Serum lipid extracts

Antonis (A3, A5) has presented an automated colorimetric procedure for the determination of acyl esters in serum lipid extracts, based on his manual method (A2). The ester groups are subjected to alkaline hy-droxylaminolysis to form hydroxamic acids, which react with ferric ion to form highly colored chelates. 

The importance of reliable methods is illustrated by the example of determining the concentration of cerebrosides in plasma. By measuring the increase in reducing power of a serum lipid extract before and after hydrolysis which was believed to be due to sugar released from cerebrosides Kirk (1938) found concentrations between 0 and 167 mg per 100 ml. With the use of purification procedures and specific reactions for sugars Svennerholm and Svennerholm (1958) were able to demonstrate a mean cerebroside content of 4.36 0.18 mg per 100 ml of plasma. 

It was demonstrated previously that allicin extracted from garlic had a lipid-lowering effect on long-term feeding to healthy rats. These studies reported a significant decrease in total serum lipids, phospholipids, and cholesterol in the animals fed allicin compared to control animals. 

Qureshi, A.A., Din, Z.Z., Abuirameileh, N., Burger, W.C., Ahmed, Y., and Elson, C.E. 1983. Suppression of avian hepatic lipid metabolism by solvent extracts of garlic impact on serum lipids. J. Nutr. 113, 1746-1755. 

The antiatherosclerotic effect of proanthocyanidin-rich grape seed extracts was examined in cholesterol-fed rabbits. The proanthocyanidin-rich extracts [0.1% and 1% in diets (w/w)] did not change the serum lipid profile, but reduced the level of the cholesteryl ester hydroperoxides (ChE-OOH) induced by 2,2/-azo-bis(2-amidinopropane-dihydrochloride (AAPH), the aortic malonaldehyde (MDA) content and severe atherosclerosis. The immuno-histochemical analysis revealed a decrease in the number of the oxidized LDL-positive macrophage-derived foam cells on the atherosclerotic lesions of the aorta in the rabbits fed the proanthocyanidin-rich extract. When the proanthocyanidin-rich extract was administered orally to the rats, proantho-cyanidin was detected in the plasma. In an in vitro experiment using human plasma, the addition of the proanthocyanidin-rich extract to the plasma inhibited the oxidation of cholesteryl linoleate in the LDL, but not in the LDL isolated after the plasma and the extract were incubated in advance. From these results, proanthocyanidins of the major polyphenols in red wine might trap ROSs in the plasma and interstitial fluid of the arterial wall, and consequently display antiatherosclerotic activity by inhibiting the oxidation of the LDL [92]. 

The direct measurement of plasma phylloquinone is probably the best indicator of vitamin K status and has been shown to correlate well with intalce. HPLC methods have been reviewed, and typically require 0.5 to 2,0mL of serum or plasma. Protein precipitation and lipid extraction (often into hexane), followed by solvent evaporation preparative HPLC (to isolate vitamin K from other lipids) reevaporation of the vitamin K-rich fraction dilution in the mobile phase and further HPLC, with either electrochemical or fluorometric detection often after postcolumn reduction, are required. Typical between-batch imprecision values are coefficient of variation (CV)s of 11% to 18% with limits of detection of lower than 50pmol/L. An external quality assessment scheme (EQAS) is avafiable in the UK. 

Antonis (A3) has estimated phospholipids by a procedure for determining fatty acids. This technique requires a total serum extract and a phospholipid-free extract for the measurement of both total and free fatty acids, the difference between them being a measure of the phospholipid content. Free fatty acids (A4) are determined on a phospholipid-free extract by a procedure based on partitioning the fatty acids as copper soaps into chloroform, and subsequent photometric determination of the copper with diethyldithiocarbamate. Phospholipids, as well as the free fatty acids present in the total lipid-extract, are measured by the same method, since they also form a complex with copper that is soluble in chloroform. A criticism of this technique is that equal response is not given by dipalmitoyl lecithin, dipalmitoyl cephalin, or beef brain sphingomyelin. 

Current interest in lipid metabolism and the relationship of serum triglycerides to atherosclerotic heart disease has also created a need for an automated direct triglyceride procedure. Manual techniques can be divided into three stages extraction of serum lipids and removal of phospholipids, saponification of triglycerides to glycerol and its oxidation to formaldehyde, and the colorimetric measurement of formaldehyde. For the present, the first step has not lent itself to automation. Currently, two semiautomated triglyceride procedures, which pertain to the last two stages of the manual technique, have been published. 

