A PCA model with oblique rotation was tested to examine the factor correlation matrix. Since none of the factor correlations were over ± 0.32, indicating factor correlations are not driven by the data, we estimated a model with orthogonal rotation to reveal a simplified structure with interpretable factor loadings. To identify metabolite patterns of interest, bivariate Pearson-correlation analysis was performed between percent changes in metabolites and clinical biomarkers of CMR and with lipoprotein particle subfraction distributions separately. A generalized estimating equation procedure determined the significance of longitudinal changes in PC scores and their contributing metabolites using age and gender as co-variates. Wald Chi-square tests determined the significance of pairwise differences in treatment responses. Statistical analyses were performed using IBM SPSS Statistics version 25 and Rstudio version 1.1.456, and p-values <0.05 were considered statistically significant for the differences in traditional biomarkers. A Bonferroni correction for multiple comparisons adjusted the p-value set as significant to <0.002 for the correlational analyses with amino acid and ceramide metabolites.The study cohort was predominantly female, 61% Hispanic , 25% Nonhispanic Black, 8% Hispanic Black. and 6% Nonhispanic White, with more Hispanic Not Black than Nonhispanic Black participants randomized by chance to the INT group . Full assessment of physical, behavioral, metabolic,25 liter plant pot and metabolomic status was conducted at baseline and study completion on the cohort of 36 participants .
There was excellent attendance in both INT and CONT groups with all Teens and PAC participating in more than 78% of group sessions and 100% of baseline and follow-up assessment visits. There was considerable prevalence of obesity, dyslipidemia, inflammation and insulin resistance in all participants and mild hypertension in adult participants at baseline . Mean baseline BMI was over 30 mg/m2 in all participants, did not differ by sex, but was significantly higher in the CONT group relative to INT. The mean CRP was over 28.6 nmol/L in all participants but higher in Teen participants assigned to the CONT group than to the Teen INT group, and between the combined CONT and INT groups. Despite these random differences in BMI and CRP, central adiposity , blood pressure, lipids, insulin resistance and inflammation were similar in both groups, suggesting closely matched CMR profiles. Although in population studies, Waist Circ is greater in males as compared with females, only one of the 18 PAC was male; while his Waist Circ fell below the adult female PAC mean, it was not the lowest recorded among PAC. The average Waist Circ for Teen males vs female Teens did not differ significantly so Waist Circ data are not reported by sex. Compliance with nutrient bar intake by self-report was 85.8 ± 11.1% and 86.7 ± 13.8% among INT group adults and Teens respectively.The quality of self-report diets assessed by Block food frequency questionnaire was poor in all participants at baseline . Modest but significant improvement in CONT PAC was evident in categories of saturated fat intake and added sugar but trends in the same direction did not reach significance in INT PAC or in Teens from either group.
Average daily servings of dietary fruit , vegetables, not including potatoes , and fiber were comparably low in both CONT and INT groups and did not change in any age subgroup . Self-report activity increased in all participants in both groups . Weight was stable, even in the INT group despite the addition of 220 kcal in two daily nutrient bars. Good compliance with nutrient bar intake was suggested by a significant increase in plasma 25 hydroxy Vitamin D in the INT group PAC and Teens, but neither CONT subgroup. There were no other consistent effects of nutrient bar supplementation evident in traditional CMR biomarkers, nor were there significant within- or between group changes in any anthropometric measures in combined CONT and INT groups. Subgroup analysis of PAC and Teens separately showed some favorable changes in both CONT and INT groups. PAC CONT participants decreased Waist Circ . Among Teen participants, CRP trended lower in CONT but did not reach significance . Among Teens, systolic blood pressure decreased significantly by 7% in INT only, trending upward in CONT Teens with a significant between-group difference.Previous bar supplementation trials showed that chronic inflammation was associated with slower response in metabolic improvement. All participants, both Teens and PACs in this study, met criteria for obesity and had baseline CRP > 14.3 nmol/L.To test the hypothesis that subtle shifts in metabolism may precede changes in traditional biomarkers, a targeted analysis of plasma ceramides and amino acids was performed. Baseline metabolite data were z-transformed and subjected to PCA analysis.
