Characterization of the urinary metabolic profile of cholangiocarcinoma in a United Kingdom population

Figures & data

Table 1 Demographics of study population

Characteristic	Benign					Malignant
	Healthy	SOD	GS	STRI	HCV	CCA	HCC	PCA	OCA	METS
Participants, n	22	8	22	9	14	10	45	22	48	11
Age (range), years	34 (24–58)	55 (30–74)	66 (24–81)	63 (45–74)	46 (29–56)	69 (57–78)	68 (48–82)	68 (51–81)	64 (45–88)	64 (38–78)
Male, %	45.4	37.5	31.8	55.5	35.7	44.4	85.7	71.4	0	63.6
BMI, kg/m^2	25 (19.5–39)	n/a	n/a	25 (20–30)*	26 (20–32)	25 (21–27)*	30 (17–50)	29 (20–76)	n/a	26 (20–29.5)
Serum biochemistry
Bilirubin, μmol/L	n/a	10.1 (4–21)	26 (5–192)	40 (10–189)	10 (3–18)	95 (20–375)	31 (4–338)	108 (7–411)	8.2 (2–30)	103 (6–443)
ALT, IU/L	n/a	53 (17–99)	79.3 (8–425)	121 (23–479)	84.9 (21–298)	181.3 (37–389)	45.8 (4–130)	155.3 (15–734)	17.1 (6–64)	98.6 (15–295)
ALP, IU/L	n/a	94 (20–184)	170 (45–376)	182(60–454)	76 (43–146)	594 (159–1242)	144 (14–550)	471 (51–1664)	85.5 (12–177)	643 (46–1905)
Albumin, g/L	n/a	27.6 (1–41)	34.3 (24–43)	37.5 (31–41)*	40.1 (34–46)	29.8 (20–49)	36 (20–46)	30.3 (17–47)	33.7 (16–44)	31.7 (18–48)
Urea, mmol/L	n/a	26.5 (3.2–109)	5.2 (2.1–14.7)	7.3(3.9–16.7)*	4.7 (2.6–6.1)*	5.2 (1.9–16)	5.5 (2.5–10.7)	4.6 (2.5–7.7)*	4.7 (1.8–18.3)	5.7 (2.6–12.6)*
Creatinine, μmol/L	n/a	66 (54–83)*	72 (47–137)	71 (3.3–131)	73 (58–93)	77 (53–146)	82 (49–204)	67 (53–101)*	71(46–132)	84 (52–121)*
Race
White	15	7	19	7	12	8	42	14	13	9
Black			1		1	1			1
Asian	2		2	1				3	2	1
Asian-Indian	3							2	4
Hispanic		1					1
Middle Eastern	2				1		1	1	3	1
Not available				1		1	1	2	25

Note: *Missing data.

Abbreviations: ALT, alanine transaminase; ALP, alkaline phosphatase; CCA, cholangiocarcinoma; GS, gallstones; HCC, hepatocellular carcinoma; HCV, noncirrhotic hepatitis C; METS, metastatic cancer of the liver; OCA, ovarian cancer; PCA, pancreatic cancer; SOD, sphincter of Oddi dysfunction; STRI, strictures; n/a, not available.

Figure 1 CCA patients vs healthy controls. PCA scores plots for (A) positive and (B) negative ion mode data of CCA patients and healthy controls. OPLS-DA scores plots showing group separation for both (C) positive and (D) negative ion mode data and the corresponding permutation tests for (E) positive and (F) negative ion mode data.Abbreviations: CCA, cholangiocarcinoma; PCA, principal component analysis; OPLS-DA, orthogonal projections to latent structures discriminant analysis.

Figure 2 CCA patients vs patients with SOD. PCA scores plots for (A) positive and (B) negative ion mode data of CCA patients and SOD cases. OPLS-DA scores plots showing group separation for both (C) positive and (D) negative ion mode data and the corresponding permutation tests for (E) positive and (F) negative ion mode data.

Abbreviations: CCA, cholangiocarcinoma; SOD, sphincter of Oddi dystfunction; PCA, principal component analysis; OPLA-DA, orthogonal projections to latent structures discriminant analysis.

Figure 3 CCA patients vs patients with gallstones. PCA scores plots for (A) positive and (B) negative ion mode data of CCA patients and gallstones cases. OPLS-DA scores plots showing group separation for both (C) positive and (D) negative ion mode data and the corresponding permutation tests for (E) positive and (F) negative ion mode data.

Abbreviations: CCA, cholangiocarcinoma; PCA, principal component analysis; OPLS-DA, orthogonal projections to latent structures discriminant analysis.

Figure 4 CCA patients vs biliary strictures patients. PCA scores plots for (A) positive and (B) negative ion mode data of CCA patients and biliary strictures patients. OPLS-DA scores plots showing group separation for both (C) positive and (D) negative ion mode data and the corresponding permutation tests for (E) positive and (F) negative ion mode data.

