Methylmalonic Acid Concentrations Research Articles

Early pregnancy folate-cobalamin interactions and their effects on cobalamin status and hematologic variables throughout pregnancy

Periconception folic acid supplementation is widespread, but how it interacts with cobalamin status is rarely considered. The aim of this study was to investigate whether first-trimester folate-cobalamin interactions affect pregnancy cobalamin status, hematologic variables, and pregnancy outcomes. In the longitudinal Reus-Tarragona Birth Cohort study from <12 gestational weeks throughout pregnancy, fasting plasma and red blood cell (RBC) folate, plasma cobalamin, holotranscobalamin (holoTC), methylmalonic acid (MMA), total homocysteine (tHcy), hemoglobin, mean cell volume (MCV), postglucose-load serum glucose, gestational hypertension, gestational age at birth, and birth weight were recorded in 563 participants. The highest plasma folate concentrations occurred in the first trimester when folic acid supplement use was extensive. Supplementation beyond the first trimester interacted with time of pregnancy on plasma folate, RBC folate, and tHcy throughout pregnancy (P-interaction<0.001). Plasma folate and RBC folate were higher and tHcy was lower in continued supplement users than in nonusers. Elevated plasma folate (≥30 nmol/L) occurred in 78.9% of women who exceeded the recommended 400µg folic acid/d. First-trimester folate-cobalamin status interactions were associated with MMA (P-interaction<0.001) throughout pregnancy. When plasma cobalamin was suboptimal (≤221 pmol/L; n=36), participants with elevated plasma folate (n=11) had higher MMA concentrations than did those with nonelevated plasma folate (n=23). First-trimester folate-MMA status interactions were associated with MCV throughout pregnancy (P-interaction<0.01) and with cord plasma holoTC (P-interaction<0.05). The mean difference (95% CI) in MCV (fL) between women with elevated and nonelevated plasma folate status was -2.12 (-3.71, -0.52) for top-quartile plasma MMA (≥0.139 µmol/L) and 0.60 (-0.39, 1.60) for plasma MMA<0.139 µmol/L. Cord plasma holoTC was higher in women with elevated compared with nonelevated plasma folate status only for MMA<0.139 µmol/L. Folate-cobalamin interactions were not associated with the other investigated outcomes. First-trimester folate-cobalamin status interactions were associated with plasma MMA and MCV throughout pregnancy. This trial was registered at www.clinicaltrials.gov as NCT01778205.

Population Reference Values for Serum Methylmalonic Acid Concentrations and Its Relationship with Age, Sex, Race-Ethnicity, Supplement Use, Kidney Function and Serum Vitamin B12 in the Post-Folic Acid Fortification Period.

Serum methylmalonic acid (MMA) is elevated in vitamin B-12 deficiency and in kidney dysfunction. Population reference values for serum MMA concentrations in post-folic acid fortification period are lacking. Aims of this study were to report the population reference values for serum MMA and to evaluate the relation between serum MMA and sex, age, race-ethnicity, kidney dysfunction and vitamin B-12. We used data from three National Health and Nutrition Examination Surveys, 1999–2000, 2001–2002 and 2003–2004 conducted after folic acid fortification commenced (n = 18,569). Geometric mean MMA was ≈22.3% higher in non-Hispanic white compared to non-Hispanic black (141.2 vs. 115.5 nmol/L) and was ≈62.7% higher in >70 years old persons compared to 21–30 years old persons (196.9 vs. 121.0 nmol/L). Median serum MMA was ≈28.5% higher in the 1st the quartile of serum vitamin B-12 than in the 4th quartile of serum vitamin B-12 and was ≈35.8% higher in the 4th quartile of serum creatinine than in the 1st quartile of serum creatinine. Multivariate-adjusted serum MMA concentration was significantly associated with race-ethnicity (p < 0.001) and age (p < 0.001) but not with sex (p = 0.057). In this large US population based study, serum MMA concentrations presented here reflect the post-folic acid fortification scenario. Serum MMA concentrations begin to rise at the age of 18–20 years and continue to rise afterwards. Age-related increase in serum MMA concentration is likely to be due to a concomitant decline in kidney function and vitamin B-12 status.

Vitamin B12 deficiency in inflammatory bowel disease: a prospective observational pilot study.

Diagnostic and management guidelines for vitamin B12 (cobalamin, Cbl) deficiency in inflammatory bowel disease (IBD) are lacking. True deficiency is defined as Cbl concentrations below reference range combined with elevated methylmalonic acid (MMA) concentrations. Studies analyzing Cbl status in IBD use only Cbl concentrations without confirmatory MMA. This study aims to determine the proportion of IBD patients with Cbl concentrations below reference range and their predisposing clinical and genetic characteristics. We then compared this to the proportion with true deficiency. In a prospective observational pilot study of adult IBD outpatients, Cbl concentrations, MMA levels, and fucosyltransferase 2 mutations were measured at clinic visits. A total of 66 Crohn's disease (CD) and 30 ulcerative colitis (UC) patients were recruited. Mean Cbl concentrations (pmol/l) in CD (253.7) were not significantly lower than UC (320.5, P=0.24). Serum Cbl below reference range (<148) was observed in 7.6 and 10% of CD and UC patients, respectively (P=0.70). True deficiency in CD and UC was 3 and 3.3%, respectively (P=1.0). Patients with ileal resections more than 30 cm had lower mean Cbl concentrations (177, P=0.02) and a trend toward higher proportions with Cbl levels below reference range (40%, P=0.06), but not increased deficiency rates (0%, P=1.0). Disease location, severity, and fucosyltransferase 2 mutations were not associated with altered Cbl status. True Cbl deficiency was rare in IBD patients in this study. A disparity in Cbl status exists when confirmatory MMA levels are used compared with Cbl concentrations alone. Asymptomatic IBD patients with low serum Cbl require confirmatory tests to guide management and avoid unnecessary treatment.