The top six PCs extracted, each composed of distinct sets of linked metabolites, explained 59.3% of the total variance in the data set . PC1, which explained 16.7% of the variance, was composed exclusively of ceramides and ceramide-1-phophates; PC2 of amino acid metabolites; PC3 of sphingomyelins; PC4 of dihydroceramide species, glucosylceramide, and one specific ceramide subspecies Cer 18:1; PC5 of biomarkers of meat consumption and transsulfuration and polyamine metabolites ; and PC6 of amino acids in antioxidant defense . Fig 2A shows bivariate correlational analysis between baseline PC1-PC6 scores with parameters of obesity, dyslipidemia, insulin resistance and inflammation. Within the full cohort, no significant correlations were observed between any PC scores and anthropometric measures of weight, BMI, WC, or waist-height ratio. In contrast, highly significant associations between specific PC scores and metabolic parameters of dyslipidemia, insulin resistance and inflammation were present. PC1 was positively correlated with fasting total cholesterol, triglycerides, and HDL , and adiponectin . PC 3 was inversely associated with CRP . PC4 was inversely associated with diastolic blood pressure and PC6 showed an inverse association with fasting insulin and HOMA . In Fig 2B positive association patterns are illustrated between PC1 scores and atherogenic lipoprotein particles, specifically very small LDLIVc , LDLIVa and all three sub-classes of VLDL . The PC1 score was also associated with very small LDL IVb and inversely with LDL diameter , both above p value threshold set at 0.01. It is also of note that the baseline relationship between PC1 Cersand HDL was driven primarily by association with the small HDL32a subclass not large HDL2b.We next determined how increased physical activity by itself or with nutrient bar supplementation modulated these baseline metabolomic parameters. Analysis of PC change was performed with adjustments of potential covariates including age, gender, and baseline CRP. While there was no change in the plasma total ceramide pool, results showed significant divergence in several key ceramide species within PC1 between CONT and INT. Results show significant increases in C14:0 , C20:0 , C22:0 and C24:1 ceramides and a nearly significant increase in C16:0 in CONT. In contrast, C24:1 ceramide significantly decreased in the INT group by 18%; all other ceramides trended lower or remained unchanged . Analysis of group by time interactions showed significant between group differences in all of these C14:0, C16:0, C20:0, C22:0 and C24:1 ceramide species. Sphinganine and sphingosine are essential substrates for ceramide synthesis through de novo synthesis and salvage pathways. In CONT only, levels of sphinganine and sphingosine increased significantly by 59% and 50%, respectively resulting in significant pairwise differences between CONT and INT groups .Sphingosine-1-phosphate is a terminal breakdown product of ceramide and an important anti-inflammatory and vascular signaling lipid mediator associated with lipoproteins, particularly HDL, in plasma. As shown in Table 5, S1P levels increased differentially in both CONT and more so INT participants. The S1P increase in the INT group differed from the degree of change in CONT at p = 0.007. Table 6 lists amino acid metabolites with significant within group and between group changes. Results show that among CONT,black plastic plant pots fasting levels of proline, ornithine, lysine, alanine and threonine trended upward. In contrast, the nutrient bar INT significantly altered amino acid metabolism, such that concentrations of several gluconeogenic, sulfur redox and urea cycle intermediates decreased, notably fasting levels of serine, proline, aspartate, cystathionine, alanine, glutamine methionine, and citrulline. A small but significant increase in histidine concentration associated with favorable nitrogen balance, improved bio-availability of arginine, and an increase in the Fischer ratio the molar ratio of BCAA and aromatic amino acids , indicative of favorable vascular and liver functions respectively, were observed in the INT group only.
