Biochemistry

Short-chain fatty acids (SCFAs), which are formed mainly by bacteria fermenting undigested carbohydrates in the colon, they are based on the number of carbon atoms in the carbon chain. Organic fatty acids with less than 6 carbon atoms are called short-chain fatty acids. SCFAs are closely related to various aspects of the human body, so more and more researchers concentrate on SCFAs. This protocol describes, a direct injection gas chromatography detection method with a pretreatment method for extracting SCFA from mice feces by combining acidification. The corresponding sample limit of quantization (LOQ) and limit of detection (LOD) are 0.8-1.0 mg/L and 0.5-0.8 mg/L, respectively. The correlation coefficient of calibration curve is greater than 0.999. The recovery rate of the spiked standard is 80%-102%. This method can be used to analyze and determine SCFAs in mice feces. Therefore, this is an economical, effective and reproducible method for SCFAs measurement in mice samples.

Mitochondrial dysfunction is a principal feature of acute pancreatitis (AP) although the underlying mechanisms are still unclear. AP precipitants induce Ca²⁺-dependent formation of the mitochondrial permeability transition pore (MPTP) in pancreatic acinar cells (PACs), leading to ATP depletion and necrosis. Evaluations of mitochondrial bioenergetics have mainly been performed in isolated PACs using confocal microscopy, with assessment of mitochondrial membrane potential, NADH/FAD⁺ and ATP levels, coupled with patch-clamp electrophysiology. These studies are technically demanding and time-consuming. Application of Seahorse flux analysis now allows detailed investigations of bioenergetics changes to be performed in cell populations using a multi-well plate-reader format; rates of oxygen consumption (OCR) and extracellular acidification (ECAR) provide important information about cellular respiration and glycolysis, respectively. Parameters such as maximal respiration, ATP-linked capacity and proton leak can be derived from application of a respiratory function “stress” test that involves pharmacological manipulation of the electron transport chain. The use of Seahorse Flux analysis therefore provides a quick, and convenient means to measure detailed cellular bioenergetics and allows results to be coupled with other plate-reader based assays, providing a fuller understanding of the pathophysiological consequences of mitochondrial bioenergetics alterations.

In mammalian organisms, fatty acids (FAs) exist mostly in esterified forms, as building blocks of phospholipids, triglycerides, and cholesteryl esters, while some exist as non-esterified free FAs. The absolute quantification of FA species in total lipids or in a specific lipid class is critical in lipid-metabolism studies. To quantify FAs in biological samples, gas chromatography–hydrogen flame ionization detection (GC-FID)-based methods have been used as highly robust and reliable techniques. Prior to GC-FID analysis, FAs need to be derivatized to volatile FA methyl esters (FAMEs). The derivatization of unsaturated FAs using classical derivatization methods that rely on high reaction temperature requires skill; consequently, the quantification results are often unreliable. The recently available FA-methylation procedure rapidly and reliably derivatizes a variety of FA species, including poly-unsaturated FAs (PUFAs). To analyze FAs in mammalian tissue samples, lipid extraction and fractionation are also critical for robust analysis. In this report, we describe a whole protocol for the GC-FID-based FA quantification of mammalian tissue samples, including lipid extraction, fractionation, derivatization, and quantification. The protocol is useful when various FAs, especially unsaturated FAs, need to be reliably quantified.

Bacteria use quorum-sensing (QS) systems to monitor and regulate their population density. Bacterial QS involves small molecules that act as signals for bacterial communication. Many Gram-negative bacterial pathogens use a class of widely conserved molecules, called diffusible signal factor (DSF) family QS signals. The measurement of DSF family signal molecules is essential for understanding DSF metabolic pathways, signaling networks, as well as regulatory roles. Here, we describe a method for the extraction of DSF family signal molecules from Xanthomonas oryzae pv. oryzae (Xoo) cell pellets and Xoo culture supernatant. We determined the levels of DSF family signals using ultra-performance liquid chromatographic system (UPLC) coupled with accurate mass time-of-flight mass spectrometer (TOF-MS). With the aid of UPLC/MS system, the detection limit of DSF was as low as 1 µM, which greatly improves the ability to detect DSF DSF family signal molecules in bacterial cultures and reaction mixtures.

Organic acids secreted from plant roots play important roles in various biological processes including nutrient acquisition, metal detoxification, and pathogen attraction. The secretion of organic acids may be affected by various conditions such as plant growth stage, nutrient deficiency, and abiotic stress. For example, when white lupin (Lupinus albus L.) is exposed to phosphorus (P)-deficient conditions, the secretion of citrate acid from its proteoid roots significantly increases (Neumann et al., 1999). This protocol describes a method for the collection and measurement of the efflux of organic acids (oxalate, malate, and citrate) from the roots of rice cultivar Nipponbare (‘Nip’) under different nitrogen forms (NH₄⁺ and NO₃^-), together with different P supply (+P and -P) conditions.

