Cbl Status Research Articles

Simultaneous estimation of total homocysteine and methylmalonic-acid using LC-MS/MS: Clinical application in adult and pediatric

Quantitative determination of biomarkers homocysteine (Hcy) and methylmalonic acid (MMA), the regulators of cobalamin (Cbl) and folate levels, together used as a biomarkers to diagnose chemical insufficiency/deficiency of Cbl and folate. We report simultaneous clinical estimation of total Hcy and MMA with efficient clean-up, sensitive and selective LC-MS/MS method. Efficient sample clean-up was achieved by a two-step extraction protocol with 100 μL serum. The validated method was applied to 893 clinical samples from 2 cohorts including pediatrics and mothers, respectively, for identifying their Cbl and folate status. The method shows excellent order of linearity for Hcy (22.2nM-3.7 μM) and MMA (42.34 nM - 5.92 μM), respectively. Complete method validation was performed where intraday-interday accuracy-precision and mean stability recovery data were found within ±15%. The validated method was extended for the quantification of serum total Hcy-MMA levels in clinical samples. The efficient extraction with negligible matrix-effect (ME) has reduced LC-MS/MS chocking and clean-up downtime. The rapid, sensitive and robust LC-MS/MS method has been successfully validated for simultaneous estimation of total Hcy and MMA using only 100 μL serum. The method was applicable to large number of clinical samples and was found to be good throughput with low contamination of mass detector, high sensitivity and selectivity.

Alterations in Sulfur Amino Acids as Biomarkers of Disease

Homocysteine (Hcy) is methylated by methionine synthase to form methionine with methyl-cobalamin as a cofactor. The reaction demethylates 5-methyltetrahydrofolate to tetrahydrofolate, which is required for DNA and RNA synthesis. Deficiency of either of the cobalamin (Cbl) and/or folate cofactors results in elevated Hcy and megaloblastic anemia. Elevated Hcy is a sensitive biomarker of Cbl and/or folate status and more specific than serum vitamin assays. Elevated Hcy normalizes when the correct vitamin is given. Elevated Hcy is associated with alcohol use disorder and drugs that target folate or Cbl metabolism, and is a risk factor for thrombotic vascular disease. Elevated methionine and cystathionine are associated with liver disease. Elevated Hcy, cystathionine, and cysteine, but not methionine, are common in patients with chronic renal failure. Higher cysteine predicts obesity and future weight gain. Serum S-adenosylhomocysteine (AdoHcy) is elevated in Cbl deficiency and chronic renal failure. Drugs that require methylation for catabolism may deplete liver S-adenosylmethionine and raise AdoHcy and Hcy. Deficiency of Cbl or folate or perturbations of their metabolism cause major changes in sulfur amino acids.

Prospective long-term evaluation of parenteral hydroxocobalamin supplementation in juvenile beagles with selective intestinal cobalamin malabsorption (Imerslund-Gräsbeck syndrome).

BackgroundProspective studies on maintenance treatment for Beagles with hereditary selective cobalamin (Cbl) malabsorption (Imerslund‐Gräsbeck syndrome, IGS) are lacking. In our experience, measurement of methylmalonic acid (MMA), a Cbl‐dependent metabolite, seems more helpful to monitor Cbl status as compared with serum Cbl concentrations.ObjectivesTo evaluate a standardized Cbl supplementation scheme in Beagles with IGS. We hypothesized that a single parenteral dose of 1 mg hydroxocobalamin (OH‐Cbl) would maintain clinical and metabolic remission for up to 2 months.AnimalsSix client‐owned juvenile Beagles with genetically confirmed IGS and 28 healthy control dogs.MethodsProspective study. Monthly IM OH‐Cbl (1 mg) supplementation was done over a median of 9 months (range, 6‐13) in 6 dogs, followed by bimonthly (every 2 months) injections in 5 dogs over a median of 6 months (range, 3‐10). Health status was assessed by routine clinical examinations at injection time points and owner observations. Voided urine samples were collected immediately before OH‐Cbl injections for measurement of MMA‐to‐creatinine concentrations using a gas‐liquid chromatography‐tandem mass spectrometry (GC‐MS) method.ResultsAll dogs were clinically healthy while receiving monthly and bimonthly OH‐Cbl supplementation. Urinary MMA results in healthy dogs ranged from 1.3 to 76.5 mmol/mol creatinine (median, 2.9). Median urinary MMA concentrations did not differ between dogs with IGS receiving monthly (n = 49; 5.3 mmol/mol creatinine; range, 2.3‐50.4) and bimonthly (n = 31; 5.3 mmol/mol creatinine; range, 1.6‐50) injections.Conclusions and Clinical ImportanceA maintenance parenteral dose of 1 mg OH‐Cbl monthly or bimonthly appears adequate in Beagles with IGS monitored by metabolic testing.

