Effect of a pulse-based diet and aerobic exercise on bone measures and body composition in women with polycystic ovary syndrome: A randomized controlled trial.

Polycystic ovary syndrome (PCOS) is the most common endocrine disorder in women of reproductive age, with clinical symptoms including menstrual dysfunction and hyperandrogenemia, as well as insulin resistance which is thought to be a key contributing factor to symptoms. Insulin is also thought to positively affect bone while oligo- and amenorrhea are known to negatively affect bone. Lifestyle modification is the first recommendation to treat symptoms of PCOS; however, little is known about the effect of lifestyle interventions on bone measures in this population. Pulses (e.g., chickpeas, beans, split peas, lentils) have been shown to lower fasting insulin, and the objective of this study was to determine the effect of a pulse-based diet compared to the therapeutic lifestyle changes (TLC) diet on bone measures and body composition in women with PCOS. Women aged 18-35 years with PCOS were randomized to either a pulse-based diet or the TLC diet for 16-weeks while following an aerobic exercise program. Thirty-one in the TLC group and 29 in the pulse group completed dual-energy X-ray absorptiometry analysis following the intervention. After 16-weeks, both groups had a lower BMI, whole body fat mass, and % fat (p < 0.005), with no difference in lean mass. In both groups, lumbar spine bone mineral content (BMC) and density were higher following the intervention (p < 0.05) while femoral neck bone mineral density (BMD) was lower (p < 0.05). Intertrochanteric section modulus improved in both groups while there was a group x time interaction in femoral shaft subperiosteal width which was more favorable in the pulse group (p < 0.05). This study demonstrates that the femoral neck may be compromised during a lifestyle intervention in women with PCOS. Research is warranted to preserve bone health during lifestyle change in women with PCOS.

A randomized controlled trial of a lifestyle intervention with longitudinal follow-up on ovarian dysmorphology in women with polycystic ovary syndrome.

OBJECTIVE: Effects of lifestyle modification on reproductive function in polycystic ovary syndrome (PCOS) remain poorly elucidated. We compared the effects of a pulse-based diet (lentils, beans, split peas and chickpeas) with Therapeutic Lifestyle Changes (TLC) diet on ultrasonographic markers of ovarian morphology, hyperandrogenism and menstrual irregularity in PCOS. DESIGN: Randomized controlled trial. PATIENTS: Women with PCOS (18-35 years). MEASUREMENTS: Thirty women randomized to the pulse-based and 31 to TLC groups completed a 16-week intervention without energy restriction. Groups performed aerobic exercise (minimum of 5 days/wk; 45 minutes/d) and received health counselling (monthly) and longitudinal follow-up. Follicle numbers per ovary (FNPO, 2-9 mm), ovarian volume (OV), free androgen index (FAI) and menstrual cycle length were measured pre- and postintervention. RESULTS: Follicle numbers per ovary (mean change ± SD, -10 ± 15), OV (-2.7 ± 4.8 mL), FAI (-3 ± 2) and menstrual cycle length (-13 ± 47 days) decreased over time in both groups (All: P < .01), without group-by-time interactions (All: P ≥ .13). Attrition rate was 33.7% and comparable between groups (P = .94). Adherence to diet intervention negatively correlated with changes in FNPO (r=-0.54), OV (r=-0.35) and FAI (r = -.29) in pooled groups (All: P ≤ .04). Groups maintained reduced OV, FNPO, FAI and menstrual cycles 6 months postintervention; however, decreased FNPO and FAI at 16 weeks tended to revert to baseline levels 12 months postintervention in both groups (All: P ≤ .05). CONCLUSIONS: Both interventions improved ovarian dysmorphology, hyperandrogenism and menstrual irregularity in PCOS, and no diet appeared to be superior at improving reproductive outcomes. Our observations highlight the importance of longitudinal surveillance for sustainable adherence to newly adopted healthy lifestyle behaviours and reproductive health in PCOS (ClinicalTrials.gov identifier, NCT01288638).

Effects of supplemental creatine and guanidinoacetic acid on spatial memory and the brain of weaned Yucatan miniature pigs.

