Sample lipid analysis

The detection and quantification of one or more of the above lipid peroxidation produas (primary and/or secondary) in appropriate biofluids and tissue samples serves to provide indices of lipid peroxidation both in ntro and in vivo. However, it must be stressed that it is absolutely essential to ensure that the products monitored do not arise artifactually, a very difiScult task since parameters such as the availability of catalytic trace metal ions and O2, temperature and exposure to light are all capable of promoting the oxidative deterioration of PUFAs. Indeed, one sensible precaution involves the treatment of samples for analysis with sufficient levels of a chainbreaking antioxidant [for example, butylated hydroxy-toluene (BHT)] immediately after collection to retard or prevent peroxidation occurring during periods of storage or preparation. 

The results for bacterial whole-cell analysis described here establish the utility of MALDI-FTMS for mass spectral analysis of whole-cell bacteria and (potentially) more complex single-celled organisms. The use of MALDI-measured accurate mass values combined with mass defect plots is rapid, accurate, and simpler in sample preparation then conventional liquid chromatographic methods for bacterial lipid analysis. Intact cell MALDI-FTMS bacterial lipid characterization complements the use of proteomics profiling by mass spectrometry because it relies on accurate mass measurements of chemical species that are not subject to posttranslational modification or proteolytic degradation. 

The potential for the preservation of lipids is relatively high since by definition they are hydrophobic and not susceptible to hydrolysis by water, unlike most amino acids and DNA. A wide range of fatty acids, sterols, acylglycerols, and wax esters have been identified in visible surface debris on pottery fragments or as residues absorbed into the permeable ceramic matrix. Isolation of lipids from these matrices is achieved by solvent extraction of powdered samples and analysis is often by the powerful and sensitive technique of combined gas chromatography-mass spectrometry (GC-MS see Section 8.4). This approach has been successfully used for the identification of ancient lipid residues, contributing to the study of artifact... 

A newer approach for lipid analysis is electrospray ionization tandem mass spectrometry (ESI-MS/MS) (Welti et al., 2002). This method requires limited sample preparation and sample size to identify and quantify lipids. Fauconnier et al. (2003) used ESI-MS/MS to analyze phospholipid and galactolipid levels during aging of potato tubers. 

In the second part of this experiment you will characterize the purified lipids (triacylgiycerols) isolated from nutmeg. The fatty acids in the triacyl-glycerols are released by saponification and their identities determined by gas chromatography. Alternatively, students may be provided various fat and oil samples for analysis. For example, the fatty acid content of triacyl-... 

This experiment provides students with the opportunity to isolate a biomolecule from its natural source, followed by its purification and identification. In addition, students will follow a procedure that is typical of the general extraction and characterization of lipids. However, unlike most lipids, the plant pigments are highly colored and may be characterized and quantified by visible spectrophotometry. Several types of plant tissue may be used. Some recommendations are fresh leaves (tree, plant, grass, spinach), green algae, or mosses. For variety, students may be asked to bring their own samples for analysis. 

Precautions should be taken to prevent oxidation during lipid analysis. Polyunsaturated fatty acids in lipid samples are easily attacked by active oxygen species (e.g., free radicals), exacerbated by the presence of strong light and metal ions. Therefore, it is arule of thumb while working with lipids that samples should be handled in a way that minimizes contact with air, light, and metals. To accomplish this, handle samples in glass vessels, use Teflon-lined or coated materials, and maintain the samples... 

This book provides a practical guide to various aspects of lipid analysis, covering topics from sample preparation (extraction, fractionation, and deri-vatization) to CC analysis. Various derivatization methods are discussed and specific procedures are given for each of them. The book provides a comprehensive overview of GC technology including instrumentation (i.e., column, oven, carrier gas, injector, and detector) and data collection. 

What factors can be used to predetermine the quality and utility of a method An analyst must consider the following questions Do I need a proximate analytical method that will determine all the protein, or carbohydrate, or lipid, or nucleic acid in a biological material Or do I need to determine one specific chemical compound among the thousands of compounds found in a food Do I need to determine one or more physical properties of a food How do I obtain a representative sample What size sample should I collect How do I store my samples until analysis What is the precision (reproducibility) and accuracy of the method or what other compounds and conditions could interfere with the analysis How do I determine whether the results are correct, as well as the precision and accuracy of a method How do I know that my standard curves are correct What blanks, controls and internal standards must be used How do I convert instrumental values (such as absorbance) to molar concentrations How many times should I repeat the analysis And how do I report my results with appropriate standard deviation and to the correct number of significant digits Is a rate of change method (i.e., velocity as in enzymatic assays) or a static method (independent of time) needed ... 

Because chlorobenzene is volatile, has limited water solubility, and has a moderate affinity for lipid tissue, chlorobenzene is easily lost from biological samples. Appropriate care must be exercised in handling and storing such samples for analysis of chlorobenzene. 

Many different types of fruit and vegetable crops and other processed foods are analysed for a variety of trace organics both for impurities (such as pesticides) and for content (e.g. amino acids, lipids, etc.). As with soil, it is important to take a representative sample for analysis. 

