Rajavel Elango

Associate Professor

Relevant Degree Programs

 

Graduate Student Supervision

Doctoral Student Supervision (Jan 2008 - May 2019)
The complexity of understanding human milk components and infant brain development (2016)

Understanding which and how human milk components contribute to infant brain development is complicated in part by their large diversity, complex arrangement in the milk matrix and potential interaction in metabolism. This research addressed the importance of studying the composition of minor milk lipids and of exploring their relationship with non-lipid milk components in infant brain development. More specifically, the milk fat globule membrane (MFGM), a complex tri-layer of cholesterol, glycoproteins, and polar lipids including ethanolamine plasmalogens (Pls-PE), naturally emulsifies milk triacylglycerols but is not currently added to infant milk substitutes. Clinical evidence suggests MFGM plays a role in cognitive function. Whether MFGM directly affects the developing brain is unclear. Due to analytical challenges, little is known about the fatty acid composition of human MFGM lipids, particularly Pls-PE. Pls-PE may be enriched in long-chain polyunsaturated fatty acids (LC-PUFA) such as docosahexaenoic acid (DHA), an important neural lipid during development. Additionally, milk contains different forms of water-soluble choline (WSC) compounds (free choline, glycerophosphocholine, phosphocholine) for which distinct biological roles are unknown, although choline as a molecule per se is an important structural component of the brain and a precursor of the neurotransmitter acetylcholine. After developing an analytical method for separation and recovery of milk Pls-PE, the first study demonstrated both human and cow milk Pls-PE are enriched in LC-PUFA including DHA compared to other phospholipids. Milk Pls-PE DHA does not seem to vary with maternal DHA intake. Using artificially-reared infant rats, the second study showed that developmental brain phospholipids and metabolites differ between rats fed formula with or without MFGM, with a closer phospholipid composition to mother-reared rats in rats fed MFGM. By analyzing human preterm and term milk samples for WSC composition using mass spectrometry in the third study, we confirmed previous findings of the wide variability in WSC total content and composition in human milk and reported no significant association between individual WSC compounds. These studies provide new knowledge that milk contains novel components potentially relevant to the brain, and, while the mechanisms for improved cognition remain unclear, MFGM affects neonatal brain phospholipid composition.

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Master's Student Supervision (2010 - 2018)
Determination of threonine requirements and the metabolic availability of threonine from food sources in healthy school-aged children (2018)

Threonine, an indispensable amino acid, is required for protein synthesis throughout the body. Due to its quantitative importance in gut mucosal proteins, consuming adequate amounts of threonine is essential for proper digestive and immune function during growth. Currently, the dietary reference intakes (DRIs) recommend an estimated average requirement (EAR), and recommended dietary allowance (RDA) of 19 and 24 mg/kg/d for threonine intake in 6-10y children based on factorial (mathematical) calculations. In addition, it is unknown to what extent dietary threonine is available for protein synthesis from food protein sources in school-aged children. The primary objective of this thesis was to determine the dietary requirement for threonine in 6-10y healthy children using the minimally invasive Indicator Amino Acid Oxidation (IAAO) method. The secondary objective was to compare threonine metabolic availability from casein (animal protein) and soy (vegetable protein) in the same set of children. Six healthy Canadian children (three boys: three girls) aged 7.5 ± 1.4 y randomly received 6-9 test threonine intakes each, ranging from 1-50 mg/kg/d, with an amino acid mixture patterned after egg protein. Study day diets were complete with protein provided at 1.5 g/kg/d, energy provided at 1.7x the resting energy expenditure. To determine threonine requirements, the oxidation of L-[1-​¹³​C]-phenylalanine to ​¹³​CO​₂ ​(F​¹³​CO​₂​) in response to the test intakes was used. To determine threonine metabolic availability, the children were tested at 0 mg/kg/d, and at 11 mg/kg/d from three different sources (threonine as crystalline amino acid, casein and soy), and the oxidation of L-[1-​¹³​C]-Phenylalanine to ​13​CO​₂ ​measured. Threonine requirement was determined to be 21.9 mg/kg/d (95% CI: 10.5 – 33.4 mg/kg/d). The metabolic availability of threonine was determined to be 96.6% and 83.4% from casein and soy, respectively. Threonine requirements in school-aged children were determined for the first time directly, and the results are ~15% higher than current DRI recommendations of EAR 19 mg/kg/d. In addition, the metabolic availability of threonine was found to be higher in casein compared to soy, which needs to be considered when making dietary recommendations.