The emergence of creatine as a potential cognitive enhancement supplement for humans prompted an investigation as to whether supplemental creatine could enhance spatial memory in young swine. We assessed memory performance and brain concentrations of creatine and its precursor guanidinoacetic acid (GAA) in 14-16-week-old male Yucatan miniature pigs supplemented for 2 weeks with either 200 mg/kg∙d creatine (+Cr; n = 7) or equimolar GAA (157 mg/kg∙d) (+GAA; n = 8) compared to controls (n = 14). Spatial memory tests had pigs explore distinct sets of objects for 5 min. Objects were spatially controlled, and we assessed exploration times of previously viewed objects relative to novel objects in familiar or novel locations. There was no effect of either supplementation on memory performance, but pigs successfully identified novel objects after 10 (p < 0.01) and 20 min (p < 0.01) retention intervals. Moreover, pigs recognized spatial transfers after 65 min (p < 0.05). Regression analyses identified associations between the ability to identify novel objects in memory tests and concentrations of creatine and GAA in cerebellum, and GAA in prefrontal cortex (p < 0.05). The concentration of creatine in brain regions was not influenced by creatine supplementation, but GAA supplementation increased GAA concentration in cerebellum (p < 0.05), and the prefrontal cortex of +GAA pigs had more creatine/g and less GAA/g compared to +Cr pigs (p < 0.05). Creatine kinase activity and maximal reaction velocity were also higher with GAA supplementation in prefrontal cortex (p < 0.05). In conclusion, there appears to be a relationship between memory performance and guanidino compounds in the cerebellum and prefrontal cortex, but the effects were unrelated to dietary supplementation. The cerebellum is identified as a target site for GAA accretion.

A pulse-based diet and the Therapeutic Lifestyle Changes diet in combination with health counseling and exercise improve health-related quality of life in women with polycystic ovary syndrome: secondary analysis of a randomized controlled trial.

Objective: A favorable dietary composition to increase health-related quality of life (HRQoL) in PCOS remains unclear. We compared changes in HRQoL of women with PCOS who participated in a low-glycemic-index pulse-based (lentils, beans, split peas, and chickpeas) or the Therapeutic Lifestyle Changes (TLC) diet intervention.Methods: Thirty women in the pulse-based and 31 in the TLC groups (18-35 years) completed a 16-week intervention without energy-restriction. Groups participated in health counseling (monthly) and aerobic exercise (5 days/week; 45 minutes/day).Results: Fifty-five (90.2%) women completed a PCOS-specific HRQoL survey. Greatest mean increases in time-effects occurred in the domains of healthy eating, PCOS knowledge, active living, healthcare satisfaction, feelings and experiences about intervention, and health concerns, respectively (p ≤ 0.02), without group-by-time interactions (p ≥ 0.13). Decreased weight (r = -0.35) and homeostatic model assessment of insulin resistance (r = -0.18) correlated with increased scores of PCOS knowledge; adherence to intervention correlated with increased scores of active living (r = 0.39) and healthy eating (r = 0.53; p ≤ 0.03).Conclusions: Both interventions improved HRQoL scores in women with PCOS without prescribed energy-restriction. Our observations add novel insights into current evidence and elucidate the need for future psychological research to target lifestyle modifications for improving HRQoL and unique psychological complications of PCOS in this high-risk population (CinicalTrials.gov identifier:NCT01288638).

Polycystic ovary syndrome is a risk factor for sarcopenic obesity: a case control study.

