Folch Method Research Articles

Modeling the processes of transendothelial transport of LDL and macrophage migration

Atherosclerosis is a multifactorial process involving various pathological mechanisms: inflammation, lipoprotein modification, cholesterol accumulation, endothelial dysfunction, oxidative stress and formation of atherosclerotic plaque. The main participants in the development of this disease are endothelial cells, leukocytes and smooth muscle cells of intima. The adult endothelium contains heterogeneous cells, including typical endothelial cells (TEC) and giant multinucleated EC variants (MVEC). The process of passing molecules is called transendothelial transport (TET). The purpose of this work is to model the processes of TET of low-density lipoproteins (LDL) and monocyte migration using various EC variants. In the study, a human EC line (EA.hy926) was used. Multicore variants of EC (MVEC) were obtained by treating EC with a 50% solution of polyethylene glycol 6000. To study LDL transcytosis and monocyte migration, tablets with inserts (0.4 or 3.0 micron pore diameter, respectively) forming a two-level system were used. The LDL transmission rate was assessed by measuring the amount of cholesterol in the upper and lower chambers every 2, 5 and 24 hours. A modified Folch method was used to measure the amount of cholesterol. The cholesterol content was adjusted according to the total protein level in the well, measured by the Lowry method. The migration of macrophages was estimated by direct counting of cells. The content of internalized cholesterol in the MVEC was statistically significantly higher than in the EC. The rate of LDL transport did not differ. The rate of passage of the endothelial barrier formed by MVEC by macrophages was higher at points 2 and 5 hours. After 24 hours the number of migrated cells did not differ. The rate of transendothelial transport for typical and multinucleated variants of EC did not statistically differ. Although the presence of MBEC does not affect the transport of lipoproteins into the subendothelial layer, the fact that multinucleated cells accumulate lipid droplets more actively than typical ECS may indicate an important role in the pathogenesis of atherosclerosis. The rate of macrophage migration after 2 and 5 hours was higher for MVEC than for TEC, whereas immature macrophages did not migrate through the endothelial barrier for 24 hours.

The Influence of Feeding Type on Quality and Nutritional Characteristics of Pork Fat

The purpose of the study was to analyze the effect of feeding type on selected quality parameters and the nutritional characteristics of pork fat. Fats were extracted by using the modified Folch method from the groin, jowl and trimming shoulder of pigs fed two types of diets: liquid and dry. The peroxide values and fatty acid profiles, as well as their distribution, in the triacylglycerol molecules were determined. Additionally, nutritional indexes and thermal properties, including the oxidative stability of pork fat, were assessed. Among the tested fats, the groin fat was characterized by the best oxidative stability which did not depend on the diet type used. In the case of jowl and trimming shoulder, a higher share of monounsaturated fatty acids was determined in fats of pigs fed a liquid diet, whereas in the groin, jowl and trimming shoulder fats of pigs fed dry feed, a higher share of polyunsaturated fatty acids was found. Fats extracted from the groin, jowl and trimming shoulder of pigs fed a liquid diet showed a more favorable ratio of n-6 to n-3 acids. Despite the different feeding systems used, tested fats were characterized by a similar distribution of fatty acids in triacylglycerol molecules with a positive effect on fat digestibility. It is worth emphasizing that especially in the case of trimming shoulder fat, the influence of a diet used in pigs’ nutrition on quality parameters was noticeable. The trimming shoulder fat of pigs fed a liquid diet was characterized by a longer induction time of oxidation, contained less saturated fatty acids and presented a more favorable fatty acid profile with a beneficial distribution of fatty acids in triacylglycerol molecules.

Microalgae as a New Source of Oxylipins: A Comprehensive LC-MS-Based Analysis Using Conventional and Green Extraction Methods.

Microalgae are promising sources of essential lipids, including omega-3 and omega-6 polyunsaturated fatty acids (n-3 and n-6 PUFA) and novel lipid metabolites like oxylipins. However, limited data exist on the oxylipin profile, its characterization, and the potential impact of the extraction process on these metabolites in microalgae. Thus, our study aimed to investigate the fatty acid and oxylipin profile of four microalgal species of interest (Microchloropsis gaditana, Tisochrysis lutea, Phaeodactylum tricornutum, and Porphyridium cruentum) while also examining the impact of the extraction method, with a focus on developing a greener process using ultrasound-assisted extraction (UAE) and ethanol. The UAE method showed similar oxylipin profiles, generally yielding concentrations comparable to those of the conventional Folch method. In total, 68 oxylipins derived from n-3 and n-6 PUFA were detected, with the highest concentrations of n-3 oxylipins found in P. tricornutum and T. lutea and of n-6 oxylipins in P. cruentum. This study provides the most extensive oxylipin characterization of these microalgae species to date, offering insights into alternative extraction methods and opening new avenues for further investigation of the significance of oxylipins in microalgae.