Association between Maternal and Infantile Markers of Cobalamin Status During the First Month Post-Delivery.

Exclusively breast-fed infants born to vitamin B12 (cobalamin, cbl)-deficient mothers can develop symptoms within a few months following delivery. The authors aimed to assess the relationship between maternal and infantile markers of cbl status. In 240 full-term infants (age, 2-30 d) admitted to Samsun Maternity and Child Health Hospital and their mothers, complete blood count testing and serum cbl, folate and plasma total homocysteine (tHcy) measurements were performed. In the mothers, serum ferritin and holotranscobalamin (holoTC) levels were measured additionally. Among the infants, 146 (60.8%) had cbl deficiency (serum cbl <259pg/mL), whereas 184 (76.7%) mothers had a low cbl level (serum cbl <300pg/mL). When cbl deficiency was defined as a serum holoTC level < 40pmol/L, 152 (63.3%) mothers were found as deficient. In addition, 147 (61.3%) infants had an elevated tHcy level (>10μmol/L), in 35 (23.8%) of these 147 infants tHcy level being markedly elevated (>20μmol/L). None of the infants had folate deficiency. In the correlational analysis between maternal and infantile markers associated with cbl status, the strongest correlation was observed between maternal holoTC and infantile tHcy (r = -0.49, p < 0.001), followed by the correlation between maternal tHcy and infantile tHcy (r = 0.47, p < 0.001). The weakest correlations were found between maternal cbl and infantile cbl (r = 0.28, p < 0.001), and between maternal cbl and infantile tHcy (r = -0.25, p < 0.001). Maternal cbl status is an important determinant of infantile cbl status. Both maternal holoTC and tHcy may assist in predicting infantile cbl status. The finding of high prevalence of maternal and infantile cbl deficiency in this study points towards the need for effective strategies to prevent cbl deficiency in women prior to getting pregnant.

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.

Tissue distribution of oral vitamin B12 is influenced by B12 status and B12 form: an experimental study in rats

PurposeHydroxocobalamin (HOCbl) is the dominating Cbl form in food, whereas cyanocobalamin (CNCbl) is common in vitamin pills and oral supplements. This study compares single-dose absorption and distribution of oral HO[57Co]Cbl and CN[57Co]Cbl in Cbl-deficient and normal rats.MethodsMale Wistar rats (7 weeks) were fed a 14-day diet with (n = 15) or without (n = 15) Cbl. We compared the uptakes of HO[57Co]Cbl (free or bound to bovine transcobalamin) and free CN[57Co]Cbl administered by gastric gavage (n = 5 in each diet group). Rats were sacrificed after 24 h. Blood, liver, kidney, brain, heart, spleen, intestines, skeletal muscle, 24-h urine and faeces were collected, and the content of [57Co]Cbl was measured. Endogenous Cbl in tissues and plasma was analysed by routine methods.ResultsMean endogenous plasma-Cbl was sevenfold lower in deficient vs. normal rats (190 vs. 1330 pmol/L, p < 0.0001). Cbl depletion increased endogenous Cbl ratios (tissue/plasma = kin/kout) in all organs except for the kidney, where the ratio decreased considerably. Twenty-four-hour accumulation of labelled Cbl showed that HOCbl > CNCbl (liver) and CNCbl > HOCbl (brain, muscle and plasma).ConclusionsThe Cbl status of rats and the administered Cbl form influence 24-h Cbl accumulation in tissues and plasma.

