Exploring the interplay between kidney function and urinary metabolites in young adults: the African-PREDICT study.

The exposure to modifiable risk factors at young ages have been linked to premature fatal and non-fatal cardiovascular and kidney outcomes. The use of urinary metabolomics has shown strong predictability of kidney function and cardiovascular disease (CVD). We therefore determined the associations between estimated glomerular filtration rate (eGFR) and urinary metabolites in young adults with and without CVD risk factors. Apparently healthy Black and White sexes were included (aged 20-30 years) and categorised by the presence or absence of risk factors, i.e., obesity, physical inactivity, smoking, excessive alcohol intake, masked hypertension, hyperglycemia, dyslipidemia and low socio-economic status, forming the CVD risk group (N = 1036), CVD risk clusters (i.e. presenting with 1 CVD risk factor (N = 344), 2 CVD risk factors (N = 360) and 3 + CVD risk factors (N = 332)) and the control group (N = 166). eGFR was calculated with CKD-EPI equations. A targeted metabolomics approach using liquid chromatography-tandem mass spectrometry was used to measure amino acids and acylcarnitines. Lower cystatin C-based eGFR were indicated in the CVD risk group, 2 and 3 + CVD risk clusters compared to the control group (all P ≤ 0.033). In the CVD risk group, eGFR associated positively with histidine, lysine, asparagine, glycine, serine, glutamine, dimethylglycine, threonine, alanine, creatine, cystine, methionine, tyrosine, pyroglutamic acid, leucine/isoleucine, aspartic acid, tryptophan, glutamic acid, free carnitine, acetylcarnitine, propionylcarnitine, isovalerylcarnitine, octanoylcarnitine and decanoylcarnitine (all P ≤ 0.044), with similar results found in the CVD risk clusters, particularly the 2 CVD risk cluster. eGFR was positively associated with metabolites linked to aromatic amino acid and branched-chain amino acid metabolism, energy metabolism and oxidative stress. These findings may indicate altered reabsorption of these metabolites or altered metabolic regulation to preserve renal health in the setting of CVD risk factors at this young age without established CVD.

Validation of the parents' version of the KINDLR and Kiddy Parents questionnaire in a South African context.

BACKGROUND: This study aimed to assess the usefulness of the parent version of the KINDLR and the additional items of the Kiddy Parents questionnaire in the South-African context and to validate it as an appropriate tool for measuring health-related quality of life (HRQoL). METHOD: The ExAMIN Youth SA study was designed to investigate lifestyle behaviours, including psychosocial factors that may adversely impact on cardiovascular health of children. Construct validity was examined by using exploratory and confirmatory factor analysis, while internal consistency was tested by Cronbach's alpha. The final factor structure was confirmed by model fit indices. RESULTS: The study included children (n = 1088) aged between 5 and 10 years in North-West, South Africa. The reliability coefficients of the original factors could not be reproduced in this data set, with the Cronbach's alphas ranging between 0.46 and 0.78. With exploratory factor analysis, including the additional items, our data supported a 7-factor structure with acceptable internal consistency (Cronbach's alpha: 0.68-0.79; Omega: 0.75-0.85) and acceptable model fit indices (CFI: 0.91; TLI: 0.90; RMSEA: 0.05; SRMR: 0.07). Two factors (emotional wellbeing and everyday functioning) further split into separate factors for positive and negative experiences related to each of these dimensions. CONCLUSION: We confirmed a new factor structure of the parent version of the KINDLR and the additional items of the Kiddy Parents questionnaire, which can be used in the African context. Although the new factor structure has great overlap with the original structure, some items did not contribute to the factors as expected. Language and cultural differences between the original German group and the current South African study group resulted in a different factor structure.

Food intake in South African children and retinal microvascular health: The ExAMIN Youth SA study.

BACKGROUND AND AIMS: Retinal arteriolar narrowing and venular widening are associated with increased cardiovascular risk, even at young ages. Whether diet contributes to early microvascular changes in children is not widely explored. We explored the associations of frequency of healthy and unhealthy food group intake with retinal vessel calibers in black and white children. METHODS AND RESULTS: This study included school-aged (5-9 years) black (N = 433, 7.46 ± 0.98 years), and white (N = 403, 7.43 ± 0.82 years) children. Anthropometric and blood pressure measurements were taken, along with retinal vessel calibers (central retinal arteriolar (CRAE) and venular (CRVE) equivalents). Frequencies of food group intake were assessed using a food-frequency questionnaire. A factor analysis was performed to describe food group patters. Independent associations between retinal vessel calibers and frequencies of food group intake and food group patters were explored. In black children, cookies, cakes, and biscuits were associated with narrower arterioles (p < 0.05). In white children, cold sweetened beverages were associated with narrower arterioles (p = 0.02), whereas salty snacks were associated with narrower arterioles (p = 0.01) and wider venules (p < 0.05). Fruits were positively associated with CRAE (p = 0.03) in white children only. CONCLUSION: A higher frequency of unhealthy food group consumption was associated with retinal arteriolar narrowing and venular widening in both black and white children. However, fruit intake was shown beneficial for retinal microvascular health in white children only. Our findings may highlight the importance of promoting healthy eating patterns from early childhood which may reduce the risk of premature cardiovascular disease development.