Maternal and neonatal vitamin B12 deficiency detected by expanded newborn screening

Infant vitamin B12 deficiency can manifest as a severe neurodegenerative disorder and is usually caused by maternal deficiency due to vegetarian diet or pernicious anaemia. Its early recognition and treatment can prevent potentially serious and irreversible neurologic damage. Biochemically, vitamin B12 deficiency leads to an accumulation of methylmalonic acid, homocysteine, and propionylcarnitine. Expanded newborn screening using tandem mass spectrometry may identify neonatal and maternal vitamin B12 deficiency by measurement of propionylcarnitine and other metabolites in the dried blood spot sample of newborns. To summarize our experiences gained by screening for vitamin B12 deficiency. Clinical and laboratory data of vitamin B12-deficient infants diagnosed in Szeged Screening Centre were retrospectively analysed. In Hungary, expanded newborn screening was introduced in 2007. Since then approximately 395 000 newborns were screened in our centre and among them, we identified four newborns with vitamin B12 deficiency based on their screening results. In three cases an elevated propionylcarnitine level and in the fourth one a low methionine level were indicative of vitamin B12 deficiency. We also detected an additional vitamin B12-deficient infant with neurological symptoms at 4 months of age, after a normal newborn screening, because of elevated urinary methylmalonic acid concentration. Vitamin B12 deficiency was secondary to maternal autoimmune pernicious anaemia in all the five infants. As a result of the recognized cases the incidence of infant vitamin B12 deficiency in the East-Hungarian region was 1.26/100 000 births, but the real frequency may be higher. Conslusions: Optimizing the cut off values of current screening parameters and measuring of methylmalonic acid and/or homocysteine in the dried blood spot, as a second tier test, can improve recognition rate of vitamin B12 deficiency. Orv Hetil. 2017; 158(48): 1909-1918.

High Serum Folate Is Associated with Brain Atrophy in Older Diabetic People with Vitamin B12 Deficiency.

Previous studies have reported the adverse cognitive effects of high folate status in older individuals with vitamin B12 (VB12) deficiency. Thus, the aim of this study was to investigate how high serum folate and VB12 deficiency could collaboratively aggravate neuronal degeneration. In total, 146 older non-demented diabetic individuals with an average age of 75 ± 3.9 were recruited. VB12 deficiency and high folate status were based on high serum methylmalonic acid (MMA) concentrations (> 0.3 μmol/L) and the serum folate concentration being in the top tertile (> 31.4 nmol/L) respectively. Among these subjects, there were 20 with elevated MMA and high folate. The structural magnetic resonance imaging data of these subjects were analyzed by performing flexible factorial analysis with the "folate level" and "MMA level" added as main effects, and the interaction effect of folate and VB12 on brain volume was evaluated. The results showed significant gray matter atrophy of the right middle occipital gyrus and the opercular part of the inferior frontal gyrus in subjects with a simultaneous high folate status and VB12 deficiency. Together with previous observational studies on cognitive function, this study lends support to the notion that high serum folate concentrations in older people with VB12 deficiency may be associated with increased neurodegeneration.

Pregnant women of South Asian ethnicity in Canada have substantially lower vitamin B12 status compared with pregnant women of European ethnicity.

Maternal vitamin B12 (B12) status has been inversely associated with adverse pregnancy outcomes and positively with fetal growth and infant development. South Asians, Canada's largest ethnic minority, are prone to B12 deficiency. Yet, data are lacking on B12 status in South Asian pregnant women in North America. We sought to determine B12 status, using multiple biomarkers, in 1st and 2nd trimester pregnant women of South Asian and, for comparison, European ethnicity living in Vancouver, Canada. In this retrospective cohort study, total B12, holotranscobalamin (holoTC), methylmalonic acid (MMA), and total homocysteine concentrations were quantified in two routinely collected (mean gestational week: 11·5 (range 8·3-13·9) and 16·5 (range 14·9-20·9)), banked serum samples of 748 healthy pregnant South Asian (n 371) and European (n 377) women. South Asian pregnant women had significantly lower B12 status than European pregnant women at both time points, as indicated by lower serum total B12 and holoTC concentrations, and higher MMA concentrations (all P≤0·001). The largest difference, which was substantial (Cohen's d≥0·5), was observed in mean serum total B12 concentrations (1st trimester: 189 (95 % CI 180, 199) v. 246 (95 % CI 236, 257) pmol/l; 2nd trimester: 176 (95 % CI 168, 185) v. 226 (95 % CI 216, 236) pmol/l). Further, South Asian ethnicity was a significant negative predictor of B12 status during pregnancy. South Asian women living in Vancouver have substantially lower B12 status during early pregnancy. Future research identifying predictors and health consequences of this observed difference is needed to allow for targeted interventions.

