Methylmalonic Acid Concentrations Research Articles

Reference interval of methylmalonic acid concentrations in dried blood spots of healthy, term newborns to facilitate neonatal screening of vitamin B12 deficiency

ObjectivesUnrecognized vitamin B12 (B-12) deficiency in early infancy can lead to poor development of the child. Methylmalonic acid (MMA) is the most specific functional biomarker of B-12 status. To facilitate timely diagnosis and treatment of neonatal B-12 deficiency, the objective of this study was to calculate a reference interval of MMA in dried blood spots (DBSs) of healthy, term newborns. Design and methodsMMA was quantified in 160 newborn DBSs, routinely collected for newborn screening, using LC–MS/MS. The reference interval of DBS MMA was calculated according to current guidelines (CLSI EP28-A3c). The effect of storage at room temperature on DBS MMA concentrations was determined. ResultsThe mean DBS MMA concentration of 160 healthy, term newborns was 16.8pmol (95% CI: 15.9–17.6pmol)/8-mm spot. The reference interval (2.5th to 97.5th percentile) for MMA was 9.89 to 29.3pmol/8-mm spot (0.450 to 1.33μmol/L whole blood). DBS MMA concentrations correlated positively (p<0.005) with storage time at room temperature. DBS MMA concentrations of children with methylmalonic acidemia (n=4) were above the upper limit of the reference interval. ConclusionsThis is the first study to present a reference interval for DBS MMA of healthy, term newborns utilizing a new highly sensitive MMA method. Analysis of DBS MMA collected during newborn screening may have the potential to identify newborns with acquired B-12 deficiency. Newborn DBS MMA concentrations increase with storage at room temperature, therefore, sample storage has to be monitored for evaluation of DBS MMA data.

Vitamin Status among Breastfed Infants in Bhaktapur, Nepal.

Vitamin deficiencies are known to be common among infants residing in low- and middle-income countries but relatively few studies have assessed several biochemical parameters simultaneously. The objective of the study was to describe the status of vitamins (A, D, E, B6, B12 and folate) in breastfed infants. We measured the plasma concentrations of trans retinol, 25 hydroxy vitamin D, α-tocopherol, pyridoxal 5′-phosphate, cobalamin, folate, methylmalonic acid, homocysteine, hemoglobin and C-reactive protein from 467 randomly selected infants. One in five (22%) was deficient in at least one vitamin. Mean (SD) plasma folate concentration was 73 (35) nmol/L, and no infant in the sample was folate deficient. Vitamin B6 deficiency and vitamin B12 deficiency was found in 22% and 17% of the infants, respectively. Elevated plasma methylmalonic acid or total homocysteine concentration was found in 82% and 62% of infants, respectively. Fifteen percent of infants were vitamin A deficient and 65% were marginally deficient in vitamin A. Fewer than 5% of infants had low plasma vitamin D concentration or vitamin E concentration (α-tocopherol <9.3 µmol/L). Our results illustrate the importance of continued supplementation campaigns and support the expansion of food fortification and dietary diversification programs that target children and women in Nepal.

Serum Cobalamin and Methylmalonic Acid Concentrations in Hyperthyroid Cats Before and After Radioiodine Treatment.

BackgroundHyperthyroidism, the most common endocrine disorder in cats, has been associated with low serum cobalamin concentrations. Whether this is a functional cobalamin deficiency of clinical importance has not been assessed.Hypothesis/ObjectivesCats with hyperthyroidism experience a functional cobalamin deficiency which correlates with their clinical catabolic state and is reversible with return of the euthyroid state.AnimalsThirty‐nine client‐owned hyperthyroid cats.MethodsProspective observational study. Serum cobalamin, methylmalonic acid, and clinical scores were determined in each hyperthyroid cat at enrollment and when euthyroid (60 days after radioiodine treatment).ResultsFive of the 39 hyperthyroid cats (13%) had a low serum cobalamin concentration ranging from <150 to 290 ng/L. Serum cobalamin concentrations normalized to >350 ng/L in 2 of the hypocobalaminemic cats once euthyroid. None of the hyperthyroid/hypocobalaminemic cats had increased serum methylmalonic acid concentrations (175–601 nmol/L). In cats with clinical and biochemical hyperthyroidism, there was no correlation between serum cobalamin concentrations with total T4 concentration (P = .12) or clinical scores including body weight (P = .11) and BCS (P = .54).Conclusions and Clinical ImportanceIn this population of hyperthyroid cats, the prevalence of hypocobalaminemia was low. Specifically, hyperthyroid cats, in which concurrent gastrointestinal disease is unlikely. Hypocobalaminemia is not a functional deficiency requiring supplementation in hyperthyroid cats without gastrointestinal disease.