Validation of a short food group questionnaire to determine intakes from healthy and unhealthy food groups in 5-9-year-old South African children.

BACKGROUND: Reliable dietary data for children are necessary to investigate associations with health outcomes. The present study aimed to develop and validate a questionnaire to determine the frequency of intakes of specific healthy and unhealthy food groups in young children. METHODS: Participants were 5-9-year-old South African children (n = 920) from 10 urban schools. Their parents completed a demographic questionnaire and the food intake questionnaire with food pictures. Based on the literature, four healthy food groups (fruits, vegetables, milk, meat/fish/poultry/eggs) and six unhealthy food groups (hot and cold sugar-sweetened beverages, candy, salty snacks, cakes and fast foods) were included, with five different frequency responses. Six experienced nutritionists assessed the face validity and content validity. After pilot testing, construct validity and homogeneity were determined in the participants. Convergent validity was determined using urinary sodium and potassium concentrations as biological intake markers. RESULTS: Nutritionists confirmed face and content validity. Caregivers confirmed understanding of the questionnaire. Three factors explained 50.2% of the variance, with most unhealthy food groups as factor 1, fruits and vegetables as factor 2, and animal source protein and milk groups clustered with sugar-sweetened beverages as factor 3. The frequency of milk group, fruits and vegetables intake correlated negatively, whereas the frequency of salty snacks and fast foods intakes correlated positively with the urinary sodium:potassium ratio. CONCLUSIONS: The healthy and unhealthy food group questionnaire has advantages of low respondent burden, as well as acceptable content and convergent validity in South African children. The questionnaire may be used to investigate associations between food intakes and health outcomes.

Identifying a urinary peptidomics profile for hypertension in young adults: The African-PREDICT study: Urinary peptidomics and hypertension: Urinary peptidomics and hypertension.

Hypertension is one of the most important and complex risk factors for cardiovascular diseases (CVDs). By using urinary peptidomics analyses, we aimed to identify peptides associated with hypertension, building a framework for future research towards improved prediction and prevention of premature development of CVD. We included 78 hypertensive and 79 normotensive participants from the African-PREDICT study (aged 20-30 years), matched for sex (51% male) and ethnicity (49% black and 51% white). Urinary peptidomics data were acquired using capillary-electrophoresis-time-of-flight-mass-spectrometry. Hypertension-associated peptides were identified and combined into a support vector machine-based multidimensional classifier. When comparing the peptide data between the normotensive and hypertensive groups, 129 peptides were nominally differentially abundant (Wilcoxon p < 0.05). Nonetheless, only three peptides, all derived from collagen alpha-1(III), remained significantly different after rigorous adjustments for multiple comparisons. The 37 most significant peptides (all p ≤ 0.001) served as basis for the development of a classifier, with 20 peptides being combined into a unifying score, resulting in an AUC of 0.85 in the ROC analysis (p < 0.001), with 83% sensitivity at 80% specificity. Our study suggests potential value of urinary peptides in the classification of hypertension, which could enable earlier diagnosis and better understanding of the pathophysiology of hypertension and premature cardiovascular disease development.

Urinary Peptidomics and Pulse Wave Velocity: The African-PREDICT Study.

Increased arterial stiffness is related to early vascular aging and is an independent predictor for cardiovascular disease and mortality. Molecular mechanisms underlying increased arterial stiffness are largely unexplored, especially at the proteome level. We aimed to explore the relationship between pulse wave velocity and urinary proteomics. We included 919 apparently healthy (no chronic illnesses) Black and White men and women (equally distributed) between 20 and 30 years from the African-PREDICT study. Capillary electrophoresis time-of-flight mass spectrometry was used to analyze the urinary proteome. We measured the carotid-femoral pulse wave velocity to estimate arterial stiffness. In the total group, pulse wave velocity correlated positively with collagen-derived peptides including collagen types I, II, III, IV, V, and IX and inversely with collagen type XI (adjusted for mean arterial pressure). Regarding noncollagen-derived peptides, pulse wave velocity positively correlated with polymeric immunoglobulin receptor peptides (n = 2) (all q-value ≤0.05). In multivariable adjusted analyses, pulse wave velocity associated positively and independently with seven urinary peptides (collagen type I, n = 5) (all p-value ≤0.05). We found significant positive and independent associations between pulse wave velocity and the collagen type I-derived peptides, suggesting that dysregulation of collagen type I in the extracellular matrix scaffold could lead to early onset of increased arterial stiffness.