Hydroxycinnamic acids, such as p-coumaric acid and ferulic acid, are a major class of compounds derived from the phenylpropanoid pathway. These compounds are widely conserved in plants and primarily accumulate in the secondary cell wall. They serve as important structural components that contribute to the overall strength and rigidity of plant cell walls and are also potent antioxidants valued for nutritional consumption. This protocol describes a method for analyzing hydroxycinnamic acids that are released after incubation under alkaline conditions.

Two enantiomers of 2-hydroxyglutarate (2HG), L (L2HG) and D (D2HG), are metabolites of unknown function in mammalian cells that were initially associated with separate and rare inborn errors of metabolism resulting in increased urinary excretion of 2HG linked to neurological deficits in children (Chalmers et al., 1980; Duran et al., 1980; Kranendijk et al., 2012). More recently, investigators have shown that D2HG is produced by mutant isocitrate dehydrogenase enzymes associated with a variety of human malignancies, such as acute myeloid leukemia, glioblastoma multiforme, and cholangiocarcinoma (Cairns and Mak, 2013; Dang et al., 2009; Ward et al., 2010). By contrast, we and others have shown that L2HG accumulates in response to cellular reductive stressors like hypoxia, activation of hypoxia inducible factors, and mitochondrial electron transport chain defects (Oldham et al., 2015; Reinecke et al., 2011; Intlekofer et al., 2015; Mullen et al., 2015). Each enantiomer is produced and metabolized in independent biochemical pathways in reactions catalyzed by separate enzymes and utilizing different cofactors with presumably different consequences for cellular metabolism (Kranendijk et al., 2012). Therefore, as research into the roles of D2HG and L2HG in human metabolism continues, it becomes increasingly important for investigators to consider each enantiomer independently (Struys, 2013). Several methods for quantification of biochemically relevant enantiomers in general have been developed and typically include enzymatic assays using enzymes specific for one enantiomeric species or the other, the use of chiral chromatography medium to facilitate chromatographic separation of enantiomers prior to spectroscopy, or the use of chiral derivatization reagents to convert a mixture of enantiomers to diastereomers with differing physical and chemical properties facilitating their chromatographic separation. In this protocol, we report the adaptation of a previously published derivatization method using diacetyl-L-tartaric anhydride (DATAN) for the quantification of 2HG enantiomers (Figure 1) (Oldham et al., 2015; Struys et al., 2004).


Figure 1. Reaction scheme for the derivatization protocol

Under certain growth conditions some microorganisms secrete organic acids into the extracellular medium to relieve the accumulation of excess energy carriers, and/or to reduce toxic concentrations of organic acids. For example, a glycogen-deficient ∆glgC mutant of the cyanobacterium Synechococcus sp. PCC 7002 secretes pyruvate, acetate, α-ketoglutarate, α-ketoisocaproate and succinate (Davies et al., 2014; Jackson et al., 2015). Secretion of these organic acids functions as a putative energy-spilling mechanism in the absence of glycogen, the major carbon and reductant sink in this organism. Identification of secreted organic acids can facilitate the design of metabolic engineering strategies that funnel over-accumulating organic acids towards metabolic pathways that make a product of interest (such as a biofuel). Here, we describe a method for analyzing secreted organic acids in the extracellular media using high-performance liquid chromatography (HPLC). This method was developed for analysis of organic acids secreted by photosynthetic microbes (cyanobacteria and algae) into media, but could be used to analyze organic acids secreted by any microorganism cultivated in liquid medium.

Citrus are among the most relevant sources of vitamin C (ascorbic acid + dehydroascorbic acid). Recent studies have revealed that it increases in the peel as fruit ripens and remains constant or even decreases in the pulp tissue. Moreover, important differences on ascorbic acid content exist among citrus varieties in both tissues. Here we describe a simple method for vitamin C analysis/quantification in the peel and pulp tissues of citrus fruit.

Intracerebral infusion of kainic acid (KA) by a microdialysis probe induces a focal swelling in the brain-perfused area which promotes inflammation (Compan et al., 2012; Oprica et al., 2003). The microdialysis technique allows the local in vivo perfusion of KA and the simultaneous collection of inflammatory mediators, and other neuroactive substances, released in the injured brain. This protocol also allows the perfusion of different solutions in each cerebral hemisphere at the same time. By perfusing KA in isotonic solution of Krebs-Ringer Bicarbonate (KRB) (280-290 mOsm) in one hippocampus and KA in hypertonic KRB solution (1,400-1,500 mOsm) in the contralateral side, we can evaluate in vivo the efficiency of hypertonic solutions in preventing inflammation induced by swelling after KA infusion. Once the inflammatory response has been induced, it is possible to infuse through the microdialysis probe a biotinylated specific inhibitor of caspase-1 allowing the detection of the brain regions and cells involved in IL-1 production in response to the injury (Oprica et al., 2003).