Gene Expression Profiling Identifies Distinct Signatures for Dysplastic and Proliferative Chronic Myelomonocytic Leukemia

Background: The 2016 revision to the World Health Organization classification of myeloid neoplasms has recommended distinction between "proliferative" (WBC ≥ 13 x 10(9)/L) and "dysplastic" (WBC < 13 X 10(9)/L) subtypes of chronic myelomonocytic leukemia (CMML). In addition, CMML is characterized by a spectrum of cytogenetic and molecular abnormalities (Patnaik Leukemia 2014). In the current study, we looked for correlations between gene expression profiles (GEP) and morphologic or molecular categories of previously untreated patients with CMML.Methods: 35 patients with CMML were included in the study. Targeted capture assays were carried out on BM DNA specimens obtained at diagnosis for 28 myeloid-relevant genes (Patnaik Blood C. J.). RNA sequencing (Stranded Total RNA- Illumina, San Diego, CA), was performed on 9 normal BM controls, and 35 CMML samples (25 peripheral blood (PB) and 10 BM). The RNA-Seq data was analyzed using MAPRSeq v.2.0. Normalization and differential expression analysis was performed using edgeR 2.6.2. We selected genes that were differentially expressed with a False Discovery Rate (FDR) <0.1 and a fold change bigger than 2. Pathway enrichment analysis was performed using Ingenuity Pathway Analysis (IPA).Results: Among the 35 study patients, 75% were males and median age was 71 years (range, 55-87). 21 (60%), 9 (25%) and 5 (15%) patients were classified as CMML-0, 1 and 2, respectively. 16 (45%) had a dysplastic while 19 (55%) had a proliferative phenotype. At a median follow-up of 16 months, 5 (14%) deaths and 4 (14%) leukemic transformations were documented. Mutational frequencies included; TET2 45%, ASXL1 45%, SRSF2 40%, NRAS 14%, SETBP1 13%, CBL 10%, JAK2 7%, RUNX1 6%, DNMT3A 6%, U2AF1 6%, SF3B1 5%, ZRSR2 4%, Tp53 4%, and IDH2 4%. Abnormal cytogenetics (n=9, 35%) included; -Y 8%, trisomy 8 6%, monosomy 7 3%, del(13q) 3% and monosomal karyotype 3%.i) Differential GEP in PB samples:25 PB CMML samples were analyzed. Unsupervised GEP demonstrated two unique clusters (figure one); cluster one was enriched with dysplastic CMML (90%) and cluster two with proliferative CMML (64%). Consistent with the difference in the distribution of morphologic categories, in comparison to cluster one, patients in cluster two had a higher white blood count (p=0.003), higher monocyte count (p=0.02) and higher risk prognostication by the Mayo Model (p=0.016); there was no significant difference between the two clusters in terms of age and gender distribution, hemoglobin level, neutrophil and platelet counts, PB or BM blast content, cytogenetic risk stratification, ASXL1, TET2, RAS, CBL and SETBP1 mutational status. Genes significantly upregulated (FDR<1e-5 and fold change ≥ 16x); in cluster two (proliferative CMML) in comparison to cluster one included; PRG2, CTSG, BEX1, CD34, CRISP2, while those significantly down-regulated included (FDR<1e-15 and fold change ≥ 5x); IL2RB, PRF1, GNLY, PDGFRB. IPA analysis identified the following pathways preferentially expressed in cluster two (proliferative CMML) 1) mitotic roles of polo-like kinase (KIF23, WEE-1, PLK1, PLK4), 2) Cell cycle: G2/M checkpoint regulation (CDC25C, WEE-1, AURKA, CDK1), 3) Cell cycle: chromosomal replication (CDC45, RPA3, MCM2, CDC7), and 4) BRCA1 in DNA damage response (BARD1, RBBP8, PLK1, RAD51). Whereas in the cluster one (dysplastic CMML), preferentially expressed pathways included; 1) T-cell receptor signalling (CD247 PRKCQ, CD4, PLCG1, CD28) and 2) STAT3 signalling (SOCS3, MAP3K9).ii) Differential GEP in BM samples:10 BM CMML samples were analyzed along with 10 normal controls. Samples and controls clustered separately and within the CMML samples two clusters were identified (figure two). These clusters had equal representations of dysplastic (n=5) and proliferative (n=5) CMML sub-types. Additional samples are being processed.Conclusions: Differential gene expression profiling in patients with CMML identifies two unique clusters, clinically demarcated, at least in PB samples, by "proliferative" and "dysplastic" CMML subtypes. The proliferative cluster is enriched in genes involved in cell cycle regulation and DNA damage response; whereas the dysplastic cluster is enriched in T-cell receptor and STAT3 signalling. The prognostic and therapeutic impact of these gene clusters remains to be assessed in a larger group of informative patients. [Display omitted] [Display omitted] DisclosuresAl-Kali:Novartis: Research Funding; Celgene: Research Funding.