BACKGROUND: Polycystic ovary syndrome (PCOS) is the most common endocrine disorder in young women and increases risk of cardiovascular and metabolic disease, and infertility. Women with PCOS share many characteristics commonly associated with aging including chronic inflammation and insulin resistance, which may be associated with "sarcopenic obesity", a term used to describe low appendicular skeletal muscle mass relative to total body mass. The purpose of this work was to determine the prevalence of sarcopenic obesity in women with PCOS. We hypothesized there would be a high prevalence of sarcopenic obesity, and that % appendicular skeletal muscle mass and markers of inflammation and insulin resistance would be inversely correlated in this population. METHODS: Dual energy X-ray absorptiometry was used to assess body composition in 68 women with PCOS aged 18-35y and 60 healthy age-matched women from the same geographic area. Sarcopenic obesity was defined as having % appendicular skeletal muscle mass 2 standard deviations below the mean for the healthy age-matched controls and a % body fat above 35%. Data were analyzed with Mann-Whitney U-tests and Spearman correlations. RESULTS: 53% of women with PCOS were classified as sarcopenic obese. Women with PCOS had a median (interquartile range) appendicular skeletal muscle mass of 23.8 (22.3-25.8)% which was lower than the control median of 30.4 (28.6-32.4)% (p < 0.0001). Among women with PCOS, there were negative correlations between % appendicular skeletal muscle mass and the homeostasis model assessment insulin resistance index (r = - 0.409; p < 0.01), high sensitivity C-reactive protein (r = - 0.608; p < 0.0001) and glycosylated hemoglobin (r = - 0.430; p < 0.0001). Furthermore, % appendicular skeletal muscle mass correlated positively with vitamin D (r = 0.398; p < 0.0001) in women with PCOS, which is thought to positively affect skeletal muscle mass. CONCLUSIONS: Women with PCOS have a high prevalence of sarcopenic obesity, which is correlated to insulin resistance and inflammation.

Comprehensive Evaluation of Type 2 Diabetes and Cardiovascular Disease Risk Profiles in Reproductive-Age Women with Polycystic Ovary Syndrome: A Large Canadian Cohort.

OBJECTIVE: This study compared the prevalence of metabolic syndrome (MetS) and characterized type 2 diabetes (DM2) and cardiovascular disease (CVD) risk profiles between Canadian women with polycystic ovary syndrome (PCOS) and healthy women recruited from the general population. Furthermore, within the PCOS cohort, the study contrasted the CVD and DM2 risk profiles of women with or without MetS. METHODS: Measures of MetS (International Diabetes Federation; National Heart, Lung, and Blood Institute; and the American Heart Association definition), DM2 (Diabetes Canada Clinical Guidelines), and CVD risk factors (Androgen Excess and Polycystic Ovary Syndrome Society statement) were evaluated for 237 women with PCOS (Androgen Excess and PCOS Society definitions) and 42 controls (aged 18-36) in a prospective observational study (Canadian Task Force Classification II-2). RESULTS: The prevalence of MetS was 29.5% in the PCOS group, which was approximately six-fold higher than age-matched controls (P < 0.001). Women with PCOS exhibited higher glucose abnormality, acanthosis nigricans, total cholesterol to high-density lipoprotein cholesterol ratio (TC/HDL-C), and lower sex hormone-binding globulin concentrations when compared with controls after accounting for differences in the BMI (P < 0.01). Further, women with PCOS and MetS exhibited exacerbated insulin and glucose responses to a 75-g oral glucose tolerance test and greater acanthosis nigricans, hirsutism, TC/HDL-C, TC, and sex hormone-binding globulin concentrations compared with their BMI-adjusted counterparts without MetS (P < 0.05). CONCLUSION: Canadian reproductive-age women with PCOS have a high prevalence of MetS and exhibit adverse cardiometabolic risk factors that warrant early screening and regular monitoring across their reproductive lifespan.

A Comparison of a Pulse-Based Diet and the Therapeutic Lifestyle Changes Diet in Combination with Exercise and Health Counselling on the Cardio-Metabolic Risk Profile in Women with Polycystic Ovary Syndrome: A Randomized Controlled Trial.