Safety of long-term restrictive diets for peroxisomal disorders: vitamin and trace element status of patients treated for Adult Refsum Disease.

Adult Refsum's Disease (ARD) is caused by defects in the pathway for alpha-oxidation of phytanic acid (PA). Treatment involves restricting the dietary intake of phytanic acid by reducing the intake of dairy-derived fat. The adequacy of micronutrient intake in patients with ARD is unknown. Patients established on the Chelsea low-PA diet had general diet macronutrients, vitamins and trace elements assessed using 7-day-weighed intakes and serial 24-h recalls. Intakes were compared with biochemical assessments of nutritional status for haematinics (ferritin), trace elements (copper, zinc, iron, selenium), water- (vitamin B6 , B12 and folate) and fat-soluble vitamins (A, D, E and K). Eleven subjects (four women, seven men) were studied. Body mass index was 27 ± 5 kg/m(2) (range 19-38). All subjects had high sodium intakes (range 1873-4828 mg). Fat-soluble vitamin insufficiencies occurred in some individuals (vitamin A, n = 2; vitamin D, n = 6; vitamin E, n = 3; vitamin K, n = 10) but were not coincident. Vitamin B6 levels were normal or elevated (n = 6). Folate and 5-methyltetrahydrofolate concentrations were normal. Metabolic vitamin B12 insufficiency was suspected in four subjects based on elevated methylmalonic acid concentrations. Low copper and selenium intakes were noted in some subjects (n = 7, n = 2) but plasma levels were adequate. Iron, ferritin and zinc intakes and concentrations were normal. Subjects with ARD can be safely managed on the Chelsea low PA without routine micronutrient supplementation. Sodium intake should be monitored and reduced. Periodic nutritional screening may be necessary for fat-soluble vitamins, vitamin B12 , copper or selenium.

Choline-phospholipids inter-conversion is altered in elderly patients with prostate cancer.

Choline is an important source of phospholipids and methyl groups in mammalian cells. High demands for methyl and phospholipids in malignant cells suggest that choline metabolism may be disturbed in patients with cancer. This case-control study investigated differences in concentrations of choline metabolites between 80 elderly men (age≥65 years) with prostate cancer (PCa) and 51 men with benign prostatic hyperplasia (BPH). Plasma/serum concentrations of free choline, betaine, dimethylglycine, folate, total homocysteine (tHcy), cystathionine, methylmalonic acid, S-adenosyl homocysteine (SAH), S-adenosyl methionine (SAM), and phospholipids were measured. Men with BPH and those with PCa showed no significant differences in the concentrations of free choline (median=9.7 vs. 10.0μmol/L), folate (17.4 vs. 19.8nmol/L), tHcy (16.0 vs. 16.2μmol/L), SAH (18.8 vs. 18.2nmol/L), and phosphatidylcholine (1634 vs. 1610μmol/L). The concentrations of methylmalonic acid were lower in men with PCa (203 vs. 228nmol/L) but the difference was not significant after adjusting for age. Sphingomyelin species (16:0, 18:0, 18:1, 20:0, 22:0, 22; 1, 23:0, 23:1, 24:0, 24:1, and 24:2) were significantly lower in men with PCa than in the controls (6-16% differences). Multiple regression analyses showed that the presence of PCa, statin use, choline, age, cystathionine, and methylmalonic acid were significant negative determinant of sphingomyelins, whereas phosphatidylcholine was a strong positive determinant. The current results support systemic alterations in phospholipids metabolism in PCa. We report on a significant decrease in plasma concentrations of sphingomyelin in elderly patients with PCa and in users of statins. The PCa-associated low sphingomyelin showed a synergy with the effect of statins. The presence of PCa was not associated with significant changes in plasma concentrations of choline or methyl metabolites. However, changes in choline absorption and tissue uptake cannot be ruled out in this study.