Ventricular-arterial coupling and cardiovascular risk among young adults: The African-PREDICT study.

Ventricular-arterial coupling (VAC) has independent diagnostic and prognostic value for cardiovascular (CV) risk stratification, but studies on its association with anthropometric and CV factors are sparse in young individuals without overt CV disease. We aim to provide descriptive data regarding VAC and its associations with CV risk factors in young adults without overt CV disease. For 631 (mean age, 24 ± 3 yr; 51% female) individuals, VAC was determined by carotid-femoral pulse wave velocity (PWV)/global longitudinal strain (GLS). Multivariable logistic and linear regression models were performed to explore the association between PWV/GLS and CV risk factors. A P-value < 0.05 was considered statistically significant. The mean PWV/GLS was 0.33 ± 0.07 m/s%. Higher ratios of PWV/GLS associated with older age, male sex, and a higher prevalence of CV risk factors (i.e., higher blood pressure, prevalent hypertension, higher waist circumference, active smoking, higher plasma triglycerides, lower high-density lipoprotein cholesterol, and an adverse urine albumin/creatinine ratio). Furthermore, higher PWV/GLS was associated with echocardiographic measures such as lower ejection fraction and higher left ventricle mass index. In expanded logistic regression models, higher ratios of PWV/GLS were significantly associated with the prevalence of active smoking [odds ratio (OR), 1.88; confidence interval (CI) 1.36-2.58, P < 0.001] and hypertension (OR 1.98; CI 1.40-2.80, P < 0.001). We demonstrated that worse VAC reflected by higher values of PWV/GLS are significantly associated with CV risk factors in young adults. The results suggest that PWV/GLS might serve as a tool to improve the profiling of cardiovascular risk in young adults.NEW & NOTEWORTHY Assessing VAC is especially useful in heart failure and valvular heart disease, but less is known about VAC in the pathophysiology of CV disease risk in younger individuals. In young individuals without overt CV disease, we showed descriptive data regarding VAC, determined by PWV/GLS ratio, and explored the associations of VAC with clinical CV disease risk factors. Worse VAC, reflected by higher values of PWV/GLS, associated with high blood pressure and smoking in young adults.

Markers of arterial stiffness and urinary metabolomics in young adults with early cardiovascular risk: the African-PREDICT study.

INTRODUCTION: Increased exposure to risk factors in the young and healthy contributes to arterial changes, which may be accompanied by an altered metabolism. OBJECTIVES: To increase our understanding of early metabolic alterations and how they associate with markers of arterial stiffness, we profiled urinary metabolites in young adults with cardiovascular disease (CVD) risk factor(s) and in a control group without CVD risk factors. METHODS: We included healthy black and white women and men (N = 1202), aged 20-30 years with a detailed CVD risk factor profile, reflecting obesity, physical inactivity, smoking, excessive alcohol intake, masked hypertension, hyperglycemia, dyslipidemia and low socio-economic status, forming the CVD risk group (N = 1036) and the control group (N = 166). Markers of arterial stiffness, central systolic blood pressure (BP) and pulse wave velocity were measured. A targeted metabolomics approach was followed by measuring amino acids and acylcarnitines using a liquid chromatography-tandem mass spectrometry method. RESULTS: In the CVD risk group, central systolic BP (adjusted for age, sex, ethnicity) was negatively associated with histidine, arginine, asparagine, serine, glutamine, dimethylglycine, threonine, GABA, proline, methionine, pyroglutamic acid, aspartic acid, glutamic acid, branched chain amino acids (BCAAs) and butyrylcarnitine (all P ≤ 0.048). In the same group, pulse wave velocity (adjusted for age, sex, ethnicity, mean arterial pressure) was negatively associated with histidine, lysine, threonine, 2-aminoadipic acid, BCAAs and aromatic amino acids (AAAs) (all P ≤ 0.044). In the control group, central systolic BP was negatively associated with pyroglutamic acid, glutamic acid and dodecanoylcarnitine (all P ≤ 0.033). CONCLUSION: In a group with increased CVD risk, markers of arterial stiffness were negatively associated with metabolites related to AAA and BCAA as well as energy metabolism and oxidative stress. Our findings may suggest that metabolic adaptations may be at play in response to increased CVD risk to maintain cardiovascular integrity.