Abbreviations: CCA, cholangiocarcinoma; PCA, principal component analysis; OPLS-DA, orthogonal projections to latent structures discriminant analysis.

Figure 5 CCA vs noncirrhotic HCV patients. PCA scores plots for (A) positive and (B) negative ion mode data of CCA patients and HCV patients. OPLS-DA scores plots showing group separation for both (C) positive and (D) negative ion mode data and the corresponding permutation tests for (E) positive and (F) negative ion mode data.

Abbreviations: CCA, cholangiocarcinoma; HCV, noncirrhotic hepatitis C; PCA, principal component analysis; OPLS-DA, orthogonal projections to latent structures discriminant analysis.

Figure 6 Box and whisker plots of compounds different between cholangiocarcinoma (CCA) cases and benign controls–carnitine species. *p 0.05, **p 0.01 and ***p 0.001. Y-axis= relative intensity.

Abbreviations: CCA, cholangiocarcinoma; GS, gallstones; SOD, sphincter of Oddi dysfunction; STRI, strictures; HCV, noncirrhotic hepatitis C.

Figure 7 Box and whisker of compounds different between cholangiocarcinoma (CCA) cases and benign controls: ESI+ metabolites. *p 0.05, **p 0.01 and ***p 0.001. Y-axis= relative intensity.

Abbreviations: CCA, cholangiocarcinoma; GS, gallstones; SOD, sphincter of Oddi dysfunction; STRI, strictures; HCV, noncirrhotic hepatitis C.

Figure 8 Box and whisker plots of compounds different between cholangiocarcinoma (CCA) cases and benign controls: ESI− metabolites. *p 0.05, **p 0.01 and ***p 0.001. Y-axis= relative intensity.

Abbreviations: CCA, cholangiocarcinoma; GS, gallstones; SOD, sphincter of Oddi dysfunction; STRI, strictures; HCV, noncirrhotic hepatitis C.

Figure 9 Box and whisker plots of compounds different between cholangiocarcinoma (CCA) cases and benign controls: ESI− metabolites. *p 0.05, **p 0.01 and ***p 0.001. Y-axis= relative intensity.

Abbreviations: CCA, cholangiocarcinoma; GS, gallstones; SOD, sphincter of Oddi dysfunction; STRI, strictures; HCV, noncirrhotic hepatitis C.

Table 2 Altered metabolites between cholangiocarcinoma patients compared to healthy controls – positive mode

m/z	RT	Tentative assignment	Adduct	VIP	Trend	p-value^†	FC	Identification*
162.113	0.49	L-carnitine (C1)	M+H	4.3	↑	NS	2.62	a
114.066	0.51	Creatinine	M+H	5.9	↓	0.043	−1.4	a
144.102	0.58	Proline betaine	M+H	4.5	↓	NS	−0.67	b
229.118	0.68	Hydroxyprolyl-proline	M+H	3.3	↑	NS	1.94	b
204.125	0.82	L-Acetylcarnitine (C2)	M+H	7.7	↑	NS	1.87	a
229.155	0.98	Leucyl-proline	M+H	5.2	↑	NS	1.45	b
169.036	1.03	Uric acid	M+H	3	↓	NS	−0.9	a
215.016	1.06	Citrate	M+Na	3.2	↓	NS	−3.58	a
188.071	2.51	Unknown	-	4.8	↑	NS	2.22	d
232.155	2.84	Butyrylcarnitine (C4)	M+H	4.4	↓	0.023	−2.22	a
296.15	3.17	Unidentified acylcarnitine	M+H	3.3	↑	NS	0.14	a
295.13	3.54	Glutamylphenylalanine	M+H	3.2	↑	<0.0001	12.194	b
246.17	3.57	Valerylcarnitine (C5)	M+H	3.4	↓	NS	−1.78	a
318.191	3.78	Acylcarnitine (C9-OH)	M+H	6.3	↑	<0.0001	9.25	b
178.05	3.8	Hippurate	M+H	8.9	↓	<0.0001	−3	a
344.207	4.4	Acylcarnitine (C10:1)	M+H	3.1	↑	0.001	4.43	b
346.223	4.57	Acylcarnitine (C10)	M+H	3.9	↑	0.0002	7.88	b
170.061	4.81	Unknown	-	5.2	↓	0.001	−3.41	d
286.201	4.92	Acylcarnitine (C8:1)	M+H	6.4	↓	NS	−0.79	b
277.142	5.12	Unknown	M+H	3.5	↑	NS	0.1	b
312.217	5.16	2-trans,4-cis-Decadienoylcarnitine (C10)	M+H	3.9	↓	NS	−5.78	b
310.202	5.19	Acylcarnitine (C10:3)	M+H	5.2	↓	NS	−1.57	b
310.202	5.35	Acylcarnitine (C10:3)	M+H	5.4	↓	0.021	−1.85	b
302.233	5.55	2,6-Dimethylheptanoyl carnitine (C9)	M+H	7.6	↓	0.001	3.65	b
314.233	5.78	Decenoylcarnitine (C10:1)	M+H	3.1	↓	NS	1.62	b
328.248	6.11	Acylcarnitine (C10:2-OH)	M+H	4	↓	NS	1.43	b

Notes: *Level of metabolite identification: (a) identified compound; (b) putitatively annotated compound; (c) putitatively characterized compound class; and (d) unknown. ^†False discovery rate (FDR) adjusted p-value (or q-value).