Peroxisome Proliferator-Activated Receptor Activation is Associated with Altered Plasma One-Carbon Metabolites and B-Vitamin Status in Rats.

Plasma concentrations of metabolites along the choline oxidation pathway have been linked to increased risk of major lifestyle diseases, and peroxisome proliferator-activated receptors (PPARs) have been suggested to be involved in the regulation of key enzymes along this pathway. In this study, we investigated the effect of PPAR activation on circulating and urinary one-carbon metabolites as well as markers of B-vitamin status. Male Wistar rats (n = 20) received for 50 weeks either a high-fat control diet or a high-fat diet with tetradecylthioacetic acid (TTA), a modified fatty acid and pan-PPAR agonist with high affinity towards PPARα. Hepatic gene expression of PPARα, PPARβ/δ and the enzymes involved in the choline oxidation pathway were analyzed and concentrations of metabolites were analyzed in plasma and urine. TTA treatment altered most biomarkers, and the largest effect sizes were observed for plasma concentrations of dimethylglycine, nicotinamide, methylnicotinamide, methylmalonic acid and pyridoxal, which were all higher in the TTA group (all p < 0.01). Hepatic Pparα mRNA was increased after TTA treatment, but genes of the choline oxidation pathway were not affected. Long-term TTA treatment was associated with pronounced alterations on the plasma and urinary concentrations of metabolites related to one-carbon metabolism and B-vitamin status in rats.

A New Approach for Fast Metabolic Diagnostics in CMAMMA.

The presence of increased urinary concentrations of both methylmalonic acid (MMA) and malonic acid (MA) is assumed to differentiate combined malonic and methylmalonic aciduria (CMAMMA), due to mutations in the ACSF3 gene, from other causes of methylmalonic aciduria (classic MMAemia). Detection of MA in urine, however, is challenging since excretion of MA can be easily missed. The objective of the study was to develop a method for quantification of MA in plasma to allow differentiation between CMAMMA and classic MMAemia. Compound heterozygosity for mutations in the ACSF3 gene was detected in two female siblings using diagnostic exome sequencing. Urine (MMA and MA) was analyzed with GC/MS, while plasma was analyzed with UPLC-MS/MS. MA/MMA ratios were calculated. Both patients had a severe psychiatric presentation (at the age of 6 years and 5.5 years, respectively) after a viral infection. MA excretion in the patients was only just above the highest control value in several samples. MA concentrations in plasma from the two patients were clearly above the highest value observed in control subjects. However, MA concentrations in plasma from patients with classic MMAemia were also elevated. Additional, calculation of MA/MMA ratio in plasma allowed to fully differentiate between CMAMMA and classic MMAemia. Calculating the MA/MMA ratio in plasma allows differentiation between CMAMMA and classic MMAemia. The full clinical spectrum of CMAMMA remains to be delineated.

Ultrasound-assisted emulsification microextraction followed by gas chromatography–flame ionization detection for urinary methylmalonic acid determination

An efficient microextraction technique followed by gas chromatography–flame ionization detector (GC–FID) analysis was developed for determination of methylmalonic acid (MMA) concentration in human urine samples. This method was based on ultrasound-assisted emulsification microextraction followed by derivatization with a low density alcoholic solvent which performs both as an extraction solvent and as a derivatization agent, simultaneously. In this procedure, 80μL of 1-heptanol was injected slowly into a 10mL acidified aqueous sample of MMA placed inside an ultrasonic water bath. The resulting emulsion was centrifuged and after derivatization, 2μL of the organic phase was injected into a GC–FID. Several factors affecting the derivatization and the extraction were optimized. Under the optimal conditions, linearity was in the range of 1 to 250 and 3 to 200μmol/L corresponding to the limits of detection (LOD) 0.8 and 2.4μmol/L in water and urine samples, respectively. The inter-day and intra-day precision of the proposed method were evaluated in terms of the relative standard deviation (RSD), which were <11% (n=4). The proposed method presented an acceptable LOD for urinary MMA analysis with satisfactory RSD.

