ISSN 2074-9414 (Print),
ISSN 2313-1748 (Online)

Comprehensive Methodological Approach to Determining Lipids in Clams

Marine bivalves are rich in polyunsaturated fatty acids. Cerastoderma glaucum is a potentially commercial sea cockle that inhabits the Mediterranean and the Black Seas. This bivalve mollusk contains omega-3, omega-6, and omega-9 fatty acids. However, its lipid composition remains understudied. When determining total lipids, their classes, and fatty acid composition, standard methods often have to be adapted to the object in hand and tools available. The research objective was to develop a complex lipid analysis method for aquatic organisms.
The study featured total soft tissues of C. glaucum harvested from the pseudolittoral zone on the sand and silt soil of the Sevastopol coast of the Black Sea. The chromatographic methods made it possible to identify total lipids and classify them into phospholipids, monoglycerides, diglycerides, sterols, and triacylglycerols. The subsequent densimetric determination involved a flatbed scanner and the TLC Manager software. The fatty acid composition for total lipids was studied using the chromatography-mass spectrometric method.
The existing methods in lipidology were adapted for determining total lipids, their classes, and the fatty acid composition of total lipids in C. glaucum. The article introduces a detailed description of the method for determining total lipids, as well as of how to prepare equipment and reagents to classify common lipids using multidimensional thin layer chromatography. It also contains an authentic scheme of chromatographic baths for stepwise separation, densitometric measurements, and examples of fatty acid chromatograms and mass spectra. The new sample preparation method for determining fatty acids in total lipids by gas chromatography demonstrated a minimal loss in native structure and proved to be less aggressive than standard methods of sample derivatization.
The new method for lipid analysis of clam tissues appeared to be economical, less time-consuming, and reagent-intensive. It can be recommended for small laboratories engaged in bioenergetics or comparative analyzes of aquatic organisms.
Total lipids, lipid classes, thin layer chromatography, densitogram, chromatogram, fatty acids, gas chromatography/mass spectrometry, shellfish
The work part of State Assignment to A.O. Kovalevsky Institute of Biology of the Southern Seas of RAS (IBSS), research topic No. 121041400077-1: Functional, metabolic, and toxicological aspects of hydrobionts and their populations in biotopes with different physical and chemical modes. The research was conducted on the premises of Core Facilities Centre of Molecular Matter Structure, Sevastopol State University.
  1. Parnova RG. Lipids as signaling platforms and signaling molecules. Journal of Evolutionary Biochemistry and Physiology. 2020;56(7):824–825. (In Russ.).
  2. Lisovaya EV, Viktorova EP, Sverdlichenko AV, Zhane MR. Effect of ultrasonic exposure on the efficiency of de-oiling fluid lecithins. Food Processing: Techniques and Technology. 2023;53(3):445–454. (In Russ.).
  3. Bakaytis VI, Golub OV, Miller YuYu. Fresh and processed wild Cantharellus cibarius L. growing in West Siberia: food value. Foods and Raw Materials. 2021;9(2):234–243.
  4. Fokina NN, Ruokolainen TR, Nemova NN, Martynova DM, Sukhotin AA. Fatty acids distribution in seston, tissues, and faecal pellets of blue mussels Mytilus edulis L. Doklady Biochemistry and Biophysics. 2020;495(1):624–631. (In Russ.).
  5. Mahony KE, Egerton S, Lynch SA, Blanchet H, Goedknegt MA, Groves E, et al. Drivers of growth in a keystone fished species along the European Atlantic coast: The common cockle Cerastoderma edule. Journal of Sea Research. 2022;179.
  6. Carss DN, Brito AC, Chainho P, Ciutat A, de Montaudouin X, Otero RMF, et al. Ecosystem services provided by a non-cultured shellfish species: The common cockle Cerastoderma edule. Marine Environmental Research. 2020;158.
  7. Kopiy VG, Bondarenko LV. Atlas of the inhabitants of the pseudo-littoral of the Sea of Azov – Black Sea coast of Crimea. Sevastopol: IBSS; 2020. 120 p. (In Russ.).
  8. Bejaoui S, Chaâbane M, Fouzai C, Chetoui I, Chalbi E, Nechi S, et al. Exploring the impacts of mercury chloride exposure on fatty acids profile, oxidative stress response and histomorphological aspect of Cerastoderma edule detoxifying organs. Ecological Indicators. 2020;118.
  9. Folch J, Lees M, Sloane Stanley CH. A simple method for the isolation and purification of total lipids from animal tissue. Journal of Biological Chemistry. 1957;226(1):497–509.
  10. Mikryakov DV, Mikryakov VR, Gordeev II. Lipid composition and oxidation processes in the blood and internal organs of the Antarctic toothfish Dissostichus mawsoni Norman, 1937 (Nototheniidae). Russian Journal of Marine Biology. 2021;47(3):160–166. (In Russ.).
  11. Kopytov YuP. A new variant of thin-layer chromatography for lipids and hydrocarbons. Ekologiya moray. 1983;13:76–80. (In Russ.).