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Genome-wide epigenetic and genetic investigation of male infertility (2018)

Genetic causes have been known to be involved in up to one-third of male infertility cases. Studies on the sperm and testis from infertile men have suggested that this population may also have higher rates of altered epigenetic modifications, particularly DNA methylation. Thus, we hypothesized that genetic and epigenetic causes may play a role together in male infertility.We used Sanger sequencing to observe the DNA methylation at imprinted differentially methylated regions– H19, IG-GTL2, and MEST – in the sperm of infertile men with oligozoospermia to assess the occurrence of alterations. We analyzed semen samples from fifty-three men (9 fertile and 44 infertile – stratified by sperm concentration). Although we observed altered cases in 13% (3/23) of men with severe oligozoospermia and none among controls, this finding was not significant. We genotyped the same men at the MTHFR C677T single nucleotide polymorphism (SNP) to determine whether this methyl supply gene is associated with DNA methylation in the sperm. We observed a trend that all altered cases had the CT genotype at this SNP and in the severe oligozoospermic subgroup, suggesting a combinatorial effect. Motivated from these findings, we conducted two genome-wide investigations to identify novel genes with DNA methylation alterations and/or SNPs relating to male infertility. We evaluated the genome-wide DNA methylation in the testis of twenty-four men (8 fertile and 16 infertile). Using predictive models, we identified 359 CpGs with altered DNA methylation among the infertile men. Of these loci and using functional analyses, we identified NDE1 which is a gene involved with cell cycle progression and centrosome formation. In the genome-wide SNP analysis of twenty men (13 fertile and 7 infertile), we identified 39 SNPs using machine learning models. Of these SNPs, candidates included MTR – a gene involved with the same folate pathway as MTHFR – and NIN which is a gene involved with proper chromosome segregation during cell division.In summary, we observed altered DNA methylation and SNPs in infertile men. We presented evidence that altered DNA methylation and changes in genetic sequence in genes involved with cell division and progression may impair spermatogenesis leading to male infertility.

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Application of metabolomics to identify metabolite patterns showing the importance of dietary carbohydrate source in neonatal rats (2016)

Lactose is a glucose and galactose disaccharide and is found exclusively in mammalian milk. The evolution of lactose as a unique component of human milk (with 50% galactose) and its metabolic advantage in infants is poorly understood. There is also a limited understanding of the potential implications of clinical nutrition support using lactose-free nutrition, such as dextrose (glucose) as the sole carbohydrate source in intravenous nutrition and glucose-corn syrup solids (CSS) (100% glucose) in formula for infants. The liver takes up 90% of galactose. In contrast, the liver clears only 30% of glucose. Glucose stimulates release of insulin from the pancreas whereas galactose does not. The goal of my thesis was to use ‘metabolomics’, in a neonatal rat model of gastrostomy feeding, to differentiate metabolic effects in rat pups fed milk formula complete in protein, fat, minerals and vitamins, but with either lactose or CSS as the carbohydrate source. I hypothesize that galactose and specific metabolites of galactose metabolism will be differentiated between lactose-fed and CSS-fed rat pups by target compound analysis; and targeted metabolomics will highlight differences in metabolite patterns in hepatic metabolism from lactose or CSS feeding. Liver, plasma and urine samples were collected at 10 and 14 days after birth. Significant differences in galactose and galactonate levels were observed in the liver at day 10 and 14, with no differences glucose. Metabolites that were different between the groups were: D-Ribose, pyrimidine, glycine and malate in rat pups at day 10 and leucine and isoleucine at day 10 and 14, which were significantly higher in the lactose-fed group. The data obtained show that early dietary lactose has definitive effects on hepatic metabolites that can be mapped to pathways of carbohydrate, protein, and fat metabolism and are different to non-lactose carbohydrate feeding. My findings suggest that utilization of galactose, as a consistent source of glucose could prove beneficial for supply to glucose-dependent organs such as the rapidly developing infant brain. Advancing the use of metabolomics to enhance understanding of the impact of diet will provide an opportunity to improve health outcome for infants.