Effects of 6Weeks of Parenteral Cobalamin Supplementation on Clinical and Biochemical Variables in Cats with Gastrointestinal Disease.

BackgroundEffects and duration of commonly used protocols for cobalamin (Cbl) supplementation on cellular Cbl deficiency have not been determined in hypocobalaminemic cats.Hypothesis/ObjectivesTo evaluate effect of Cbl supplementation on clinical signs, serum and urine methylmalonic acid (MMA) concentrations over 16 weeks.AnimalsTwenty client‐owned hypocobalaminemic cats with enteropathy.MethodsProspective study. Serum Cbl and serum and urine MMA concentrations were determined prospectively in cats at enrollment (t0), immediately before (t6), and 4 (t10) and 10 weeks (t16) after 6th Cbl injection (250 μg, IM q 7 days). Clinical signs severity (activity, appetite, vomiting, diarrhea, body weight) graded at each time point and expressed as clinical disease activity score.ResultsClinical disease activity score decreased during supplementation and increased after treatment discontinuation. Median serum Cbl concentration increased significantly from t0 (111 pmol/L, range 111–212) to t6 (2,332.5 pmol/L, range 123–22,730) (P < 0.01). Values at t10 were 610.5 pmol/L (range, 111–2,527) and 180.5 pmol/L (range, 111–2,262) at t16 (P < 0.01). Median baseline serum MMA concentration (372 μmol/L, range 0.39–147,000) decreased significantly to 1.62 μmol/L (range, 0.18–806) at t6 (P < 0.01) and gradually increased to 5.34 μmol/L (range, 0.13–1,730) at t10 and 189 μmol/L (range, 0.4–983) at t16. Similar, nonsignificant, pattern observed for urine MMA concentration. Serum and urine MMA concentrations had not normalized in 12 and 6 cats, respectively, at t6.Conclusion and Clinical ImportanceThe Cbl supplementation protocol used here did not lead to complete normalization of cellular Cbl deficiency in all examined cats, and biochemical improvements were transient.

Whole-body propionate and glucose metabolism of multiparous dairy cows receiving folic acid and vitamin B12 supplements

This study was undertaken to evaluate the effect of supplementation of folic acid and vitamin B12 on glucose and propionate metabolism. Twenty-four multiparous cows were assigned according to a complete block design in a 2 × 2 factorial arrangement to one of the following treatments: (1) saline 0.9% NaCl, (2) 320 mg of folic acid, (3) 10 mg of vitamin B12, or (4) 320 mg of folic acid and 10 mg of vitamin B12. Intramuscular injections were given weekly from 3 wk before the expected calving date until 9 wk postpartum. At 63 d in milk, d-[6,6-2H2]-glucose (16.5 mmol/h; jugular vein) and [1-13C]-sodium propionate (13.9 mmol/h; ruminal vein) were simultaneously infused for 4 h; blood samples were collected from 2 to 4 h of the infusion period. Liver biopsies were carried out the following day. Supplements of folic acid and vitamin B12 respectively increased folate and vitamin B12 concentrations, both in milk and liver. Although dry matter intake was unaffected by treatments, milk and milk lactose yields tended to be lower by 5.0 and by 0.25 kg/d, respectively, for cows receiving the folic acid supplement. Plasma β-hydroxybutyrate concentration with the folic acid supplement followed the same tendency. Hepatic gene expression of methylmalonyl-CoA mutase and S-adenosylhomocysteine hydrolase was higher for cows receiving the combined folic acid and vitamin B12 supplement compared with cows receiving only the supplement of folic acid, whereas no treatment effect was noted for cows not receiving the folic acid supplement. Whole-body glucose rate of appearance and the proportion of whole-body glucose rate of appearance secreted in milk lactose decreased by 229 g/d and 5%, respectively, for animals receiving the folic acid supplement, concomitant with the lower milk lactose synthesis in these cows, indicating that supplementary folic acid may alter energy partitioning in cows. The absence of treatment effect on plasma concentrations of methylmalonic acid as well as on the proportion of glucose synthesized from propionate, averaging 60%, supports the fact that vitamin B12 supply was sufficient in control cows in the current study. Our results suggest that the folic acid supplement reduced glucose-derived lactose synthesis by redirecting glucose for other metabolic activity in the mammary gland or in other tissues.

A Black Cohosh Extract Causes Hematologic and Biochemical Changes Consistent with a Functional Cobalamin Deficiency in Female B6C3F1/N Mice.