We compared the effects of a low-glycemic index pulse-based diet, containing lentils, beans, split peas, and chickpeas, to the Therapeutic Lifestyle Changes (TLC) diet on cardio-metabolic measures in women with polycystic ovary syndrome (PCOS). Ninety-five women (18⁻35 years) enrolled in a 16-week intervention; 30 women in the pulse-based and 31 in the TLC groups completed the study. Women participated in aerobic exercise training (minimum 5 days/week for 45 min/day) and were counselled (monthly) about PCOS and lifestyle modification. Women underwent longitudinal follow-up post-intervention. The pulse-based group had a greater reduction in total area under the curve for insulin response to a 75-g oral glucose tolerance test (mean change ± SD: -121.0 ± 229.9 vs. -27.4 ± 110.2 µIU/mL × min; p = 0.05); diastolic blood pressure (-3.6 ± 6.7 vs. -0.2 ± 6.7 mmHg, p = 0.05); triglyceride (-0.2 ± 0.6 vs. 0.0 ± 0.5 mmol/L, p = 0.04); low-density lipoprotein cholesterol (-0.2 ± 0.4 vs. -0.1 ± 0.4 mmol/L, p = 0.05); total cholesterol/high-density lipoprotein cholesterol (TC/HDL-C; -0.4 ± 0.4 vs. 0.1 ± 0.4, p < 0.001); and a greater increase in HDL-C (0.1 ± 0.2 vs. -0.1 ± 0.2 mmol/L, p < 0.01) than the TLC group. Decreased TC/HDL-C (p = 0.02) at six-month and increased HDL-C and decreased TC/HDL-C (p ≤ 0.02) at 12-month post-intervention were maintained in the pulse-based group. A pulse-based diet may be more effective than the TLC diet at improving cardio-metabolic disease risk factors in women with PCOS. TRIAL REGISTRATION: CinicalTrials.gov identifier, NCT01288638.

Betaine or folate can equally furnish remethylation to methionine and increase transmethylation in methionine-restricted neonates.

Methionine partitioning between protein turnover and a considerable pool of transmethylation precursors is a critical process in the neonate. Transmethylation yields homocysteine, which is either oxidized to cysteine (i.e., transsulfuration), or is remethylated to methionine by folate- or betaine- (from choline) mediated remethylation pathways. The present investigation quantifies the individual and synergistic importance of folate and betaine for methionine partitioning in neonates. To minimize whole body remethylation, 4-8-d-old piglets were orally fed an otherwise complete diet without remethylation precursors folate, betaine and choline (i.e. methyl-deplete, MD-) (n=18). Dietary methionine was reduced from 0.3 to 0.2 g/(kg∙d) on day-5 to limit methionine availability, and methionine kinetics were assessed during a gastric infusion of [13C1]methionine and [2H3-methyl]methionine. Methionine kinetics were reevaluated 2 d after pigs were rescued with either dietary folate (38 µg/(kg∙d)) (MD + F) (n=6), betaine (235 mg/(kg∙d)) (MD + B) (n=6) or folate and betaine (MD + FB) (n=6). Plasma choline, betaine, dimethylglycine (DMG), folate and cysteine were all diminished or undetectable after 7 d of methyl restriction (P<.05). Post-rescue, plasma betaine and folate concentrations responded to their provision, and homocysteine and glycine concentrations were lower (P<.05). Post-rescue, remethylation and transmethylation rates were~70-80% higher (P<.05), and protein breakdown was spared by 27% (P<.05). However, rescue did not affect transsulfuration (oxidation), plasma methionine, protein synthesis or protein deposition (P>.05). There were no differences among rescue treatments; thus betaine was as effective as folate at furnishing remethylation. Supplemental betaine or folate can furnish the transmethylation requirement during acute protein restriction in the neonate.

Women With Polycystic Ovary Syndrome Have Comparable Hip Bone Geometry to Age-Matched Control Women.

