Laboratory for lipid analysis and determination of lipid status

Objectives:

  • To determine lipid status – total cholesterol, HDL- and LDL-cholesterol and triglyceride level and to assess the risk of cardiovascular disease (CVD).
  • To determine the fatty acids profile in plasma and/or erythrocyte phospholipids and to investigate relations between fatty acid profiles and various disease including CVD, cancer, obesity, metabolic syndrome and chronic inflammation.

Methods:

1) Total lipids extraction from:
Serum – Red blood cells – Liver – Heart

Total lipid extracts are prepared using organic solvent mixture chloroform-methanol according to the method by Folch et al. (1957) and the modifications of this method (Harth et al, 1978)

2) TLC lipids separation
– One-dimensional thin-layer chromatography (TLC) on silica gel G layer in a neutral solvent system n-hexane: diethyl ether: acetic acid (87:12:1, v / v / v) for the separation of lipid classes: phospholipids, diacylglycerols, free fatty acids, cholesterol , triglycerides and cholesterol esters
TLC separation of phospholipid fractions on silica gel H with the addition of florisil:
a) one-dimensional chromatography, using alkaline solvent mixture chloroform-methanol-20% ammonium hydroxide (65:30:5, v / v / v)
b) two-dimensional chromatography, for the second dimension the solvent mixture is chloroform-acetone-methanol-acetic acid-water (70:17,5:12,5:10:4,4 v / v / v / v / v)
From the total lipid extract we can separate the following fractions by TLC: lysolecithin, phosphatidylcholine, sphingomyelins, phosphatidylethanolamine, diphosphatidylglicerol, phosphatidylinozitola, phosphatidylserine, phosphatidic acid.

3) Fatty acid methylation
Derivatization of fatty acid methyl esters (FAMEs) affects the quality of the chromatographic separation by gas-liquid chromatography since FAMEs are more volatile than the fatty acids. FAMEs are prepared using methanol at 85 ˚C in the presence of sodium hydroxide in the first step, and sulfuric acid in the second step.

4) Separations of FAMEs by gas liquid chromatography (GLC)
For analysis of FAMEs we use the gas chromatograph Shimadzu 2014 (Kyoto, Japan)
equipped with a capillary column (Rtx 2330, RESTEK, Bellafonte, Pa., USA). The individual FA methyl esters are identified from the retention times of authentic standard mixtures (Sigma Chemical Co., St. Louis, Mo., USA) and (or) PUFA-2 standard mixture (Supelco, Inc., Belleforte, Pa., USA). The results are expressed as the percentage of total identified FA.

New Methods:

1) Determination of trans fatty acids in foods
Trans fatty acids (TFA) are rarely present in nature (naturally occurring ruminant TFA) but arise in the production and processing of food (TFA of industrial origine – iTFA). Because of their adverse effects on blood lipoprotein profile, and cardiovascular disease risk, dietary intake is recommended to be less than 1 g/day. In Canada and the United States the label on products containing TFA is obligatory, and many European countries have imposed legislative limits to the presence of TFA in food. One of the validate techniques currently used for measuring TFA in food is gas chromatography. The introduction of new methods for identification and quantification of TFA would enable us to achieve cooperation with industry, but it would be useful for scientific research as well. We could study the influence of TFA intake on development of different pathologies (cardiovascular disease, insulin resistance, liver disease, cancer …).

2) A new method for the separation of lipid classes
The separation of lipid classes using SPE chromatography would enable us to obtain pure fractions of triglycerides and cholesterol esters, whose fatty acid composition could be determined by the gas-liquid chromatography. SPE separation is a quick and simple method, which has been increasingly used. Our preliminary experiments showed satisfactory separation of lipid classes.

Scientific results:

Our results showed significantly altered fatty acid profile in patients with CVD, cancer, diabetes and metabolic syndrome. Furthermore, abnormal profiles were also found in pregnant women with preeclampsia and in babies small for gestational age. In addition, different status of fatty acid was detected in elite athletes, but the changes are related to type of sport. These results have been published in a number of international journals.

Projects:

  • Biological effects, nutritional intake and status of folate and polyunsaturated fatty acids: The improvement of nutrition in Serbia”, 2011-2014, III 41030, Ministry of Science, Republic of Serbia: FOLOMEGA
  • Development of new therapeutic procedures in prevention and cure of liver disease – the role and mechanism of polyunsaturated fatty acids, 2006-2010, No 145071.

GUIDE TO HYPERLIPIDEMIA DIAGNOSIS


Dr Maria Glibetic

SOULFOOD Serbia

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