Black cohosh rhizome, available as a dietary supplement, is most commonly marketed as a remedy for dysmenorrhea and menopausal symptoms. A previous subchronic toxicity study of black cohosh dried ethanolic extract (BCE) in female mice revealed a dose-dependent ineffective erythropoiesis with a macrocytosis consistent with the condition known as megaloblastic anemia. The purpose of this study was to investigate potential mechanisms by which BCE induces these particular hematological changes. B6C3F1/N female mice (32/group) were exposed by gavage to vehicle or 1,000 mg/kg BCE for 92 days. Blood samples were analyzed for hematology, renal and hepatic clinical chemistry, serum folate and cobalamin, red blood cell (RBC) folate, and plasma homocysteine and methylmalonic acid (MMA). Folate levels were measured in liver and kidney. Hematological changes included decreased RBC count; increased mean corpuscular volume; and decreased reticulocyte, white blood cell, neutrophil, and lymphocyte counts. Blood smear evaluation revealed increased Howell-Jolly bodies and occasional basophilic stippling in treated animals. Plasma homocysteine and MMA concentrations were increased in treated animals. Under the conditions of our study, BCE administration caused hematological and clinical chemistry changes consistent with a functional cobalamin, and possibly folate, deficiency. Further studies are needed to elucidate the mechanism by which BCE causes increases in homocysteine and MMA.

South Asian Ethnicity Is Related to the Highest Risk of Vitamin B12 Deficiency in Pregnant Canadian Women.

Vitamin B12 (B12) adequacy during pregnancy is crucial for maternal health and optimal fetal development; however, suboptimal B12 status has been reported in pregnant Canadian women. Methylmalonic acid (MMA) is a sensitive indicator of B12 status. Since few studies have measured MMA during pregnancy in Canadian women, the objective of this study was to evaluate B12 status in pregnant women living in Metro Vancouver, using both plasma total B12 and MMA. We recruited a convenience sample of 320 pregnant women between 20 and 35 gestational weeks from local healthcare facilities. Plasma total B12 concentrations indicative of deficiency (<148 pmol/L) and suboptimal B12 status (148–220 pmol/L) were found in 18% and 33% of the women, respectively. Normal plasma MMA concentration (<210 nmol/L) was observed in 82% of all women. Gestational age was a strong predictor of plasma total B12 and MMA concentration, and South Asian ethnicity of B-12 deficiency and MMA concentrations. Overall, there was a high discrepancy between the prevalence of B12 inadequacy depending on the biomarker used. Independently, however, South Asian women were at particular risk for B12 deficiency, likely due to lower animal source food intake. Further study of this vulnerable group and performance testing of B12 biomarkers is warranted.

Vitamin B-12–fortified toothpaste improves vitamin status in vegans: a 12-wk randomized placebo-controlled study ,

Background: The oral application of vitamin B-12 may prevent its deficiency if the vitamin is absorbed via the mucosal barrier.Objectives: We studied the effect of the use of a vitamin B-12–fortified toothpaste on vitamin-status markers in vegans and assessed the efficiency of markers in the identification of vitamin-augmentation status.Design: In this 12-wk, double-blinded, randomized, placebo-controlled study, 76 vegans received either a placebo (n = 34) or vitamin B-12 (n = 42) toothpaste. Sixty-six subjects (n = 30 in the placebo arm; n = 36 in the vitamin B-12 arm) completed the intervention. Serum and plasma concentrations of vitamin B-12, holotranscobalamin, total homocysteine (tHcy), and methylmalonic acid (MMA) were measured before and after the intervention.Results: Both postintervention concentrations of vitamin B-12 and holotranscobalamin and their changes over 12 wk were higher in the vitamin B-12 group (mean ± SD change: 81 ± 135 pmol/L for vitamin B-12 and 26 ± 34 pmol/L for holotranscobalamin) than in the placebo group (−27 ± 64 and −5 ± 17 pmol/L, respectively) after adjustment for baseline concentrations. Postintervention concentrations of MMA and their changes differed significantly between groups (MMA changes: −0.169 ± 0.340 compared with −0.036 ± 0.544 μmol/L in vitamin B-12 and placebo groups, respectively; P < 0.001). After adjustment for baseline tHcy, postintervention concentrations of tHcy tended to be lower (P = 0.051), and the changes in tHcy (−0.7 ± 4.4 compared with 2.0 ± 5.6 μmol/L, respectively) were greater in the vitamin B-12 group than in the placebo group. Changes in vitamin B-12 markers were more prominent in vegans who reported that they had not taken vitamin B-12 supplements.Conclusion: Vitamin B-12 that is applied to the oral cavity via toothpaste enters the circulation and corrects the vitamin B-12 markers in the blood of vegans who are at higher risk of vitamin B-12 deficiency. This trial was registered at clinicaltrials.gov as NCT02679833.