Figure 3.1 IR spectra from 950 to 1800 and from 2750 to 3600cm (A-E) and Raman spectra from 400 to 1800cm (F-J) of the protein concanavalin A (A, F), the protein bovine serum albumin (B, G), DNAfrom calf thymus (C, H), a lipid extract (D, I) and cholesterol (E, J). The wavenumber scale of the IR spectra in the interval 2750-3600cm is twofold compressed.

Yamaguchi Y, Kagota S, Nakamura K, Shinozuka K, Kunitomo M. Inhibitory effects of water extracts from fruiting bodies of cultured Cordyceps sinensis on raised serum lipid peroxide levels and aortic cholesterol deposition in atherosclerotic mice. Phytother Res 14(8) 650-652, 2000b. 

One milhliter serum, or a suitable aliquot of lipoprotein solution, was extracted with chloroform-methanol (2 1) by a modification of the procedure of Sperry and Brand (22). The total lipid extract was freed of solvents by evaporation under nitrogen and weighed on a semimicro balance. Infrared analysis of the total lipid by the method of Freeman... 

Many investigators have shown that nonspecific reagents as diverse as calcium phosphate gel, EDTA, histidine, and nonspecific proteins activate succinoxidase preparations in otherwise unfavorable environments. The mechanism of the activation is not established, but it has been repeatedly suggested that the activators in some manner influence the steric orientation of components of the particulate succinoxidase. Another component of electron-transport systems has been implicated by Nason and Lehman. DPNH oxidation by a particulate fraction of rat muscle was found to be decreased by extraction of 10 per cent of the lipid with isooctane the activity was restored by addition of a-tocopherol (vitamin E) or the lipid extracted from muscle or bovine serum albumin. These lipids are able to reverse the inhibition of cytochrome c reduction caused by antimycin A. It has not been determined whether the tocoph-... 

Numerous extraction procedures are available for lipids, and this subject has been reviewed by Phillips and Privett (1979), Christie (1984), and Fried (1993). Of all extraction procedures, that of Folch et al. (1957) is used most frequently. This procedure involves extracting fresh animal or plant material with chloroform-methanol (2 1), usually in a volume ratio of 20 parts of the solvent to 1 part tissue or fluid. There are many variations of the Folch et al. (1957) procedure, and for descriptions of these variations, see Christie (1982, 1984). Experiment 4 involves the use of the Folch et al. (1957) procedure on 100 mg of animal tissue and 100 pi of serum. The Folch et al. (1957) procedure extracts nonlipid material along with lipids. Nonlipids are usually removed by an aqueous wash with either water or a dilute salt solution. Phillips and Privett (1979) devised an extraction procedure for brain tissue in which nonlipid material is first removed with dilute acetic acid. The brain tissue is then treated with chloroform-methanol essentially as described in Folch et al. (1957) to get a relatively pure lipid fraction. Because so many different lipid extraction procedures have been used prior to TLC analysis, different results for a given separation may reflect technique differences rather than acmal differences in lipid constituents of a sample. 

Resolution of hydroxylated metabolites of vitamin D and their isolation from total lipid extracts (removal of interferences) as part of GC or radioligand assays of these metabolites in human plasma or serum... 

Daily administration of curcuminoids (0.5 g) to healthy human volunteers produced a 33% reduction in blood lipid peroxide levels (Soni and Kuttan 1992). This was accompanied by an increase in HDL cholesterol and a decrease in total serum cholesterol as a result of curcumin administration (500 mg/day for 7 days) (Quiles etal. 2002). The reduction in serum lipid peroxides and cholesterol suggests the potential of curcumin against arterial diseases. Supplementation with turmeric extract reduced oxidative stress and attenuated the development of atherosclerotic fatty streaks in rabbits fed on a high-cholesterol diet (Quiles etal. 2002). 

Avigan, j. Modification of human serum lipoprotein fractions by lipide extraction. J. biol. Chem. 226, 957—964 (1957). 

For determination of total fatty acids lipids must be split by alkaline hydrolysis. Hydrolysis of lipids from purified lipid extracts is preferable to hydrolysis of tissues or serum directly. After extraction of unsaponifiable matter fatty acids are recovered from their soaps by treatment with acids and can subsequently be assayed by titration. During hydrolysis high concentrations of alkali must be avoided to prevent isomerization of unsaturated fatty acids. A suitable method for quantitation of plasma total fatty acids was described by Albrink (1959/1960). 

Gjone, E., j. F. Berry, and D. A. Turner The isolation and identification of lysolecithin from lipid extracts of normal human serum. J. Lipid Res. 1, 66 (1959/60). 

A variety of interfering constituents were removed by filtering the lipid extracted serum through an ion exchange bed consisting of a Dowex-50 (acidic) and Biorex-5 (basic) resins placed into a small Pasteur pipette, one on top of the other (acidic on top). 

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