Genetic analysis of impaired trimethylamine metabolism using whole exome sequencing.

BACKGROUND: Trimethylaminuria (TMAU) is a genetic disorder whereby people cannot convert trimethylamine (TMA) to its oxidized form (TMAO), a process that requires the liver enzyme FMO3. Loss-of-function variants in the FMO3 gene are a known cause of TMAU. In addition to the inability to metabolize TMA precursors like choline, patients often emit a characteristic odor because while TMAO is odorless, TMA has a fishy smell. The Monell Chemical Senses Center is a research institute with a program to evaluate people with odor complaints for TMAU. METHODS: Here we evaluated ten subjects by (1) odor evaluation by a trained sensory panel, (2) analysis of their urine concentration of TMA relative to TMAO before and after choline ingestion, and (3) whole exome sequencing as well as subsequent variant analysis of all ten samples to investigate the genetics of TMAU. RESULTS: While all subjects reported they often emitted a fish-like odor, none had this malodor during sensory evaluation. However, all were impaired in their ability to produce >90% TMAO/TMA in their urine and thus met the criteria for TMAU. To probe for genetic causes, the exome of each subject was sequenced, and variants were filtered by genes with a known (FMO3) or expected effect on TMA metabolism function (other oxidoreductases). We filtered the remaining variants by allele frequency and predicated functional effects. We identified one subject that had a rare loss-of-function FMO3 variant and six with more common decreased-function variants. In other oxidoreductases genes, five subjects had four novel rare single-nucleotide polymorphisms as well as one rare insertion/deletion. Novel in this context means no investigators have previously linked these variants to TMAU although they are in dbSNP. CONCLUSIONS: Thus, variants in genes other than FMO3 may cause TMAU and the genetic variants identified here serve as a starting point for future studies of impaired TMA metabolism.

The effects of normalizing hyperhomocysteinemia on clinical and operative outcomes in patients with critical limb ischemia.

PURPOSE: To assess the outcome of patients with medically treated hyperhomocysteinemia (HHC) requiring intervention for critical limb ischemia (CLI). METHODS: A parallel observational study was conducted to compare the clinical and revascularization outcomes of CLI patients who received standardized treatment for HHC preoperatively (folic acid and vitamin B12) vs. contemporaneous patients with normal homocysteine levels. The threshold for HHC diagnosis was 13.0 µmol/L. From 2009 to 2011, 169 patients underwent revascularization procedures for CLI. Of these, all 66 patients (40 men; mean age 69.6 ± 11.2 years) with HHC (mean 17.3 µmol/L, range 13.5-34.9) were treated to normalize the homocysteine level prior to lower limb revascularization. The remaining 103 patients (58 men; mean age 72.7±8.1 years) had normal homocysteine levels (8.2 µmol/L, range 5-12.3) before revascularization. The primary endpoint was symptomatic and hemodynamic improvement in the treated HHC group. The secondary endpoints were all-cause survival, binary restenosis, reintervention, amputation-free survival, and major adverse events. The treated HHC cohort was compared to an age/sex-matched historical group of patients with untreated HHC from 2002 to 2006 before HHC pretreatment became routine. All interventions (endovascular, hybrid, and open) were performed by the same surgeon, and the groups were evenly matched. RESULTS: Patients with HHC were treated for a mean 12.2 days, which significantly reduced their mean homocysteine level after 3 weeks to 10.1 µmol/L (range 6.2-14.4, p<0.05). After revascularization, immediate clinical improvement was similar between normal homocysteine and medically corrected HHC groups. There was no significant difference in time to binary restenosis (p=0.822). Secondary endpoints and all-cause mortality were similar. Multivariate logistic regression showed that untreated HHC was a significant factor for graft occlusion and limb loss (p<0.0001), but medically corrected HHC was no longer predictive of adverse operative outcome. CONCLUSION: Patients with medically corrected HHC have similar outcomes compared to those with normal homocysteine levels. Thus, aggressively treating HHC with folic acid and vitamin B12 may help enhance the clinical outcome of CLI patients undergoing revascularization.

Effects of a 16-month randomized controlled exercise trial on body weight and composition in young, overweight men and women: the Midwest Exercise Trial.

