Features of the mobile pool of fatty acids (lipoproteins and free fatty acids) in adolescents with different body weights

Introduction

In recent years, increasing attention has been paid to the identification and study of early clinical markers of overweight and obesity, as shown by a literature review [1, 2].

From this standpoint, lipids, both endogenous and dietary, are of particular interest, probably playing a decisive role in the prevention and treatment of obesity [3].

It is known that fat accumulation is associated with the quality and quantity of fatty acids (FA) from the diet, and also adipose tissue secretes a wide range of substances that affect the development of obesity and related pathologies. Therefore, lipidomics can play a key role in describing molecular signaling scenarios, providing important information on the various stages of weight gain, from overweight to obesity [4, 5].

Lipidomics allows the identification of various types of lipids present in cells, tissues, biological fluids or throughout the body, reflecting lipid metabolism, including the early phase of pathophysiological changes associated with the development of the obesity phenotype. FA analysis, including membrane analysis, has reached a high level of technological maturity. Simple, relatively inexpensive and robust analytical methods with high resolution have been developed and demonstrated in several studies [2, 6].

Various compartments can be used to assess the lipid composition of blood. FA in plasma or serum are widely analyzed, reflecting short-term changes associated with fat intake. In this regard, the analysis of the lipid composition of mature red blood cell membranes has an advantage over plasma analysis, since it maintains a more stable FA composition and better reflects the long-term effects of FA due to a longer circulation in the blood [2, 7].

Membrane lipids determine the qualitative characteristics of the lipid bilayer (fluidity, permeability, thickness) and its lipid signaling through the FA residues of membrane phospholipids. This suggests that FA may describe the predisposition of cells to respond to various stimuli from the extracellular environment [2, 8]. Thus, the balance of FA composition of membranes can affect the balance of functions in each individual cell and, consequently, in tissues and in the entire body [9, 10].

The goal of this study was to investigate the characteristics of the mobile pool of FA (including membrane indices) in adolescents with different body weights. This direction is of particular interest for a deeper understanding of the mechanisms of obesity phenotype formation in adolescents.

Material and Methods

Study subjects

A single-center comparative study was conducted in parallel groups from September 2023 to February 2024 at the Children’s Clinic (Director: MD D.V. Kozyritskaya) of the Siberian State Medical University of the Russian Federation Ministry of Healthcare, where overweight and obese children were examined and treated. Healthy schoolchildren of the Municipal Educational Institution, Perspektiva School, in Tomsk (Principal: I.E. Sakharova) were invited to participate in the study.

A total of 75 individuals were examined. The main group (n=45) comprised children and adolescents, including 22 boys (48.9%) and 23 girls (51.1%); their median age was 14.7 [11.3; 16.2] years with Grade 1 or 2 exogenous constitutional obesity. The control group consisted of 30 healthy children and adolescents, including 14 boys (46.7%) and 16 girls (53.3%), their median age was 15.1 [11.8; 16.5] years. The control and main groups were divided into subgroups based on gender (Figure 1).

Figure 1. Flow chart illustrating the distribution of study participants among groups and subgroups.

The inclusion criteria for the main group were as follows: children and adolescents aged 10 to 17 years Grade 1 or 2 exogenous constitutional obesity (standard deviation score, SDS<3.0; body mass index, BMI³2.0) who signed informed consent to participate in the study (approval by the Ethics Committee of Siberian State Medical University No. 8459/2 of October 28, 2020).

We employed the following exclusion criteria:

1. Monogenic obesity

2. Type 1 and Type 2 diabetes mellitus

3. Severe or unstable somatic diseases

4. Traumatic brain injury in anamnesis

5. History of alcoholism or drug addiction

Anthropometric measurements

Anthropometric assessment included height measurement to the nearest 0.1 cm, body weight to the nearest 0.1 kg without shoes and outerwear on the scale integrated into the InBody 770 analyzer (InBody Co., Ltd, Republic of Korea). The body mass index standard deviation score (BMI SDS) and SDS of height were calculated using the software developed by the World Health Organization (WHO): WHO AnthroPlus (for children aged 6 to 19 years) (URL: www.who-anthroplus.informer.com).

Analyzing mobile pool of fatty acids (lipoproteins and free fatty acids)

The mobile pool of FA (lipoproteins and free fatty acids, FFA) in serum was analyzed on an Agilent 7000B chromatograph mass spectrometer (Agilent Technologies, USA). The sample volume was 2 μl, injected with a split ratio of 1:5.