Clinical characteristics of hemolytic uremic syndrome secondary to cobalamin C disorder in Chinese children.

The present study was undertaken to investigate the clinical characteristics of hemolytic uremic syndrome (HUS) secondary to cobalamin C disorder (cbl-C disorder). We reviewed retrospectively the medical records of 3 children with HUS secondary to cbl-C disorder who had been treated between April 1, 2009 and October 31, 2013. The 3 patients with HUS secondary to cbl-C disorder presented with progressive hemolytic anemia, acute renal failure, thrombocytopenia, poor feeding, and failure to thrive. Two of the 3 patients once had high blood pressure. The mutations of c.609G>A (p.W203X), c.217C>T (p.R73X) and c.365A>T (p.H122L) in the methylmalonic aciduria (cobalamin deficiency) cbl-C type, with homocystinuria gene were detected in the 3 patients. In these patients the levels of lactate dehydrogenase and homocysteine in serum were elevated and the level of methylmalonic acid (MMA) in urine was also elevated. After treatment with hydroxocobalamin, 2 patients were discharged with no obvious abnormal growth and neurological development and 1 patient died of multiple organ failure. The results of this study demonstrated that cbl-C disorder should be investigated in any child presenting with HUS. The high concentrations of homocysteine and MMA could be used for timely recognization of the disease. Once the high levels of plasma homocystein and/or plasma or urine MMA are detected, the treatment with parenteral hydroxocobalamin should be prescribed immediately. The early diagnosis and treatment would contribute to the good prognosis of the disease.

Vitamin B12 Status in a Cohort of Canadian Pregnant Women and Newborn Infants

Low maternal concentrations of vitamin B12 (B12) adversely affect fetal development. In Canada, low serum B12 concentrations (&lt;148 pmol/L) are prevalent among women of reproductive age. We measured concentrations of serum B12 and its functional biomarkers, plasma total homocysteine (tHcy) and methylmalonic acid (MMA), in 368 pregnant women at 12‐16 wk gestation, at delivery and in cord blood. We also determined the effect of major fetal genetic variants of B12 metabolism on cord plasma concentrations of B12, tHcy and MMA. The prevalence of low serum B12 concentrations at recruitment and delivery was 17% and 37%, respectively. Elevated plasma MMA concentrations (&gt;270 nmol/L) were only observed in 2% and 5% of women at recruitment and delivery, respectively, and no women had plasma tHcy concentrations &gt;13 μmol/L. During pregnancy, mean serum B12 concentrations decreased by 23%, while plasma tHcy and MMA increased by 20% and 24%, respectively (p&lt;0.0001). Mean cord blood concentrations of B12, tHcy and MMA were 1.9, 0.8 and 2.3 times maternal concentrations at delivery (p&lt;0.0001). Cord plasma MMA concentrations were significantly altered by the MTR rs1805087, MUT rs1141321 and MMAA rs2270655 genotypes (p&lt;0.04), while cord serum B12 and plasma tHcy concentrations were altered by the TCN1 rs34324219 genotype (p=0.04). Our data suggest that although “suboptimal” serum B12 levels are prevalent in Canadian pregnant women, they are unlikely functionally significant as evidenced by MMA and tHcy status. Fetal genetic variants appear to influence cord blood concentrations of B12, MMA and tHcy. Funded by CIHR MOP#106446

Vision Issues and Space Flight: Evaluation of One‐Carbon Metabolism Polymorphisms