Fasting time and vitamin B12 levels in a community-based population

ObjectivesVitamin B12, also known as cobalamin (Cbl), is an essential vitamin that manifests with numerous severe but non-specific symptoms in cases of deficiency. Assessing Cbl status often requires fasting, although this requirement is not standard between institutions. This study evaluated the impact of fasting on Cbl levels in a large community-based cohort in an effort to promote standardization of Cbl testing between sites. Design and methodsLaboratory data for Cbl, fasting time, patient age and sex were obtained from laboratory information service from Calgary Laboratory Services (CLS) for the period of April 2011 to June 2015. CLS is the sole supplier of laboratory services in the Southern Alberta region in Canada (population, approximately 1.4 million). To investigate potential sex-specific effects of fasting on Cbl levels, males and females were analyzed separately using linear regression models. ResultsA total of 346,957 individual patient results (196,849 females, 146,085 males) were obtained. The mean plasma Cbl level was 386.5 (±195.6) pmol/L and 412.0 (±220.8) pmol/L for males and females, respectively. Linear regression analysis showed fasting had no significant association with Cbl levels in females; however a statistically significant decrease of 0.9pmol/L/hour fasting (p<0.001) was noted in males. ConclusionsThe broad population variance in Cbl suggests the slight gender-specific differences noted in this study are insignificant. Despite this, fasting has the potential to contribute to higher rates of Cbl deficiency in men. Together, these data suggest fasting should be excluded as a requirement for evaluating plasma Cbl.

Correlation of Tc-99 m ethyl cysteinate dimer single-photon emission computed tomography and clinical presentations in patients with low cobalamin status.

BackgroundCobalamin (Cbl) deficiency has been associated with various neuropsychiatric symptoms of different severities. While some studies dedicated in structural neuroimaging credibly address negative impact of low Cbl status, functional imaging reports are limited. We herein retrospectively review the correlation of Tc-99 m ethyl cysteinate dimer single-photon emission computed tomography (Tc-99 m-ECD SPECT) and clinical presentations among patients with low serum cobalamin (Cbl) status (<250 pg/ml).MethodsTwelve symptomatic patients with low serum Cbl status were enrolled. Clinical presentations, Tc-99 m-ECD SPECT, and neuropsychological tests were reviewed.ResultsDysexecutive syndrome (67 %), forgetfulness (50 %), attention deficits (42 %), and sleep disorders (33 %) constituted the major clinical presentations. All patients (100 %) had temporal hypoperfusion on the Tc-99 m-ECD SPECT. Five patients (42 %) had hypoperfusion restricted within temporal regions and deep nuclei; seven patients (58 %) had additional frontal hypoperfusion. In patients with hypoperfusion restricted within temporal regions and deep nuclei, psychiatric symptoms with spared cognition were their main presentations. Among patients with additional frontal hypoperfusion, six of seven patients (86 %) showed impaired cognitive performances (two of them were diagnosed as having dementia). Among ten patients who finished neuropsychological tests, abstract thinking (70 %) was the most commonly affected, followed by verbal fluency (60 %), short-term memory (50 %), and attention (50 %). Anxiety and sleep problems were the major clinically remarkable psychiatric features (33 % both). Four Tc-99 m-ECD SPECT follow-up studies were available; the degree and extent of signal reversal correlated with cognitive changes after Cbl replacement therapy.ConclusionsOur TC-99 m-ECD SPECT observations provide pivotal information of neurobiological changes within basal ganglia and fronto-temporal regions in conjunction with disease severity among patients with Cbl deficiency. Hypoperfusion within thalamus/basal ganglia and temporal regions may be seen in the earlier state of Cbl deficiency, when psychiatric symptoms predominate. Hypoperfusion beyond thalamus/basal ganglia and involving frontal regions appears when cognitive problems, mostly dysexecutive syndrome, are manifested. Symmetric hypofrontality of SPECT in the context of dysexcutive syndrome serves as a distinguishing feature of non-amnestic mild cognitive impairment attributed to Cbl deficiency. Concordant with TC-99 m-ECD SPECT findings, the psychiatric symptoms and dysexcutive syndrome undergird impaired limbic and dorsolateral prefrontal circuits originating from basal ganglia respectively.