Abbreviations: FC, fold change; RT, retention time; VIP, variable importance in projection score.

Table 3 Altered metabolites between cholangiocarcinoma patients compared to healthy controls – negative mode

m/z	RT	Tentative assignment	Adduct	VIP	Trend	p-value^†	FC	Identification*
96.962	0.60	Sulfate	M-H	3.8	↓	0.025	−4.83	b
243.061	0.85	Uridine/pseudouridine	M-H	3.1	↑	0.0002	2.24	b
191.018	0.94	Isocitrate	M-H	5.8	↓	0.023	−1.58	b
167.02	1.03	Uric acid	M-H	3.1	↑	0.011	1.7	a
191.018	1.06	Citrate	M-H	4.7	↓	NS	−1.93	a
227.996	2.65	Sulfated compound		3.2	↓	0.005	−6.12	d
188.985	3.13	Pyrocatechol sulfate	M-H	4.5	↓	0.03	−2.66	b
172.991	3.45	Phenyl sulfate	M-H	6.8	↓	0.039	−3.44	b
203.001	3.75	O-methoxycatechol-O-sulfate	M-H	3.0	↓	0.014	−2.45	b
178.049	3.80	Hippurate	M-H	8.5	↓	0.006	−2.2	a
263.102	3.84	Phenylacetylglutamine	M-H	4.4	↑	NS	1.11	b
212.001	3.87	Indoxylsulfate	M-H	9.1	↓	0.021	−1.93	a
241.118	3.92	L-gamma-glutamyl-L-isoleucine	M-H2O-H	3.2	↑	0.008	3.14	b
287.022	4.13	DHPV O-sulfate^‡	M-H	4.2	↓	0.018	−6.32	b
243.134	4.18	Hydroxyprolyl-isoleucine	M-H	2.9	↑	0.002	3.24	b
187.005	4.41	p-cresol sulfate	M-H	17	↓	0.051	−1.85	b
352.085	5.88	Fatty acid		3.6	↓	0.004	−5.31	d
465.248	6.37	Steroid glucuronide (C25H38O8)	M-H	3.0	↓	0.001	3.9	b
419.228	6.48	Steroid compound		3.1	↑	0.005	15.3	d
528.263	6.76	Glycochenodeoxycholate-N-sulfate	M-H	4.4	↑	0.011	18.3	b

Notes: *Level of metabolite identification: (a) identified compound; (b) putatively annotated compound; (c) putatively characterized compound class; and (d) unknown. ^†False discovery rate (FDR) adjusted p-value (or q-value). ^‡5ʹ-(3ʹ,4ʹ-dihydroxyphenyl)-gamma-valerolactone sulfate.

Abbreviations: FC, fold change; RT, retention time; VIP, variable importance in projection score.

Figure 10 Urine metabolites associated with the metabolic signatures of the six distinct disease conditions – positive mode. (A) Scores plot of the principal component analysis (PCA) analysis, R2Y=41% and Q2Y=16% from 2 PC model, p-value=0.01 from permutation test and (B) important metabolites selected on the basis of VIP score.

Abbreviations: CCA, cholangiocarcinoma; GS, gallstones, HCV, noncirrhotic hepatitis C; SOD, sphincter of Oddi dysfunction; STRI, strictures.

Figure 11 CCA patients vs HCC patients. PCA scores plots for (A) positive and (B) negative ion mode data of CCA patients and HCC patients. OPLS-DA scores plots showing group separation for both (C) positive and (D) negative ion mode data and the corresponding permutation tests for (E) positive and (F) negative ion mode data. Abbreviations: CCA, cholangiocarcinoma; HCC, hepatocellular carcinoma; PCA, principal component analysis; OPLA-DA, orthogonal projections to latent structures discriminant analysis.

Figure 12 CCA patients vs ovarian cancer. PCA scores plots for (A) positive and (B) negative ion mode data of CCA patients and OCA patients. OPLS-DA scores plots showing group separation for both (C) positive and (D) negative ion mode data and the corresponding permutation tests for (E) positive and (F) negative ion mode data. Abbreviations: PCA, principal component analysis; CCA, cholangiocarcinoma; OCA, ovarian cancer; OPLS-DA, orthogonal projections to latent structures discriminant analysis.