Effects of intramuscular injections of folic acid, vitamin B12, or both, on lactational performance and energy status of multiparous dairy cows

The purpose of this experiment was to gain understanding on changes in energy partitioning when folic acid and vitamin B12 supplements, alone or combined, were given by weekly intramuscular injections from 3 wk before the expected calving date until 7 wk postpartum. Twenty-four multiparous cows were assigned to 6 blocks of 4 cows each according to previous 305-d lactation yield to either 0 or 320 mg of folic acid and 0 or 10 mg of vitamin B12 in a 2 × 2 factorial arrangement. Plasma concentration of folates was increased by folic acid supplement, and this increase was greater with the combined supplement. Vitamin B12 supplement increased plasma concentration of vitamin B12. Even though postpartum energy balance was similar among treatments, postpartum body condition score was higher for cows receiving folic acid supplement compared with cows that did not. Milk yield of cows receiving folic acid supplement reached a plateau earlier than for cows that did not. Fat and protein, as well as total solid concentrations and yields, were unaffected by treatments. Postpartum plasma concentrations of glucose and insulin were higher and postpartum plasma concentration of nonesterified fatty acids was lower for cows that received weekly folic acid supplement compared with cows that did not. Plasma concentration of methylmalonic acid was low and unaffected by treatments, suggesting that vitamin B12 supply was not limiting, even for unsupplemented cows. Postpartum plasma concentrations of Cys, His, Phe, and Tyr were increased, whereas plasma concentration of Gly was decreased, by folic acid supplement. In the present study, supplementary folic acid altered energy partitioning in early lactation as suggested by similar milk total solid yield and postpartum energy balance, lower plasma nonesterified fatty acid concentration and body condition score losses, and higher plasma glucose and insulin concentrations for cows receiving folic acid supplement compared with cows that did not.

Folate and Vitamin B12-Related Biomarkers in Relation to Brain Volumes.

Aim: We investigated cross-sectional associations between circulating homocysteine, folate, biomarkers of vitamin B12 status and brain volumes. We furthermore compared brain volumes of participants who received daily folic acid and vitamin B12 supplementation with participants who did not. Methods: Participants of the B-PROOF study (n = 2919) were assigned to 400 µg folic acid and 500 µg vitamin B12, or a placebo. After two years of intervention, T1-weighted magnetic resonance imaging (MRI) scans were made in a random subsample (n = 218) to obtain grey and white matter volume, and total brain volume (TBV). Plasma homocysteine, serum folate, vitamin B12, holotranscobalamin, and methylmalonic acid concentrations were measured. Results: Multiple linear regression analyses showed inverse associations between plasma homocysteine with TBV (β = −0.91, 95% CI −1.85–0.03; p = 0.06) and between serum folate and TBV (β = −0.20, 95% CI −0.38, −0.02; p = 0.03). No significant associations were observed for serum vitamin B12 and holotranscobalamin. Fully adjusted ANCOVA models showed that the group that received B-vitamins had a lower TBV (adjusted mean 1064, 95% CI 1058–1069 mL) than the non-supplemented group (1072, 95% CI 1067–1078 mL, p = 0.03). Conclusions: Results were contradictory, with higher Hcy levels associated with lower TBV, but also with higher folate levels associated with lower TBV. In addition, the lack of a baseline measurement withholds us from giving recommendations on whether folic acid and vitamin B12 supplementation will be beneficial above and beyond normal dietary intake for brain health.

A Prospective Study on Serum Methylmalonic Acid and Homocysteine in Pregnant Women.

This study aimed to investigate serum methylmalonic acid (MMA) and homocysteine levels and to assess their effects on pregnancy and neonatal outcomes. Serum MMA and homocysteine levels in 278 pregnant Korean women, determined by liquid chromatography–tandem mass spectrometry in each trimester, were compared with those of previous studies in other ethnic groups. We investigated the association between MMA and homocysteine status with pregnancy and neonatal events: gestational diabetes, preeclampsia, gestational age at delivery, preterm birth, small for gestational age, neonatal birth weight, and congenital abnormalities. The median (range) MMA level was 0.142 (0.063–0.446) µmol/L and homocysteine level was 10.6 (4.4–38.0) µmol/L in pregnant women. MMA levels were significantly higher in the third trimester than during other trimesters (p < 0.05), while homocysteine levels were not. No significant association was observed between MMA or homocysteine levels and any of the maternal or neonatal outcomes examined. Future studies are needed to assess the associations among maternal serum concentrations of MMA and homocysteine, and maternal and neonatal outcomes.

Application of a Multi-Marker Approach to Assess Vitamin B12 Status at the End of the First Trimester of Pregnancy