Fatty acids and chlorogenic acid content in Plectranthus edulis root tubers.

This study quantified the fatty acid profile and total chlorogenic acid content of various Ethiopian cultivars of the Plectranthus edulis tuber, traditionally known as 'Agew Dinich'. Lipid extraction utilized the Folch method and the acid-catalyzed derivatization method to derivatize the fatty acids into fatty acid methyl ester (FAME) were used. Whereas maceration was used to extract chlorogenic acid from the fresh and freeze- dried tuber samples. Analysis revealed a total of thirteen fatty acids in all P. edulis samples, with nine classified as saturated and four as unsaturated. Palmitic acid was the most abundant fatty acid in P. edulis and accounted for 40.57%-50.21% of the total fatty acid content. The second and third most abundant fatty acids in the P. edulis sample were stearic and linoleic acids, which accounted for 8.38%-12.92% and 8.12%-11.28%, respectively. We reported chlorogenic acid for the first time in this potato species and found it to contain a concentration of 211± 4.2-300±24.7 mg/100g of dry weight basis when the determination was made using fresh samples. On the other hand, these samples yielded a chlorogenic acid concentration ranging from 115 ±8.6 mg/100g-175±3.9 mg/100g of freeze-dried powder samples. These findings suggest that P. edulis tubers could represent a significant dietary source of both chlorogenic acid and fatty acids.

Lipidomics reveals ceramide biomarkers for detecting central precocious puberty in girls

BackgroundPubertal timing is modulated by complex interactions between the pituitary and gonadal sex steroid hormones. Evidence indicates that sphingolipids are involved in the biosynthesis of steroid hormones at multiple levels. MethodThis study recruited adolescent female patients from pubertal and pediatric endocrine clinics in Northern and Southern Taiwan from the Taiwan Puberty Longitudinal Study. A total of 112 plasma samples (22 healthy control, 29 peripheral precocious puberty (PPP), and 61 CPP samples) were collected. We extracted lipids from the plasma samples using the modified Folch method. The un-targeted ultrahigh-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) was employed for the lipid analysis. ResultsWe identified sphingolipid-linked metabolites, including Cer(18:0/15:0), Cer(18:1/16:0), and Cer(18:1/26:0) as candidate biomarkers for distinguishing girls with CPP from the control group by using an excellent discrimination model (AUC = 0.964). Moreover, Cer(18:0/22:0) and Cer(d18:0/18:1) were identified as potential biomarkers of PPP, with an AUC value of 0.938. Furthermore, CerP(18:1/18:0) was identified as the sole candidate biomarker capable of differentiating CPP from PPP. ConclusionsThe biomarkers identified in this study can facilitate the accurate detection of CPP in girls, provide insights into lipid-linked pathophysiology, and present a novel method of monitoring the progression of this disorder.

Assessment of Heavy Metal Contamination and Potential Health Risk of Selected Fish Species in Dhaka District

