Fatty matrices

G. R. van der Hoff, R. A. Baumann, P. van Zoonen and U. A. Th. Brinkman, Determination of organocWoiine compounds in fatty matrices application of normal-phase PC clean-up coupled on-line to GC/ECD , 7. High Resolut. Chromatogr. 20 222-226(1997). 

LC-GC, therefore, shows promise for forensic science applications, reducing sample handling and preparation steps by essentially using an on-line LC column in place of one or more extraction steps. This is followed by a traditional high resolution GC analysis. The methods described here for pesticides and hormones could be readily adapted to a variety of analyses, especially those involving fatty matrices. Such as tissues, food or blood. 

The rationale of validation experiments with fatty matrices is the high amount of fat extracted with many organic solvents. If analytes are not fat soluble and extraction is performed with water or aqueous buffer solutions, the troublesome fat is not extracted together with the analyte. Such extractions are typical for, e.g., the class of sulfonylurea herbicides. Examples exist where in such cases the applicability of an analytical method to fatty matrices was accepted by the authority without particular validation. 

GR Van der Hoof, AC Van Beuzekom, UAT Brinkman, RA Baumann, P Van Zoonen. Determination of the organochlorine compounds in fatty matrices application of rapid off-line normal-phase liquid chromatographic clean-up. J Chromatogr A 754 487-496, 1996. 

Figure 10.13 GC chromatogram obtained after on-line LC-GC(ECD) of a human milk sample analysed for PCBs (attenuation X 64). Peak identification is as follows (1) PCB 28 (2) PCB 118 (3) PCB 153 (4) PCB 138 (5) PCB 180 (6) PCB 170 (7) PCB 207. Reprinted from Journal of High Resolution Chromatography, 20, G. R. van der Hoff et al., Determination of organochlorine compounds in fatty matrices application of normal-phase LC clean-up coupled on-line to GC/ECD , pp. 222-226, 1997, with permission from Wiley-VCH.

Juhler, R. K. (1997). Optimized method for the determination of organophosphoms pesticides in meat and fatty matrices. J. Chmmatogr. A 786, 145-153. 

Blagdon PA, Morgan R. Methods of encapsulating liquids in fatty matrices, and products thereof. US patent 5204029, 1993. 

Taking into account these results, our objective was to develop a SPME procedure that improved the release of PCBs from the sample to the fiber coating irrespective of the fat content of the samples. Saponification of fats to their corresponding glycerols and carboxylates facilitates the release of PCBs from fatty matrixes and also can... 

Saponification of fats... facilitates the release of RGBs from fatty matrixes. (From Llompart et ah, 2001). 

The next example (excerpt 5A) returns us to the analysis of PCBs in milk. Recall that Elompart et al. (2001) (excerpt 4C) used HSSPME techniques to detect PCBs in milk. Their stumbling block was the fat content. The original method worked fine on skim milk, but the fat contained in nonskim milk trapped the PCBs in the liquid phase (those pesky matrix effects ), rendering headspace (i.e., gas-phase) analysis of the PCBs disappointing at best. More promising results were obtained when the milk fat was first saponified with base (NaOH), a process that helps to release the PCBs from the fatty matrix. 

In a number of indications once-daily dosages have proved to be beneficial, and therefore attempts have been made to develop once-daily formulations of opioids. In the case of morphine, a step in this direction was achieved with MST continus long, which has been approved for once or twice-daily use, by incorporating the active substance in a purely fatty matrix to produce granules with a particle size of about 1 mm, which are then filled into single-dose sachets. Other morphine solutions have been incorporated in prolonged-release pellets with a release rate of about 24 h (e.g. Morphelan developed by Elan). 

In Great Britain tramadol is available as once-daily tablets made up of granules in a purely fatty matrix (Zydol XL), as described above for morphine, which again reduces the release rate compared with the granules. 

As the surface concentration of enhancer in the adhesive coating increased, the mechanism of skin permeation enhancement showed some changes as reflected in the relative contribution of fatty matrix and protein gel pathways (Table VII ). The behavior for azone and oleic acid at high concentration was observed to be identical to that at low concentration (compare the data in... 

Tables VI and VII ). On the other hand, capric acid and decylmethyl sulfoxide showed a dual effect on the hydrophilic protein gel and also on the lipophilic fatty matrix. In the case of capric acid, the overall enhancement in the permeation of progesterone was increased by 354%, in which the protein gel pathway and fatty matrix pathway contribute approximately equally (with enhancement factor of 15.3 vs. 13.0). In the case of decylmethyl sulfoxide, the overall enhancement was improved by 515% (40.2 vs. 7.8).