Polycystic ovary syndrome (PCOS) is an endocrine disorder affecting women of reproductive age manifesting with polycystic ovaries, menstrual irregularities, hyperandrogenism, hirsutism, and insulin resistance. The oligomenorrhea and amenorrhea characteristic to PCOS are associated with low bone mineral density (BMD); conversely, the hyperandrogenism and hyperinsulinemia may elicit a protective effect on BMD. As bone geometric properties provide additional information about bone strength, the objective of this study was to compare measures of hip geometry in women with PCOS to a healthy female population. Using dual-energy X-ray absorptiometry, BMD and measures of hip geometry were determined in women with PCOS (n = 60) and healthy controls (n = 60) aged 18-35 years. Clinical biochemical measures were also determined in women with PCOS. Measures of hip geometry, including cross-sectional area, cross-sectional moment of inertia, subperiosteal width (SPW), and section modulus, were similar between groups following correction for body mass index (BMI) (all p > 0.05) with intertrochanter SPW significantly lower in women with PCOS (p < 0.05). BMI-corrected whole body BMD as well as the lumbar spine and regions of proximal femur were also comparable between groups. In women with PCOS, BMI-corrected correlations were found between insulin and femoral shaft SPW (r = 0.322, p < 0.05), glucose and femoral neck (r = 0.301, p < 0.05), and trochanter BMD (0.348, p < 0.05), as well as between testosterone and femoral neck BMD (0.376, p < 0.05) and narrow neck cross-sectional area (0.306, p < 0.05). This study demonstrates that women with PCOS may have compromised intertrochanter SPW while oligomenorrhea appears to have no detrimental effect on bone density or geometry in women with PCOS.

The role of a pulse-based diet on infertility measures and metabolic syndrome risk: protocol of a randomized clinical trial in women with polycystic ovary syndrome.

BACKGROUND: Polycystic Ovary Syndrome (PCOS) is an endocrine disorder in women of reproductive age with an estimated prevalence of 5-20% of premenopausal women. The clinical symptoms common to PCOS include menstrual dysfunction, hyperandrogenemia, hirsutism, polycystic ovaries, insulin resistance, and hyperinsulinemia. Women with PCOS are at an increased risk of infertility, obesity and type 2 diabetes mellitus. Insulin resistance and hyperinsulinemia are believed to be key contributing factors to the pathogenesis of PCOS; excessive amounts of insulin are directly associated with the increased ovarian production of androgens and metabolic features of PCOS. Pulse-based diets (e.g., beans, chickpeas) are associated with improved glycemic control and have insulin lowering effects. The purpose of this study is to determine whether a pulse-based diet is more effective than the diet recommended by the National Cholesterol Education Program. The primary outcomes of this study are disease measures related to PCOS, with secondary outcomes including measures related to metabolic syndrome. METHODS: Women with symptoms of PCOS will be recruited for the study and a diagnosis of PCOS will be determined by an obstetrician-gynecologist. Women with PCOS will be randomly assigned to receive either a pulse-based diet or the National Cholesterol Education Program therapeutic lifestyle changes (TLC) diet for 16 weeks while participating in an aerobic exercise program. One hundred participants will be required (drop-out rate of 32%) for recruitment to provide 80% power for detecting a significant difference in fasting glucose (p < 0.05). Measures related to infertility, metabolic syndrome, quality of life, dietary intake and physical activity will be assessed pre- and post-intervention with follow up assessment at 6- and 12-months post-intervention. DISCUSSION: Polycystic ovary syndrome is the most common endocrine disorder in women of reproductive age and there is currently no recommended diet for this population of women. The multidisciplinary nature of this study, including determination of measures related to metabolic syndrome, infertility and physical activity provide a comprehensive assessment of any benefits associated with a pulse-based diet in women with PCOS. The results of this study will help in providing evidence-based recommendations for the optimum diet to reduce symptoms associated with PCOS. TRIAL REGISTRATION: NCT01288638. Trial registered January 13, 2011.

Restriction of dietary methyl donors limits methionine availability and affects the partitioning of dietary methionine for creatine and phosphatidylcholine synthesis in the neonatal piglet.

