NMR-based Stable Isotope Resolved Metabolomics in systems biochemistry

Abstract

Metabolism is the basic activity of live cells, and monitoring the metabolic state provides a dynamic picture of the cells or tissues, and how they respond to external changes, for in disease or treatment with drugs. NMR is an extremely versatile analytical tool that can be applied to a wide range of biochemical problems. Despite its modest sensitivity its versatility make it an ideal tool for analyzing biochemical dynamics both in vitro and in vivo, especially when coupled with its isotope editing capabilities, from which isotope distributions can be readily determined. These are critical for any analyses of flux in live organisms. This review focuses on the utility of NMR spectroscopy in metabolomics, with an emphasis on NMR applications in stable isotope-enriched tracer research for elucidating biochemical pathways and networks with examples from nucleotide biochemistry. The knowledge gained from this area of research provides a ready link to genomic, epigenomic, transcriptomic, and proteomic information to achieve systems biochemical understanding of living cells and organisms.

Keywords: Isotope editing; Isotopomer distribution analysis; Stable Isotope Resolved Metabolomics (SIRM).

Figure 1. Biochemical network involving glucose metabolism

Glucose is oxidized to pyruvate (glycolysis). Several intermediates are involved in parallel and intersecting pathways: glycogen metabolism, pentose phosphate pathway, serine pathways, lipid biosynthesis and the Krebs Cycle. G6P: glucose-6-phosphate; F6P fructose-6-phosphate; F1,6BP fructose-1,6-bisphosphate; GAP glyceraldehyde-3-phosphate; DHAP dihydroxyacetone phosphate; 1,3bisPG 1,3-bisphosphoglycerate; 2PGA 2-phosphogycerate; PEP phosphoenolpyruvate; Pyr pyruvate; OAA oxaloacetate; AcCoA acetyl CoA; Lac lactate; Ru5P ribose-5-phosphate; HK hexokinase; G6Pase glucose-6-phosphatase; PGI phosphoglucose isomerase; PFK1 phosphofructokinase 1; FBPase fructose 1,6 bisphosphatase; ALD aldolase; GAPDH glyceraldehyde-3- phosphate dehydrogenase; PGK phosphoglycerate kinase; PGM phosphoglycerate mutase; ENO enolase; PK pyruvate kinase; LDH lactate dehydrogenase; ALT alanine transaminase; PC pyruvate carboxylase; PDH pyruvate dehydrogenase; PPPox oxidative branch of the pentose phosphate pathway; PPPnx non-oxidative branch of the pentose phosphate pathway. Adapted from [12] “With permission of Springer”.

Figure 2. Glu isotopomers from Krebs cycling

Isotopomers created via Krebs cycle activity by: (A) ¹³C₆ glucose. Black dots are ¹²C. ¹³C₆ glucose (red dots) produces ¹³C₃ pyruvate which can enter the Krebs cycle either via PDH (2 ¹³C) or PCB (3 ¹³C, shown as green dots), which give rise to different isotopomers of Krebs cycle intermediates and anabolic products such as uracil and its precursor Asp, distinguishable by NMR as ¹³C_1,2 + ¹³C_3,4 via PDH in the forward direction, and ¹³C_1,2,3 Asp via PCB (B) ¹³C₅,¹⁵N₂ Gln plus unlabeled glucose. Red dots are ¹³C atoms from Gln, blue dots are the two nitrogen atoms. Open circles are ¹²C. The anaplerotic input of fully labeled Gln produces the fully labeled Krebs cycle intermediates. Fully labeled OAA from Gln condenses with glucose-derived AcCoA to produce quadruple labeled citrate, which becomes via the Krebs cycle triply labeled αKG, and doubly labeled succinate. AST will transaminate OAA with Glu, transferring the amino nitrogen to Asp. The isotopomers produced evolve on further cycles, and differ with other inputs to the cycle. Isotopomer analysis is needed to sort out the resulting complex patterns.

Figure 3. ¹⁵N incorporation from Glutamine in purine nucleotides

Spectral editing via ¹H{¹⁵N}-HSQC detects ¹⁵N-isotopomers of nucleotides. A549 cells were grown for 24 h in the presence of 5 mM unlabeled glucose and 4 mM [U–¹³C,¹⁵N]-glutamine. The spectra of the polar extract were recorded at 14.1 T at 20 °C with acquisition times of 0.15 s in t₂ and 0.021 s in t₁ and a recycle time of 1.6 s. The value of the INEPT transfer delay was set to 10 ms. A. 1D ¹H{¹⁵N}-HSQC spectra at different time points. B. 2D HSQC shows editing of the complex proton spectrum. The long range ¹H-¹⁵N couplings shows utilization of both the amido (blue) and amino N of Gln. Adapted from ref. [2] “With permission of Springer”

Figure 4. Nucleotide analysis by ¹H NMR

¹H NMR spectra of polar extracts of cells or tissues. A. 1H NMR spectrum at 600 MHz of an extract of BEAS-2B cells (an immortalized bronchial epithelial cell line) showing the spectral region containing the resonances of the nicotinamide subunits of NAD⁺ (N) and NADP⁺ (NP). The spectrum was recorded at 288 K with an acquisition time of 2 s and a recycle time of 6 s (512 transients). The free induction decays were zero-filled once and apodized using an unshifted Gaussian function and a 05 Hz line broadening exponential. B. ¹H NMR spectrum of an extract of mouse heart showing the Adenine nucleotide region (C8-H) resonances) of different nucleotides including oxidized and reduced forms of NAD+ and NADP+. From [1]