Early vascular ageing phenotypes and urinary targeted metabolomics in children and young adults: the ExAMIN Youth SA and African-PREDICT studies.

Some individuals are susceptible to accelerated biological ageing, resulting in premature alterations in arterial structure and function. Identifying early-onset vascular ageing characterised by arterial stiffening is vital for intervention and preventive strategies. We stratified and phenotyped healthy children (5-9 yrs) and young adults (20-30 yrs) into their vascular ageing extremes established by carotid-femoral pulse wave velocity (cfPWV) percentiles (i.e., healthy vascular ageing (HVA) and early vascular ageing (EVA)). We compared anthropometric, cardiovascular, and metabolomic profiles and explored associations between cfPWV and urinary metabolites. Children and adults in the EVA groups displayed higher levels of adiposity, cardiovascular, and lifestyle risk factors (adults only) (all p ≤ 0.018). In adults, several urinary metabolites were lower in the EVA group (all q ≤ 0.039) when compared to the HVA group, with no differences observed in children. In multiple regression analysis (adults only), we found inverse associations between cfPWV with histidine (adj. R2 = 0.038; ß = -0.192; p = 0.013) and beta-alanine (adj. R2 = 0.034; ß = -0.181; p = 0.019) in the EVA group, but with arginine (adj. R2 = 0.021; ß = -0.160; p = 0.024) in the HVA group. The inverse associations of beta-alanine and histidine with cfPWV in the EVA group is suggestive that asymptomatic young adults who present with an altered metabolomic and less desired cardiovascular profile in combination with unfavourable lifestyle behaviours may be predisposed to early-onset vascular ageing. Taken together, screening on both a phenotypic and metabolic level may prove important in the early detection, prevention, and intervention of advanced biological ageing.

Using urinary metabolomics to identify metabolic pathways linked to cardiac structural alterations in young adults: The African-PREDICT study.

BACKGROUND AND AIMS: Risk factor exposure from young ages was shown to contribute to cardiovascular events - cardiac hypertrophy, which may be accompanied by an altered metabolism. To determine how early metabolic alterations associate with myocardial structural changes, we profiled urinary metabolites in young adults with cardiovascular disease (CVD) risk factor(s) and a control group without CVD risk factors. METHODS AND RESULTS: We included healthy adults (N = 1202), aged 20-30 years, stratified based on risk factors, i.e., obesity, physical inactivity, elevated blood pressure (BP), hyperglycemia, dyslipidemia, low socio-economic status, smoking and excessive alcohol use - forming the CVD risk group (N = 1036) and the control group (N = 166). Relative wall thickness (RWT) and left ventricular mass index (LVMi) were measured using echocardiography. Targeted metabolomics data were obtained using a liquid chromatography-tandem mass spectrometry method. Clinic systolic BP, 24 h BP and RWT were higher in the CVD risk group compared to the control group (all P ≤ 0.031). Exclusively in the CVD risk group, RWT associated with creatine and dodecanoylcarnitine; while LVMi associated with glycine, serine, glutamine, threonine, alanine, citrulline, creatine, proline, pyroglutamic acid and glutamic acid (all P ≤ 0.040). Exclusively in the control group, LVMi associated with propionylcarnitine and butyrylcarnitine (all P ≤ 0.009). CONCLUSION: In young adults without CVD, but with CVD risk factors, LVMi and RWT associated with metabolites linked energy metabolism (shifting from solely fatty acid oxidation to glycolysis, with impaired creatine kinase activity) and oxidative stress. Our findings support early onset metabolic changes accompanying cardiac structural alterations due to lifestyle and behavioural risk factors.

Genetically predisposed and resilient animal models of depression reveal divergent responses to early-life adversity.