BACKGROUND: In light of the current obesity epidemic, treatment models are needed that can prevent weight gain or provide weight loss. We examined the long-term effects of a supervised program of moderate-intensity exercise on body weight and composition in previously sedentary, overweight and moderately obese men and women. We hypothesized that a 16-month program of verified exercise would prevent weight gain or provide weight loss in the exercise group compared with controls. METHODS: This was a randomized controlled efficacy trial. Participants were recruited from 2 midwestern universities and their surrounding communities. One hundred thirty-one participants were randomized to exercise or control groups, and 74 completed the intervention and all laboratory testing. Exercise was supervised, and the level of energy expenditure of exercise was measured. Controls remained sedentary. All participants maintained ad libitum diets. RESULTS: Exercise prevented weight gain in women and produced weight loss in men. Men in the exercise group had significant mean +/- SD decreases in weight (5.2 +/- 4.7 kg), body mass index (calculated as weight in kilograms divided by the square of height in meters) (1.6 +/- 1.4), and fat mass (4.9 +/- 4.4 kg) compared with controls. Women in the exercise group maintained baseline weight, body mass index, and fat mass, and controls showed significant mean +/- SD increases in body mass index (1.1 +/- 2.0), weight (2.9 +/- 5.5 kg), and fat mass (2.1 +/- 4.8 kg) at 16 months. No significant changes occurred in fat-free mass in either men or women; however, both had significantly reduced visceral fat. CONCLUSIONS: Moderate-intensity exercise sustained for 16 months is effective for weight management in young adults.

Biochemical and clinical aspects of the human flavin-containing monooxygenase form 3 (FMO3) related to trimethylaminuria.

Trimethylaminuria is a rare metabolic disorder that is associated with abnormal amounts of the dietary-derived trimethylamine. Excess unmetabolized trimethylamine in the urine, sweat and other body secretions confers a strong, foul body odor that can affect the individual's ability to work or engage in social activities. This review summarizes the biochemical aspects of the condition and the classification of the disorder into: 1) primary genetic form, 2) acquired form, 3) childhood forms, 4) transient form associated with menstruation, 5) precursor overload and 6) disease states. The genetic variability of the flavin-containing monooxygenase (form 3) that is responsible for detoxication and deodoration of trimethylamine is discussed and put in context with other variant forms of the flavin-containing monooxygenase (forms 1-5). The temporal-selective expression of flavin-containing monooxygenase forms 1 and 3 is discussed in terms of an explanation for childhood trimethylaminuria. Information as to whether variants of the flavin-containing monooxygenase form 3 contributes to hypertension and/or other diseases are presented. Discussion is provided outlining recent bioanalytical approaches to quantify urinary trimethylamine and trimethylamine N-oxide and plasma choline as well as data on self-reporting individuals tested for trimethylaminuria. Finally, trimethylaminuria treatment strategies and nutritional support are described including dietary sources of trimethylamine, vitamin supplementation and drug treatment and issues related to trimethylaminuria in pregnancy and lactation are discussed. The remarkable progress in the biochemical, genetic, clinical basis for understanding the trimethylaminuria condition is summarized and points to needs in the treatment of individuals suffering from trimethylaminuria.

Transplacental carbohydrate and sugar alcohol concentrations and their uptakes in ovine pregnancy.

The concentrations of glucose, fructose, sorbitol, glycerol, and myo-inositol in sheep blood and tissues have been reported previously (1--5). However, the other polyols that are at low concentrations have not been investigated in pregnant sheep due to technical difficulties. By using HPLC and gas chromatography-mass spectrometry, seven polyols (myo-inositol, glycerol, erythritol, arabitol, sorbitol, ribitol, and mannitol) and three hexoses (mannose, glucose, and fructose) were identified and quantified in four blood vessels supplying and draining the placenta (maternal artery, uterine vein, fetal artery, and umbilical vein). Uterine and umbilical blood flows were measured, and uptakes of all the polyols and hexoses in both maternal and fetal circulations were calculated. There was a significant net placental release of sorbitol to both maternal and fetal circulations. Fructose was also taken up significantly by the uterine circulation. Maternal plasma mannose concentrations were higher than fetal concentrations, and there was a net umbilical uptake of mannose, characteristics that are similar to those of glucose. Myo-inositol and erythritol had relatively high concentrations in fetal plasma (697.8 plus minus 53 microM and 463.8 plus minus 27 microM, respectively). The ratios of fetal/maternal plasma arterial concentrations were very high for most polyols. The concentrations of myo-inositol, glycerol, and sorbitol were also high in sheep placental tissue (2489 plus minus 125 microM/kg wet tissue, 2119 plus minus 193 microM/kg wet tissue, and 3910 plus minus 369 microM/kg wet tissue), an indication that these polyols could be made within the placenta.

Individuals reporting idiopathic malodor production: demographics and incidence of trimethylaminuria.

