Lipid classes, quantitation

Each phospholipid class in a given tissue has a characteristic fatty acid composition. Though the same fatty acid may be present in a number of lipids, the quantitative fatty acid composition is different for each class of lipids and remains fairly constant during the growth and development of the brain. A typical distribution profile of the major fatty acids in rat brain phospholipids is given in Table 3.1. Not only do the phosphoglycerides differ in the structure of the polar head groups, or phospholipid... 

Recently, a quantitative electrospray ionization/mass spectrometry method (ESI/MS) has been developed to analyze the molecular profile, or hpidome of different lipid classes in very small samples. In this method, total lipid extracts from tissues or cultured cells can be directly analyzed. By manipulating the ionization method, the mass spectrographs of polar or even non-polar lipids can be obtained [8]. This method and the use of lipid arrays allow precise and quantitative identification of the lipid profile of a given tissue, and map functional changes that occur. 

D1.6 Quantitation of Lipid Classes by Thin-Layer Chromatography with Flame Ionization Detection... 

Support Protocol Quantitation of Lipid Classes After Hydrogenation Dl.6.9... 

Accurate determination of lipids in foods is required for nutritional labeling, certification, or for evaluation of standard of identity and uniformity, as well as examination of their effects on functional and nutritional properties of foods. Following lipid extraction and precise quantitative analysis, lipids so obtained may be used for analysis of other lipid characteristics and properties provided that nondestructive and mild extraction procedures are employed that retain the integrity of lipids. Thus, determination of lipid classes, fatty acid composition (unit du), and oxidative state of lipids (Chapter D2), amongst others, may be pursued following the extraction process. 

Thin-layer chromatography (TLC) on silica gel is well known for its separation power for lipids and related compounds. The flame ionization detector (FID) is a universal analytical instrument that offers high sensitivity and linearity for carbon-containing organic compounds. The combination of TLC and FID led to the wide use of the Iatroscan TLC-FID for the analysis of lipid classes. The adoption of the Iatroscan TLC-FID in both academia and industry has generated sufficient data to indicate that TLC-FID is currently one of the most efficient tools for the quantitation of lipids classes (Ackman et al., 1990 Hammond, 1993). 

Chromarod FID peaks of sterols, diglycerides, monoglycerides, and polar lipids are narrower and sharper than peaks of triglycerides and free fatty acids when analyzed using either method described in this unit (see Basic Protocol and Alternate Protocol). Hydrogenation of total lipids (see Support Protocol) results in much sharper and narrower peaks, which in turn substantially improves the resolution between lipid classes. The accuracy and precision in quantitating lipid classes of vegetable oils and animal fats are expected to be better than those from marine lipids. 

One of the disadvantages of using TLC-FID in the quantitation of lipid compounds is the low precision in spotting fixed volumes of sample solution onto the Chromarods, which results in variations in the quantitation of lipid classes. This problem can be minimized if all calibration standards are dissolved in one solution and if calibration is performed using the same set of Chromarods used for the lipid samples. 

The accurate quantitation of minor lipid classes depends not only on the concentration of total lipids in the sample, but also on the profile of lipid classes in the sample. If one lipid class (e.g., triglycerides) dominates all others, it would be difficult to simultaneously quantify the minor lipid classes due to the overloading problem of the dominating lipid class. 

It is not known whether hydrogenation in the presence of catalyst (see Support Protocol) causes side reactions other than the saturation of double bonds. Therefore, this method should only be used if the accurate quantitation of lipid classes cannot be achieved using the Basic Protocol or Alternate Protocol. 

Neutral lipid classes, TLC-FID quantitation, 492-498 Neutral sugars... 

As mentioned above, two different mass spectrometric approaches to lipid analysis exist The first one is performed directly from lipid extracts without prior chromatographic separation and is referred to as shotgun lipidomics. Here, lipid classes are separated in the ion source according to their intrinsic electrical properties (24). Detailed and unambiguous structural and quantitative analysis of individual species is obtained by means of multiplexed mass spectrometry using NL, PIS, and... 

The amount of data obtained by mass spectrometry is enormous, particularly when many lipid classes or several lipid species are investigated simultaneously. As such, manual management of the different data sets is impractical. For this reason, various attempts have been initiated to create software that is capable of evaluating and handling the generated data sets in a qualitative, quantitative, and comparative manner. One example is the software called Lipid Profiler, which has been used to achieve automated identification, deconvolution, and absolute quantification... 

The fatty acids of the bloodstream forms of salivarian trypanosomes appear, on the most part, to be qualitatively similar to their host, although the quantitative FA composition of some lipid classes is much different. The Afriean trypanosome seems to possess the ability to rearrange the FA composition of individual complex lipid elasses and, through chain elongation and desaturation to modify host-acquired FA. 

Several studies have shown that different mixmres of solvents used to extract the sample give different total lipid contents. Different results are also obtained if different extraction techniques are applied to the same sample. The chloroform/methanol mixture that quantitatively extracted all lipid classes has produced the highest lipid yields. ... 

The separated lipid classes can then be collected as discrete fractions in solvent. This is particularly useful for either the direct detection and quantitation of isolated lipid fractions or for further derivatization and analysis of lipid components. For example, acylglycerols, wax esters, and phospholipid classes can be separated by column chromatography and each fraction reacted as in Section 9.3.2 to analyze the FA present in each lipid class. 

The analysis (separation, identification, and quantitation) of lipid classes from total lipid extracts is of prime importance in many food industry applications. While the chemical and chromatographic approaches outlined are elegant, well-established, and robust, they are relatively time-consuming and rely on separate stages of separation, identification, and quantitation. 

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