OBJECTIVE: Early-life adversity (ELA) is one of the strongest predictors of childhood depression that may be exacerbated by a genetic predisposition to develop depression. We therefore investigated the bio-behavioural effects of an early-life stressor in an accepted rodent model of depression. METHODS: The Flinders sensitive line (FSL) and resistant line (FRL) rats were subjected to an early-life stressor, whereafter their bio-behavioural response during pubertal onset was evaluated. Male and female pups were maternally separated for 3 h per day from postnatal day 02 (PND02) to 17, when they were also weaned. Control animals were left undisturbed, until weaning on PND21. Depressive-like behaviour was analysed on PND21 and reassessed on PND36. Hippocampal monoamine levels, markers of oxidative stress and metabolic markers implicating mitochondrial function were also measured. RESULTS: On PND21, the non-maternal separation and early weaning (non-MSEW) FSL rats spent 10% more time mobile than their FRL controls in the tail suspension test (TST) yet displayed increased depressive-like behaviour in the forced swim test (FST) on PND36. This depressive-like behaviour coincided with increased hippocampal norepinephrine levels, serotonin turnover and a dysfunctional redox state. Maternal separation and early weaning (MSEW) appeared to initially reduce early-life (PND21) depressive-like behaviour in the TST but then induced depressive-like behaviour on PND36 and increased norepinephrine levels more profoundly in the FRL rats. CONCLUSION: These findings highlight the need to further investigate the stress response pathway in these animals and that the absence or presence of genetic susceptibility may influence the presentation of ELA effects.

Running from depression: the antidepressant-like potential of prenatal and pre-pubertal exercise in adolescent FSL rats exposed to an early-life stressor.

OBJECTIVE: We aimed to answer the questions of whether early-life (perinatal and/or juvenile) exercise can induce antidepressant-like effects in a validated rodent model of depression, and whether such early-life intervention could prevent or reverse the adverse effects of early-life stress in their offspring. METHODS: Male and female Flinders sensitive line rats born to a dam that exercised during gestation, or not, were either maternally separated between PND02 and 16 and weaned on PND17 or not. Half of these animals then underwent a fourteen-day low-intensity exercise regimen from PND22. Baseline depressive-like behaviour was assessed on PND21 and then reassessed on PND36, whereafter hippocampal monoamine levels, redox state markers and metabolic markers relevant to mitochondrial function were measured. RESULTS: Pre-pubertal exercise was identified as the largest contributing factor to the observed effects, where it decreased immobility time in the FST by 6%, increased time spent in the open arms of the EPM by 9%. Hippocampal serotonin and norepinephrine levels were also increased by 35% and 26%, respectively, whilst nicotinic acid was significantly decreased. CONCLUSION: These findings suggest that pre-pubertal low-intensity exercise induces beneficial biological alterations that could translate into antidepressant behaviour in genetically susceptible individuals.

Specific and sensitive GC-MS analysis of hypusine, Nε-(4-amino-2-hydroxybutyl)lysine, a biomarker of hypusinated eukaryotic initiation factor eIF5A, and its application to the bi-ethnic ASOS study.

Hypusination is a unique two-step enzymatic post-translational modification of the Nε-amino group of lysine-50 of the eukaryotic initiation factor 5A (eIF5A). We developed a specific and sensitive gas chromatography-mass spectrometry (GC-MS) method for the measurement of biological hypusine (Hyp), i.e., Nε-(4-amino-2-hydroxybutyl)lysine. The method includes a two-step derivatization of Hyp: first esterification with 2 M HCl in CH3OH (60 min, 80 °C) to the methyl ester (Me) and then acylation with penta-fluoro-propionic (PFP) anhydride in ethyl acetate (30 min, 65 °C). Esterification with 2 M HCl in CD3OD was used to prepare the internal standard. The major derivatization product was identified as the un-labelled (d0Me) and the deuterium-labelled methyl esters (d3Me) derivatives: d0Me-Hyp-(PFP)5 and d3Me-Hyp-(PFP)5, respectively. Negative-ion chemical ionization generated the most intense ions with m/z 811 for d0Me-Hyp-(PFP)5 and m/z 814 for the internal standard d3Me-Hyp-(PFP)5. Selected-ion monitoring of m/z 811 and m/z 814 was used in quantitative analyses. Free Hyp was found in spot urine samples (10 µL) of two healthy subjects at 0.60 µM (0.29 µmol Hyp/mmol creatinine) in the female and 1.80 µM (0.19 µmol Hyp/mmol creatinine) in the male subject. The mean accuracy of the method in these urine samples spiked with 1-5 µM Hyp was 91-94%. The limit of detection (LOD) of the method is 1.4 fmol Hyp. The method was applied to measure the urinary excretion rates of Hyp in healthy black (n = 38, age 7.8 ± 0.7 years) and white (n = 41, age 7.7 ± 1.0 years) boys of the Arterial Stiffness in Offspring Study (ASOS). The Hyp concentrations were 3.55 [2.68-5.31] µM (range 0.54-9.84 µM) in the black boys and 3.87 [2.95-5.06] µM (range 1.0-11.7 µM) in the white boys (P = 0.64). The creatinine-corrected excretion rates were 0.25 [0.20-0.29] µmol/mmol (range 0.11-0.36 µmol/mmol) in the black boys and 0.26 [0.21-0.30] µmol/mmol (range 0.10-0.45 µmol/mmol) in the white boys (P = 0.82). These results suggest that there is no ethnic-related difference in the ASOS population in the eIF5A modification. Remarkable differences were found between black and white boys with respect to correlations of urinary Hyp with amino acids and advanced glycation end-products of Lys, Arg and Cys. Deoxyhypusine, formally the direct precursor of Hyp, seems not to be excreted in the urine by healthy subjects.

