In a subsequent 2 mo study of the effects of the nutrient bar on CMR markers in individuals across a range of BMIs, only those with low inflammation at baseline as assessed by high sensitivity C-reactive protein < 14.3 nmol/L responded comparably to those in the earlier trial, with not only increased HDL cholesterol and large HDL particles but also a trend toward increased high molecular weight adiponectin and a decrease in other CMR factors at 2 weeks, sustained at 2 months . In particular, a shift in low density lipoprotein particle subfractions toward a less atherogenic profile was evident in the non-inflamed group . Although the participants with overweight or obesity and CRP > 14.3 nmol/L did not show this response, they did experience an upward trend in adiponectin by 2 months. These results suggest that there may be a continuum of metabolic responsiveness to this nutrient supplement that is slowed in the face of the chronic low-level inflammation commonly observed with obesity and insulin resistance. A 6 month study of this nutrient bar in obese adolescents with non-eosinophilic asthma showed improved lung function at 2 months, but favorable movement in cardiometabolic biomarkers only began to emerge at 6 months.It is not known whether more sensitive biomarkers of early metabolic change may be capable of detecting short-term effects of the nutrient bar supplementation in persons at CMR.
Past metabolomics studies in obese adolescents and adults have identified strong positive associations between baseline levels of branched chain, aromatic, sulfur, and gluconeogenic amino acid metabolites and parameters of inflammation and insulin resistance. Similarly, planting gutter elevated levels of specific ceramide species have been shown to associate with inflammation, dyslipidemia and insulin resistance in both adult and adolescent obesity. The relative sensitivity of these biomarkers to reflect moderate changes in dietary intake of polyphenols, essential lipids, fiber and vitamin/minerals, remains incompletely understood. In the present randomized, controlled, non-blinded trial, a two month intervention with exercise and nutrition counseling alone or with nutrient bar supplementation was performed in a high CMR cohort of adolescent /parent adult caretaker family units to determine 1) cross-sectional relationships in both adolescents and adults between traditional CMR biomarkers and amino acid and ceramide metabolites and 2) longitudinal changes within groups in the same CMR biomarkers following the lifestyle +/- nutrient bar intervention.Anthropometric and clinical evaluation: All anthropometric measures were performed in duplicate in the clinical research center and if not within 10% agreement, were repeated a third time. The reported measure is an average of the two closest numbers. Height was measured with a stationary stadiometer.
Weight was measured using a digital electronic scale and the BMI and waist to height ratio were calculated. Waist circumference was measured at end expiration to the nearest mm with a Gulick II Plus tape midway between the lowest border of the rib cage and the upper border of the iliac crest.Blood pressure and Resting Heart Rate: Each was measured in triplicate after 5 minutes sitting quietly with readings taken at least one minute apart. An automatic digital blood pressure monitor was used with cuff size adjusted for arm size. Traditional CMR biomarkers: Fasting blood samples were drawn and processed in the UCSF Benioff Children’s Hospital Oakland Clinical Research Center and sent to ARUP Diagnostic Laboratories for: standard lipid profile [total triglyceride, total cholesterol, and cholesterol within HDL and LDL , glucose, insulin, 25 hydroxy Vitamin D level and CRP. TG to HDL ratio and non-HDL were calculated. Fasting insulin and glucose were used to calculate the Homeostasis Model Assessment of Insulin Resistance Index according to the formula: fasting insulin x fasting glucose /22.5. Lipoprotein particle sub-classes were analyzed by an ion mobility procedure that sensitively and directly measures concentrations of lipoprotein particle subfractions. High molecular weight adiponectin was measured by solid-phase sandwich ELISA . Metabolomic analyses: 1) Targeted analyses of 42 amine-containing metabolites consisting of 20 major amino acids, and secondary metabolites of arginine and cysteine whose levels are sensitive to inflammation and oxidative stress were performed on stored samples preserved at -70o Fahrenheit. Briefly, plasma was acidified with 5% perchloric acid containing 8 stable isotope internal standards. Acid-soluble supernatant was used for strong-cation exchange solidphase extraction to capture cationic amine- containing metabolites. Extracted metabolites were further derivatized with isopropylchloroformate.
Derivatives of metabolites were resolved using Agilent 1260 ultra-high pressure liquid chromatography and eluted with a gradient of water and isopropanol . An Agilent 6490 triple quadrupole mass spectrometer was used to detect resolved analytes and quantify them using authenticated external and internal standards. 2) Sphingolipidomics by electrospray tandem mass-spectrometry by validated techniques was used to identify sphingolipid metabolites, including ceramides.De-identified baseline and study completion data points, paired by participant study ID, were entered into SPSS . Descriptive analyses of the study cohort were summarized and results for Teens and PACs, as well as for combined CONT and INT groups, were compared by unpaired Student t-test. Continuous physical and metabolic variables were tested for normality by examining the skewness, kurtosis and the Shapiro Wilk tests and transformed as necessary before analysis. Most of the variables in our data were normally distributed. Log transformations were conducted for the continuous physical and metabolic variables that were skewed to make them as normal as possible. The Shapiro-Wilk tests show that all of the transformed variables except two are approximately normal. The measures of metabolite concentrations used for principal component analysis were Z-transformed to render them normally distributed on the same scale with mean of zero and standard deviation of one. Pre-post change in absolute metabolite levels were compared by repeated samples paired t-test. Baseline z-scores of metabolites were subjected to principal component analysis without rotation. PCA is an unsupervised analysis that aims to decrease the complexity of data by reducing variables to a smaller number of principal components . Each of the PCs were vectors of metabolite contributions. A direct oblimin rotation was used and 6 factors before the bend in the scree plot , and eigenvalues >1 were retained. Component scores for each participant were calculated with a standardized scoring coefficient. 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, gutter berries with more Hispanic Not Black than Nonhispanic Black participants randomized by chance to the INT group . Full assessment of physical, behavioral, metabolic, 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. 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 .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.