Specific urinary metabolites in canine mammary gland tumors

Marcela Valko-Rokytovská; Peter Očenáš; Aneta Salayová; Radka Titková; Zuzana Kostecká

doi:10.4142/jvs.2020.21.e23

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Valko-Rokytovská, Očenáš, Salayová, Titková, and Kostecká: Specific urinary metabolites in canine mammary gland tumors

Original Article

J Vet Sci 2020;21(2):e23.

Published online: 4 March 2020

DOI: https://doi.org/10.4142/jvs.2020.21.e23

Specific urinary metabolites in canine mammary gland tumors

Marcela Valko-Rokytovská^1,^*, Peter Očenáš^1,, Aneta Salayová^1,, Radka Titková², Zuzana Kostecká¹

¹Department of Chemistry, Biochemistry and Biophysics, University of Veterinary Medicine and Pharmacy in Košice, 041 81 Košice, Slovakia

²Section of Surgery, Orthopaedics, Roentgenology and Reproduction, Small Animal Clinic, University Veterinary Hospital, University of Veterinary Medicine and Pharmacy in Košice, 041 81 Košice, Slovakia

^*Corresponding authors: Marcela Valko-Rokytovská Department of Chemistry, Biochemistry and Biophysics, University of Veterinary Medicine and Pharmacy in Košice, Komenského 73, 041 81 Košice, Slovakia. E-mail: marcela.valko.rokytovska@gmail.com

Peter Očenáš Department of Chemistry, Biochemistry and Biophysics, University of Veterinary Medicine and Pharmacy in Košice, Komenského 73, 041 81 Košice, Slovakia. E-mail: peter.ocenas@uvlf.sk

Aneta Salayová Department of Chemistry, Biochemistry and Biophysics, University of Veterinary Medicine and Pharmacy in Košice, Komenského 73, 041 81 Košice, Slovakia. E-mail: aneta.salayova@uvlf.sk

Conflict of Interest The authors declare no conflicts of interest.

Received 23 July 2019 Revised 28 November 2019 Accepted 30 November 2019

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

The identification of biomarkers that distinguish diseased from healthy individuals is of great interest in human and veterinary fields. In this research area, a metabolomic approach and its related statistical analyses can be useful for biomarker determination and allow non-invasive discrimination of healthy volunteers from breast cancer patients. In this study, we focused on the most common canine neoplasm, mammary gland tumor, and herein, we describe a simple method using ultra-high-performance liquid chromatography to determine the levels of tyrosine and its metabolites (epinephrine, 3,4-dihydroxy-L-phenylalanine, 3,4-dihydroxyphenylacetic acid, and vanillylmandelic acid), tryptophan and its metabolites (5-hydroxyindolacetic acid, indoxyl sulfate, serotonin, and kynurenic acid) in canine mammary cancer urine samples. Our results indicated significantly increased concentrations of three tryptophan metabolites, 5-hydroxyindolacetic acid (p < 0.001), serotonin, indoxyl sulfate (p < 0.01), and kynurenic acid (p < 0.05), and 2 tyrosine metabolites, 3,4-dihydroxy-L-phenylalanine (p < 0.001), and epinephrine (p < 0.05) in urine samples from the mammary gland tumor group compared to concentrations in urine samples from the healthy group. The results indicate that select urinary tyrosine and tryptophan metabolites may be useful as non-invasive diagnostic markers as well as in developing a therapeutic strategy for canine mammary gland tumors.

Keywords: Canine mammary cancer, urine metabolites, UHPLC

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Fig. 1.

Urinary metabolite to creatinine ratios in mammary gland cancer and healthy control dogs. Horizontal lines represent median values. TYR, tyrosine; E, epinephrine; L-DOPA, 3,4-dihydroxy-L-phenylalanine; DOPAC, 3,4-dihydroxyphenylacetic acid; VMA, vanillylmandelic acid; TRP, tryptophan; 5-HIAA, 5-hydroxyindolacetic acid; IS, indoxyl sulfate; 5-HT, serotonin; KYNA, kynurenic acid.

Table 1.

