Cell Metabolism

Cellular metabolism is characterized as the summation of changes that take place within the cell, through which energy and components are used for various biochemical processes. Cellular respiration, a function of cell metabolism, breaks down sugars and other monosaccharides and chemically harnesses the produced ATP to create energy.

Mainly, cellular respiration involves three steps; glycolysis, the citric acid cycle, and oxidative phosphorylation. These processes together form various building blocks that are used in the creation of many essential molecules including proteins, lipids, nucleic acids and carbohydrates. The disposal of waste is an essential function of cell metabolism that allows for the functional excretion of potentially toxic components including nitrogenous compounds, phosphates, and sulfates.

These processes together form various building blocks that are used in the creation of many essential molecules including proteins, lipids, nucleic acids and carbohydrates. The disposal of waste is an essential function of cell metabolism that allows for the functional excretion of potentially toxic components including nitrogenous compounds, phosphates, and sulfates. As cellular metabolism is associated with many known biological pathways (amino acid metabolism and transport, urea cycle, carbohydrate tolerance, glycogen storage, lipid metabolism, fatty acid oxidation, and mitochondrial activity to name a few) the importance of understanding its roles in cell physiology, disease pathology, and etiology should not be understated.

 

Methods and Analysis

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One of the most common techniques of detecting and assessing cellular metabolism activity is through colorimetric or fluorometric assays. These assays target specific analytes known to associated pathways; for example detection of glucose, hexokinase, pyruvate, pyruvate kinase, lactate, and glycogen may be chosen when identifying the glycolysis pathway. In fatty acid metabolism, triglyceride, free fatty acids, and pyrophosphate may be targeted. Additionally, urea, glutamate, or L-arginine may be chosen when looking at urea metabolism, and ATP, NAD, NADH, NADP, or NADPH (or a combination thereof) may be targeted for overall metabolic activity. These assays are commercially available as kits, and though technical specifications of each differ slightly, many steps in each process are similar.



First, samples must be prepared, and various cells, tissues or other biological fluids may be used as starting material. Processes are specific to the sample and assay, though if samples contain enzymes that might interfere with analysis, a deproteinization step is critical. Next, if necessary, a standard curve must be prepared and samples may be loaded into wells with associated reagents. After an incubation step, protected from light, the samples can be read on a suitable microplate reader at a wavelength outlined in the kit protocol.

Once data has been collected, the duplicate or triplicate readings from each standard or sample should be averaged. To correct the absorbance for each well, the zero value as well as the background control should both be subtracted from each optical density (OD) readout. These values can then be plotted as a function of the final concentration, and normally a compatible plate reading software is used to help interpret and graph the data. Usually, analytes are targeted for their concentration or activity that helps define the extent of metabolic processes within the cell. Equations for measuring both are listed below.

Measuring Substrate Concentration	Measuring Substrate Activity
(x÷v) × D	(X÷(ΔT × V)) × D

X = Amount of analyte in sample found from standard curve (nmol)
V = Sample volume added in wells (μL)
D = Dilution factor used
ΔT = Reaction time between two time points (T2 - T1)
ΔY = Activity of the substrate in the test sample from two time points (Y2 - Y1)

Kits for detecting and analyzing analytes through luminescence follow very similar processes to colorimetric and fluorometric assays, though these involve chemical bioluminescence. The analyte is first digested by its associated enzyme to release NADH, which is readily taken in by pro-luciferin to form luciferin. Luciferin reacts with ATP and its associated oxidative enzyme, luciferase, which produces luminescence measured in relative light units (RLU). Protocols may also include the construction of a standard curve to aid in analysis, and data may be assessed by looking at RLU across a number of variables including substrate concentration, time in culture, or by comparing specific compositions of each well.



Nuclear magnetic resonance (NMR) based metabolomics may also be used to study the physicochemical extracts of live samples. There are many pros to using NMR in aqueous solution, versus in in vivo or ex vivo techniques, in that the number of detectable, and therefore quantifiable metabolites, is greatly increased. The increased spectral resolution also significantly improves the distinguishability of metabolite signals, which NMR metabolomics offers better sensitivity to from concentrated samples.