Fish is a rich source of quality protein and fatty acids. Bangladesh's average daily fish consumption is 67.8 grams per people. Fish species are also prone to heavy metal contamination from their surrounding environment. The main objective of this research was to assess the degree of risk associated with the consumption of various fish species in Dhaka, Bangladesh. Three fish species—Tilapia (Oreochromis mossambicus), Shorputi (Systomus sarana), and Mola (Amblypharyngodon mola)—from three local markets in Savar Union, Dhaka, Bangladesh, were selected for analysis. The Kjeldahl technique, Folch method, and several formulae were used to determine the protein, fat, ash, and moisture content for the proximate analysis. The concentration of heavy metals was determined using Atomic Absorption Spectrophotometry (AAS). The estimated daily intake (EDI), target hazard quotient (THQ), total target hazard quotient (TTHQ), hazard index (HI), and cancer risk (CR) were used to evaluate the danger to human health. Tilapia had the highest moisture and lipid content, whereas Shorputi and Mola had the highest protein and ash contents, ranging from 20.77% to 18.16%, respectively. The mean concentration of heavy metals in fish was determined to be Pb>Ni>Cr>Cd in this study. Every fish species had extremely high levels of lead (Pb), with the Shorputi fish having the highest levels. Fish from the Shorputi species had the highest EDI value. Pb>Ni>Cr>Cd was the trace element EDI for adults. Among all species, only Pd had a THQ value higher than 1. The CR value across all species were within E-3 and E-6 range, while the HI value was >1. In conclusion, the highest average Pb concentrations were detected in Shorputi, while the highest amounts of Cd, Cr, and Ni were observed in Tilapia. The findings of this study recommended that the Bangladeshi government regularly assess the levels of dangerous heavy metal and metalloid contamination in the daily meals of its citizens in order to enforce regulatory limits and determine the likelihood of long-term exposure. Bioresearch Commu. 10(2): 1524-1531, 2024 (July)

Зміни жирнокислотного складу проростків кукурудзи за впливу мікробіологічного комплексу

Orientation of agricultural production to ecological expediency leads to interest in the use of microbial preparations that are able to increase the resistance of plants to negative environmental factors and activate germination processes. This study was conducted with the aim to investigate the influence of a microbiological complex on the sowing properties of seeds and the fatty acid composition of seedlings of different breeding samples of corn. Methods. Corn seeds of two lines 1-Ak026 and 2-Ak033 and their simple interline hybrid, which is the parent component of the three-line hybrid NUBiSe included in the State Register of plant varieties suitable for distribution in Ukraine, were used for the research. Seed inoculation was carried out with a microbial complex, which contained biological preparations Sporazyn, Avercom, and Phytovit. Seedlings from the seeds of the corresponding corn samples not treated with the microbial complex served as controls. The fatty acid composition of lipids was determined in the biological material of corn seedlings of different variants of the experiment. The Folch method was used to extract fats from plant material. Hydrolysis and methylation of fatty acids of lipids obtained from corn seedlings was carried out according to DSTU ISO 5509-2002. Fatty acids were detected and identified by gas chromatography. The quantity of fatty acids was calculated by the method of internal normalization in percentages. Results. The influence of the microbiological complex on the quantitative and qualitative composition of fatty acids of the lipid complex of corn seedlings was established, which depended on the breeding sample. In the process of research, 22 fatty acids were detected and quantitatively identified in corn seedlings. In the seedlings of lines 1-Ak033 and 2-Ak033, 20 and 19 of them were found, respectively, and in the hybrid – 18. The highest percentage content (more than 90 %) was revealed for palmitic (C16:0), oleic (C18:1n9c), and linoleic (C18:2ω6c) fatty acids. The use of the microbial complex made it possible to detect the fatty acids in line 1 and the hybrid that were not detected in the samples without inoculation. Conclusions. The study of the fatty acid composition of the microbial preparation Sporazyn revealed the highest content of unsaturated fatty acids (67.47 %) and the ratio of saturated to unsaturated (0.48). The use of microbiological preparations on corn seeds increased the activity of the germination processes, which led to the improvement of sowing properties and changes in the fatty acid composition of the lipid complex of seedlings of various breeding forms of Zea mays.

Unlocking functional lipid ingredients from algae by food-grade biosolvents and ultrasound-assisted extraction for nutritional applications

Algae are a sustainable source of healthy and functional lipids. The production of extracts suitable for food and nutraceutical industries requires food-grade solvents, which may have low lipid extraction yield and/or purity. This work aimed to assess the efficiency of biosolvents ethanol (E) and ethyl acetate (EA) in obtaining food-grade lipid ingredients from an algae blend, Fucus vesiculosus, Ulva sp., and Chlorella vulgaris, in comparison with the Folch method. Ultrasound (UAE) was assayed to enhance the biosolvent extraction. The results showed that the different extraction approaches can significantly influence the extraction yield, lipid purity, and lipid composition, including fatty acid (FA) content. EA and EA + UAE produced high lipid purity (>74%) extracts with high polyunsaturated FA (≤77.5%) and n-3 FA (≤74.4%) contents, despite the lower extraction yield. E + UAE may be used to obtain lipid-rich ingredients from algae blend (57%) and C. vulgaris (91%). All extracts showed anti-inflammatory and antioxidant properties. This study provides relevant information for the production of food-grade lipid ingredients from algae.