  12. Murzina SA, Pekkoeva SN, Churova MV, Nefedova ZA, Filippova KA, Falk-Petersen S, et al. Daily dynamics of lipids and fatty acids and the activity of enzymes of the energy and carbohydrate metabolism in young fish of the daubed shanny Leptoclinus maculatus (Fries, 1838) at different developmental stages during polar night. Ontogenez. 2020;51(2):143–153. (In Russ.).
  13. Merdzhanova A, Panayotova V, Dobreva D, Bratoeva K, Makedonski L. Health-beneficial properties of Black Sea shellfish for the Bulgarian consumers. Proceedings of the Nutrition Society. 2020;79.
  14. Eibler D, Krüger S, Skírnisson K, Vetter W. Combined thin layer chromatography and gas chromatography withmass spectrometric analysis of lipid classes and fatty acids inmalnourished polar bears (Ursus maritimus) which swam to Iceland. Journal of Chromatography B. 2017;1046:138–146.
  15. Voronin AV. The densitometric quantitation of some drugs in whole blood. Bashkortostan Medical Journal. 2018;13(2):40–43. (In Russ.).
  16. Renkevich AYu, Kulikov AYu. Developing and validating a quantitative determination method for 4-aminobutanoic acid in sodium alendronate tablets using micellar thin layer chromatography. Methods and Objects of Chemical Analysis. 2013;8(4):199–206. (In Russ.).
  17. Zakharenko AM, Kirichenko KYu, Vakhniuk IA, Golokhvast KS. Supercritical extraction technology of obtaining polyunsaturated acids from starfish (Lysastrosoma anthosticta Fisher, 1922). Food Processing: Techniques and Technology. 2021;51(4):753–758.
  18. Chen C, Li R, Wu H. Recent progress in the analysis of unsaturated fatty acids in biological samples by chemical derivatization-based chromatography-mass spectrometry methods. Journal of Chromatography B. 2023;1215.
  19. Juarez M, Polvillo O, Contò M, Ficco A, Ballico S, Failla S. Comparison of four extraction/methylation analytical methods to measure fatty acid composition by gas chromatography in meat. Journal of Chromatography A. 2008;1190(1–2):327–332.
  20. Irwinda R, Hiksas R, Siregar AA, Saroyo YB, Wibowo N. Long-chain polyunsaturated fatty acid (LC-PUFA) status in severe preeclampsia and preterm birth: A cross sectional study. Scientific Reports. 2021;11.
  21. Qiu J, Ji Y, Fang Y, Zhao M, Wang S, Ai Q, et al. Response of fatty acids and lipid metabolism enzymes during accumulation, depuration and esterification of diarrhetic shellfish toxins in mussels (Mytilus galloprovincialis). Ecotoxicology and Environmental Safety. 2020;206.
  22. Panayotova V, Merdzhanova A, Stancheva R, Dobreva DA, Peycheva K, Makedonski L. Farmed mussels (Mytilus galloprovincialis) from the Black Sea reveal seasonal differences in their neutral and polar lipid fatty acids profile. Regional Studies in Marine Science. 2021;44.
  23. Hernando M, de Troch M, de la Rosa F, Giannuzzi L. Fatty acid response of the invasive bivalve Limnoperna fortuneifed with Microcystis aeruginosa exposed to high temperature. Comparative Biochemistry and Physiology Part C: Toxicology and Pharmacology. 2021;240.
  24. Fiorini R, Ventrella V, Trombetti F, Fabbri M, Pagliarani A, Nesci S. Lipid-protein interactions in mitochondrial membranes from bivalve mollusks: Molecular strategies in different species. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology. 2019;227:12–20.
  25. Anganea M, Gupta S, Fletcher GC, Summers G, Hedderley DI, Quek SY. Effect of air blast freezing and frozen storage on Escherichia coli survival, n-3 polyunsaturated fatty acid concentration and microstructure of Greenshell™ mussels. Food Control. 2020;115.
  26. Peñuela-Jiménez JH, Guevara M, Saucedo PE, Núñez MP, Troccoli L, Freites L. Influence of contrasting environmental variables on the fatty acid profile of the winged oyster Pteria colymbus. Regional Studies in Marine Science. 2021;41.
  27. Chaâbanea M, Bejaoui S, Trabelsi W, Telahigue K, Chetoui I, Chalghaf M, et al. The potential toxic effects of hexavalent chromium on oxidative stress biomarkers and fatty acids profile in soft tissues of Venus verrucosa. Ecotoxicology and Environmental Safety. 2020;196.
  28. Almutairi AW, El-Sayed AE-KB, Reda MM. Combined effect of salinity and pH on lipid content and fatty acid composition of Tisochrysis lutea. Saudi Journal of Biological Sciences. 2020;27(12):3553–3558.
How to quote?
Borodina AV, Veliaev YuO, Osokin AR. Comprehensive Methodological Approach to Determining Lipids in Clams. Food Processing: Techniques and Technology. 2023;53(4):662–671. (In Russ.). 
About journal