The utility of biomarkers in hepatocellular carcinoma: review of urine-based ¹H-NMR studies – what the clinician needs to know

Figures & data

Table 1 Reasons for using TSP as a reference standard during ¹H-NMR spectroscopy

• Reference peak with chemical shift frequency is far from that of signals from metabolites of interest

• Resonance of TSP does not overlap with sample components

• TSP is chemically stable

• TSP does not interact with sample components

• Shifts from TSP are precisely characteristic of certain nuclei within a metabolite and are very sensitive to environmental changes such as pH and temperature, although there is minimal variation when prepared accurately

• TSP dissolves well in aqueous solvents used in metabonomics, unlike tetramethylsilane (TMS), another commonly used reference standard in ^1H-NMR

• TSP generates a single signal peak, unlike TMS which can give more, thereby obstructing the metabolic profile of the spectra

• TSP can be used as a concentration reference to calculate concentration of metabolites

Note: Data from Holzgrabe et al⁴¹ and Stella et al.⁴²

Abbreviations: TSP, 3-(trimethylsilyl)-2,2′,3,3′-tetradeuteropropionic acid; ¹H-NMR, proton nuclear magnetic resonance.

Table 2 Previously published studies aiming to identify HCC biomarkers through analysis of urine by ¹H-NMR spectroscopy

Studies	Year	Country	Main HCC etiology	Significant metabolites discriminatory for HCC vs controls	↓/↑
Shariff et al^Citation54	2010	Nigeria	HBV	Creatinine	↓
				Carnitine	↑
Shariff et al^Citation64	2011	Egypt	HCV	Glycine, TMAO, hippurate, citrate	↓
				Creatine	↑
Ladep et al^Citation38	2014	Nigeria	HBV	Hippurate, TMAO, pyruvate, citrate	↓
				Acetylcarnitine, dimethylglycine, carnitine, indole-3-acetate, creatine, methionine, unknown spectral signal putatively assigned to N-acetylated amino acid, 2-oxoglutarate	↑
Cox et al^Citation49	2016	Bangladesh	HBV	Creatinine, hippurate, TMAO	↓
				Carnitine	↑

Notes: Statistically significant metabolites discriminatory for HCC vs controls are shown and arrows indicate whether increased or decreased in HCC.

Abbreviations: HCC, hepatocellular carcinoma; HBV, hepatitis B virus; HCV, hepatitis C virus; TMAO, trimethylamine-N-oxide; ¹H-NMR, proton nuclear magnetic resonance.

Figure 1 (A) Unedited ¹H-NMR spectra example. Different colors correspond to individual samples overlaid. (B) Median spectra of unedited data for ppm values ranging from above 4.5 to below 9.5. (C) Median spectra of unedited data for ppm values ranging from 1 to 4.5. Numbers indicate the following commonly reported metabolites – 1: lactate; 2: alanine; 3: acetoacetate; 4: carnitine; 5: citrate; 6: creatinine; 7: creatine; 8: TMAO; 9: glycine; 10: hippurate; 11: 1-methylnicotinamide; 12: water; 13: urea; 14: formate.

Abbreviations: ¹H-NMR, proton nuclear magnetic resonance; δ, chemical shift; PPM, parts per million; TMAO, trimethylamine-N-oxide; TSP, 3-(trimethylsilyl)-2,2′,3,3′-tetradeuteropropionic acid.

Figure 2 Example of univariance scaled PCA scores plot after data processing using the first and third principal components.

Abbreviations: PCA, principal component analysis; HCC, hepatocellular carcinoma; PC, principal component.

Figure 3 Loading plot from an OPLS-DA model built using HCC and healthy control as disease classes pairwise.

Notes: (A) Loading plot ranging from 7 to 8 ppm, with labelled peaks signifying hippurate metabolites. (B) Loading plot ranging from 3 to 4.2 ppm, with labelled peaks signifying creatinine and hippurate metabolites. Significant signals (pFDR<0.05) are shown in colors corresponding to the correlation coefficient (|r|). Hippurate and creatinine metabolites are labeled.
Abbreviations: HCC, hepatocellular carcinoma; OPLS-DA, orthogonal partial least squares discriminant analysis; pFDR, false discovery rate; δ, chemical shift; PPM, parts per million.

Figure 4 Identification of peaks from the same compound (hippurate) using STORM.⁵⁰ Significant signals (pFDR<0.05) are shown in colors corresponding to the correlation coefficient (|r|).

Abbreviations: STORM, subset optimization by reference matching; δ, chemical shift; PPM, parts per million; pFDR, false discovery rate.

Figure 5 Example of 2D JRES image (blue) with 1D NOESY spectra overlaid (red) used to verify multiplicity.

Notes: F1 axis shows the proton-proton coupling. F2 axis shows the chemical shift.
Abbreviations: JRES, J-resolved spectroscopy; 2D, two-dimensional; 1D, one-dimensional; NOESY, nuclear Overhauser effect spectroscopy; ppm, parts per million.

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