Modulación por Passiflora edulisde triglicéridos y colesterol intracelulares en líneas celulares de cáncer colorrectal SW480 y sus derivadas metastásicas (SW620)

Cancer cells modify lipid metabolism to proliferate, Passiflora edulis (P. edulis) fruit juice (ZuFru) has antitumor activity, but whether a mechanism is through modulation of cell lipids is unknown. To establish if ZuFru modifies cholesterol and triglycerides in SW480 and SW620. ZuFru composition was studied by phytochemical march; antiproliferative activity by sulforhodamine B, cholesterol, and triglycerides by Folch method. Zufru contains anthocyanins, flavonoids, alkaloids, and tannins. Cell lines showed differences in their growth rate (p=0.049). At 39.6 μg/mL of ZuFru, cell viability was decreased: SW480 (45.6%) and SW620 (45.1%). In SW480, cholesterol (44.6%) and triglycerides (46.5%) decreased; In SW620, cholesterol decreased 14.8% and triglycerides increased 7%,with significant differences for both lines. Antiproliferative activity of ZuFru could be associated with the inhibition of intracellular biosynthesis of cholesterol and triglycerides in SW480. Action mechanisms need to be further investigated

RAPID DETECTION OF RAT MEAT ADULTERATION IN BEEF SAUSAGES USING FTIR‑ATR SPECTROSCOPY AND CHEMOMETRICS FOR HALAL AUTHENTICATION

Objective: The objective of this study was to employ Fourier Transform Infrared-Attenuated Total Reflectance (FTIR‑ATR) spectroscopy in combination with chemometrics for the analysis of rat meat adulteration in beef sausages.
 Methods: Lipid components in sausages were extracted using three extraction methods, namely Bligh and Dyer, Folch, and Soxhlet methods. The lipid components extracted were then analysed using FTIR‑ATR spectroscopy, and their spectra obtained were used as variables during chemometrics modeling. Samples were prepared by mixing beef with adulterant of rat meat in the concentration range of 0-100% of rat meat. Each sample was scanned using FTIR-Attenuated Total Reflectance (ATR) spectroscopy in three replicates at 4000-650 cm-1 wavenumber region.
 Results: The absorbance values at wavenumbers regions of 3100-700 cm-1 were used to discriminate lipid components extracted by the Bligh Dyer, Folch, and Soxhlet Method with an accuracy level of 100%. The prediction of rat sausages was successfully determined using multivariate calibrations of Partial Least Square (PLS) and Principle Component Regression (PCR) using optimised conditions.
 Conclusion: FTIR-ATR spectroscopy coupled with chemometrics is a rapid and accurate method for detecting and quantifying rat meat in beef sausages for halal authentication.

Effects of lipid extraction on stable isotope ratios of carbon and nitrogen in muscles of freshwater fish

ABSTRACT The extraction of lipids by the Folch method from the muscles of all the fish studied led to statistically significant differences in the values of δ 15N. At the same time, lipid extraction led to a statistically significant increase in δ 13C in pike and roach, and to a statistically insignificant decrease in δ 13C in perch and bream. Thus, lipid extraction cannot serve as a universal method of sample preparation for the analysis of the isotopic composition of carbon (13C/12C) and nitrogen (15N/14N) in fish muscles. The differences between the δ 13C values in the samples before and after lipid extraction were statistically investigated by different models. It is shown that mathematical correction method models can be used, but the results are depending on the fish types.

Development of FTIR Spectroscopy and multivariate calibration for authentication beef meatballs from pork meat

