¹H-NMR-based metabolomics for cancer targeting and metabolic engineering –A review

Highlights

• Metabolomics is used to assess metabolic phenotyping and metabolic engineering

• Metabolomics finds therapeutic liabilities in cancer microenvironments

• NMR technologies have used to profiling to metabolite imaging

• The pattern-recognition method gives the best discrimination between cells

Abstract

Nuclear magnetic resonance (NMR) spectroscopy acts as the best tool that can be used in tissue engineering scaffolds to investigate unknown metabolites. Moreover, metabolomics is a systems approach for examining in vivo and in vitro metabolic profiles, which promises to provide data on cancer metabolic alterations. However, metabolomic profiling allows for the activity of small molecules and metabolic alterations to be measured. Furthermore, metabolic profiling also provides high-spectral resolution, which can then be linked to potential metabolic relationships. An altered metabolism is a hallmark of cancer that can control many malignant properties to drive tumorigenesis. Metabolite targeting and metabolic engineering contribute to carcinogenesis by proliferation, and metabolic differentiation. The resulting the metabolic differences are examined with traditional chemometric methods such as principal component analysis (PCA), and partial least squares-discriminate analysis (PLS-DA). In this review, we examine NMR-based activity metabolomic platforms that can be used to analyze various fluxomics and for multivariant statistical analysis in cancer. We also aim to provide the reader with a basic understanding of NMR spectroscopy, cancer metabolomics, target profiling, chemometrics, and multifunctional tools for metabolomics discrimination, with a focus on metabolic phenotypic diversity for cancer therapeutics.

Introduction

In general, nuclear magnetic resonance (NMR) signals are generated based on the behavior of atoms that have been subjected to a strong and uniform magnetic field. However, NMR acts as powerful electromagnetic radiation for material testing. Additionally, NMR spectral intensity is an analytically traceable and well-resolved [1,2]. In small molecules,1-dimensional (1D) nuclei of ¹H, ¹³C, ³¹P, and ¹⁹F and 2D nuclei such as ¹H-¹H, ¹H-¹³C can be characterized by NMR spectroscopy. NMR plays a valuable tool that can be used for quantitative fingerprinting to identify different metabolites within tissue engineering scaffolds. Moreover, NMR can also be exploited for targeted and untargeted human metabolic phenotype diversity. Proton (¹H)-NMR has been employed to characterize molecules, drugs, and toxic substances in plasma samples [[3], [4], [5]]. ¹H-NMR-based metabolomics profiling first became a popular technology in the early 2000s because it could be used to identify organic compounds within biological fluids. Moreover, newly captured NMR data can be compared with legacy or archived NMR data as long as the experimental parameters (i.e., solvent, time(s), temperature, pH, angles, hardware and, software) are kept the same. These parameters may result in oscillated spectral shifts that may not be present in all peaks [6,7]. The second most critical alternative nuclei, ¹³C-NMR, acts as an alternative for analyzing the carbon framework of molecules. However, ¹³C-NMR capture took more time than ¹H-NMR capture [7]. In physical organic chemistry, nuclei of ¹⁴N-NMR [8,9], ²³Na-NMR [10], and ³¹P-NMR [11,12] are also frequently measured to functional changes.

Conventional 1D and 2D NMR examinations provide structural validation and conformational information. Additionally, 1D-NMR spectroscopy contains regular ¹H, ¹³C, ¹⁴N, ²³Na, and ³¹P, as well as the spectra of other nuclei that may also be regular and decoupled. Furthermore, 2D-NMR provides two different fingerprinting tools for homonuclear correlations (i.e., ¹H-¹H correlational spectroscopy (COSY) and ¹H-¹H total correlational spectroscopy (TOCSY)) and heteronuclear single quantum coherence spectroscopy (HSQC; ¹H-¹³C). The 2D-NMR technique is sensitive and provides ¹H scalar coupling (i.e., atom bonding connectivity) [13,14]. HSQC of ¹H-¹³C has been delivered the signals and reveal correlations between two nuclei that chemically bonded each other (¹H-¹³C single bond) [[15], [16], [17]].

As frequently, an emergence of low-frequency benchtop machines has allowed for the advent of 60 MHz NMR spectroscopy, which is convenient, easy to use, and requires very little bench space. These low-frequency machines also deliver accurate data in a timely manner [18,19]. The 1000 MHz NMR engine provides a higher magnetic force up to 23.5 Tesla than benchtop machines. At the same time, for metabolomics profiling, higher field increases the signal separation and sensitivity. Usually, 5 mm and 4 mm NMR tubes have used for solution-state NMR and solid-state NMR experiments, separately. The key drawbacks of using such machines include low sensitivity and ¹H signal overlapping. However, overlapping ¹H signals can be controlled when using a high-level magnetic NMR system [20]. The liquid- helium is used to maintain NMR probes as cold as possible, as this results in excellent performance and clean data. Additionally, cryogenically cooled NMR probes have been shown to reduce the thermal signal to noise ratio (S/N) and improve the sensitivity of electromagnetic signals. Shimming and locking are used to control magnetic homogeneity to parts per billion (ppb) [19,21].

The concept of “perfect echo” includes the three NMR pulse sequences that are used for different applications; 1) t₂-filtering CPMG spin-echo pulse sequences (RD-90˚-[τ-180˚-τ-]_n) for metabolites observations; 2) for the nuclear overhauser effect spectroscopy (NOESY) pulse sequences, which yields a signal for metabolites and high molecular weight molecules; 3) the DIFFUSION-EDITED sequence that gives specific observation of macromolecular components in solutions containing small molecule metabolites [3,22,23]. Chemical signals from free induction decay (FID) have low an S/N but can increase the average of repeated acquisitions. The t₂-filtering such as spin-spin relaxation time effectively limits transverse magnetization [2,24]. Among these processes, ¹H-splitting patterns, such as singlets (s), doublets (d), triplets (t), doublets of doublets (dd), multiples (m) and so on, of small molecules and macromolecules are divided according to Pascal’s triangle laws.

Generally, 128 repeats are acquired for excellent ¹H-NMR data with a data acquisition time of 5–10 minutes. The 3-(trimethylsilyl) propionic acid-d₄ sodium salt (TSP-d₄), contains deuterated methylene groups that act as a concentration reference that has been aligned at δ-0.0 ppm [5,25,26]. Additionally, deuterochloroform (CDCl₃), deuterium oxide/heavy water (D₂O), dimethyl sulfoxide (DMSO), and 2,2-dimethyl-2-silapentane-5-sulfonate sodium salt (DSS) are used as standard references in characterization experiments [27]. Moreover, deuterated benzene acts as a basic solvent. During the chemical signals process, carbon nuclei require more time for relaxation compared to hydrogen nuclei. As a result, the heavy-element spectral acquisition is more time consuming (i.e., from minutes to hours) [16,28]. The ¹H-NMR Carr-Purcell-Meiboom-Gill (CPMG) stacked spectra (δ 0.5-5.5 ppm) of head and neck squamous cell carcinoma cancer (HNSCC) patients undergoing radio-/chemo-radiotherapy (RT/CHRT) were acquired by 400 MHz spectrometers (Fig. 1a). The key metabolites and their 1D signal intensity are shown in Fig. 1b [22].