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