The range of specific parameters for the pool of FA included their following groups:

·          Saturated fatty acids (SFA) with decanoic (10:0), lauric (12:0), myristoleic (14:0), palmitic (16:0), stearic (18:0), arachidic (20:0), behenic (22:0), and lignoceric (24:0) FA;

·          Monounsaturated fatty acids (MUFA) with omega-5, 7 and 9 (ω-5, ω-7, ω-9): myristoleic (14:1n5), palmitoleic (16:1n7), oleic (18:1n9), erucic (22:1n9), nervonic (24:1n9), and eicosatrienoic FA (Mead acid) (20:3n9);

·          Polyunsaturated fatty acids (PUFA) with omega-3 and 6 (ω-3, ω-6): linolenic (ALA 18:3n3), docosapentaenoic (DPA 22:5n3), docosahexaenoic (DHA 22:6n3), linoleic (LA 18:2n6), gamma-linolenic (GLA 18:3n6), dihomo-gamma-linolenic (DGLA 20:3n6), arachidonic (AA 20:4n6), and docosatetraenoic FA (adrenic acid) (DTA 22:4n6);

·          Trans fatty acids (TFA): elaidic (ELA 18:1n9t) and linolelaidic FA;

·          Odd-chain MUFA and SFA: pentadecanoic (15:0), margaric (heptadecanoic) (17:0), heneicosylic (21:0), tricosylic (23:0), and heptadecenoic (17:1n7).

·          Multi-methyl-branched fatty acids: phytanic (3,7,11,15-tetramethylhexadecanoic) acid.

Additionally, we determined the relative content of FA in the groups and calculated the following indices and ratios: ω-3 FA and ω-6 FA in % of SFA; PUFA, MUFA, SFA, TFA in % of the total amount of FA; whole blood ω-3 index, erythrocyte membrane ω-3 index, ω-6/ω-3 FA ratio, and ω-6 desaturase activity index.

Statistical analyses were performed using IBM SPSS Statistics v.20 software. Frequency analysis was employed for qualitative data, and the results were presented as counts ​​and percentages. Nominal data comparisons between groups were conducted using the Pearson’s chi-squared test. In cases where the expected frequency in any cell of the 4-row/4-column table was less than 10, Fisher’s exact test was used to assess the significance of differences.

The subintimal inflammatory response risk index was elevated in both groups, but it was significantly higher among adolescents with normal body weight: 51.320 [38.368; 57.260] % of FA and 81.450 [63.120; 210.860] % of FA, respectively. The PUFA/SFA ratio exhibited levels of 0.400 [0.375; 0.558] % of FA and 0.740 [0.630; 0.990] % of FA, respectively, in the main and control groups (see reference ranges ​​in Table 4).

Girls

Analysis of the FA pool concentration, which yielded statistically significant differences in the groups of girls, when compared with laboratory reference ranges, revealed that the level of arachidonic acid representing the ω-6 PUFA family (3.880 [3.800; 4.470] % of FA) was statistically significantly lower in obesity group vs. the control group and fell below the lower limit of the reference range. Among the ω-9 MUFA, all FA, except oleic acid, exhibited an increase above the upper limit of the reference range in obesity. However, a similar trend was observed for stearic, behenic and lignoceric acids in individuals with normal weight. More details can be found in Table 5.

Table 5. Comparison of the parameters of membrane and mobile (lipoproteins and free fatty acid) pools of fatty acids (FA) in whole blood of girls in the study groups (Me [Q1; Q3]) with reference laboratory values

We noted that the levels of ω-3 FA (% of SFA) (1.680 [1.365; 1.910] % of FA) and ω-6 FA (% of SFA) (21.050 [19.130; 23.100] % of FA) were below the lower limit of the reference range. PUFA (% of total FA) were lower vs. the control and vs. reference values: 22.380 [20.790; 24.990] % of FA and 35.460 [30.290; 42.950] % of FA, respectively. SFA (% of total FA) exceeded the upper reference limit in both study groups: 55.240 [52.470; 56.185] % of FA and 45.900 [38.160; 50.290] % of FA, respectively (Table 5).

The subintimal inflammatory response risk index exhibited an increase in both the obesity group and control group (it was even higher in the latter): 57.090 [49.290; 82.385] % of FA and 136.990 [63.120; 210.860] % of FA, respectively). PUFA/SFA ratio showed levels of 0.400 [0.390; 0.485] % of FA and 0.810 [0.630; 1.160] % of FA, correspondingly, in the main and control groups.