Development, validation of a GC-MS method for the simultaneous measurement of amino acids, their PTM metabolites and AGEs in human urine, and application to the bi-ethnic ASOS study with special emphasis to lysine.

A gas chromatography-mass spectrometry (GC-MS) method was developed and validated in relevant concentration ranges for the simultaneous measurement of L-lysine (Lys, L) and its Nε- and Nα-methylated (M), Nε- and Nα-acetylated (Ac), Nε-carboxymethylated (CM) and Nε-carboxyethylated (CE) metabolites in human urine. Analyzed Lys metabolites were the post-translational modification (PTM) products Nε-mono-, di- and trimethyllsine, Nε-MML, Nε-DML, Nε-TML, respectively, Nα-ML, Nε-AcL, Nα-AcL, and its advanced glycation end-products (AGEs) Nε-CML, Nε-CM-[2,4,4-2H3]Lys (d3-CML), Nε-CEL and furosine. AGEs of arginine (Arg) and cysteine (Cys) were also analyzed. De novo synthesized trideutero-methyl esters (R-COOCD3) from unlabelled amino acids and derivatives were used as internal standards. Native urine samples (10 µL aliquots) were evaporated to dryness under a stream of nitrogen. Analytes were esterified using 2 M HCl in methanol (60 min, 80 °C) and subsequently amidated by pentafluoropropionic anhydride in ethyl acetate (30 min, 65 °C). The generated methyl ester-pentafluoropropionyl (Me-PFP) derivatives were reconstituted in borate buffer and extracted immediately with toluene. GC-MS analyses were performed by split-less injection of 1-µL aliquots, oven-programmed separation and negative-ion chemical ionization (NICI). Mass spectra were generated in the scan mode (range, m/z 50-1000). Quantification was performed in the selected-ion monitoring (SIM) mode using a dwell time of 50 or 100 ms for each ion. The GC-MS method was suitable for the measurement of Lys and all of its metabolites, except for the quaternary ammonium cation Nε-TML. The Me-PFP derivatives of Lys, Arg and Cys and its metabolites eluted in the retention time window of 9 to 14 min. The derivatization of Nε-CML, d3-CML and Nε-CEL was accompanied by partial Nε-decarboxylation and formation of the Me-PFP Lys derivative. The lowest derivatization yield was observed for Nε-DML, indicating a major role of the Nε-DML group in Lys derivatization. The GC-MS method enables precise (relative standard deviation, RSD < 20%) and accurate (bias, < ± 20%) simultaneous measurement of 33 analytes in human urine in relevant concentration ranges. We used the method to measure the urinary excretion rates of Lys and its PTM metabolites and AGEs in healthy black (n = 39) and white (n = 41) boys of the Arterial Stiffness in Offspring Study (ASOS). No remarkable differences were found indicating no ethnic-related differences in PTM metabolites and AGEs except for Nε-monomethyllysine and S-(2-carboxymethylcysteine).

Determining Underlying Mechanisms of Early Vascular Ageing by Clustered Analysis: The African-PREDICT Study.

OBJECTIVE: Identifying individuals at increased risk of early vascular ageing (EVA) is paramount to inform intervention and prevention strategies and curb the increasing burden of cardiovascular disease. METHODS: We stratified and phenotyped pre-screened young apparently healthy South African adults (20-30 yrs) (n=1,041) into vascular ageing profile groups based on carotid femoral pulse wave velocity (cfPWV) percentiles (healthy vascular ageing [HVA]; average vascular ageing [AVA] and EVA). We further compared various anthropometric, cardiovascular (CV), oxidative stress and lifestyle risk factors and determined factor scores to explore associations between CV measures and factor clusters to explore associations in those at risk of EVA. RESULTS: Young adults in the EVA group displayed marked phenotypic characteristics in terms of anthropometry, CV, and lifestyle risk factors, even though cfPWV were within healthy ranges. Blood pressure (brachial and central) and cfPWV were all incrementally higher across all three vascular ageing groups (p-trend ≤0.011). Hypertension, lifestyle risk factors such as self-reported smoking and alcohol consumption were all highest in the EVA group (p-trend ≤0.046). Additionally, in the EVA group only, cfPWV (adj. R2=0.028; ß=0.171; p=0.042) associated positively with Factor 2 (oxidative stress and antioxidant capacity). No associations existed between Factor 1 (basic lipids) and any anthropometric or CV measures (p>0.050). CONCLUSION: Young adults with higher cfPWV presented with a less favourable vascular profile and more unhealthy lifestyle behaviours compared to groups with lower cfPWV. In the EVA group, cfPWV positively associated with a cluster of oxidative stress and antioxidant capacity. Early lifestyle behaviours may have the ability to modify the balance between oxidants and antioxidants, potentially contributing to early onset arterial stiffness.