Methionine is required for protein synthesis and provides a methyl group for >50 critical transmethylation reactions including creatine and phosphatidylcholine synthesis as well as DNA and protein methylation. However, the availability of methionine depends on dietary sources as well as remethylation of demethylated methionine (i.e., homocysteine) by the dietary methyl donors folate and choline (via betaine). By restricting dietary methyl supply, we aimed to determine the extent that dietary methyl donors contribute to methionine availability for protein synthesis and transmethylation reactions in neonatal piglets. Piglets 4-8 days of age were fed a diet deficient (MD-) (n=8) or sufficient (MS+) (n=7) in folate, choline and betaine. After 5 days, dietary methionine was reduced to 80% of requirement in both groups to elicit a response. On day 8, animals were fed [(3)H-methyl]methionine for 6h to measure methionine partitioning into hepatic protein, phosphatidylcholine, creatine and DNA. MD- feeding reduced plasma choline, betaine and folate (P<.05) and increased homocysteine ~3-fold (P<.05). With MD- feeding, hepatic phosphatidylcholine synthesis was 60% higher (P<.05) at the expense of creatine synthesis, which was 30% lower during MD- feeding (P<.05); protein synthesis as well as DNA and protein methylation were unchanged. In the liver, ~30% of dietary label was traced to phosphatidylcholine and creatine together, with ~50% traced to methylation of proteins and ~20% incorporated in synthesized protein. Dietary methyl donors are integral to neonatal methionine requirements and can affect methionine availability for transmethylation pathways.

Methionine metabolism in Yucatan miniature swine.

Methionine is an essential amino acid which when not incorporated into protein, can be converted to S-adenosylmethionine, the universal methyl donor in over 200 transmethylation reactions, which include creatine and phosphatidylcholine (PC) synthesis, as well as deoxyribonucleic acid (DNA) methylation. Following transmethylation, homocysteine is formed, which can be converted to cysteine via transsulfuration or remethylated to methionine by receiving a methyl group from folate or betaine. Changes to methyl group availability in utero can lead to permanent changes in epigenetic patterns of DNA methylation, which has been implicated in "fetal programming", a phenomenon associated with poor nutrition during fetal development that results in low birth weight and disease in later life. It has been shown that programming can also occur in the neonate. Our global objective was to understand how the variability of nutrients involved in methionine metabolism can affect methionine and methyl group availability. We hypothesize that nutrients that converge on methionine metabolism can affect methionine availability for its various functions. In this thesis, we used intrauterine growth restricted (IUGR) piglets to investigate whether a global nutritional insult in utero can lead to a perturbed methionine metabolism. Our results demonstrate that IUGR piglets have a lower capacity to dispose of homocysteine via both transsulfuration and remethylation pathways, as well as a lower incorporation of methyl groups into PC. The second objective of this thesis was to determine whether variation in methionine supply and demand can affect methionine availability. We demonstrated that stimulating either acute or chronic creatine synthesis leads to lower methyl incorporation into protein and PC in pigs. Furthermore, when methionine is limiting, supplementation with either folate or betaine leads to higher methionine availability for protein synthesis. Finally, because creatine is increasingly being utilized as an ergogenic and neuroprotective supplement, we wanted to determine whether provision of the creatine precursor, guanidinoacetate (GAA), could effectively increase tissue creatine stores. We showed that 2.5 weeks of supplementation with GAA is more effective than creatine at increasing hepatic and muscle creatine stores. The results of this thesis demonstrate that the presence of IUGR, an increased demand for creatine synthesis, or the supplementation with remethylation nutrients can each affect methionine availability; all are important when considering neonatal nutrient requirements. Furthermore, although GAA is effective at increasing levels of tissue creatine, higher GAA methylation can limit methionine availability for growth and synthesis of PC.

The dynamics of methionine supply and demand during early development.

Methionine is an indispensable amino acid that, when not incorporated into protein, is converted into the methyl donor S-adenosylmethionine as entry into the methionine cycle. Following transmethylation, homocysteine is either remethylated to reform methionine or irreversibly trans-sulfurated to form cysteine. Methionine flux to transmethylation and to protein synthesis are both high in the neonate and this review focuses on the dynamics of methionine supply and demand during early development, when growth requires expansion of pools of protein and transmethylation products such as creatine and phosphatidylcholine (PC). The nutrients folate and betaine (derived from choline) donate a methyl group during remethylation, providing an endogenous supply of methionine to meet the methionine demand. During early development, variability in the dietary supply of these methionine cycle-related nutrients can affect both the supply and the demand of methionine. For example, a greater need for creatine synthesis can limit methionine availability for protein and PC synthesis, whereas increased availability of remethylation nutrients can increase protein synthesis if dietary methionine is limiting. Moreover, changes to methyl group availability early in life can lead to permanent changes in epigenetic patterns of DNA methylation, which have been implicated in the early origins of adult disease phenomena. This review aims to summarize how changes in methyl supply and demand can affect the availability of methionine for various functions and highlights the importance of variability in methionine-related nutrients in the infant diet.