The Relationship of Serum Cobalamin to Methylmalonic Acid Concentrations and Clinical Variables in Cats

Serum cobalamin concentration [CBL] suggests CBL deficiency in cats but serum methylmalonic acid concentration [MMA] more accurately indicates CBL deficiency. To examine the ability of [CBL] to predict CBL deficiency defined by increased [MMA], and relationships of [CBL] and [MMA] with select clinical and clinicopathological variables. One hundred sixty-three client-owned cats with [CBL] measurements, 114 cats with simultaneous [MMA] measurements; 88 cats with medical information. Prospectively collected [CBL] and [MMA] were compared using scatter plots, receiver operating characteristic and correlative analyses with historical [CBL] thresholds and those identified in the study. [CBL] and [MMA] were compared retrospectively to specific clinical and clinicopathological variables. [CBL] correlated negatively with [MMA] (τ = -0.334, P < .0001). [MMA] ≥ 1,343 nmol/L identified CBL deficiency. [CBL] = 209 pg/mL optimized sensitivity (0.51), specificity (0.96), PPV (0.89), and NPV (0.74) for detecting [MMA] ≥ 1,343 nmol/L. Prevalence of CBL deficiency was 42% (48/114) when defined by [MMA] ≥ 1,343 nmol/L versus 23% (27/114) by [CBL] ≤ 209 pg/mL. Unexpectedly, 23 and 45% of 48 cats with [MMA] ≥ 1,343 nmol/L had [CBL] > 900 pg/mL and 290 pg/mL (historical thresholds). [CBL] correlated with mean corpuscular volume (τ = -0.199, P = .013) and [MMA] with hematocrit (τ = -0.28, P = .006). Cobalamin deficiency ([MMA] ≥ 1,343 nmol/L) occurred in 42% of cats and is predicted with high specificity by [CBL] ≤ 209 pg/mL. CBL status correlates with microcytosis and anemia. Discordance between [CBL] and [MMA] cautions against relying on any single marker for determining CBL status.

Vitamin B12 dependent changes in mouse spinal cord expression of vitamin B12 related proteins and the epidermal growth factor system

Chronic vitamin B12 (cobalamin) deficiency in the mammalian central nervous system causes degenerative damage, especially in the spinal cord. Previous studies have shown that cobalamin status alters spinal cord expression of epidermal growth factor (EGF) and its receptor in rats. Employing a mouse model of cobalamin-depletion and loading, we have explored the influence of Cbl status on spinal cord expression of cobalamin related proteins, as well as all four known EGF receptors and their activating ligands. Following four weeks of osmotic minipump infusion (n=7 in each group) with cobinamide (4.25nmol/h), saline or cobalamin (1.75nmol/h) the spinal cords were analyzed for cobalamin and for the mRNA levels of cobalamin related proteins and members of the EGF system using quantitative reverse transcription PCR. The median spinal cord cobalamin content was 17, 32, and 52pmol/gr of tissues in cobinamide, saline, and cobalamin treated animals, respectively. Both cobinamide and cobalamin induced a significant decrease in the expression of the lysosomal membrane cobalamin transporter. All four EGF receptors and their activating ligands, except for EGF, were expressed in the spinal cord. Notably, the expression of one of the EGF receptors, HER3, and the ligands heparin-binding EGF-like growth factor, transforming growth factor-α, and neuregulins 1α was increased in cobalamin treated mice. Our studies show that four weeks treatment of mice with cobinamide induces spinal cord cobalamin depletion and that cobalamin loading induces an altered expression pattern of the EGF system thus confirming a spinal cord cross talk between Cbl and the EGF system.

Transcobalamin Polymorphism 67A->G, but Not 776C->G, Affects Serum Holotranscobalamin in a Cohort of Healthy Middle-Aged Men and Women