Unusual Derivatization of Methylmalonic Acid with Pentafluorobenzyl Bromide to a Tripentafluorobenzyl Derivative and Its Stable-Isotope Dilution GC-MS Measurement in Human Urine.

Methylmalonic acid (MMA) is a very short dicarboxylic acid (methylpropanedioic acid; CH3CH(COOH)2; pKa1, 3.07; pKa2, 5.76) associated with vitamin B12 deficiency and many other patho-physiological conditions. In this work, we investigated several carboxylic groups-specific derivatization reactions and tested their utility for the quantitative analysis of MMA in human urine and plasma by gas chromatography-mass spectrometry (GC-MS). The most useful derivatization procedure was the reaction of unlabeled MMA (d0-MMA) and trideutero-methyl malonic acid (d3-MMA) with 2,3,4,5,6-pentafluorobenzyl bromide (PFB-Br) in acetone. By heating at 80 °C for 60 min, we observed the formation of the dipentafluorobenzyl (PFB) ester of MMA (CH3CH(COOPFB)2). In the presence of N,N-diisopropylamine, heating at 80 °C for 60 min resulted in the formation of a tripentafluorobenzyl derivative of MMA, i.e., CH3CPFB(COOPFB)2). The retention time was 5.6 min for CH3CH(COOPFB)2 and 7.3 min for CH3CPFB(COOPFB)2). The most intense ions in the negative-ion chemical ionization (NICI) GC-MS spectra of CH3CH(COOPFB)2 were mass-to-charge (m/z) 233 for d0-MMA and m/z 236 for d3-MMA. The most intense ions in the NICI GC-MS spectra of CH3CPFB(COOPFB)2 were mass-to-charge (m/z) 349 for d0-MMA and m/z 352 for d3-MMA. These results indicate that the H at C atom at position 2 is C-H acidic and is alkylated by PFB-Br only in the presence of the base N,N-diisopropylamine. Method validation and quantitative analyses in human urine and plasma were performed by selected ion monitoring (SIM) of m/z 349 for d0-MMA and m/z 352 for the internal standard d3-MMA in the NICI mode. We used the method to measure the urinary excretion rates of MMA in healthy black (n = 39) and white (n = 41) boys of the Arterial Stiffness in Offspring Study (ASOS). The creatinine-corrected excretion rates of MMA were 1.50 [0.85-2.52] µmol/mmol in the black boys and 1.34 [1.02-2.18] µmol/mmol in the white boys (P = 0.85; Mann-Whitney). The derivatization procedure is highly specific and sensitive for MMA and allows its accurate and precise measurement in 10-µl of human urine by GC-MS.

Socioeconomic inequalities, modifiable lifestyle risk factors, and retinal vessel calibers: The African-PREDICT Study.

OBJECTIVE: Lifestyle risk factors vary between socioeconomic status (SES) groups and may influence cardiovascular function differently. The retinal microvasculature allows for monitoring early changes in cardiovascular health, and therefore, we investigated whether retinal vessel calibers associate differently with modifiable risk factors in different SES groups. METHODS: We included 1064 young adults (aged 20-30 years) grouped by low and high SES. The central retinal artery and vein equivalents (CRAE, CRVE) were determined from fundus images captured using the Dynamic Retinal Vessel Analyzer (Imedos Systems GmbH, Jena, Germany). We collected anthropometry, self-reported alcohol consumption, and biochemical data. RESULTS: Retinal vessel calibers did not differ between SES groups (p ≥ .80) after adjusting for sex and ethnicity. Unique independent associations were observed in the low SES group, where CRAE (ß = 0.08, p = .042) and CRVE (ß = .14, p = .001) associated positively with cotinine and body mass index, respectively. In the high SES group, CRAE (ß = -0.09, p = .027) associated negatively with alcohol consumption. CONCLUSION: At young ages, retinal vessel calibers associated differently with modifiable lifestyle risk factors within each SES group. Our data highlight the importance of detecting adverse lifestyle risk factors among young adults from diverse socioeconomic settings to improve prevention of cardiovascular disease.

