Research Article: Metabolomic study of human tissue and urine in clear cell renal carcinoma by LC-HRMS and PLS-DA

Date Published: April 16, 2018

Publisher: Springer Berlin Heidelberg

Author(s): Joanna Nizioł, Vincent Bonifay, Krzysztof Ossoliński, Tadeusz Ossoliński, Anna Ossolińska, Jan Sunner, Iwona Beech, Adrian Arendowski, Tomasz Ruman.


Renal cell carcinoma (RCC) is the most prevalent and lethal malignancy of the kidney. Despite all the efforts made, no tissue biomarker is currently used in the clinical management of patients with kidney cancer. A search for possible biomarkers in urine for clear cell renal cell carcinoma (ccRCC) has been conducted. Non-targeted metabolomic analyses were performed on paired samples of surgically removed renal cancer and normal tissue, as well as on urine samples. Extracts were analyzed by liquid chromatography/high-resolution mass spectrometry (LC-HRMS). Hydroxybutyrylcarnitine, decanoylcarnitine, propanoylcarnitine, carnitine, dodecanoylcarnitine, and norepinephrine sulfate were found in much higher concentrations in both cancer tissues (compared with the paired normal tissue) and in urine of cancer patients (compared with control urine). In contrast, riboflavin and acetylaspartylglutamate (NAAG) were present at significantly higher concentrations both in normal kidney tissue as well as in urine samples of healthy persons. This preliminary study resulted in the identification of several compounds that may be considered potential clear cell renal carcinoma biomarkers.

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Biomarkers provide a powerful approach to understanding diseases with applications in epidemiology, clinical trials, screening, diagnosis, and prognosis. Defined as alterations in the constituents of tissues or body fluids, they often offer the means for classification of a disease and can extend our knowledge about the underlying pathogenesis of disease. Theoretically, efficient biomarkers can also reflect the entire spectrum of disease from the earliest manifestation to the terminal stage. The development of cancer therapies is increasingly dependent on the understanding of tumor biology, and biomarkers are becoming essential tools in the field of medicine.

Examples of total ion chromatograms for cancer and normal tissue extracts are shown in the ESM (Fig. S1). Similar comparison of chromatograms for cancer patient urine and control urine samples is shown in the ESM (Fig. S2). For water-extract-based analysis, a total of 4040 features were detected in the set of tissue samples and a total of 3368 in the set of urine samples. Each feature is associated with an exact mass, a retention time, and average abundancies in cancer and normal tissue, as well as with urine samples from patients with and without renal cancers. Results for cancer tissue as compared with normal tissue or in cancer urine as opposed to normal urine, are listed in Table 2.Table 2List of features that are over-abundant in either cancer (1–4, 14–20) tissue or normal (5–13, 21–23) tissue or over-abundant either in urine from cancer patients (2, 4, 6, 10, 15–18, 19–22, 23–28) or from control patients (3, 9, 11, 13, 17)No.FormulaMassaMass error (ppm)Retention time (min)Putative metaboliteAverage abundancesTissueUrineNormalCancerControlCancer1C12H20O10324.10590.70.81Bis-fructose 2′,1:2,1′-dianhydride11634,973––2C8H11NO6S249.03090.80.98Norepinephrine sulfate1749993806303C5H10N2O3146.06941.50.80Glutamine23,47131,94415,54212,2124C5H9NO4147.05320.20.94Glutamate170,025196,812145116035C10H21NOS203.1335− 4.50.85Methylthiononanaldoxime46,6176349––6C9H17NO3187.1156− 0.22.52N-Heptanoylglycine8801113351310217C9H19NO2173.14181.53.27Amino-nonanoic acid6276427––8C33H40O22788.20302.47.87Quercetin sophoroside glucoside3509231––9C17H20N4O6376.13840.44.16Riboflavin11,837682581296610C13H15NO5265.0955− 5.73.70N-Phenylacetylglutamic acid23071192457699311C12H21NO5259.14283.21.82N-(3-oxooctanoyl)homoserine2039404461432312C11H14N2O3S254.07281.04.21Alanyl-α-thiophenylglycine148029––13C11H16N2O8304.0905− 0.51.02N-Acetylaspartylglutamate (NAAG)1546–117667514C9H17NO4203.11643.11.05Acetylcarnitine1317119,400––15C17H33NO4315.24131.28.51Decanoylcarnitine127772724217716C10H19NO4217.13192.51.40Propanoylcarnitine394813,560568410,61917C10H19NO5233.1257− 2.813.90Hydroxypropionylcarnitine59218396303139818C11H21NO5247.14252.01.07Hydroxybutyrylcarnitine984122,738203611,53819C7H15NO3161.10551.90.85Carnitine120,165194,04153,16577,35820C19H35NO4341.25691.36.622-Dodecenoylcarnitine62198800417172821C18H35NO4329.25701.19.524,8-Dimethylnonanoylcarnitine29822875147110622C13H25NO4259.17902.74.66Hexanoylcarnitine46422592––23C13H23NO6289.15270.61.923-Methylglutarylcarnitine1888328476616,40024C11H19NO4229.13192.11.67Butenylcarnitine––1109338325C14H27NO4273.19472.45.36Heptanoylcarnitine––1734317726C16H31NO4301.22591.97.172,6-Dimethylheptanoylcarnitine––14,60659,51527C14H25NO6303.16851.12.78Pimelylcarnitine––203613,59828C19H35NO6373.2459− 1.36.78Dodecanedioylcarnitine––2942114For each pair (tissue, normal or cancer and urine, control or cancer), the high abundance values are set in italics. Putative identifications are given if mentioned in the text, if they are known human metabolites or otherwise may seem relevant to include“–” peak not detectedaExperimental monoisotopic neutral mass

Liquid chromatography/high-resolution mass spectrometry analysis of extracts from cancer and healthy tissue regions allowed the identification of up- and downregulated compounds that could potentially serve as renal cancer biomarkers, ccRCC. Similar analyses of urine from cancer patients and from a healthy control group yielded additional putative biomarkers. The putative identifications of compounds were based on exact mass and on data base hits on important human metabolites, known to be relevant for cancer. Cross-comparison of two sets of results allowed the identification of four kidney cancer biomarkers that are either over- or under-expressed in both cancer tissue and urine from cancer patients. Hydroxybutyrylcarnitine, decanoylcarnitine, propanoylcarnitine, carnitine, dodecanoylcarnitine, and norepinephrine sulfate were found in distinctly higher concentrations in both cancer tissues and in urine of cancer patients compared with controls. In contrast, feature assigned to riboflavin and NAAG were present at significantly higher concentrations both in normal kidney tissue as compared with renal cancer tissue and in urine samples of healthy persons than in urine from the cancer patients. All eight mentioned compounds may be considered potential clear cell renal carcinoma biomarkers. Preliminary research presented in this work will be followed in the future with larger-scale study based on higher amount of patients.




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