To establish the clinical significance of differences in metabolism detected among INT participants, bivariate correlations between percent changes in ceramide and amino acid metabolites and clinical biomarkers of dyslipidemia and insulin resistance were considered . Positive correlations were observed between changes in TG and changes in ceramides composed of C14:0 , C16:0 , C24:1 and C26:1 acyl chain lengths . Specific associations were evident between percent changes in C18:1 ceramide and changes in very small LDL IVc and IVb particles . In addition, changes in fasting blood glucose were positively associated with percent changes in citrulline and cystathionine , and the arginine bioavailability index , assessed by ratio of arginine to sum of citrulline + ornithine. Cysth change also correlated with change in insulin , and accordingly with HOMA . A strong positive correlation emerged for ornithine alone with change in CRP . Consistent with the baseline analysis, changes in amino acids did not associate significantly with lipid parameters.Improved nutrition represents a lifestyle modification target to lower the growing global cardiometabolic disease burden, especially among youth and their families struggling with socioeconomic disparity and the many associated barriers to a healthy diet. Even without economic barriers, lifestyle adherence is challenging. Furthermore, strict dietary counseling and adherence for the management of hyperlipidemia has been associated with increased anxiety and depression in youth. The nutrient bar strategy utilized in this study was developed to enable research on the impact of nutrition on CMR, by facilitating daily delivery of nutrients from foods missing from a standard American diet, essential fibers, minerals, vitamins, omega-3 fatty acids, and polyphenols, in a readily consumed, low-calorie, palatable nutrient bar. Results herein show that a 2 month nutrient bar intervention together with increased physical activity improved systolic blood pressure in Teens only without other significant changes observed in traditional biomarkers, but was associated with meta bolomicshifts in the full INT PAC and Teen group that correlate with favorable changes in cardiometabolic disease risk biomarkers. Baseline metabolomic analysis identified ceramides as unique biomarkers of atherogenic dyslipidemia in both Teens and adults at heightened CMR . Although the metabolomic panel included BCAA and AAA previously implicated in CMR, only PC1, composed primarily of ceramide metabolites, associated with baseline TG levels and TG-rich VLDL subspecies, suggesting that ceramides may contribute to TG homeostasis. Fatty-acyl CoA is a substrate for both ceramide and TG biosynthesis and thus, excess ceramide and TG synthesis may occur under conditions of abundant fatty acid availability through both high fat diets or high refined carbohydrate diets and de novo lipogenesis. In contrast to TGs that serve a passive fatty acid storage function, ceramides have unique signaling roles in mediating insulin resistance and mitochondrial bioenergetics. Ceramides may therefore act as cellular energy sensors to regulate tissue fuel uptake and storage via insulin signaling. In obesity with chronic inflammation, excess ceramide, particularly C16:0 Cer, is associated with mitochondrial dysfunction, impaired sphingolipid cell signaling and insulin resistance. Decreasing tissue ceramide through pharmacological or genetic manipulations can reverse or attenuate insulin resistance, providing support for a direct role of ceramides as critical part of fuel sensing circuitry in the body. Ceramides may represent a sensitive biomarker of metabolic dysregulation when energetic supply chronically exceeds storage capacity. The current findings suggest that this tipping point may be influenced not only by absolute caloric intake but also by the dietary nutrient value. Intensive lifestyle modification has been shown to improve CMR biomarkers in the obese. In this trial, despite excellent participation in weekly exercise sessions, self-report of increased exercise between sessions in all subjects and both subjective and objective evidence of compliance with nutrient bar intake in the INT group, there were minimal changes in traditional parameters of dyslipidemia, inflammation, and insulin resistance . Changes in the plasma metabolome were however able to signify highly significant differences between CONT and INT . Contrary to the expectation that physical activity would lead to lower ceramide formation by improving lipid oxidation capacity, significant increases in C14:0, C16:0, C20:0 and C22:0 and overall increasing trends with all ceramide species were observed in CONT participants. A study by Bergman and co-workers similarly found that acute exercise transiently increases serum ceramides in obese untrained subjects.