In several countries, especially Indonesia, meatballs are one of the favorite foods made from meat. The high price of beef is a reason for adulterated beef meat with pork to gain a significant profit. All food products must not contain pig derivatives such as pork in Muslim societies. This study aimed to develop Fourier Transform Infrared (FTIR) spectroscopy in combination with linear discriminant analysis (LDA) for classifying pork in beef meatballs and multivariate calibration for qualitative and quantitative analysis methods using partial least square regression (PLSR) and principal component regression (PCR). The lipid component from meatballs containing pork and beef in different concentrations was obtained by employing three lipid extraction methods, namely BlighDyer, Folch, and Soxhlet methods, and subjected to attenuated total reflectance (ATR) spectral measured at wavenumbers of 4000-650 cm-1 . LDA at 3800-800 cm-1 was able to differentiate between beef meatballs and meatballs containing pork meat (PM). Additionally, PLSR using second derivative FTIR spectra at wavenumbers of 3100-900 cm-1 delivered great region for predicting pork levels in beef meatballs extracted using Folch methods with coefficient determination (R2 ) values was > 0.99 and 0.02% for root mean square error of calibration (RSMEC) and root mean square error of prediction (RSMEP) values, respectively. PCR using absorbance values of 1st derivative FTIR spectra at wavenumbers 1400-800 cm-1 for Bligh-Dyer method and 3800-800 cm-1 for Soxhlet was offered the R2 value was> 0.99 with low RSMEC and RSMEP values for prediction of pork fat in beef meatballs, respectively. It can be concluded that FTIR spectroscopy on the Savitzy-Golay derivatization method in combination with chemometrics was an accurate and quick approach for authenticating pork adulteration in beef meatballs.

Changes in vaginal secretion lipidom as a result of therapy in patients with vulvovaginal atrophy

Introduction. Vulvovaginal atrophy is a chronic progressive disease that includes physiological and anatomical changes as a result of a decrease in the level of sex hormones. VVA affects approximately 50% of middle-aged and older women and has a detrimental effect on interpersonal relationships, sexual health, and overall quality of life. Dynamic quadripolar radiofrequency (DRF) is a non-invasive procedure that may be a potential treatment for vulvar diseases.Aim. To study the metabolites of vaginal swabs in postmenopausal women treated with DRF.Materials and methods. The study included 180 women divided into 3 groups: treatment with DRF; treatment with a combination of DRF and estradiol, and estradiol treatment. In all patients, vaginal swabs were taken before the start of treatment and a month after treatment, and clinical parameters were assessed at 6 time points. Lipids were extracted from the cervico-vaginal fluid by the Folch method followed by liquid chromatography-mass spectrometric analysis. A comparative analysis of the clinical data of each of the groups and lipid profiles after treatment with a zero time point and their changes and a correlation analysis of changes in lipid levels and changes in clinical parameters before treatment and after 1 month was carried out.Results. There was a statistically significant reduction in the severity of VVA symptoms one month after treatment in all groups. The most pronounced effect of the therapy was observed in groups with the use of DRF with the preservation of the effect up to 12 months. In the first and second groups, a statistically significant difference was found in the levels of 2 lipids, in the third – in 4. In the first group, a correlation was found with lipids related with pain and lubrication, in the second – with vaginal pH, in the third – with the index of vaginal health, orgasm, satisfaction and pain.Conclusions. VVA therapy by DRF and local hormonal treatment triggers an inflammatory cascade in tissues. The use of the strategy of treatment with physical methods of influence has a mechanism of influence similar to the local application of estrogen in VVA. DRF therapy leads to the normalization of the vaginal microbiota and can compete with the use of estrogen in this therapeutic effect.

Optimizing MS-Based Multi-Omics: Comparative Analysis of Protein, Metabolite, and Lipid Extraction Techniques.

Multi-omics integrates diverse types of biological information from genomic, proteomic, and metabolomics experiments to achieve a comprehensive understanding of complex cellular mechanisms. However, this approach is also challenging due to technical issues such as limited sample quantities, the complexity of data pre-processing, and reproducibility concerns. Furthermore, existing studies have primarily focused on technical performance assessment and the presentation of modified protocols through quantitative comparisons of the identified protein counts. Nevertheless, the specific differences in these comparisons have been minimally investigated. Here, findings obtained from various omics approaches were profiled using various extraction methods (methanol extraction, the Folch method, and Matyash methods for metabolites and lipids) and two digestion methods (filter-aided sample preparation (FASP) and suspension traps (S-Trap)) for resuspended proteins. FASP was found to be more effective for the identification of membrane-related proteins, whereas S-Trap excelled in isolating nuclear-related and RNA-processing proteins. Thus, FASP may be suitable for investigating the immune response and bacterial infection pathways, whereas S-Trap may be more effective for studies focused on the mechanisms of neurodegenerative diseases. Moreover, regarding the choice of extraction method, the single-phase method identified organic compounds and compounds related to fatty acids, whereas the two-phase extraction method identified more hydrophilic compounds such as nucleotides. Lipids with strong hydrophobicity, such as ChE and TG, were identified in the two-phase extraction results. These findings highlight that significant differences among small molecules are primarily identified due to the varying polarities of extraction solvents. These results, obtained by considering variables such as human error and batch effects in the sample preparation step, offer comprehensive and detailed results not previously provided by existing studies, thereby aiding in the selection of the most suitable pre-processing approach.