The processing scheme for the 53-jLtm Nitex samples was similar to that described previously (10) where each filter was transferred to the PVC stand, rinsed with deionized water under suction to reduce sea salt, folded once onto itself to prevent loss of large particles, and then dried at 60 °C for >24 h. Where samples larger than 53 pm have been required for lipid analysis, precombusted, 53-pm stainless steel mesh of identical specifications to the 53-jLtm Nitex was used. In this case a subsample was dried for the regular chemical analysis the remaining sample was specially preserved. 

The MQ-filter sandwich was similarly transferred to the filter-rinsing stand, the top 149-jLtm Nitex support was folded back, and three sharpened acrylic tubes were pressed into the filter pair. The MQ-filter area not enclosed by the acrylic tubes was rinsed with several aliquots of deionized water (totaling 200-300 mL) with suction alternately applied from below to reduce sea salt by an order of magnitude this process was required for accurate dry-weight and major-ion determinations. The acrylic tubes were subsequently removed, and the fresh, unrinsed subsamples were preserved for lipid analysis. The remaining filter material was dried at 60 °C for >24 h. Both the MQ and 53-jitm Nitex samples were stored and transported flat in individual polyethylene bags to prevent loss of material. 

The analysis of simple lipids can be done with good results using common analytical methods without any need for decreasing the molecular weight of the sample by techniques such as pyrolysis. HPLC, SFC or GC procedures were applied for simple lipid analysis, and even the mass spectra of some simple triglycerides are known. As an example, Figure 8.1.1 shows the El mass spectrum of tripalmitin (standard ionization condition). 

Today, HPLC is the dominant analytical technique used for the analysis of most classes of compounds. The analyses can be carried out at room temperature and the collection of fractions for reanalysis or further manipulation is straightforward. The main reason for the slow acceptance of the HPLC technique for Upid analysis has been the detection system. Traditionally, HPLC used ultraviolet/visible (UV/vis) detection, which requires the presence of a chromophore in the analyte. Most lipid molecules do not contain chromo-phores and therefore would not be detected by UV/vis. Modern HPLC detection techniques, such as the use of a mass spectrometer as the detector, derivatization techniques to introduce chromophores, and the availability of pure solvents to reduce interference, have allowed HPLC to compete with and/or complement GC and other traditional methods of lipid analysis. In addition to analytical HPLC, preparative HPLC has been used extensively to collect pure samples of the lipids for the derivatization or synthesis of new compounds. 

The total volumes of both aspirates were measured and samples were collected for lipase, pH, PEG and lipid analysis. The single lumen gastric tube was used to administer the meal and to collect gastric samples every 15 minutes as well as the total residual gastric contents at the end of the experiment. 

Much of the work on lipids has continued to rely on the application of conventional high-resolution methods. Many general papers are available, tackling both sampling and analysis issues (Table 4). 

Route B does not strictly represent a fractionation protocol but is worthy of highlighting in the instances where an alternative rapid procedure is preferred for the analysis of total FA. Here, an acid-catalyzed transesterification can be undertaken, converting total ester-finked acyl residues directly to their methyl esters. As a byproduct, nonsaponifiable lipids are also retained in this fraction (hydrocarbons, isoprenoids) and analyzed simultaneously by chromatographic techniques. While more rapid, the technique yields a more complex sample for analysis. 

Phospholipid fatty acid analysis (PLFA) is based on the determination of signature lipid biomarkers from the cell membranes and walls of microorganisms. Phospholipids are an essential part of intact cell membranes, and information from the lipid analysis provides quantitative insight into three important attributes of microbial communities viable biomass, community structure, and nutritional status. Phospholipid fatty acid prohles have been used to show the response of the microbial community to phosphorus availability (Keinanen et ah, 2002). Signature lipid biomarker analysis may not detect every species of microorganism in an environmental sample accurately, because many species have similar PLFA patterns. 

Several components of chlordane trans- and c/s-chlordane, trans- and c/s-nonachlor, heptachlor, gamma-chlordene) were detected in the skin lipids of humans (Sasaki et al. 1991b). The samples were taken by swabbing the face with cotton soaked with 70% ethanol 3-4 hours after the face was washed with soap. Because all samples from inhabitants of an area known to be contaminated with chlordane contained chlordane residues, and because the profile of chlordane components in skin lipids closely resembled those in technical chlordane, the authors suggested that skin lipid analysis is a satisfactory indicator of dermal exposure to airborne chlordane, such as occurs in homes treated for termites. Oxychlordane in the skin lipids was positively correlated (correlation coefficient = 0.68, p<0.01) with concentrations in internal adipose tissue. The authors concluded that the concentration of oxychlordane in skin lipids was a satisfactory indicator of body accumulation of chlordane. 

Serum Lipid Analysis. Blood samples were withdrawn from the marginal ear-vein after overnight food deprivation every 2 wk until the termination of the experimental periods. Total cholesterol, high density lipoprotein cholesterol (HDL-C), and tri-acylglycerol (TG) concentrations were determined using enzymatic methods. Low density lipoprotein cholesterol (LDL-C) was calculated according to Friedewald et al. (14). 

Serum Lipid Analysis. Blood samples from subjects who had fasted overnight were collected from each volunteer before breakfast at the beginning and the end of each trial pe-... 

---