Intermediates of the one‐carbon metabolic pathway are altered in astronauts who experience vision‐related issues during and after space flight. Serum concentrations of homocysteine, cystathionine, 2‐methylcitric acid, and methylmalonic acid were higher in astronauts with ophthalmic changes than in those without (Zwart et al., J Nutr, 142:427‐31, 2012). These differences existed before, during, and after flight. Potential confounding factors did not explain the differences. Genetic polymorphisms could contribute to these differences, and could help explain why crewmembers on the same mission do not all have ophthalmic issues, despite the same environmental factors (e.g., microgravity, exercise, diet). A follow‐up study was conducted to evaluate 5 polymorphisms of enzymes in the one‐carbon pathway, and to evaluate how these relate to vision and other ophthalmic changes after flight. Preliminary evaluations of the genetic data indicate that all of the crewmembers with the MTRR GG genotype had vision issues to one degree or another. However, not everyone who had vision issues had this genetic polymorphism, so the situation is more complex than the involvement of this single polymorphism. Metabolomic and further data analyses are underway to clarify these findings, but the preliminary assessments are promising. This study was funded by the Human Health Countermeasures Element of the NASA Human Research Program.

Long-term treatment for hyperphenylalaninemia and phenylketonuria: a risk for nutritional vitamin B12 deficiency?

The objective of the study was to determine the incidence of vitamin B12 deficiency in patients under long-term treatment for phenylketonuria (PKU) and hyperphenylalaninemia (HPA), as well as its associations with B12 vitamin parameters (holotranscobalamin - active vitamin B12, serum folate, total plasma homocysteine, and plasma methylmalonic acid concentration). The group consisted of 51 PKU (n=29) and HPA (n=22) patients aged 3-48 years (28 children, 23 adults). A significant difference in serum folate levels was discovered between adult HPA patients and PKU patients (p=0.004, Mann-Whitney U-test). A significant difference in plasma homocysteine concentrations within the normal levels (p=0.032, χ2-test) was detected between adult HPA and PKU patients. In the group of adults, we also found significant differences in serum holotranscobalamin concentrations regarding both concentration levels and the proportion of patients with concentrations within the normal levels (p=0.031, Mann-Whitney U-test; p=0.006, χ2-test). We have proven that adult patients with PKU and HPA are at risk of vitamin B12 nutritional deficiency. The most effective parameter for these adults is the monitoring of holotranscobalamin in the serum.

Serum folate, cobalamin, homocysteine and methylmalonic acid concentrations in pigs with acute, chronic or subclinical Lawsonia intracellularis infection

Lawsonia intracellularis is the causative agent of porcine proliferative enteropathy. The clinical presentation can be acute (i.e. proliferative hemorrhagic enteropathy, PHE), chronic (i.e. porcine intestinal adenomatosis, PIA) or subclinical. In humans with chronic enteropathies, low serum folate (vitamin B9) and cobalamin (vitamin B12) concentrations have been associated with increased serum concentrations of homocysteine and methylmalonic acid (MMA), which reflect the availability of both vitamins at the cellular level. The aim of this study was to evaluate serum folate, cobalamin, homocysteine and MMA concentrations in serum samples from pigs with PHE, PIA or subclinical L. intracellularis infection, and in negative controls. Serum folate, cobalamin, homocysteine and MMA concentrations differed significantly among pigs in the PHE, PIA, subclinical and negative control groups. Serum folate concentrations in the PHE and PIA groups were lower than in the subclinical and negative control groups, while serum cobalamin concentrations were lower in the PIA group than in other groups. Serum concentrations of homocysteine were higher in the PHE, PIA and subclinical groups than in the negative control group. Serum concentrations of MMA were higher in the subclinical and PIA groups than in the control group. These data suggest that pigs infected with L. intracellularis have altered serum cobalamin, folate, homocysteine and MMA concentrations.

Micronutrient status in female university students: iron, zinc, copper, selenium, vitamin B12 and folate.