Two polymorphic variants in the gene coding for transcobalamin II (TCN2),TCN2776C- > G andTCN267A- > G, may alter serum holotranscobalamin (holoTC), which in turn may affect cellular uptake of cobalamin (Cbl) and thereby Cbl status indicators. We studied the effects ofTCN2776C- > G andTCN267A- > G on blood concentrations of holoTC, Cbl, methylmalonic acid (MMA), and total homocysteine (tHcy) in 2411 individuals (50–64 y) that had been selected on the basis of theseTCN2genotypes from 10601 Norwegian inhabitants. The serum holoTC concentration was lower inTCN267AG (55 ± 0.75 pmol/L) and 67GG (48 ± 2.14 pmol/L) than in 67AA (62 ± 0.67 pmol/L) (P< 0.001) but did not differ amongTCN2776C- > G genotypes. The polymorphisms interacted as serum holoTC determinants (P= 0.001) and the presence ofTCN267AG and GG in strata of 776CC and CG, but not 776GG, increased the risk of having serum holoTC < 45.6 pmol/L [tertile 1 vs. tertiles 2 and 3: OR = 2.5 (95% CI 1.8–3.5) for 67AG; OR = 5.7 (95% CI 3.5–9.1) for 67GG in 776CC; OR = 2.1 (95% CI 1.6–2.9) for 67AG; and OR = 4.5 (95% CI 2.4–8.2) for 67GG in 776CG; all P< 0.001]. Plasma MMA, tHcy, and Cbl were not affected by either polymorphism. In summary, serum holoTC, but not plasma Cbl, MMA, or tHcy, varied according toTCN267A- > G genotypes. It remains to be determined whether this polymorphic effect on serum holoTC alters its diagnostic utility as Cbl status indicator.

Clinical presentation and metabolic consequences in 40 breastfed infants with nutritional vitamin B 12 deficiency – What have we learned?

Background Maternal vitamin B 12 (Cbl) deficiency causes nutritional Cbl deficiency in breastfed infants. Aims To analyse clinical presentation and metabolic consequences in 40 breastfed infants with Cbl deficiency. Methods Cbl levels in serum and breast milk were determined by an electrochemiluminescence immunoassay, methylmalonic acid level by GC/MS, plasma homocysteine by HPLC and propionylcarnitine by MS/MS. Profound Cbl deficiency was found in 17 children (69 ± 17 ng/l, controls 200–900), and milder Cbl deficiency in 23 children (167 ± 40 ng/l). Maternal Cbl deficiency was mostly caused by insufficient Cbl absorption. Only six mothers were vegetarian. Results The average age at diagnosis was 4.4 ± 2.5 months. Clinical symptoms included failure to thrive (48% of children), hypotonia (40%), developmental delay (38%) and microcephaly (23%). 63% of children had anaemia (megaloblastic in 28% of all children). All but one patient had methylmalonic aciduria, 80% of patients had hyperhomocysteinemia and 87% had increased aminotransferases. Propionylcarnitine was elevated in two out of 25 infants. Comparing groups with severe and mild Cbl deficiency, a marked difference was found in severity of clinical and laboratory changes. Conclusion Maternal Cbl status and diagnostic delay are the major factors influencing severity and progression of Cbl deficiency in breastfed infants. In our cohort, propionylcarnitine was not sufficiently sensitive marker of Cbl deficiency. Although symptoms are reversible on Cbl substitution, permanent neurological damage can result. Selective screening for Cbl deficiency is indicated in all breastfed infants with failure to thrive, hypotonia, developmental delay, microcephaly or megaloblastic anaemia. The best prevention in future could be the screening of all pregnant women.

Long-term Effects of Folic Acid Fortification and B-vitamin Supplementation on Total Folate, Homocysteine, Methylmalonic Acid and Cobalamin in Older Adults

To investigate the long-term effects of the Canadian folic acid fortification program in older adults' whole blood cell folate (folate) and cobalamin (Cbl) status, including homocysteine (tHcy) and methylmalonic acid (MMA), with and without voluntary B-vitamin intake, from 1997 to 2004. Cohort of community-dwelling volunteer older adults. Clinical and biochemical data, including intake of B-vitamin supplements, were obtained at 2- to 2.5-year intervals and divided in 4 periods. Random coefficients (mixed effects) models were used to estimate the linear trend in folate and to compare levels of biochemical parameters between periods. All models were estimated by restricted maximum likelihood as implemented in PROC MIXED of SAS V8.2. Folate levels increased continuously at a yearly rate of 234 ng/mL (95% CI 213-254; p < 0.001) and had not plateaued by the last period when 84% of subjects without B-vitamins had elevated folate. Homocysteine did not remain suppressed. Elevated tHcy was as prevalent in the last study period as in the first. No significant deficits of Cbl or increases of MMA were observed, but MMA levels tended to increase with time in subjects without B-vitamins. B-vitamin supplements significantly affected all results, reducing tHcy and MMA levels. In this population, fortification with folic acid has resulted in cumulative increases of folate with no long-term reduction in tHcy or changes in Cbl or MMA. Possible deleterious effects of cumulative increases of folate, and beneficial effects of B-vitamin supplements in reducing tHcy and MMA, should be investigated.