BACKGROUND: Individuals with the metabolic disorder trimethylaminuria may sporadically produce malodors despite good hygiene. The psychosocial impact of trimethylaminuria can be considerable. However, trimethylaminuria is difficult to diagnose without specialized tests, in part because odor production is diet-dependent, and malodors may not be present during medical examinations. Thus, the prevalence and demographics of trimethylaminuria remain unclear. METHODS: We tested 353 patients who had unexplained (idiopathic) malodor production for trimethylaminuria using a standard choline challenge. We also collected basic demographic information. RESULTS: Approximately one third of patients (118) tested positive for trimethylaminuria. Consistent with previous reports, women, particularly African American women, were significantly overrepresented among trimethylaminuria-positive patients. Of note, the same pattern was seen among trimethylaminuria-negative patients. Also consistent with previous reports, trimethylaminuria-positive women who were still menstruating tended to produce higher levels of trimethylamine within ± 7 days of menses, although this trend was statistically marginal (P = .07). CONCLUSION: If our patient sample is representative of patients with idiopathic malodor, demographic information (race and gender) may not be useful in a differential diagnosis of trimethylaminuria. However, undiagnosed cases of trimethylaminuria may be fairly common among patients with idiopathic malodor. If so, choline challenge testing should be indicated for all such patients because trimethylaminuria is responsive to dietary and other treatments. We speculate that testing also might reveal cases of trimethylaminuria among those diagnosed with certain psychologic disorders, including olfactory reference syndrome.

Ingested fat oxidation contributes 8% of 24-h total energy expenditure in moderately obese subjects.

The role of ingested fat in the etiology of obesity is controversial. The aims of this study were to determine the contributions of ingested fat oxidation to: 1) 24-h total energy expenditure (TEE), and 2) substrate oxidation during acute stationary cycle exercises in adult humans. Healthy, moderately obese (n = 18; BMI = 31 +/- 1 kg/m2) subjects (8 men; 10 women) were each studied in a whole-room calorimeter for 24 h. They were fed mixed meals (55, 30, and 15% as energy from carbohydrate, fat and protein, respectively) to maintain energy balance. Each subject performed 1255-kJ cycle exercises at 50% VO2max in the calorimeter. Study test meal fat was labeled with carbon-13 (13C). Ingested fat oxidation was estimated from breath 13CO2 excretion and the subject's chamber CO2 production. Total fat and carbohydrate oxidations were estimated from nonprotein respiratory quotient (NP-RQ) values. Endogenous fat oxidation was estimated as the difference between total fat and ingested fat oxidations. TEE was estimated from gas exchanges; 28 +/- 3% of ingested fat was oxidized and it provided 8 +/- 1% of 24-h TEE. During cycle exercises, ingested fat provided 50% of total fat oxidized and 13.0 +/- 2% of energy expended. Endogenous fat oxidation contributed 10.4 +/- 3% of energy expenditure during cycle exercises. This study extended to 24-h observations of previous studies that lasted 6-9 h on ingested fat oxidation in humans. Understanding the factors that promote ingested fat oxidation could lead to more effective obesity intervention programs.

Folate cofactors regulate serine metabolism in fetal ovine hepatocytes.

The fetal liver is the primary site of fetal serine production. The regulation of this unique fetal hepatic serine production is unknown. We hypothesized that serine production would be responsive to folate cofactor supply or hormonal regulation. To test this hypothesis, we determined the effect of key folate cofactors and insulin and glucagon on serine and glycine metabolism in primary culture of fetal ovine hepatocytes. Hepatocytes were cultured in serum-free, low-folate media [5 nM 5-methyl-tetrahydrofolate (THF)] with or without 50 nM 5,10-methylene-THF (MTHF) or 5-formyl-THF (FTHF). Serine and glycine production (P) and utilization (U) were determined by stable isotope dilution with [1-(13)C]serine and [1-(13)C]glycine for 24 h. The effect of insulin (1 microM) or glucagon (1 micro M) was determined in a similar manner. Under basal conditions, serine P (43.2 +/- 5.1 micromol/mg DNA per 24 h) is greater than serine U (24.1 +/- 3.1 micromol/mg DNA per 24 h), whereas glycine U (27.3 +/- 3.0 micromol/mg DNA per 24 h) exceeds glycine P (16.7 +/- 1.9 micromol/mg DNA per 24 h). MTHF results in a significant decrease in serine U (16.0 +/- 2.7 micromol/mg DNA per 24 h; p = 0.02 versus low folate), with no change in serine P. FTHF reduces serine P (36.2 +/- 4.9 micromol/mg DNA per 24 h; p = 0.01), but does not alter serine U. There were no effects on glycine metabolism with 50 nM MTHF or FTHF. Serine P and U were inversely correlated whereas glycine P and U were directly correlated with the media concentration of MTHF or FTHF. Glucagon treatment increased serine U by 260 +/- 65% versus low folate (p = 0.0004) but did not change serine P. Insulin treatment led to parallel increases in both serine P and U. Both folate cofactor availability and hormone concentrations regulate serine metabolism in the fetal liver. We speculate that serine metabolism may be a marker of fetal hepatic folate cofactor supply.

Effect of low versus high intravenous amino acid intake on very low birth weight infants in the early neonatal period.