Background:During pregnancy, vitamin B12 (B12) utilisation increases, with deficiency being associated with adverse outcomes (Murphy 2007, VanderJagt 2011). It remains unclear however at what level vitamin B12 status becomes detrimental, and the correlation with fetal harm.Establishing B12 status during pregnancy is complicated by physiological changes such as haemodilution due to expanded blood volume, altered renal function, alterations in the vitamin B12 -binding proteins and transfer of materno-fetal B12 yet laboratories typically rely on the total abundance of B12 in serum (serum B12) asthe sole laboratory status indicator, interpreting results against reference range cut offs derived from non-pregnant populations, including men.Serum B12 assays measure the sum of holohaptocorrin and holotranscobalamin (holoTC). Other markers of B12 status are also available and are increasingly being adopted: holoTC (active B12) accounts 6-20% of bound B12, which is the only form of B12 presented for cellular uptake and used to satisfy metabolic demand; methylmalonic acid (MMA) is a by-product of methylmalonyl-CoA metabolism, the concentration of which in serum correlates inversely with tissue B12 utilisation.Methods:Over a 2 monthperiod in 2015 we used an extended panel of laboratory tests consisting of serum B12, holoTC, and MMA to determine B12 status, and assess appropriateness of our assay reference ranges, in 100 pregnant women with normal renal function at the end of the first trimester.Laboratory AnalysisSerum B12 and holoTC were determined by an Abbott Architect analyzer. The serum B12 assay has a reference range of 187 - 883 ng/L. The holoTC assay has ²10% cross-reactivity from B12-binding proteins haptocorrin and apotranscobalamin. Values >128 pmol/L are above the dynamic assay range. In our laboratory holoTC results <25 pmol/L are defined as deficient and those >70 pmol/L as replete. Results 25-70 pmol/L are classified as indeterminate and a serum MMA assay subsequently performed (Sobczyńska-Malefora A et al 2014). We determined serum MMA using a Gerstel Multipurpose Sampler coupled to a liquid chromatography tandem mass spectrometer with electrospray ionization with results >270 nmol/L considered indicative of suboptimal B12 status (Harrington DJ 2016).Results:All three assays, figure 3, were performed for 92 women (insufficient sample available n=8).HoloTCand MMA results are shown in Table 1. The distribution ofholoTCand MMA concentrations are shown in Figure 1.One woman had a serum B12 below the reference range with a corresponding indeterminate holoTC and normal MMA. This set of results suggests questionable abundance of B12 that has not translated to functional deficiency.One woman had a holoTCconcentration <25pmol/L, however the corresponding serum B12 and MMA were not indicative of sub optimal B12 status. Fourteen women had aholoTC result above the dynamic assay range.Two women had an elevated MMA concentration with corresponding holoTCresults indicating questionable abundance of B12 but serum B12 within our reference limit. This triad of values indicates possible emerging suboptimal B12 status that would have been overlooked if serum B12 had been used in isolation.Discussion:B12 requirement increases in pregnancy and diet alone is unlikely to meet requirements (Murphy et al 2007). Deficiency has a detrimental impact on maternal and infant development and therefore it is useful to explore the relationship between different laboratory markers of B12 status in pregnancy (Ramirez-Velez et al 2016, Siddiqua et al 2014). The comparison of serum B12,holoTC and MMA levels at the end of the first trimester with reference ranges derived from non-pregnant adults suggests that they are broadly applicable.Serum B12 is the least sensitive marker and its use in isolation to assess B12 status is limiting. The application of holoTC and MMA in tandem can reveal previously missed deficient states. Using these markers two women in this small study were identified as possibly deficient although the true significance of a mildly elevated MMA in pregnancy requires further investigation. These data highlight the benefit of adopting a panel of markers ahead of a single test to assess B12 status. Identifying women at the end of the first trimester that may benefit from B12 replacement potentially protects from deficient states. [Display omitted] [Display omitted] [Display omitted] DisclosuresRobinson:Pharmacosmos: Honoraria; Amgen: Honoraria; Novartis: Honoraria.

Homocysteine metabolism and the associations of global DNA methylation with selected gene polymorphisms and nutritional factors in patients with dementia

Epigenetics (particularly DNA methylation) together with environmental and genetic factors, are key to understanding the pathogenesis of many diseases including dementia. Disturbances in DNA methylation have already been implicated in dementia. Homocysteine metabolism, with folate and vitamin B12 as essential cofactors, is integral to methylation processes.We evaluated in a case-control study the association of global DNA methylation, homocysteine, folate and vitamin B12 status with dementia. Selected polymorphisms of genes previously associated with dementia development and the influence of various factors on DNA methylation were also investigated.102 patients with dementia (53 with Alzheimer's disease, 17 with vascular dementia and 32 with mixed dementia) were recruited. The non-demented controls consisted of 45 age-matched subjects without dementia and 47 individuals with mild cognitive impairment. Global DNA methylation was determined by Imprint Methylated DNA Quantification Kit MDQ1 (Sigma-Aldrich, Gillingham, Dorset, UK). Plasma homocysteine, serum folate and vitamin B12 were determined by chemiluminescence. Plasma and erythrocyte 5-methyltetrahydrofolate and plasma methylmalonic acid (markers of folate and vitamin B12 status) were measured by HPLC. APOE, PON1 p.Q192R, MTHFR 677C>T (c.665C>T) and IL1B-511C>T polymorphisms were identified using PCR-RFLP methods.Patients with dementia had significantly higher concentrations of homocysteine (p=0.012) and methylmalonic acid (p=0.016) and lower folate (p=0.002) and plasma 5-methyltetrahydrofolate (p=0.005) than non-demented subjects. There was no difference in DNA methylation between patients and controls. A non-significant tendency to higher DNA methylation in patients with vascular dementia (p=0.061) was observed. Multivariate regression analysis of all recruited individuals demonstrated a significant positive association between DNA methylation and folate (p=0.013), creatinine (p=0.003) concentrations and IL1B-511T (p=0.002) and PON1 192R (p=0.049) alleles and negative association with fasting glucose (p=0.004).The biochemical results showed significantly lower folate and vitamin B12 status in demented patients than controls. Global DNA methylation was associated with markers of folate status, creatinine, glucose and PON1 and IL1B polymorphisms.