Growth differentiating factor-15 and adiposity in young adults: The African-PREDICT study.

BACKGROUND AND AIMS: Circulating growth differentiation factor-15 (GDF-15) is a stress-responsive cytokine that increases in older individuals with established cardiovascular disease (CVD) and obesity. To address potential targets in primary prevention, we aimed to determine whether body weight, waist circumference, waist/height ratio, body mass index (BMI), body surface area (BSA) and leptin associate with GDF-15 in young underweight, lean and overweight/obese (ow/ob) adults. METHODS AND RESULTS: We included 1189 adults aged 20-30 years. We grouped participants as underweight (BMI ≤ 18 kg/m2, n = 59), lean (BMI > 18 kg/m2 and ≤25 kg/m2; n = 616) or ow/ob (BMI ≥ 25 kg/m2; n = 514) and determined serum GDF-15 and leptin levels. Body composition measurements, leptin and blood pressure readings were higher in the ow/ob group compared to the underweight and lean groups (all p < 0.0001). GDF-15 was higher in the underweight group compared to the lean and combined ow/ob groups (p = 0.041), and higher in obese (BMI ≥ 30 kg/m2) compared to overweight (p = 0.002) individuals. In multiple regression analysis, we found positive associations (all p ≤ 0.020) of body weight (adj. R2 = 0.398; ß = 0.11), waist circumference (adj. R2 = 0.271; ß = 0.11), waist/height ratio (adj. R2 = 0.168; ß = 0.14), BMI (adj. R2 = 0.263; ß = 0.14), BSA (adj. R2 = 0.508; ß = 0.083) and leptin (adj. R2 = 0.622; ß = 0.10) with GDF-15 in the ow/ob group. However, waist circumference (adj. R2 = 0.536; ß = -0.45), waist/height ratio (adj. R2 = 0.471; ß = -0.51) and leptin (adj. R2 = 434; ß = -0.25) associated inversely with GDF-15 in the underweight group (all p < 0.050). CONCLUSION: Our findings may suggest that in young adults with either underweight or excess adiposity, increased GDF-15 levels may contribute to the development of future cardiovascular health risks associated with pro-inflammation.

Ethnicity and Arterial Stiffness.

Early vascular aging reflects increased arterial stiffness of central blood vessels at young chronological ages and powerfully predicts cardiovascular events and mortality, independent of routine brachial blood pressure and other risk factors. Since ethnic disparities exist in routine blood pressure, in hypertension and cardiovascular outcomes, this review evaluates major studies comparing arterial stiffness through the life course between different ethnic groups or races (which have no biological definition)-in children, adolescents, young, and middle-aged adults and the very elderly. Most report that compared with white European-origin samples, populations of black African descent have increased central arterial stiffness throughout different life stages, as well as a more rapid increase in arterial stiffness at young ages. Exceptions may include African Caribbean origin people in Europe. Differences in vascular structure and function are clearest, where obesity, socioeconomic, and psychosocial factors are most marked. Few studies evaluate a wider spectrum of ethnic groups or factors contributing to these ethnic disparities. Genetic effects are not obvious; maternal risk and intergenerational studies are scarce. Nevertheless, across all ethnic groups, for given levels of blood pressure and age, some people have stiffer central arteries than others. These individuals are most at risk of vascular events and mortality and, therefore, may benefit from early, as yet untested, preventive action and treatment.

Racial differences of early vascular aging in children and adolescents.

The prevalence of non-communicable disease (NCDs) is rising globally, with a large burden recorded in sub-Saharan countries and populations of black race/ethnicity. Accelerated vascular deterioration, otherwise known as early vascular aging (EVA), is the underlying factor for highly prevalent NCDs such as hypertension. The etiology of EVA is multifactorial with a central component being arterial stiffness with subsequent development of hypertension and cardiovascular complications. Although arterial stiffness develops with increasing age, many children and adolescents are subjected to the premature development of arterial stiffness, due to genetic or epigenetic predispositions, lifestyle and behavioral risk factors, and early life programming. Race/ethnic differences in pediatric populations have also been reported with higher aortic stiffness in black (African American) compared with age-matched white (European American) counterparts independent of blood pressure, body mass index, or socioeconomic status. With known evidence of race/ethnic differences in EVA, the pathophysiological mechanisms underlying graded differences in the programming of EVA are still sparse and rarely explored. This educational review aims to address the early life determinants of EVA in children and adolescents with a particular focus on racial or ethnic differences.