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Application of stable isotope tracers to examine phenylalanine metabolism and protein requirements in children with phenylketonuria (pku) (2015)

Phenylketonuria (PKU) is an inherited inborn error of phenylalanine (PHE) metabolism caused by deficiency of hepatic enzyme phenylalanine hydroxylase (PAH). Therefore, PHE accumulates in plasma leading to mental retardation and developmental delay. Kuvan® (Sapropterin dihydrochloride), a synthetic form of tetrahydrobiopterin (BH₄), has been shown to reduce plasma PHE levels in PKU, but not all patients respond to sapropterin treatment. The major mode of treatment remains nutritional management with dietary restriction of PHE and provision of sufficient protein. The dietary protein requirement in children with PKU remains unknown. Therefore the objectives of the current thesis were: 1) to identify sapropterin responsiveness in PKU children using a minimally invasive L-[1-¹³C] phenylalanine breath test (¹³C-PBT), and 2) to determine protein requirements in PKU children using the indicator amino acid oxidation (IAAO) technique.Experiment 1- Nine children with PKU (5-18y) underwent ¹³C-PBT tracer protocols twice, once before and once after 1-2 weeks of sapropterin therapy. ¹³CO₂ was measured using isotope ratio mass spectrometer (IRMS). The study protocol was tested in healthy children (n= 6) as proof of principle. Experiment 2- Four PKU children (5-18y) were recruited to participate in test protein intakes (ranging from deficiency to excess 0.2 – 3.2 g/kg/d) with the IAAO protocol using L-[1-¹³C] leucine, followed by collection of breath and urine samples over 8 hours. Results 1- ¹³CO₂ productions in all children with PKU pre-sapropterin treatment were low, except in one child (PKU04). Five children with PKU showed a significantly higher peak enrichment after sapropterin treatment at 20min. Three PKU children had no change in ¹³CO₂ production post sapropterin therapy. Results 2- The mean protein requirement, identified using 2-phase linear regression analysis was determined to be 1.85 g/kg/d. This result is significantly higher than the most recent PKU recommendations (2014) (1.14 – 1.33g/kg/d, based on 120-140% above current recommended dietary allowance RDA). These findings show that the ¹³C-PBT can be a minimally invasive method to examine in vivo PHE metabolism in PKU children responsive to sapropterin therapy. Also, current recommendations for optimal protein intake may be underestimated.

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Nonessential amino acid metabolism in humans (2015)

Nutritionally, there is a dietary requirement for the essential amino acids (EAA) but also a requirement for nitrogen (N) intake for the de novo synthesis of the nonessential amino acids (NEAA). It has been suggested that some NEAA may be more metabolically important than others. The first study (Glutamate Requirement Study) aims to examine the application of the indicator amino acid oxidation (IAAO) technique to determine if a dietary requirement for glutamate exists in adult humans. The second study (NEAA Study) aims to determine the metabolic demand of nine of the NEAA (Ala, Arg, Asn, Asp, Gln, Glu, Gly, Pro, Ser) as an ideal N source using the IAAO technique. Seven subjects were maintained on an adaptation diet for 2 days prior to each test day. Each subject participated in two or eleven test diet intakes, assigned randomly, in the glutamate study and the NEAA study, respectively. In the glutamate study, the diets corresponded to the amino acid pattern present in egg protein, in which all glutamate and glutamine was present as glutamate, or removed, with serine used to make the diets isonitrogenous. In the NEAA Study, one test intake was a base diet consisting of only the EAA provided at the recommended dietary allowance. All other test intakes involved the base diet with the addition of one NEAA to meet a 50:50 ratio of EAA: NEAA on a N basis. Each study day followed the IAAO protocol using L-[1-¹³C]-Phenylalanine as the indicator. Breath and urine samples were collected at baseline and isotopic steady state. Enrichments of ¹³C in breath were analyzed by isotope ratio mass spectrometry to calculate F¹³CO₂. In the glutamate study, a paired-samples t-test did not find a significant difference between the F¹³CO₂ in response to the two glutamate intakes. In the NEAA study, repeated measures ANOVA with post hoc multiple comparisons showed that seven of the nine NEAA decrease IAAO significantly. Thus the results suggest that in healthy adults, there is no dietary requirement for glutamate, and that most NEAA are good N sources, in the presence of adequate EAA.