The criteria for the choice of the CRM are not different from the criteria to select the material for the preparation of a laboratory reference material for method development, statistical control charts etc. The difference lies in the availability of adequate CRMs from reliable suppliers and the level of compromise which the analyst must make between an ideal situation and the reality of what is on offer. Massart and co-workers have proposed a principle component analysis to help select the best adapted CRMs available on the market to verify AAS analysis of foodstuffs [10], Their approach took into account the analytes as well as the matrix composition. Besides the fact that they highlighted a lack of sorts of CRM, in particular those having a fatty matrix, they demonstrated that such a statistical approach can help in the most appropriate selection of materials. Boenke also proposed a systematic approach for the choice of materials to be certified for mycotoxins [11] and which could be followed by potential users. The selection of the CRM by the analyst should include a certain number of parameters this can cover the following properties to fulfil the intended purpose level of concentration of the analytes ... 

Matrix solid-phase dispersion (MSPD) is an SPE variant where samples are ground and mixed with a support. In the initial application, samples were placed in a disposable column previously packed with Florisil, which trapped the fat from the sample and allowed the compounds of interest to be eluted. This has successfully been applied to the determination of lipophilic pesticides from both fatty and non-fatty matrixes. Recently, an orthogonal technique, dispersive solid-phase extraction, for the isolation and analysis of a variety of pesticides on numerous food matrixes has been introduced. The technique is called QuEChERS, which stands for quick, easy, cheap, effective, rugged, and safe. The technique offers advantages in time and solvent usage since it uses approximately 10 ml of solvent per sample when compared to the potentially hundreds of milliliters of solvent used for more standard extraction and isolation protocols. It uses a combination of MgS04 and primary secondary amine (PSA) sorbent not only to remove water and non-target compounds, but also isolate the compounds of interest. 

Polymers for a fatty matrix glycerol behenate, glycerol palmitostearate, waxes, cetyl alcohol... 

The space inside the inner mitochondrial membrane is called the matrix, and it contains most of the enzymes of the TCA cycle and fatty acid oxidation. (An important exception, succinate dehydrogenase of the TCA cycle, is located in the inner membrane itself.) In addition, mitochondria contain circular DNA molecules, along with ribosomes and the enzymes required to synthesize proteins coded within the mitochondrial genome. Although some of the mitochondrial proteins are made this way, most are encoded by nuclear DNA and synthesized by cytosolic ribosomes. 

This is a crucial point because (as we will see) proton transport is coupled with ATP synthesis. Oxidation of one FADHg in the electron transport chain results in synthesis of approximately two molecules of ATP, compared with the approximately three ATPs produced by the oxidation of one NADH. Other enzymes can also supply electrons to UQ, including mitochondrial 5w-glyc-erophosphate dehydrogenase, an inner membrane-bound shuttle enzyme, and the fatty acyl-CoA dehydrogenases, three soluble matrix enzymes involved in fatty acid oxidation (Figure 21.7 also see Chapter 24). The path of electrons from succinate to UQ is shown in Figure 21.8. 

COMPARTMENTALIZED PYRUVATE CARBOXYLASE DEPENDS ON METABOLITE CONVERSION AND TRANSPORT The second interesting feature of pyruvate carboxylase is that it is found only in the matrix of the mitochondria. By contrast, the next enzyme in the gluconeogenic pathway, PEP carboxykinase, may be localized in the cytosol or in the mitochondria or both. For example, rabbit liver PEP carboxykinase is predominantly mitochondrial, whereas the rat liver enzyme is strictly cytosolic. In human liver, PEP carboxykinase is found both in the cytosol and in the mitochondria. Pyruvate is transported into the mitochondrial matrix, where it can be converted to acetyl-CoA (for use in the TCA cycle) and then to citrate (for fatty acid synthesis see Figure 25.1). /Uternatively, it may be converted directly to 0/ A by pyruvate carboxylase and used in glu-... 

All of the other enzymes of the /3-oxidation pathway are located in the mitochondrial matrix. Short-chain fatty acids, as already mentioned, are transported into the matrix as free acids and form the acyl-CoA derivatives there. However, long-chain fatty acyl-CoA derivatives cannot be transported into the matrix directly. These long-chain derivatives must first be converted to acylearnitine derivatives, as shown in Figure 24.9. Carnitine acyltransferase I, located on the outer side of the inner mitochondrial membrane, catalyzes the formation of... 