DISORDERS OF THE FATTY ACID SPECTRUM OF ERYTHROCYTES AND BLOOD PLASMA OF ADOLESCENTS WITH RECURRENT CEPHALGIA ASSOCIATED WITH HYPERTENSION. APPROACHES TO CORRECTION

The spectrum of fatty acids (FA) in erythrocytes and blood plasma reflects the characteristics of lipid metabolism in diseases of vascular origin, including cephalalgia associated with arterial hypertension (AH). Timely diagnosis and correction of violations of this spectrum is important for the prevention of diseases of the cardiovascular and nervous system in subsequent age periods of life. Objective. To study the characteristics of the FA spectrum of erythrocyte membranes and blood plasma in adolescents with recurrent cephalalgia associated with AH. Material and methods. The composition of FA of erythrocytes and blood plasma was studied in 51 adolescents aged 12–18 years with recurrent cephalgia associated with AH and without AH. Determination of the FA composition of erythrocytes and blood plasma was carried out by gas-liquid chromatography-mass spectrometry with preliminary lipid extraction according to the Folch method. The analysis of FA methyl esters was carried out on a gas chromatograph with a GCD Plus mass spectrometric detector (Hewlett-Packard, USA). Results. The features of the structural redistribution of individual FA in adolescents with cephalgia and AH in the form of a predominance of saturated and monoenic FA in erythrocytes with a simultaneous decrease in their content in blood plasma, as well as a tendency to a decrease in the concentration of polyunsaturated FA in erythrocytes and their increase in plasma. Conclusion. Early detection and timely correction of disorders of the FA spectrum of erythrocyte membranes and blood plasma is necessary, starting from childhood and adolescence, in order to subsequently reduce the risk of developing diseases of the cardiovascular and nervous system associated with these disorders.

The interactive impact of sex and APOE genotype on post‐mortem brain fatty acids profile in Alzheimer’s Disease, Results from the UK BrainBank.

AbstractBackgroundTwo‐thirds of Alzheimer’s Disease (AD) patients are women, with the risk becoming higher if carrying the APOE4 genotype. Omega‐3 fatty acids status is associated with reduced risk of AD. Little is known about the interactive impact of sex and APOE genotype on brain fatty acids status. In this study, fatty acids were measured in post‐mortem brain tissue of AD patients and stratified according to sex and APOE genotype.MethodFatty acids were extracted from the hippocampus and frontal cortex of AD cases (n = 25, 11 are women) and controls (n = 25, 12 are women) using Folch method (from total lipids). Fatty acids were analysed using gas chromatography and flame ionization detection (GC‐FID). Samples were requested and granted from the UK Brain Bank. Adjustment for age and post‐mortem delay was done.ResultArachidonic acid (ARA) and n‐6DPA were higher in APOE4 women with AD compared to control (P‐Disease*sex*APOE = 0.01 and <0.001 respectively).These results were more evident in the hippocampus than the frontal cortex. n‐3DPA/ARA ratio (indicator of inflammatory status) was significantly higher in APOE4 women control compared to AD in both the hippocampus and frontal cortex (P‐disease*sex*APOE = 0.028 and <0.001 respectively). Similar trends were observed for EPA/ARA ratio. There was no impact of sex or APOE on BRAAK stage.ConclusionAPOE4 women with AD show a pro‐inflammatory brain fatty acid profile compared to controls, with increased n‐6 PUFAs (ARA and DPA) and reduced n‐3DPA/ARA ratios. Further extraction and analysis of fatty acids‐derived oxylipins and SPMs will be carried out.