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Determination of lysine requirements in healthy pregnant women using the indicator amino acid oxidation technique (2014)

Perinatal nutrient status influences the health of both mother and child. Lysine, an essential amino acid found mainly in animal derived products, is the first limiting amino acid in plant proteins. Inadequate lysine intakes during pregnancy may impact foetal health in both the short and long-term. Current Dietary Reference Intakes (DRI) recommendations extrapolate lysine requirements during pregnancy from non-pregnant adult data and may underestimate true requirements. Moreover, these recommendations remain constant throughout pregnancy and do not reflect the dynamic metabolic adaptations that occur during gestation. This study aims to define a quantitative lysine requirement in healthy pregnant women and to determine whether lysine requirements vary between phases of gestation. Two phases of pregnancy (early gestation, 12-19 weeks; late gestation, 33-39 weeks) were studied using the indicator amino acid oxidation (IAAO) technique. Pregnant women (22-36y) consumed a diet containing a random lysine intake (range = 6 - 86 mg/kg/d) in a crystalline amino acid mixture based on egg protein profile. Diets were isonitrogenous with caloric and protein intakes maintained at 1.7 x resting energy expenditure and 1.5g/kg/d, respectively. Breath and urine samples were collected at baseline and isotopic steady state. Lysine requirements were determined by measuring oxidation of L-[1-¹³C]-phenylalanine to ¹³CO₂ (F¹³CO₂). Bi-phase linear regression crossover analysis was used to determine a breakpoint (estimated average requirement, EAR) in F¹³C₂ data. The breakpoint in early gestation (n=27) was determined to be 36.6 mg/kg/d (r² = 0.484, Upper 95% CI = 46.2), similar to current non-pregnant recommendations of 41mg/kg/d. The breakpoint in late gestation (n=36) was determined to be 50.3 mg/kg/d (r² = 0.664, Upper 95% CI = 60.4), and 25% higher than current DRI recommendations of 41 mg/kg/d. Urinary phenylalanine flux did not change in both early and late gestation due to lysine intake. These data are the first to directly define a quantitative requirement for lysine during human pregnancy and to account for gestational age, describing an increase in requirement as pregnancy progresses. We expect these results will have significant implications for setting recommendations globally where plant based diets are the primary source of protein and amino acids.

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Determination of protein requirements of healthy pregnant women during early and late gestation using the indicator amino acid oxidation technique (2014)

Adequate maternal dietary protein (PRO) intake is necessary to support rapid tissue accretion during a healthy pregnancy. Both insufficient and excessive maternal PRO intake during pregnancy is associated with intrauterine growth restriction (IUGR) of the fetus. IUGR increases the risk of neonatal morbidity and mortality, and is associated with an increased risk of future health problems, including cardiovascular disease, kidney disease, obstructive airway disease, and obesity. However, current PRO intake recommendations for healthy pregnant women are based on factorial calculations of nitrogen balance data derived from non-pregnant adults. Thus, an estimate of PRO requirements based on pregnancy-specific data is needed. PRO requirements of healthy pregnant women at 11-20 (early) and 31-38 (late) weeks gestation were determined using the indicator amino acid oxidation (IAAO) method. Twenty-nine healthy women (age 24-37) each randomly received a different test PRO intake (range = 0.22-2.56 g/kg/day) during each study day in early (n=37) and late (n=44) gestation. The diets were isocaloric and provided energy at 1.7 X resting energy expenditure (REE). PRO was given as a crystalline amino acid mixture based on egg PRO composition, except phenylalanine and tyrosine, which were maintained constant across intakes. PRO requirements were determined by measuring the oxidation of L-[1-¹³C]phenylalanine to ¹³CO₂ (F¹³CO₂). Breath and urine samples were collected at baseline and isotopic steady state. Linear regression crossover analysis identified a breakpoint (requirement) at minimal F¹³CO₂ in response to different PRO intakes. The estimated average requirement (EAR) for PRO in early and late gestation was determined to be 1.22 and 1.52 g/kg/d, respectively. Both of these estimates are significantly greater than the EAR of 0.88 g/kg/d currently recommended by the Dietary Reference Intakes (DRI 2005). Our results indicate increased demand for PRO before 20 weeks gestation (on a gram per kilogram body weight basis), a consideration that has not been addressed by current DRI recommendations. This study is the first to directly estimate gestational PRO requirements in a population composed solely of healthy pregnant women, and suggests that current recommendations based on the nitrogen balance method and factorial calculations underestimate PRO requirements.

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