Succinyl-CoA derived from propionyl-CoA can enter the TCA cycle. Oxidation of succinate to oxaloacetate provides a substrate for glucose synthesis. Thus, although the acetate units produced in /3-oxidation cannot be utilized in glu-coneogenesis by animals, the occasional propionate produced from oxidation of odd-carbon fatty acids can be used for sugar synthesis. Alternatively, succinate introduced to the TCA cycle from odd-carbon fatty acid oxidation may be oxidized to COg. However, all of the 4-carbon intermediates in the TCA cycle are regenerated in the cycle and thus should be viewed as catalytic species. Net consumption of succinyl-CoA thus does not occur directly in the TCA cycle. Rather, the succinyl-CoA generated from /3-oxidation of odd-carbon fatty acids must be converted to pyruvate and then to acetyl-CoA (which is completely oxidized in the TCA cycle). To follow this latter route, succinyl-CoA entering the TCA cycle must be first converted to malate in the usual way, and then transported from the mitochondrial matrix to the cytosol, where it is oxida-... 

The acetyl-CoA derived from amino acid degradation is normally insufficient for fatty acid biosynthesis, and the acetyl-CoA produced by pyruvate dehydrogenase and by fatty acid oxidation cannot cross the mitochondrial membrane to participate directly in fatty acid synthesis. Instead, acetyl-CoA is linked with oxaloacetate to form citrate, which is transported from the mitochondrial matrix to the cytosol (Figure 25.1). Here it can be converted back into acetyl-CoA and oxaloacetate by ATP-citrate lyase. In this manner, mitochondrial acetyl-CoA becomes the substrate for cytosolic fatty acid synthesis. (Oxaloacetate returns to the mitochondria in the form of either pyruvate or malate, which is then reconverted to acetyl-CoA and oxaloacetate, respectively.)... 

Margarine is an example of a solid sample where the materials of interest are soluble in one solvent (in this case methanol) whereas the matrix materials, largely triglycerides, are not. As a consequence, the sample preparation procedure is relatively simple. The chromatographic separation is achieved by using the dispersive interactions between the hydrocarbon chains of the fatty acids and the hydrocarbon chains of a reversed phase. 

Transport of Fatty Acid Acyl Groups into the Mitochondrial Matrix 113... 

Figure 3. Mitochondrial fatty acid oxidation. Long-chain fatty acids are converted to their CoA-esters as described in the text, and their fatty-acyl-groups transferred to CoA in the matrix by the concerted action of CPT 1, the acylcarnitine/carnitine exchange carrier and CPT (A) as described in the text. Medium-chain and short-chain fatty acids (Cg or less) diffuse directly into the matrix where they are converted to their acyl-CoA esters by a acyl-CoA synthase. The mechanism of p-oxidation is shown below (B). Each cycle of P-oxidation removes -CH2-CH2- as an acetyl unit until the fatty acids are completely converted to acetyl-CoA. The enzymes catalyzing each stage of P-oxidation have different but overlapping specificities. In muscle mitochondria, most acetyl-CoA is oxidized to CO2 and H2O by the citrate cycle (Figure 4) some is converted to acylcamitine by carnitine acetyltransferase (associated with the inner face of the inner membrane) and exported from the matrix. Some acetyl-CoA (if in excess) is hydrolyzed to acetate and CoASH by acetyl-CoA hydrolase in the matrix. Enzymes ...

Stable under the electron beam. These results assume that in the case of sample preparation according to the procedure (b) or (c) the removal of the fatty acid matrix was not complete. The residual fatty acid molecules resulted in the decrease in sample stability. 

Several enzymes, known collectively as fatty acid oxidase, are found in the mitochondrial matrix or inner membrane adjacent to the respiratory chain. These catalyze the oxidation of acyl-CoA to acetyl-CoA, the system being coupled with the phosphorylation of ADP to ATP (Figure 22-3). 

Besides water, the diet must provide metabolic fuels (mainly carbohydrates and lipids), protein (for growth and turnover of tissue proteins), fiber (for roughage), minerals (elements with specific metabolic functions), and vitamins and essential fatty acids (organic compounds needed in small amounts for essential metabolic and physiologic functions). The polysaccharides, tri-acylglycerols, and proteins that make up the bulk of the diet must be hydrolyzed to their constituent monosaccharides, fatty acids, and amino acids, respectively, before absorption and utilization. Minerals and vitamins must be released from the complex matrix of food before they can be absorbed and utifized. 

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