SAT040 Administration Of A DHCR24 Inhibitor Reduces The Excessive Adrenal Steroidogenesis In db/db Mice Through Effects The Alteration Of Intracellular Cholesterol Metabolism

Abstract Disclosure: R. Kamigaki: None. H. Kameda: None. Y. Shibayama: None. H. Nomoto: None. K. Cho: None. A. Nakamura: None. S. Jin: None. K. Matoba: None. H. Miyoshi: None. T. Atsumi: None. Background and purpose: The circulating cortisol concentrations of patients with type 2 diabetes are high, suggesting that cortisol may be involved in diabetic complications. It has also been reported that the db/db mouse, a model of obesity and type 2 diabetes, has high circulating concentrations of steroid hormones. However, the mechanisms involved are unclear. In our previous study, we found that the expression of DHCR24, an enzyme that converts desmosterol (DES) to cholesterol (Chol) as part of de novo cholesterol synthesis, is upregulated in the adrenal glands of db/db mice. In the present study, we aimed to determine the effects of a DHCR24 inhibitor on adrenal steroidogenesis in db/db mice. Methods: Eight-week-old male db/db and db/+ mice were allocated to two groups, which were administered a weekly intraperitoneal injection of a DHCR24 inhibitor (U18666A; 10 mg/kg; (U) or PBS (P). Blood samples and their adrenal glands were collected 4 weeks later. We then compared the blood glucose, body mass, adrenal DES content, adrenal Chol content, and serum corticosterone (B) between the two groups. Sterols were extracted from adrenal tissue using the Folch method, and their concentrations were measured following picolinyl derivatization using liquid chromatography tandem-mass spectrometry. Results: There were no significant differences in the blood glucose concentrations or body masses of the P and U groups of both db/+ and db/db mice. The adrenal DES/adrenal mass ratio was higher in the db/db U group than in the db/db P group (db/db P vs. db/db U vs. db/+ P vs. db/+ U: 16.0±4.3 vs. 98.4±73.2 vs. 42.9±8.6 vs. 124.9±64.6 ng/mg, respectively; p<0.05). The adrenal Chol/adrenal mass and serum B concentration were lower in the db/db U and db/+ P groups than in the db/db P group (db/db P vs. db/db U vs. db/+ P vs. db/+ U: 2.1±0.2 vs. 1.3±0.2 vs. 1.1±0.6 vs. 1.3±0.2 μg/mg; and 232.8±20.1 vs. 189.6±13.1 vs. 126.3±20.6 vs. 111.2±25.8 ng/mL; respectively; all p<0.05). Conclusion: DHCR24 inhibition reduces cholesterol production and steroid hormone synthesis in the adrenal glands of db/db mice. This implies that the abnormal adrenal cholesterol metabolism in db/db mice plays a role in the greater adrenal steroidogenesis in type 2 diabetes, and that adrenal cholesterol metabolism may represent a novel therapeutic target for the affected patients. Presentation: Saturday, June 17, 2023