Betaine is as effective as folate at re-synthesizing methionine for protein synthesis during moderate methionine deficiency in piglets.

PURPOSE: Both folate and betaine (synthesized from choline) are nutrients used to methylate homocysteine to reform the amino acid methionine following donation of its methyl group; however, it is unclear whether both remethylation pathways are of equal importance during the neonatal period when remethylation rates are high. Methionine is an indispensable amino acid that is in high demand in neonates not only for protein synthesis, but is also particularly important for transmethylation reactions, such as creatine and phosphatidylcholine synthesis. The objective of this study was to determine whether supplementation with folate, betaine, or a combination of both can equally re-synthesize methionine for protein synthesis when dietary methionine is limiting. METHODS: Piglets were fed a low methionine diet devoid of folate, choline, and betaine, and on day 6, piglets were supplemented with either folate, betaine, or folate + betaine (n = 6 per treatment) until day 10. [1-13C]-phenylalanine oxidation was measured as an indicator of methionine availability for protein synthesis both before and after 2 days of supplementation. RESULTS: Prior to supplementation, piglets had lower concentrations of plasma folate, betaine, and choline compared to baseline with no change in homocysteine. Post-supplementation, phenylalanine oxidation levels were 20-46 % lower with any methyl donor supplementation (P = 0.006) with no difference among different supplementation groups. Furthermore, both methyl donors led to similarly lower concentrations of homocysteine following supplementation (P < 0.05). CONCLUSIONS: These data demonstrate an equal capacity for betaine and folate to remethylate methionine for protein synthesis, as indicated by lower phenylalanine oxidation.

Guanidinoacetate is more effective than creatine at enhancing tissue creatine stores while consequently limiting methionine availability in Yucatan miniature pigs.

Creatine (Cr) is an important high-energy phosphate buffer in tissues with a high energy demand such as muscle and brain and is consequently a highly consumed nutritional supplement. Creatine is synthesized via the S-adenosylmethionine (SAM) dependent methylation of guanidinoacetate (GAA) which is not regulated by a feedback mechanism. The first objective of this study was to determine the effectiveness of GAA at increasing tissue Cr stores. Because SAM is required for other methylation reactions, we also wanted to determine whether an increased creatine synthesis would lead to a lower availability of methyl groups for other methylated products. Three month-old pigs (n = 18) were fed control, GAA- or Cr-supplemented diets twice daily. On day 18 or 19, anesthesia was induced 1-3 hours post feeding and a bolus of [methyl-3H]methionine was intravenously infused. After 30 minutes, the liver was analyzed for methyl-3H incorporation into protein, Cr, phosphatidylcholine (PC) and DNA. Although both Cr and GAA led to higher hepatic Cr concentration, only supplementation with GAA led to higher levels of muscle Cr (P < 0.05). Only GAA supplementation resulted in lower methyl-3H incorporation into PC and protein as well as lower hepatic SAM concentration compared to the controls, suggesting that Cr synthesis resulted in a limited methyl supply for PC and protein synthesis (P < 0.05). Although GAA is more effective than Cr at supporting muscle Cr accretion, further research should be conducted into the long term consequences of a limited methyl supply and its effects on protein and PC homeostasis.

Partitioning of [methyl-3H]methionine to methylated products and protein is altered during high methyl demand conditions in young Yucatan miniature pigs.

