Lipids quantification

Briefly, liposomes (10mM) were incubated for 30minutes at 37°C for egg phosphatidylcholine (EPC) and at 60°C for HSPC-based liposomes with 50 X 10 dpm of methylamine (1 x 10 dpm/mole). At the end of incubation an aliquot of this mixture was passed down a Sephadex G-50 minispin column equilibrated in 10 mM histidine-sucrose buffer 10%, pH 6.7 buffer. Liposomes were eluted at the column void volume and separated from the unencapsulated methylamine. The concentration of liposomes in the original liposomal dispersion and in the void volume fraction was determined from the organic phosphorus (phospholipid) concentration (see section Lipid Quantification and Chemical Stability above) (10,49,53). 

In many cases, sample preparation is mandatory prior to lipid extraction. This crucial process might include tissue homogenization, determination of dry weights, cell numbers, protein content, or DNA content for normalization purposes. The addition of internal standards is not only used to control extraction efficiency, but also is required for lipid quantification by mass spectrometric analysis, in which an ion current is translated into a lipid concentration. Many standard lipids that can be distinguished from endogenously occurring lipids by using rare... 

Dairy lipids Quantification of triglycerides and fatty acids in dairy products 1999(232)... 

HPLC-TS-MS was used by Kuypers, Biitikofer and Shackleton (1991) for the determination of glycerobenzoate derivatives of diradylglycerols of phospholipids they did not determine intact phospholipids or classes of polar lipids. Quantification was by HPLC with UV detection, with subsequent identification of individual molecular species by TS. They describe the possibility of quantification of molecular species as well as positional isomers of individual molecular species by HPLC-TS-MS. They also discussed the possibility of determining positional isomers, that is the snA or sn-2 position of a fatty acyl group. The method was applied to lipids from human erythrocytes. 

This experiment is based on the use of a Kontes fiber optics densitometer (K-49500) with baseline corrector and strip-chart recorder. The description given in the second paragraph of Experiment 8, Section 8 is based on the use of that equipment. Readers will have to modify their approaches to in situ densitometry based on the available instrumentation in their laboratories. In our laboratory, we no longer use the K-49500 model and do most of our lipid in situ densitometry with either a Kontes Model 800 densitometer equipped with a Hewlett-Packard Model 3992A integrator/recorder (Morris et al., 1987) or a Shimadzu CS-930 computerized TLC densitometer operated in the single-beam, reflectance mode. [See Morris et al. (1987), Park et al. (1991), and Masterson et al. (1993) for further information on the use of these instruments in lipid quantification by TLC-densitometry.]... 

These principles of shotgun lipidomics can only be achieved in conjunction with the major feature of direct infusion, that is, ESI-MS analysis of lipids is conducted at a constant concentration of the solution. This feature in shotgun lipidomics provides many advantages for lipid analysis, particularly for the quantification of individual lipid species. Some of these advantages are as follows. First, constant interactions between lipid species are maintained under a constant concentration condition therefore, contribution of individual lipid species to the ion current in an ESI source is constant, thereby leading to a constant ratio of ion peak intensities between lipid species of a class. Such a constant ratio can be achieved under different experimental conditions (see Chapter 4), on different MS instruments, and in different laboratories. Second, also due to the constant interactions between lipid species under the condition, ion suppression between each other within a lipid class or between lipid classes is constant. Third, lipid aggregation, which is a big concern for lipid quantification, can be well controlled and minimized. 

The amount of individual internal standard should be optimized to make the relative intensity of the internal standard peak in the range of >20-<5(X)% in comparison to the ion peak corresponding to the most abundant species in the class. When the relative peak intensity of a selected internal standard is lower than 20% in comparison to the base peak, the experimental error is greatly amplified. Addition of too much internal standard could lead to an ion suppression effect, making the endogenous lipid species close to the baseline. Accordingly, the optimal amounts of internal standards necessary for lipid quantification could vary largely for different kinds of samples. 

Quantification of lipids by ESI-MS through a lipidomic approach is an interdisciplinary task that largely determines the amounts of intact individual species in a biological sample based on the selected internal standard(s) and a normalizer (although other relative measurements are also used (see below)). Unfortunately, the measurements of the contents of both the internal standard and the normalizer contain experimental errors. These errors impact the lipid quantification. Collectively, by this approach for lipidomic analysis, the amounts of individual lipid species in a selected sample size can be determined if appropriate internal standards are added prior to extraction with a tolerant experimental error, in which correction for any bias in extraction recovery, molecular species-dependent ionization efficiencies, and other factors is considered within a variation of 10%. 