Greater protein intakes are required than have been commonly used to achieve fetal in utero protein accretion rates in preterm neonates. To study the efficacy and safety of more aggressive amino acid intake, we performed a prospective randomized study in 28 infants [mean wt, 946 +/- 40 g (SEM)] of 1 (low amino acid intake, LAA) versus 3 g.kg(-1).d(-1) (high amino acid intake, HAA) at 52.0 +/- 3.0 h of life. After a minimum of 12 h of parenteral nutrition, efficacy was determined by protein balance and was significantly lower in the LAA versus HAA groups by both nitrogen balance (-0.26 +/- 0.11 versus 1.16 +/- 0.15 g.kg(-1).d(-1), p < 0.00005) and leucine stable isotope (0.184 +/- 0.17 versus 1.63 +/- 0.20 g.kg(-1).d(-1), p < 0.0005) methods. Leucine flux and oxidation and nonoxidative leucine disposal rates were all significantly higher in the HAA versus LAA groups (249 +/- 13 versus 164 +/- 8, 69 +/- 5 versus 32 +/- 3, and 180 +/- 10 versus 132 +/- 8 micro mol.kg(-1).h(-1), respectively, p < 0.005), but leucine appearance from protein breakdown was not (140 +/- 15 in HAA versus 128 +/- 8 micro mol.kg(-1).h(-1)). In terms of possible toxicity with HAA, there were no significant differences between groups in the amount of sodium bicarbonate administered, degree of acidosis as determined by base deficit, or blood urea nitrogen concentration. Parenteral HAA versus LAA intake resulted in increased protein accretion, primarily by increasing protein synthesis versus suppressing protein breakdown, and appeared to be well tolerated by very preterm infants in the first days of life.

Ovine placental and fetal arginine metabolism at normal and increased maternal plasma arginine concentrations.

Arginine (A) may play a significant role in fetal growth, by stimulating insulin secretion and as a precursor for both polyamine synthesis and nitric oxide production. To determine whether increased maternal plasma A concentrations can enhance delivery of A to the fetus, uterine, umbilical, and net uteroplacental (UP) A uptake rates were measured in 12 pregnant ewes at 129.6 +/- 0.4 d gestation (mean +/- SEM) during normal and after 3 h of increased maternal plasma A concentrations. With a 2.7-fold increase in maternal plasma A concentrations (p < 0.001), there were significant increases in uterine A uptake (13.8 +/- 1.0 to 41.3 +/- 7.7 micromol/min, p < 0.005), umbilical A uptake (3.3 +/- 0.5 to 5.2 +/- 0.8 micromol.min(-1).kg(-1) fetus, p < 0.005), UP A uptake (17.8 +/- 6.2 to 89.2 +/- 20.3 micromol.min(-1).kg(-1) placenta, p < 0.01), fetal arterial A concentration (98.7 +/- 6.3 to 137.1 +/- 9.9 microM, p < 0.001), maternal A disposal rate (143.7 +/- 9.4 to 217.0 +/- 6.7 micromol/min, p < 0.001), fetal A disposal rate (7.9 +/- 0.8 to 9.9 +/- 1.1 micromol.min(-1).kg(-1), p < 0.05), fetal A oxidation rate (1.31 +/- 0.24 to 1.84 +/- 0.36 micromol.min(-1).kg(-1), p < 0.05), and plasma insulin concentration in both fetus (16 +/- 2 to 20 +/- 2 microU/mL, p < 0.001) and mother (24 +/- 3 to 32 +/- 4 microU/mL, p < 0.001). Thus, increased maternal plasma A concentration increases maternal, UP, and fetal A net uptake, and increases insulin secretion in mother and fetus. The 4.2-fold larger increase in UP than net fetal A uptake could represent preferential UP A metabolism relative to fetal A metabolism, relatively limited placental-fetal A transport capacity compared with uterine A uptake capacity, or both; responsible mechanisms remain unknown.

Multi-point estimation of total energy expenditure: a comparison between zinc-reduction and platinum-equilibration methodologies.

Reducing water to hydrogen gas by zinc or uranium metal for determining D/H ratio is both tedious and time consuming. This has forced most energy metabolism investigators to use the "two-point" technique instead of the "Multi-point" technique for estimating total energy expenditure (TEE). Recently, we purchased a new platinum (Pt)-equilibration system that significantly reduces both time and labor required for D/H ratio determination. In this study, we compared TEE obtained from nine overweight but healthy subjects, estimated using the traditional Zn-reduction method to that obtained from the new Pt-equilibration system. Rate constants, pool spaces, and CO2 production rates obtained from use of the two methodologies were not significantly different. Correlation analysis demonstrated that TEEs estimated using the two methods were significantly correlated (r=0.925, p=0.0001). Sample equilibration time was reduced by 66% compared to those of similar methods. The data demonstrated that the Zn-reduction method could be replaced by the Pt-equilibration method when TEE was estimated using the "Multi-Point" technique. Furthermore, D equilibration time was significantly reduced.