Methionine is the main source of methyl groups that are partitioned to synthesize various methylated products including creatine, phosphatidylcholine (PC), and methylated DNA. Whether increased methylation of 1 product can divert methionine from protein synthesis or other methylation products was the aim of this experiment. We used an excess of guanidinoacetate (GAA) to synthesize creatine to create a higher demand for available methyl groups in normal-weight (NW) (n = 10) and intrauterine growth-restricted (IUGR) (n = 10) piglets. Anesthetized piglets (15-18 d old) were intraportally infused with either GAA or saline for 2 h. A bolus of l-[methyl-(3)H]methionine was intraportally infused at 1 h, and hepatic metabolites were analyzed for methyl-(3)H incorporation 1 h later. Overall, 50-75% of label was recovered in creatine and PC with negligible amounts in DNA. In both NW and IUGR piglets, excess GAA led to an ≈ 80-120% increase in methyl incorporation into creatine (P < 0.05) with a concomitant decrease by ≈ 75-85% in methyl incorporation into PC (P < 0.05) as well as a 40% decrease in methyl incorporation into protein (P < 0.05), suggesting methyl groups were limited for PC synthesis and that methionine was diverted from protein synthesis. Compared with NW piglets, IUGR piglets had lower methyl incorporation into PC (P < 0.05), but not DNA or protein, suggesting IUGR affects methyl metabolism and could potentially impact lipid metabolism. The partitioning of methionine is sensitive to methyl supply in neonates, which has implications in infant diet composition and growth.

The nutritional burden of methylation reactions.

PURPOSE OF REVIEW: Methyl group metabolism is a metabolically demanding process that has significant nutritional implications. Methionine is required not only for protein synthesis but also as the primary source of methyl groups. However, demethylated methionine can be remethylated by methyl groups from methylneogenesis (via folate) and betaine (synthesized from choline). This review discusses the impact of methylation precursors and products on the methionine requirement. RECENT FINDINGS: Recent evidence has clearly demonstrated that transmethylation reactions can consume a significant proportion of the flux of methionine. In particular, synthesis of creatine and phosphatidylcholine consume most methyl groups and their dietary provision could spare methionine. Importantly, methionine can become limiting for protein and phosphatidylcholine synthesis when creatine synthesis is upregulated. Other research has shown that betaine and choline seem to be more effective than folate at reducing hyperhomocysteinemia and impacting cardiovascular outcomes suggesting they may be limiting. SUMMARY: It appears that methyl groups can become limiting when dietary supply is inadequate or if transmethylation reactions are upregulated. These situations can impact methionine availability for protein synthesis, which can reduce growth. The methionine requirement can likely be spared by methyl donor and methylated product supplementation.

Intrauterine growth restriction leads to changes in sulfur amino acid metabolism, but not global DNA methylation, in Yucatan miniature piglets.

Intrauterine growth restriction (IUGR), in both animals and humans, has been linked to metabolic syndrome later in life. There has been recent evidence that perturbations in sulfur amino acid metabolism may be involved in this early programming phenomenon. Methionine is the precursor for cellular methylation reactions and for the synthesis of cysteine. It has been suggested that the mechanism behind the "fetal origins" of adult diseases may be epigenetic, involving DNA methylation. Because we have recently demonstrated the fetal origins phenomenon in Yucatan miniature swine, we hypothesized that sulfur amino acid metabolism is altered in IUGR piglets. In this study, metabolites and the activities of sulfur amino acid cycle enzymes were analyzed in liver samples of 3- to 5-day-old runt (IUGR: 0.85±0.13 kg) and large (1.36±0.21 kg) Yucatan miniature pig littermates (n=6 pairs). The IUGR piglets had significantly lower specific and total activities of betaine-homocysteine methyltransferase (BHMT) and cystathionine γ-lyase (CGL) than larger littermates (P<.05). Expression of CGL (but not BHMT) mRNA was also lower in IUGR piglets (P<.05). This low CGL reduced cysteine and taurine concentrations in IUGR pigs and led to an accumulation of hepatic cystathionine, with lower homocysteine concentrations. Methylation index and liver global DNA methylation were unaltered. Reduced prenatal growth in Yucatan miniature piglets impairs their remethylation capacity as well as their ability to remove cystathionine and synthesize cysteine and taurine, which could have important implications on long-term health outcomes of IUGR neonates.