Association between decreased vitamin levels and MTHFR, MTR and MTRR gene polymorphisms as determinants for elevated total homocysteine concentrations in pregnant women

To examine the association between methylenetetrahydrofolate reductase (MTHFR) (C677T and A1298C), methionine synthase (MTR) A2756G and methionine synthase reductase (MTRR) A66G gene polymorphisms and total homocysteine (tHcy), methylmalonic acid (MMA) and S-adenosylmethionine/S-adenosylhomocysteine (SAM/SAH) levels; and to evaluate the potential interactions with folate or cobalamin (Cbl) status. Two hundred seventy-five healthy women at labor who delivered full-term normal babies. Cbl, folate, tHcy, MMA, SAM and SAH were measured in serum specimens. The genotypes for polymorphisms were determined by PCR-restriction fragment length polymorphism (RFLP). Serum folate, MTHFR 677T allele and MTR 2756AA genotypes were the predictors of tHcy levels in pregnant women. Serum Cbl and creatinine were the predictors of SAM/SAH ratio and MMA levels, respectively. The gene polymorphisms were not determinants for MMA levels and SAM/SAH ratios. Low levels of serum folate were associated with elevated tHcy in pregnant women, independently of the gene polymorphisms. In pregnant women carrying MTHFR 677T allele, or MTHFR 1298AA or MTRR 66AA genotypes, lower Cbl levels were associated with higher levels of tHcy. Lower SAM/SAH ratio was found in MTHFR 677CC or MTRR A2756AA genotypes carriers when Cbl levels were lower than 142 pmol/l. Serum folate and MTHFR C677T and MTR A2576G gene polymorphisms were the determinants for tHcy levels. The interaction between low levels of serum Cbl and MTHFR (C677T or A1298C) or MTRR A66G gene polymorphisms was associated with increased tHcy.

The return of the Scarlet Pimpernel: cobalamin in inflammation II - cobalamins can both selectively promote all three nitric oxide synthases (NOS), particularly iNOS and eNOS, and, as needed, selectively inhibit iNOS and nNOS.

The up-regulation of transcobalamins [hitherto posited as indicating a central need for cobalamin (Cbl) in inflammation], whose expression, like inducible nitric oxide synthase (iNOS), is Sp1- and interferondependent, together with increased intracellular formation of glutathionylcobalamin (GSCbl), adenosylcobalamin (AdoCbl), methylcobalamin (MeCbl), may be essential for the timely promotion and later selective inhibition of iNOS and concordant regulation of endothelial and neuronal NOS (eNOS/nNOS.) Cbl may ensure controlled high output of nitric oxide (NO) and its safe deployment, because: (1) Cbl is ultimately responsible for the synthesis or availability of the NOS substrates and cofactors heme, arginine, BH4 flavin adenine dinucleotide/flavin mononucleotide (FAD/FMN) and NADPH, via the far-reaching effects of the two Cbl coenzymes, methionine synthase (MS) and methylmalonyl CoA mutase (MCoAM) in, or on, the folate, glutathione, tricarboxylic acid (TCA) and urea cycles, oxidative phosphorylation, glycolysis and the pentose phosphate pathway. Deficiency of any of theNOS substrates and cofactors results in ‘uncoupled’ NOS reactions, decreasedNO production and increased or excessive O2−, H2O2, ONOO− and other reactive oxygen species (ROS), reactive nitric oxide species (RNIS) leading to pathology. (2) Cbl is also the overlooked ultimate determinant of positive glutathione status, which favours the formation of more benign NO species, s-nitrosothiols, the predominant form in which NO is safely deployed. Cbl status may consequently act as a ‘back-up disc’ that ensures the active status of antioxidant systems, as well as reversing and modulating the effects of nitrosylation in cell signal transduction.New evidence shows that GSCbl can significantly promote iNOS/ eNOS NO synthesis in the early stages of inflammation, thus lowering high levels of tumour necrosis factor-a that normally result in pathology, while existing evidence shows that in extreme nitrosative and oxidative stress, GSCbl can regenerate the activity of enzymes important for eventual resolution, such as glucose 6 phosphate dehydrogenase, which ensures NADPH supply, lactate dehydrogenase, and more; with human clinical case studies of OHCbl for cyanide poisoning, suggesting Cbl may regenerate aconitase and cytochrome c oxidase in the TCA cycle and oxidative phosphorylation. Thus, Cbl may simultaneously promote a strong inflammatory response and the means to resolve it.

