The Plasma and Serum Metabotyping of Hepatocellular Carcinoma in a Nigerian and Egyptian Cohort using Proton Nuclear Magnetic Resonance Spectroscopy

Abstract

Background/aims: Previous studies have observed disturbances in the ¹H nuclear magnetic resonance (NMR) blood spectral profiles in malignancy. No study has metabotyped serum or plasma of hepatocellular carcinoma (HCC) patients from two diverse populations. We aimed to delineate the HCC patient metabotype from Nigeria (mostly hepatitis B virus infected) and Egypt (mostly hepatitis C virus infected) to explore lipid and energy metabolite alterations that may be independent of disease aetiology, diet and environment.

Methods: Patients with HCC (53) and cirrhosis (26) and healthy volunteers (19) were recruited from Nigeria and Egypt. Participants provided serum or plasma samples, which were analysed using 600 MHz ¹H NMR spectroscopy with nuclear Overhauser enhancement spectroscopy pulse sequences. Median group spectra comparison and multivariate analysis were performed to identify regions of difference.

Results: Significant differences between HCC patients and healthy volunteers were detected in levels of low density lipoprotein (P = 0.002), very low density lipoprotein (P < 0.001) and lactate (P = 0.03). N-acetylglycoproteins levels in HCC patients were significantly different from both healthy controls and cirrhosis patients (P < 0.001 and 0.001).

Conclusion: Metabotype differences were present, pointing to disturbed lipid metabolism and a switch from glycolysis to alternative energy metabolites with malignancy, which supports the Warburg hypothesis of tumour metabolism.

Keywords: 1-D, One-dimensional; 1H NMR, proton nuclear magnetic resonance; AFP, α-fetoprotein; ALP, Alkaline phosphatase; ALT, Alanine transaminase; CT, Computed Tomography; EDTA, Ethylenediaminetetraacetic acid; ELISA, Enzyme-linked immunosorbent assay; Egypt; FID, Free induction decays; HBV, Hepatitis B virus; HBsAg, Hepatitis B surface antigen; HCC, Hepatocellular carcinoma; HCV, Hepatitis C virus; IDL, Intermediate density lipoprotein; IQR, Interquartile ranges; JUTH, Jos University Teaching Hospital; LDL, Low density lipoprotein; MRI, Magnetic resonance imaging; NOESY, Nuclear Overhauser enhancement spectroscopy; Nigeria; PC, Principal component; PCA, Principal components analysis; PLS-DA, Partial least squared discriminant analysis; PPARα, Peroxisome proliferator-activated receptor α; RD, Relaxation delay; US, Ultrasonography; VLDL, Very low density lipoprotein; WHO, World Health Organisation; hepatocellular carcinoma; ppm, Parts per million; proton nuclear magnetic resonance spectroscopy; serum metabotype; tm, Mixing time.

Figure 1

Representative plasma spectrum with EDTA exclusion. Key: (1) and (2) LDL/VLDL; (3) lactate (CH₃); (4) alanine; (5) N-acetylglycoproteins; (6) acetoacetate; (8) and (9) citrate; (10) creatinine; (11) glucose resonances; (12) lactate (CH); (13) albumin and albumin-bound fatty acids (Nicholson et al., 1995). Purple bars indicate areas of EDTA resonance exclusion.

Figure 2

Multivariate analyses of combined Nigerian and Egyptian samples. (A) PCA scatter plot of all groups; (B) PLS-DA scatter plot of HCC and healthy volunteer samples; (C) PLS-DA scatter plot of HCC and cirrhosis samples.

Figure 3

Multivariate analysis plots of Nigerian and Egyptian data. (A) and (B) PCA and PLS-DA loadings plot of Nigerian data; (C) and (D) PCA and PLS-DA loadings plot of Egyptian data.

Figure 4

Principal components analysis of male volunteer samples.

Figure 5

Univariate analysis of discriminatory metabolites. Key:P1 = P-value of HCC versus healthy control analyses; P2 = P-value of HCC versus cirrhosis analyses. Mann–Whitney tests of significance used for generation of P-values.