Young women are at an increased risk of micronutrient deficiencies, particularly due to higher micronutrient requirements during childbearing years and multiple food group avoidances. The objective of this study was to investigate biomarkers of particular micronutrients in apparently healthy young women. Female students (n = 308; age range 18–35 year; Body Mass Index 21.5 ± 2.8 kg/m2; mean ± SD) were recruited to participate in a cross-sectional study. Blood samples were obtained from participants in the fasted state and analysed for biomarkers of iron status, vitamin B12, folate, homocysteine, selenium, zinc, and copper. The results show iron deficiency anaemia, unspecified anaemia, and hypoferritinemia in 3%, 7% and 33.9% of participants, respectively. Low vitamin B12 concentrations (<120 pmol/L) were found in 11.3% of participants, while 4.7% showed sub-clinical deficiency based on serum methylmalonic acid concentrations >0.34 μmol/L. Folate concentrations below the reference range were observed in 1.7% (serum) or 1% (erythrocytes) of participants, and 99.7% of the participant had erythrocyte-folate concentrations >300 nmol/L. Serum zinc concentrations <10.7 μmol/L were observed in 2% of participants. Serum copper and selenium concentrations were below the reference range in 23% and 11% of participants, respectively. Micronutrient deficiencies including iron and vitamin B12, and apparent excess of folate are present in educated Australian female students of childbearing age, including those studying nutrition. The effects of dietary behaviours and food choices on markers of micronutrient status require further investigation.

Suitability of methylmalonic acid and total homocysteine analysis in dried bloodspots

Methylmalonic acid (MMA) and total homocysteine (tHCYS) concentrations are used to detect acquired and inborn errors of cobalamin (vitamin B12, Cbl) metabolism and to evaluate the effect of therapeutic interventions. Dried blood spot sampling offers a patient-friendly and easy alternative to plasma sampling. However, dried blood spot concentrations are not necessarily equal to plasma concentrations. Therefore, the objective of this work was to establish the relationship between MMA and tHYS dried blood spot and plasma concentrations to facilitate clinical implementation of dried blood spot sampling. MMA and tHCYS in both plasma and DBS were validated on ultra performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS). While position of the punch (in DBS) did affect tHCYS concentration, no influence of hematocrit (Ht) and blood volume on both MMA and tHCYS concentrations was observed. The plasma assay performed better than the DBS assay by most criteria. However, the DBS matrix was superior for tHCYS stability. Paired plasma and DBS samples were obtained from patients suspected for Cbl deficiency and from patients with a known inborn error of metabolism affecting MMA or tHCYS concentration. Based on the strong correlation of tHCYS in both matrices (y=0.46±1.12 (r2=0.91)), determination of tHCYS in plasma can be replaced by tHCYS in DBS. However, for MMA, a correlation in the higher (pathological) range of MMA exist, but no correlation was observed in the lower ranges. Therefore the added value of MMA concentrations in DBS is currently unknown and should be further investigated.

Relationship between cobalamin-dependent metabolites and both serum albumin and alpha1 -proteinase inhibitor concentrations in hypocobalaminemic dogs of 7 different breeds.

Increased serum concentrations of homocysteine (HCY) and methylmalonic acid (MMA), the 2 main cobalamin-dependent metabolites, as well as decreased serum albumin and canine alpha1 -proteinase inhibitor (cα1 -PI) concentrations have previously been described in hypocobalaminemic dogs with gastrointestinal disease. However, no studies have been conducted to evaluate potential relationships between these serum biomarkers. The aim of this study was to evaluate the relationship between HCY and MMA, 2 cobalamin-dependent metabolites, and both serum albumin and cα1 -PI concentrations in hypocobalaminemic dogs. Serum samples from 285 dogs including 7 different breeds (Beagle, Boxer, Cocker Spaniel, German Shepherd, Labrador Retriever, Chinese Shar-Pei, and Yorkshire Terrier) with hypocobalaminemia were used. Serum HCY, MMA, albumin, and cα1 -PI concentrations were determined. There was a significant correlation between serum HCY and albumin concentrations, as well as serum HCY and cα1 -PI concentrations (ρ=0.62 and ρ=0.37, respectively; P<.0001). No correlations were observed between serum MMA and albumin concentrations, or cα1 -PI concentrations (ρ=0.01 and ρ=0.08, respectively; P>.05). In addition, significant breed-specific correlations were observed between serum MMA and albumin concentrations in German Shepherds, and serum HCY and MMA concentrations in Chinese Shar-Peis with hypocobalaminemia. This study shows a correlation between serum albumin and cα1 -PI and HCY concentrations, but not with serum MMA concentration in dogs with hypocobalaminemia. In addition, significant breed-specific correlations were observed between serum MMA and albumin concentrations in German Shepherds, as well as serum HCY and MMA concentrations in Chinese Shar-Peis, emphasizing the unique metabolic interactions in those dog breeds affected by hypocobalaminemia.