Low Serum Vitamin B-12 Concentrations Are Prevalent in a Cohort of Pregnant Canadian Women

Background: Among Canadian women of reproductive age, 5% and 20% have serum vitamin B-12 concentrations indicative of deficiency (<148 pmol/L) and marginal status (148–220 pmol/L), respectively. Given the association between suboptimal vitamin B-12 and adverse pregnancy outcomes, an understanding of vitamin B-12 status during pregnancy, and factors that influence it, is required.Objective: This prospective analysis from the PREFORM (PREnatal FOlic acid exposuRe on DNA Methylation in the newborn infant) study investigated 1) vitamin B-12 status in a cohort of Canadian pregnant women and their newborns, 2) the association of maternal dietary vitamin B-12 intake with maternal and cord blood concentrations of vitamin B-12 and its biomarkers, and 3) the association of fetal genetic polymorphisms with cord blood concentrations of vitamin B-12 and its biomarkers.Methods: In pregnant Canadian women (n = 368; mean ± SD age: 32 ± 5 y), vitamin B-12 intakes were assessed in early (0–16 wk) and mid- to late (23–37 wk) pregnancy. Serum vitamin B-12 and plasma total homocysteine (tHcy) and methylmalonic acid (MMA) in maternal blood at 12–16 wk of pregnancy and at delivery (28–42 wk) and in cord blood were measured and compared by using regression analyses. The associations of 28 fetal genetic variants in vitamin B-12 metabolism and cord blood vitamin B-12, tHcy, and MMA concentrations were assessed by using regression analysis, with adjustment for multiple testing.Results: A total of 17% and 38% of women had deficient and 35% and 43% had marginal serum vitamin B-12 concentrations at 12–16 wk of pregnancy and at delivery, respectively. Only 1.9–5.3% had elevated MMA (>271 nmol/L), and no women had elevated tHcy (>13 μmol/L). Maternal dietary vitamin B-12 intake during pregnancy was either weakly associated or not associated with maternal and cord blood vitamin B-12 (r2 = 0.17–0.24, P < 0.0008), tHcy (P = NS) and MMA (r2 = 0.05–0.11, P < 0.001). Fetal genetic polymorphisms were not associated with cord blood concentrations of vitamin B-12 and its biomarkers.Conclusions: Deficient and marginal serum vitamin B-12 concentrations are prevalent in Canadian pregnant women with the use of traditional cutoffs, despite supplement use. Given the growing interest among women to adhere to a vegetarian diet that may be lower in vitamin B-12, and vitamin B-12’s importance in pregnancy, the functional ramifications of these observations need to be elucidated. This trial was registered at clinicaltrials.gov as NCT02244684.

353. 1-13C-Propionate Oxidation as a Measure of Methylmalonyl-CoA Mutase (MUT) Activity in Methylmalonic Acidemia

Isolated methylmalonic acidemia (MMA) is a grave autosomal recessive inborn error of metabolism, caused by a defect in methylmalonyl-CoA mutase (MUT). In some severe patients, liver (LT), kidney (KT), or combined liver and kidney (LKT) transplantation has been used as a surgical treatment for MMA. Previous studies have shown that 1-13C-propionate oxidation can be used to measure whole body MUT activity in gene therapy treated and transgenic MMA mice and have encouraged translation to patients, with the aim of using this approach as an outcome measure in future gene therapy clinical trials.We therefore investigated 1-13C-propionate oxidation in 29 patients with isolated MMA (mut0 N=17, mut− N=6, cblA N=5, cblB N=1, age range: 4-41 years), including 3 KT, 1 LT, and 4 LKT recipients, as well as one patient who received a heterotopic liver allograft post KT (hLKT). In addition, 7 healthy volunteers (3 males, 4 females, age range: 21-44 years) and 8 heterozygote controls (age range:35-62 years) were tested to establish test reproducibility. Baseline CO2 production of the participants was measured using an indirect calorimetry cart before sodium 1-13C-propionate was administered as an oral or G-tube bolus, and breath samples were collected serially over 2 hours via disposable breath collection kits to measure 13CO2 enrichment and subsequently propionate oxidation.Propionate oxidation was decreased in all non-transplanted MMA patients compared with controls (p<0.0001). Both LT and LKT recipients demonstrated complete restoration of oxidation rates to control levels (p=NS), while only minimal oxidation was observed in the single hLKT recipient. KT recipients responsive to vitamin B-12 supplementation (N=2) with cblA MMA showed oxidation rates similar to controls. However, the remaining KT recipient, a severely affected mut0 patient carrying two stop mutations, showed almost no metabolism of label.Repeat testing conducted both on healthy volunteers in triplicate and 7 MMA patients (6 mut, 1 cblA) in duplicate supported excellent reproducibility. Notably, similar oxidation rates were observed in 2 patients despite markedly different plasma methylmalonic acid concentrations between one assessment and the next (1741 vs. 2246 and 2260 vs. 719 µmol/L). This suggests that measured 1-13C-propionate oxidation may be a more consistent and reproducible clinical biomarker than plasma metabolites that depend, in part, on protein intake and kidney function.Our results demonstrate that 1-13C-propionate oxidation, measured using breath testing, has great potential as an outcome measure for therapeutic interventions aimed at increasing hepatic MUT activity, such as gene or cell therapy. It is safe, non-radioactive, minimally invasive, well tolerated by very young patients, and can be frequently repeated over the course of treatment to evaluate the magnitude and sustainability of whole body in vivo MUT activity, with every patient's baseline values serving as an individual pre-treatment control. Furthermore, the method is readily applicable to the study of other inborn errors of metabolism affecting propionate oxidation, such as propionic acidemia.