Content of fatty acids in lipids of adipose derived mesenchymal stem cells

The content of fatty acids in the lipids of mesenchymal stem cells of dog adipose tissue culture was studied. Mesenchymal stem cells of dog adipose tissue culture were obtained by culturing the primary material in a CO2 incubator with a content of 5 % CO2, at a temperature of 37 °C in DMEM medium with the addition of 10–15 % fetal bovine serum and 1 % antibiotic-antimycotic. When the confluency of the monolayer reached 70–80 %, the cells were transferred to a suspension and subcultivated in order to reduce the heterogeneity of the culture and obtain a sufficient amount of biological material. The lipids of the obtained stem cells were analyzed for the content of fatty acids by the method of thin-layer gas-liquid chromatography. Determination of the content of lipids of fatty acids in FSK of a cat was carried out by the Folch method. A mixture of fatty acid methyl esters was analyzed on a Trace GC Ultra gas chromatograph with a flame ionization detector on a capillary column SPTM –2560, 100 m x 0.25 mm ID, 0.20 μm film (Supelco). Identification of fatty acids was carried out using a standard sample of Supelco 37 Сomponent FAME Mix. Quantitative assessment of the LC spectrum was carried out by the method of normalization of the peak planes of methylated LC derivatives and their content was determined as a percentage of the total content of all LC. The conducted study of the content of fatty acids in lipids made it possible to reveal certain features of the lipid metabolism of mesenchymal stem cells cultured in dog adipose tissue. A high content of oleic acid, characteristic of cells resistant to apoptosis and with high proliferative potential, was determined; a high ratio of unsaturated linoleic to saturated stearic acid (С18:1/С18.0), which reflects the high activity of the stearoyl-coenzyme-desaturase enzyme and, indirectly, the active state of the Wnt/β-catenin signaling pathway; inability to lengthen the chain of saturated fatty acids; lack or low activity of de novo synthesis of omega-6 polyunsaturated fatty acids. 18 fatty acids were found in the composition of lipids of fetal stem cells of a cat, of the saturated ones - the most palmitic acid (33.70 ± 0.02 %), of the monounsaturated ones – oleic acid (21.63 ± 0.03 %), of the polyunsaturated ones – linoleic acid (6.45 ± 0.07 %). The least amount of cis-,11,14-eicosadienoic acid (0.04 ± 0.01 %) was found in the composition of cell lipids. The total amount of saturated fatty acids in dog mesenchymal stem cell lipids was 65.65 ± 0.02 %), unsaturated fatty acids – 34.35 ± 0.02 %. Monoene fatty acids were determined in the amount of 24.46 ± 0.02 %, and polyene – 9.89 ± 0.02 %. The ratio index of polyunsaturated fatty acids ω 3 to ω 6 is 0.40. Lipids of mesenchymal stem cells of adipose tissue culture were characterized by a lower content of monoene unsaturated fatty acids 24.46 ± 0.02; (P < 0.05), with a higher content of ω3 fatty acids 3.04 ± 0.02 %; (P < 0.05), with a lower content of ω6 fatty acids 6.86 ± 0.02 %; (P < 0.05) in contrast to lipids of red bone marrow stem cells.

Untargeted hair lipidomics: comprehensive evaluation of the hair-specific lipid signature and considerations for retrospective analysis.

Lipidomics investigates the composition and function of lipids, typically employing blood or tissue samples as the primary study matrices. Hair has recently emerged as a potential complementary sample type to identify biomarkers in early disease stages and retrospectively document an individual's metabolic status due to its long detection window of up to several months prior to the time of sampling. However, the limited coverage of lipid profiling presented in previous studies has hindered its exploitation. This study aimed to evaluate the lipid coverage of hair using an untargeted liquid chromatography-high-resolution mass spectrometry lipidomics platform. Two distinct three-step exhaustive extraction experiments were performed using a hair metabolomics one-phase extraction technique that has been recently optimized, and the two-phase Folch extraction method which is recognized as the gold standard for lipid extraction in biological matrices. The applied lipidomics workflow improved hair lipid coverage, as only 99 species could be annotated using the one-phase extraction method, while 297 lipid species across six categories were annotated with the Folch method. Several lipids in hair were reported for the first time, including N-acyl amino acids, diradylglycerols, and coenzyme Q10. The study suggests that hair lipids are not solely derived from de novo synthesis in hair, but are also incorporated from sebum and blood, making hair a valuable matrix for clinical, forensic, and dermatological research. The improved understanding of the lipid composition and analytical considerations for retrospective analysis offers valuable insights to contextualize untargeted hair lipidomic analysis and facilitate the use of hair in translational studies.

A Comparative Analysis on the Environmental Impact of Selected Methods for Determining the Profile of Fatty Acids in Cheese

The fatty acid profile of cheese influences its sensory parameters, such as color, texture, or flavor. Examining the fatty acid profile also helps to assess the nutritional value of the cheese that is being tested. However, the determination of fatty acids in cheese samples is a multi-stage and time-consuming task. In addition, large amounts of toxic organic solvents are used to prepare samples for analysis purposes. This paper presents the results of a study to determine the fatty acid profile of yellow cheese samples. Six different methods of sample preparation were compared for analysis purposes. The profile of fatty acids was determined using gas chromatography with flame ionization detection (GC-FID). The study showed significant differences (p > 0.05) in the resulting fatty acid profile between the methods used. It was found that the most reliable fatty acid profile results were obtained using methods derived from the Folch method. In addition, tools such as the Analytical Eco-Scale tool and the Analytical Greenness Metric for Sample Preparation (AGREEprep) tool were used to assess the greenness of the methods used. In the case of the Analytical Eco-Scale tool, all six methods scored ‘acceptable green analysis’ with scores ranging from 61 to 73. However, an evaluation of methods using the AGREEprep metric showed that the results of the methods (0.13–0.27) did not show the “greenness” of the analytical methods.