411 412 413 immediately via rapid freezing or chemical extraction, but practical considerations involved in the collection of material from patients can sometimes make rapid quenching impossible. Therefore, focusing analysis on only those metabolites that are less sensitive to change due to delays in processing time may be important to gain biological insight. 414 415 Sequential metabolomic analyses of the same type of biological material from a patient can explore how metabolite levels vary over time. It is interesting that, when measured, many metabolites are found to be relatively stable. However, the extensive variability exhibited by some metabolites indicates that findings involving those metabolites should be interpreted with caution. 416 417 Finally, the method of sample processing can affect which metabolites are extracted from the material and thus influence what is measured. 418 419 420 ## METABOLOMICS TECHNOLOGIES 421 422 Metabolomics relies heavily on the intersection of instrumentation, software, and statistical and computational approaches for measurement of metabolite levels and downstream data analysis. While the development of new and emerging techniques to assess the metabo-lome is ongoing, the current, clinically applicable approaches can be separated into two broad categories: nuclear magnetic resonance (NMR)–based approaches and chromatography/mass spectrometry (MS)–based approaches. Each of these two approaches has its own set of advantages and disadvantages. 423 424 425 ## NUCLEAR MAGNETIC RESONANCE 426 427 NMR is a technique that, at its core, exploits intrinsic magnetic properties of atomic nuclei to generate data. Nuclei with an odd total number of protons and neutrons (such as 1H, 13C, 15N, and 31P) have a non-zero spin, and this spin generates a magnetic field that can interact with externally applied electromagnetic fields. NMR places compounds into a magnetic field that induces the smaller magnetic fields to align with the larger one. Samples are then exposed to a perpendicular electromagnetic field; the frequency of electromagnetic radiation needed to flip the spin of a nucleus in the exact opposite direction represents the frequency at which an atom "resonates" and can be measured. The resonance frequency of a given atom is affected by adjacent atoms and is ultimately unique for a given arrangement of atoms (i.e., each metabolite). This distribution or "spectrum" of signals is measured and recorded in an NMR experiment. 428 429 With respect to clinical applications, the primary benefits of NMR-based approaches are that they are nondestructive and can be performed on living samples, such as patients, cells, or tissues. They are also highly reproducible and require minimal sample preparation. Measurements are necessarily quantitative as the signal measured directly reflects concentration. These features ensure that multiple, comparable measurements can be made in a given sample either at a single point in time or across time. In addition, given that spins of different elements require sufficiently disparate resonance-inducing radiofrequencies in order to be entirely distinguishable, multiple ele-ments can be assessed in a sample; this feature allows multidimensional 430 431 *TABLE S02-1 Comparison of Nuclear Magnetic Resonance (NMR)- 432 Based and Mass Spectrometry (MS)-Based Approaches to Metabolomic 433 Analyses* 434 435 436 < [... Output truncated due to length. 6157 tokens/28505 characters removed ...]

FEATURE	NMR	MS
Reproducibility	High	Lower
Sensitivity	Low (low μM)	High (low nM)
Selectivity	Untargeted	Targeted >> untargeted
Sample preparation	Minimal	Complex
Sample measurement	Simple: single prep	Multiple preps
Metabolites per sample	50–200	>1000
Identification	Easy, using one- or two-dimensional databases	Complex; need standards and additional analyses
Quantitation	Inherently quantitative; intensity proportional to concentration	Requires standards because of varying ionization efficiency