Methylmalonic Acid Quantified in Dried Blood Spots Provides a Precise, Valid, and Stable Measure of Functional Vitamin B-12 Status in Healthy Women

Methylmalonic acid (MMA) is a sensitive and specific functional biomarker of vitamin B-12 status, commonly assessed in plasma or serum. Dried blood spots (DBSs) allow simpler and more cost-efficient blood sampling than plasma. To facilitate convenient testing for vitamin B-12 deficiency in large-scale surveys and in population groups from remote areas, we developed a method for MMA quantification in DBSs and tested its applicability as well as the long-term stability of MMA in DBSs at various temperatures. MMA was extracted from an 8-mm DBS punch with water:methanol (95:5, v:v) and methyl-d3-malonic acid as the internal standard. After sample cleanup by ultrafiltration and hexane extraction, MMA was quantified by using reversed-phase LC-tandem mass spectrometry. Extraction conditions were optimized to maximize the detection signal and achieve DBS extract concentrations above the lowest limit of quantification (signal-to-noise ratio ≥ 10) of 10 nmol/L. Recovery was between 93% and 96%. Intra- and interassay variation (CV%) for DBS MMA was 0.49% and 2.3%, respectively. Calibrators showed linearity (R2 = 0.998) between 10 and 10,000 nmol/L. In 94 healthy women, MMA concentrations in DBS extract (min-max: 10.2–80.5 nmol/L) and plasma (min-max: 68–950 nmol/L) were correlated (ρ = 0.90) (P < 0.001). MMA concentrations in DBSs were stable at room temperature for 1 wk, in the refrigerator for 8 wk, and at −80°C for at least 1 y. This simple and robust method allows quantification of MMA in DBSs of healthy individuals. The linear relation between plasma and DBS MMA suggests that DBS MMA could predict plasma MMA, the current reference indicator for functional vitamin B-12 deficiency. With the advantages of minimally invasive specimen collection and no need for laborious blood processing steps, this method has the potential to be a reliable, convenient, and field-applicable alternative for assessment of vitamin B-12 status.

PO-0136 Increased Risk Of Vitamin B12 Nutritional Deficiency In Long-term Treated Patients With Phenylketonuria And Hyperfenylalaninemia

The aim of this study was to assess the prevalence of nutritional deficiency of vitamin B12 in long-term treated patients with phenylketonuria (PKU) and hyperfenylalaninemia (HPA), together with parameters of vitamin B12 and metabolic control. Methods In 51 patients aged 3–48 years (28 children, 23 adults) was examined levels of active vitamin B12 in serum, folate concentration in blood, plasma homocysteine and methylmalonic acid concentrations in urine. Results We found a statistically significant difference between the levels of folate in the blood among patients with PKU and HPA (p = 0.046, Mann Whitney U test). This difference was also statistically significant for adults with HPA and PKU (p = 0.004, Mann Whitney U test). There was a statistically significant difference in the proportion of normal homocysteine concentrations in plasma in the overall evaluation of both groups (p = 0.023, chi -square test). This difference was also statistically significant in adults with HPA and PKU (p = 0.032, chi -square test). In the group of adults we detected a statistically significant difference in the concentrations of active vitamin B12 in the blood as in the evaluation of the concentration and the proportion of patients with normal levels (p = 0.031, Mann Whitney U test, p = 0.006, chi -square test). Conclusions In the analysed group of patients we demonstrated that our patients are at risk of vitamin B12 nutritional deficiency and the risk increases with age.