Morphological characteristics of mammary gland tumors in patients (n = 14)

Histopathological type	Stage	Grade	Size (cm)	Consistency	Localization	Age (yr)
Adenocarcinoma	I	II	3 × 4 × 3	Solid	M3, 4 – right side	13
Adenocarcinoma	I	III	3 × 2 × 2	Solid	M3, 4, 5 – bilateral	16
Adenocarcinoma	I	III	3 × 2 × 2	Solid	M4, 5 – bilateral	9
Adenocarcinoma	I	III	3 × 2 × 2	Solid	M3, 4, 5 – bilateral	11
Adenocarcinoma	II	III	2 × 3 × 2	Solid	M4, 5 – bilateral	9
Adenocarcinoma	II	III	1 × 1 × 1	Solid	M2 – left side	14
Adenocarcinoma	III	III	10 × 5 × 6	Solid	M3, 4, 5 – bilateral	6
Cystic papillar adenocarcinoma	I	I	1 × 1 × 1	Solid	M3 – left side	10
Cystic papillar adenocarcinoma	I	III	0.5 (diameter)	Solid	M4 – left side	7
Fibrosarcoma	III	III	10 × 10 × 10	Solid	M4 – left side	10
Low differentiated carcinoma	III	III	10 × 10 × 3	Solid	M5 – right side	7
Mixed carcinoma (with osteoid and myxoid tissue)	II	III	2 × 2 × 2	Elastic	M5 – right side	9
Mixed tumor	II	II	2 × 2 × 2	Elastic	M2 – right side	6
Necrotizing carcinoma	III	III	3 × 4 × 4	Solid	M3, 4, 5 – bilateral	6

Table 2.

Chromatographic parameters for urine metabolites derived via DAD or FLD

Detection	Metabolite	Retention time (min)	Regression equation	Standard curve linearity (R²)
DAD	Creatinine	0.717	y = 0.3889x	0.9997
	DOPAC	6.917	y = 0.6198x	0.9997
	KYNA	9.427	y = 1.0333x	0.9998
FLD	E	1.039	y = 38550.0444x	0.9991
	L-DOPA	1.599	y = 16747.3489x	0.9991
	TYR	2.425	y = 4590.4682x	0.9988
	VMA	3.762	y = 2118.4295x	0.9985
	5-HT	4.139	y = 658758.7562x	0.9994
	IS	6.752	y = 183019.6362x	0.9993
	TRP	7.485	y = 241663.4940x	0.9990
	5-HIAA	8.665	y = 17127.7056x	0.9993

DAD, diode array detector; FLD, fluorescence detector; DOPAC, 3,4-dihydroxyphenylacetic acid; KYNA, kynurenic acid; E, epinephrine; L-DOPA, 3,4-dihydroxy-L-phenylalanine; TYR, tyrosine; VMA, vanillylmandelic acid; 5-HT, serotonin; IS, indoxyl sulfate; TRP, tryptophan; 5-HIAA, 5-hydroxyindolacetic acid.

Table 3.

Levels of metabolites in the urine of study groups as determined by UHPLC and presented as median and interquartile range values (expressed as µmol/mmol creatinine)

Metabolite	Canine mammary gland tumor (n = 14)		Healthy control (n = 16)
Metabolite	Median	Interquartile range	Median	Interquartile range
TYR	7.85	7.32	5.50	4.98
	p = 0.1079
E	30.47	34.27	0.37	0.35
	p = 0.0303^*
L-DOPA	40.96	32.95	3.53	3.74
	p = 0.0003^***
DOPAC	2.39	6.61	4.78	5.78
	p = 0.4848
VMA	2.50	3.35	4.27	3.07
	p = 0.5167
TRP	1.20	1.08	1.51	0.82
	p = 0.3540
5-HIAA	9.83	18.87	2.02	4.11
	p = 0.0003^***
IS	31.68	16.08	9.39	11.99
	p = 0.0091^**
5-HT	0.45	15.24	0.02	0.06
	p = 0.0019^**
KYNA	47.97	66.89	26.99	41.40
	p = 0.0227^*

The p value of Mann-Whitney U test of urine metabolites in canine mammary gland tumor patients versus healthy control. UHPLC, ultra-high-performance liquid chromatography; TYR, tyrosine; E, epinephrine; L-DOPA, 3,4-dihydroxy-L-phenylalanine; DOPAC, 3,4-dihydroxyphenylacetic acid; VMA, vanillylmandelic acid; TRP, tryptophan; 5-HIAA, 5-hydroxyindolacetic acid; IS, indoxyl sulfate; 5-HT, serotonin; KYNA, kynurenic acid.

^* Correlation is significant at the 0.05 level;

^** Correlation is significant at the 0.01 level;

^*** Correlation is significant at the 0.001 level.

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