A simple high-throughput method for the determination of plasma methylmalonic acid by liquid chromatography-tandem mass spectrometry

Cobalamin (Cbl) deficiency is a common clinical phenomenon, in particular among the elderly and possibly also among infants. Methylmalonic acid (MMA) is the most sensitive and specific marker of intracellular Cbl status, but its application is hindered by limited methods available for accurate and high-throughput MMA determination. We developed a non-laborious method for determination of MMA without the need for prior derivatization using HPLC combined with liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Stable isotope-labeled methyl-d(3)-malonic acid (MMA-d(3)) was added to 100 microL of plasma as an internal standard. After deproteinization by ultrafiltration, an acidified aliquot of the eluate was injected into the HPLC system and analyzed by LC-ESI-MS/MS monitoring of the carbonyl loss of MMA and MMA-d(3). Calibrations between 0.1 and 1.0 microM exhibited consistent linearity and reproducibility. The lower limit of detection for plasma MMA was 0.1 microM (signal-to-noise ratio > or = 10). The intra- and inter-assay CVs of ten determinations of a plasma sample were 1.5% and 6.7%, respectively, at a mean concentration of 0.29 microM. Inter-assay CVs for 25 determinations of low, medium and high concentrations (0.22, 0.45 and 0.94 microM MMA) were 8.3%, 5.9% and 4.6%, respectively. The mean recovery of MMA added to plasma was 100%. By avoiding derivatization, we developed a new, non-laborious, simple and reliable high-throughput method for the determination of MMA that is suitable for automation.

Cobalamin Status (Holo-Transcobalamin, Methylmalonic Acid) and Folate as Determinants of Homocysteine Concentration

Concern has emerged in America about subtle cobalamin (Cbl; vitamin B12) deficiency, especially in at-risk population groups such as the elderly and vegetarians (1). An optimal test to diagnose vitamin B12 deficiency is still not available (2)(3)(4). The determination of total serum vitamin B12 has a low diagnostic accuracy (2)(5). Measurements of homocysteine (HCY) and methylmalonic acid (MMA) have shown more specificity and sensitivity for subnormal Cbl status, but have disadvantages. HCY, for example, is also increased in folate and vitamin B6 deficiencies, as well as in renal insufficiency (4); in addition, MMA is expensive to measure, and it, too, increases in renal insufficiency (2)(4)(5). To improve specificity and sensitivity in diagnosis of vitamin B12 deficiency, holo-transcobalamin (holoTC) assays have been introduced (6). Because only transcobalamin II promotes the specific cellular uptake of Cbl, the Cbl subfraction attached to transcobalamin II represents the biologically active vitamin B12 fraction (6)(7)(8). Our previous observations in Syrian individuals revealed a high prevalence of Cbl deficiency (≈49%) when we used MMA as a metabolic marker for Cbl status (9). The present work …

Further evidence for the involvement of epidermal growth factor in the signaling pathway of vitamin B12 (cobalamin) in the rat central nervous system.

In order to get further evidence for a mandatory involvement of epidermal growth factor (EGF) in the neutrophic action of vitamin B12 (cobalamin (Cbl)) in the central nervous system (CNS) of the rat, we observed the effects of repeated intracerebroventricular (ICV) microinjections of EGF in rats made Cbl-deficient through total gastrectomy. Morphometric analysis demonstrated a significant reduction in both intramyelinic and interstitial edema in the white matter of the spinal cord (SC) of totally gastrectomized (TGX) rats after treatment. Intramyelinic and interstitial edema are characteristic of Cbl-deficient central neuropathy in the rat. Similar lesions were also present in SC white matter of rats treated with repeated ICV microinjections of specific anti-EGF antibodies without any modification in their Cbl status. These results, together with those of a previous study showing the cessation of EGF synthesis in the CNS of TGX rats, demonstrate that: a) EGF is necessarily involved in the signaling pathway of Cbl in the rat CNS; and b) the lack of a neurotrophic growth factor EGF, and not the mere withdrawal of Cbl, causes or at least contributes to neurodegenerative Cbl-deficient central neuropathy.