An LC-MS/MS method for serum methylmalonic acid suitable for monitoring vitamin B12 status in population surveys.

Methylmalonic acid (MMA), a functional indicator of vitamin B12 insufficiency, was measured in the US population in the National Health and Nutrition Examination Survey (NHANES) from 1999 to 2004 using a GC/MS procedure that required 275 μL of sample and had a low throughput (36 samples/run). Our objective was to introduce a more efficient yet highly accurate LC-MS/MS method for NHANES 2011-2014. We adapted the sample preparation with some modifications from a published isotope-dilution LC-MS/MS procedure. The procedure utilized liquid-liquid extraction and generation of MMA dibutyl ester. Reversed-phase chromatography with isocratic elution allowed baseline resolution of MMA from its naturally occurring structural isomer succinic acid within 4.5 min. Our new method afforded an increased throughput (≤160 samples/run) and measured serum MMA with high sensitivity (LOD = 22.1 nmol/L) in only 75 μL of sample. Mean (±SD) recovery of MMA spiked into serum (2 d, 4 levels, 2 replicates each) was 94 % ± 5.5 %. Total imprecision (41 d, 2 replicates each) for three serum quality control pools was 4.9 %-7.9 % (97.1-548 nmol/L). The LC-MS/MS method showed excellent correlation (n = 326, r = 0.99) and no bias (Deming regression, Bland-Altman analysis) compared to the previous GC/MS method. Both methods produced virtually identical mean (±SD) MMA concentrations [LC-MS/MS: 18.47 ± 0.71 ng/mL (n = 17), GC/MS: 18.18 ± 0.67 ng/mL (n = 11)] on a future plasma reference material compared with a GC/MS method procedure from the National Institute of Standards and Technology [18.41 ± 0.70 ng/mL (n = 15)]. No adjustment will be necessary to compare previous (1999-2004) to future (2011-2014) NHANES MMA data.

Vitamin B-12 Supplementation during Pregnancy and Early Lactation Increases Maternal, Breast Milk, and Infant Measures of Vitamin B-12 Status

Pregnant women in resource-poor areas are at risk of multiple micronutrient deficiencies, and indicators of low vitamin B-12 status have been associated with adverse pregnancy outcomes, including anemia, low birth weight, and intrauterine growth retardation. To evaluate whether daily oral vitamin B-12 supplementation during pregnancy increases maternal and infant measures of vitamin B-12 status, we performed a randomized, placebo-controlled clinical trial. Pregnant women <14 wk of gestation in Bangalore, India, were randomly assigned to receive daily oral supplementation with vitamin B-12 (50 μg) or placebo through 6 wk postpartum. All women were administered iron and folic acid supplements throughout pregnancy. One hundred eighty-three women were randomly assigned to receive vitamin B-12 and 183 to receive placebo. Compared with placebo recipients, vitamin B-12–supplemented women had significantly higher plasma vitamin B-12 concentrations at both the second (median vitamin B-12 concentration: 216 vs. 111 pmol/L, P < 0.001) and third (median: 184 vs. 105 pmol/L, P < 0.001) trimesters. At 6 wk postpartum, median breast milk vitamin B-12 concentration was 136 pmol/L in vitamin B-12–supplemented women vs. 87 pmol/L in the placebo group (P < 0.0005). Among vitamin B-12–supplemented women, the incidence of delivering an infant with intrauterine growth retardation was 33 of 131 (25%) vs. 43 of 125 (34%) in those administered placebo (P = 0.11). In a subset of infants tested at 6 wk of age, median plasma vitamin B-12 concentration was 199 pmol/L in those born to supplemented women vs. 139 pmol/L in the placebo group (P = 0.01). Infant plasma methylmalonic acid and homocysteine concentrations were significantly lower in the vitamin B-12 group as well. Oral supplementation of urban Indian women with vitamin B-12 throughout pregnancy and early lactation significantly increases vitamin B-12 status of mothers and infants. It is important to determine whether there are correlations between these findings and neurologic and metabolic functions. This trial was registered at clinicaltrials.gov> as NCT00641862.