Discussion

It is known that in adults, excess visceral fat is associated with changes in the composition of circulating FA and impaired adipose tissue function [11]. The opinion on the contribution of FA to the development of obesity, insulin resistance and other metabolic abnormalities in pediatric practice remains controversial.

The study by L.M. Beccarelli et al. (2018) showed that, regardless of obesity, changes in FA and lipid metabolism associated with the development of metabolic disorders can be observed. In contrast, the study by M.C. Hua et al. (2021) demonstrated that elevated palmitoleic acid levels were associated with increased metabolic risk in obese and overweight children, as well as with the accumulation of visceral fat [13]. In this study, adolescents in the main group had statistically significantly higher levels of palmitoleic acid compared with the control group.

Similar results were obtained in the study by S. Bonafini et al. (2020) [14]. Dietary intake does not significantly affect the level of palmitoleic acid, which is formed as a result of palmitic acid metabolism by stearoyl-CoA desaturase-16 (SCD-16) [15]. In this study, palmitoleic acid levels and estimated SCD-16 activity were associated with obesity markers (BMI, waist-to-height ratio) and blood pressure, most pronounced in the obesity group. These results indicate that not only genetically determined increased SCD-16 activity but also other exogenous factors (including excessive palmitic acid intake) may support this association, leading to a metabolic response.

In addition, higher levels of arachidonic acid were noted in children with normal body weight, which contradicts the data on its adverse role in the development of insulin resistance. Several studies in both pediatric and adult populations demonstrated positive associations with insulin resistance and metabolic syndrome [2, 16, 17].

However, this study yielded reverse findings: non-obese adolescents had statistically significantly higher arachidonic FA levels. Similar results were obtained by S. Marth et al. (2020), who found an inverse relationship between arachidonic acid levels and HOMA-IR, with higher arachidonic acid levels in non-obese children [18]. The authors suggest that such conflicting data may be due to differences in the methods used and desaturase activity between the studied populations.

Both the main and control groups exhibited higher values ​​of the subintimal inflammatory response risk index and the PUFA/SFA index vs. the control group. These parameters reflect the viscosity, fluidity, and permeability of the cell membrane. However, among adolescents with normal body weight, their levels were statistically significantly higher. These findings require further study, including dietary habits, which, according to publications, have a more substantial impact on the FA pool, including more steady red blood cell indices [2, 6].

In this study, obese adolescents had statistically significantly lower values ​​of the whole blood ω-3 index and the erythrocyte membrane ω-3 index compared with the control group. Such results suggest an increased risk of cardiovascular disease.

Similar values ​​of the erythrocyte membrane ω-3 index in studies involving adults were associated with a higher risk of coronary artery disease and were proposed as an addition to traditional scales (Framingham and GRACE) for predicting fatal cardiovascular events. They also correlated with the severity of insulin resistance [19, 20].

However, our obtained data cannot be extrapolated to the pediatric population, and changes in the FA profile associated with growth and development require further study. For example, I. Jauregibeitia et al. (2021) conducted a comparative analysis of erythrocyte FA indices in children and adults, demonstrating that obese children had higher levels of ω-6 PUFA (primarily, linoleic acid; p<0.01) and lower values ​​of ω-3 FA (primarily, DHA; p<0.001) vs. adults [21].

The results of this study show significant changes in the mobile (lipoproteins and FFA) pool of FA in obese adolescents, suggesting that they are involved in the development of overweight and obesity and, therefore, can be considered as potential parameters for early clinical diagnosis.

Study limitations

Potential limitations of our study include the small sample size and data collection at a single center. Furthermore, adolescents with SDS BMI SDS>3 were excluded from the analysis, thereby making it difficult to compare the results with published data due to differences between cohorts. It is also important to note that the study did not assess dietary habits, which could have influenced the results.

Conclusion

All the indices examined in children (whole blood ω-3 index, erythrocyte membrane ω-3 index, subintimal inflammatory response risk index, cell membrane viscosity, fluidity and permeability index) exhibited statistically significant changes in the groups of adolescents with obesity. These changes were associated not only with increased cardiovascular risk, progression of chronic inflammation, but also with the nature of children’s growth and development.

Conflict of interest

The authors declare no conflicts of interest.

Funding

The study was supported by the Russian Science Foundation, project No. 23-75-01034, Using lipidomics profiles to create a predictive model for the realization of the obesity phenotype in children and adolescents, (agreement of August 14, 2023).