Importantly, the combination of ESI-MS detection with high-performance liquid chromatography (HPLC) separation makes this approach a potential choice for lipid quantification in lipidomics if one of the following conditions can be met. 

This outcome was consistent with a hypothesis that structural deterioration could have been a byproduct of microorganism activity. The higher lipid content in the poorly preserved tissue suggests that those lipids are primarily extrinsic, that is, that they were produced by bacteria and/or fungi. As the food source for such microorganisms, the protein within the bone may have been substantially altered in concert with the microstructure deterioration. The quantification of the changes to the organic fraction became our next focus of research. 

Alkaline hydrolysis (saponification) has been used to remove contaminating lipids from fat-rich samples (e.g., pahn oil) and hydrolyze chlorophyll (e.g., green vegetables) and carotenoid esters (e.g., fruits). Xanthophylls, both free and with different degrees of esterification with a mixture of different fatty acids, are typically found in fruits, and saponification allows easier chromatographic separation, identification, and quantification. For this reason, most methods for quantitative carotenoid analysis include a saponification step. 

Two-dimensional development is commonly used in analytical TLC for the separation, identification, and sometimes, quantification of complex lipid species especially when one-dimensional development does not provide satisfactory separation where some individual hpids coelute in a number of common solvent systems. 

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. 

Lipids are generally identified in paint samples by evaluating characteristic ratio values of FA amounts and comparing them with naturally or artificially aged reference paint layers. Molar or weight contents are obtained after quantification based on calibration curves. 

The products formed during lipid peroxidation include unsaturated aldehydes, such as 4-hydroxynonenal. Their quantification is of great interest because of their extremely reactive and cytotoxic properties. This extreme reactivity and metabolic conversion, however, may make them unsuitable as test analytes for in vivo antioxidant activity studies except at high levels of oxidative stress. Furthermore, simple chemical tests such as the TBARS (thiobarbituric acid reactive substances) and LPO-586 (colorimetric... 

The determination of F2-isoprostanes, oxidation products of arachidonic acid, has been proposed as a more reliable index of oxidative stress in vivo, overcoming many of the methodological problems associated with other markers. The isoprostanes have emerged as a most effective method of quantifying the potential of antioxidants to inhibit lipid peroxidation. However, one drawback of this method is that quantification of F2-iP requires sophisticated techniques, in particular GC/MS and HPLC/MS... 

The photochemiluminiscence (PCL) assay was initially used by Popov and others (1987). Popov and Lewin (1994 1996) have extensively studied this technique to determine water-soluble and lipid-soluble antioxidants. The PCL assay measures the antioxidant capacity, toward the 02 radical, in lipidic and water phase. This method allows the quantification of both the antioxidant capacity of hydrophilic and/or lipophilic substances, either as pure compounds or complex matrices from different origin synthetic, vegetable, animal, human, etc. The PCL method is based on an approximately 1,000-fold acceleration of the oxidative reactions in vitro by the presence of an appropriate photosensitizer. The PCL is a very quick and sensitive method. Chua and others (2008) used this assay to determine the antioxidant potential of Cin-namomum osmophloeum, whereas Kaneh and Wang and others (2006) determined the antioxidant capacity of marigold flowers. The antioxidant activity of tree nut oil extracts was also assessed by this method (Miraliakbari and Shahidi 2008). 

Quantification Lipid Membrane and Binding of Membrane Proteins... 

Charters, S., Evershed, R. P., Goad, L. J., et al. (1993). Quantification and distribution of lipid in archaeological ceramics implications for sampling potsherds for organic residue analysis and the classification of vessel use. Archaeometry 35 211-223. 

Lieser B, Liebisch G, Drobnik W, Schmitz G. 2003. Quantification of sphingosine and shinganine from crude Hpid extracts by HPLC electrospray ionization tandem mass spectrometry. J Lipid Res 44 2209. 

A colorimetric estimation of inserted PEG-phospholipids was developed based on the two-phase system used to quantify phospholipids (49). The formation of a complex between the phospholipids and Fe(SCN)3 transfers the chromophore Fe(SCN)3 to the organic phase, allowing quantification of the phospholipids present in the solution. This system was applied to PEG-phospholipids (50). It is quite sensitive but obviously limited to PEG-phospholipids. The PEG-lipids, which bear no phosphate group, cannot be quantified by this method. 

Quantification of lipids with irregular signal patterns in muscle spectra 33... 

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