In NMR, a wide variety of samples may also be used, including plasma, serum, saliva, etc., and these must be deproteinized prior to analysis through precipitation or extraction to weaken the intensity of interfering resonances. After derivatization and buffering the samples undergo analysis to produce a specific NMR profile, constitutive of many peaks that represent various molecules within the sample. For NMR, various nuclei spectras may be used, including 1H, 13C, 15N and 31P, and the profile can be interpreted using multivariate statistical analysis with pattern recognition software.

Common types of analysis include those that quantify principal components, associate hierarchical clusters, create partial least squares, discriminant function, or even form artificial neural networks. Collectively, this analysis helps identify and discriminate the function of the metabolites in the sample, where databases can secondarily be used to validate specific pathway activity.

Mass Spectrometry (MS)

Similar to NMR, mass spectrometry (MS) easily distinguishes the masses of molecular components, or more specifically, ionized molecules and fragments.

1. Samples are prepared accordingly then undergo a rapid quenching step to quickly stop any metabolic activity within the cell. If required, samples must undergo a purification step to separate the cellular phase from the extracellular medium.
2. The sample will undergo an extraction phase that aims to remove and dissolve the maximum amount of original sample metabolites as possible. There is no one-size-fits-all for extraction, as these steps may be variable and must be empirically determined for each sample and reagent combination.
3. Samples are concentrated to partially, if not totally, remove leftover solvents.

Usually, samples will undergo another technique for better resolution prior to MS analysis.

Liquid Chromatography (LC)

Liquid chromatography (LC) is most commonly used to separate unneeded metabolites according to a column and eluent, though gas chromatography (GC) can be used to separate volatile and non-volatile metabolites as well.

Capillary Electrophoresis (CE)

Alternatively, capillary electrophoresis (CE) techniques may be used to separate polar metabolites if only a small testing sample volume is preferred. The samples will then undergo MS analysis that specifically identifies metabolites using their molecular mass and fragmentation pattern. The data will be representative of a peaked chart that graphs the mass:charge ratio of ions against their relative abundance in the sample.

Comparing & Contrasting Methodologies

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Colorimetric, fluorometric, and luminescent methods are quantitative, commercially available in many kit forms, low cost, and offer quick experimental times. Care must be taken in the construction of the standard curve, as this is what the data is referenced against. In some instances, samples that produce signals greater than the linear range of the highest standard should be further diluted and retested; conversely the opposite applies to samples that produce signals lower than the linear range of the lowest standard.

Though techniques are similar, it is important to note that calorimetry requires clear plates, fluorometry requires plates that have black wells and clear bottoms, while luminescent assays require white plates.

	White Solid	Black solid	Black, clear bottom
Luminescence	xx
Fluorescence		xx	x
Absorbance			xx
BRET	xx
FRET		xx	x
AlphaScreen®	xx
Fluorescence Polarization (FP)		xx
HTRF®	xx	x

Method Selection Criteria

Troubleshooting may be necessary, though methods are so widely used that helpful techniques are commonly available with many kit protocols. Though a single statistical analysis method can be used, it is normal to use multiple together to provide a more well-rounded explanation of metabolite concentration and activity for the sample. Laboratory capability and personnel should ultimately decide what the best technique (or techniques) for assessing cell metabolism activity actually is.

• NMR is non-destructive, non-invasive, highly reproducible, and can provide data in real-time. NMR is not just quantitative, but additionally qualitative. NMR, however, is not well-suited to distinguish metabolites that are not soluble in water (lipids) as produced images make identification, quantification, or classification of these nearly impossible. Samples with high protein content, too, may obscure images of smaller molecules in the sample, which is why a deproteinization step is often necessary.

• MS offers higher sensitivity, high throughput capabilities, and less cost than NMR, though steps involved, namely extraction and concentration, are often time consuming. Another consideration of this technique should be that though necessary, inherently, extraction will always cause sample loss.

• In order to use GC-MS, samples must be volatile to separate on the column, which many naturally occurring metabolizes are not. Therefore a derivatization step is required. It is important to note that GC-MS is also not well-suited to analyze heat labile compounds. Technically, CE-MS is quicker and potentially simpler than GC-MS, however these methods require a specific interface and technology so are not as widely used.

• LC-MS is most often the method of choice as it can provide information on many chemically diverse, heat labile analytes that can be tricky to separate using other methods.

Product Ordering Information

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