357. A Mouse Model of Cobalamin A (cblA) Class Isolated Methymalonic Academy (MMA) Provides Unique Platform for Testing Gene and Cell Therapies

Isolated Methylmalonic Acidemia (MMA) comprises a relatively common and heterogeneous group of inborn errors of metabolism. Most affected individuals display severe multisystemic disease with episodes of metabolic instability, chronic renal disease, and neurological complications. The most severe clinical presentations are typically associated with complete loss of methylmalonyl-CoA mutase (MUT) enzymatic activity. MUT converts methylmalonyl-CoA to succinyl-CoA within mitochondria but defects in the transport or synthesis of its cobalamin co-factor also variably impair MUT activity. Previous mouse models of Mut MMA generated by our group have been critical to developing gene therapy for MMA but have either displayed neonatal lethality or required concomitant transgenesis for viability. Thus, the need for ameliorated models to further investigate disease-associated pathophysiology and facilitate gene therapy studies exists. We have therefore constructed a mouse model of an attenuated form of isolated MMA, the cblA subtype, by using homologous recombination to create a deletion allele of Mmaa, the enzyme that performs the gated transfer of adenosylcobalamin to Mut and protects Mut from oxidative inactivation. Patients with the cblA subtype of MMA can have variable presentations, spanning the full spectrum of MMA associated symptoms and pathology, yet always harbor an element of clinical and biochemical responsiveness to injectable B12.Mmaa-/- mice were born in mendelian proportions, exhibited decreased survival after weaning (P<0.0001), weighed 50% less than littermates at 6 months (P<0.0001), lacked immunoreactive Mmaa and Mmaa mRNA in multiple tissues, including the liver, and manifested severe metabolic perturbations. Plasma methylmalonic acid concentrations ranged between 63.88-1641.91 µM, representing a 62-fold or more elevation over age matched littermates. Mmaa-/- mice also display diminished 1-C-13 propionate oxidative capacity (P<0.0001) and hepatic complex IV activity (P=0.002). Electron microscopy confirmed the presence of hepatic megamitochondria and abnormal proximal renal tubular mitochondria. The glomerular filtration rate, measured using FITC-sinistrin decay, showed that Mmaa-/- mice have diminished kidney function compared to heterozygous littermates. Following hydroxycobalamin administration, Mmaa-/- mice had improved survival and increased weight (P=0.0003), which was associated with improved 1-13C-propionate oxidative capacity.This new mouse model fully recapitulates the clinical and biochemical features seen in cblA patients, and provides a robust platform for testing gene and cell therapies for MMA because affected mice are relatively severe but survive until weaning. These animals also offer the opportunity to longitudinally examine disease biomarkers and more easily assay vectors across a wide age range.

A Common Polymorphism in HIBCH Influences Methylmalonic Acid Concentrations in Blood Independently of Cobalamin

Methylmalonic acid (MMA) is a by-product of propionic acid metabolism through the vitamin B12 (cobalamin)-dependent enzyme methylmalonyl CoA mutase. Elevated MMA concentrations are a hallmark of several inborn errors of metabolism and indicators of cobalamin deficiency in older persons. In a genome-wide analysis of 2,210 healthy young Irish adults (median age 22 years) we identified a strong association of plasma MMA with SNPs in 3-hydroxyisobutyryl-CoA hydrolase (HIBCH, p = 8.42× 10(-89)) and acyl-CoA synthetase family member 3 (ACSF3, p = 3.48× 10(-19)). These loci accounted for 12% of the variance in MMA concentration. The most strongly associated SNP (HIBCH rs291466; c:2T>C) causes a missense change of the initiator methionine codon (minor-allele frequency = 0.43) to threonine. Surprisingly, the resulting variant, p.Met1?, is associated with increased expression of HIBCH mRNA and encoded protein. These homozygotes had, on average, 46% higher MMA concentrations than methionine-encoding homozygotes in young adults with generally low MMA concentrations (0.17 [0.14-0.21] μmol/L; median [25(th)-75(th) quartile]). The association between MMA levels and HIBCH rs291466 was highly significant in a replication cohort of 1,481 older individuals (median age 79 years) with elevated plasma MMA concentrations (0.34 [0.24-0.51] μmol/L; p = 4.0× 10(-26)). In a longitudinal study of 185 pregnant women and their newborns, the association of this SNP remained significant across the gestational trimesters and in newborns. HIBCH is unique to valine catabolism. Studies evaluating flux through the valine catabolic pathway in humans should account for these variants. Furthermore, this SNP could help resolve equivocal clinical tests where plasma MMA values have been used to diagnose cobalamin deficiency.