Fig 1 - uploaded by Per M Ueland
Content may be subject to copyright.
Source publication
Newborn screening for total homocysteine (tHcy) in blood may identify babies with vitamin B12 (B12) deficiency or homocystinuria, but data on the causes of increased tHcy in screening samples are sparse.
Serum concentrations of tHcy, cystathionine, methionine, folate, and B12 and the methylenetetrahydrofolate reductase (MTHFR) 677C > T polymorphism...
Contexts in source publication
Context 1
... intention with newborn screening is to identify babies with serious and treatable conditions before symptoms arise (1 , 2 ) . Newborn-screening programs differ among countries, but most programs screen for phenylketonuria and congenital hypothyroidism and represent an important investment for the prevention of death and disability (1 ) . Newborn screening for homocystinuria attributable to cystathionine  -synthase (CBS) 6 deficiency is carried out in several countries and in some regions of the United States (1 , 3 ) . CBS deficiency is an autosomal recessively inherited disorder of the transsulfuration pathway (Fig. 1). On the basis of data from metabolic newborn screening, the worldwide birth prevalence of CBS deficiency is ϳ 1 in 300 000, but with marked regional differences, being more common in Ireland and New South Wales ( ϳ 1:60 000) (3 ) . Recent data based on mutation analyses in newborn samples suggest that it may be far more common, i.e., 1:20 000 or even higher (4 – 6 ) . To date, more than 130 disease-associated mutations in the CBS gene have been identified (7 ) . CBS deficiency leads to markedly increased concentrations of homocystine in urine and total homocysteine (tHcy) and methionine in ...
Context 2
... , 8 ) . In addition to a greatly increased risk of thrombo- embolic events, clinical signs and symptoms include mental retardation, psychiatric disorders, ectopia lentis, and skeletal abnormalities such as osteoporosis and marfanoid stature (3 ) . Approximately 50% of patients respond to pyridoxine with a marked decrease in tHcy. Independent of pyridoxine responsiveness, treatment from infancy with tHcy-lowering agents prevents premature vascular disease and mortality (9 , 10 ) . Homocystinuria may also be caused by severe deficiencies of methylenetetrahydrofolate reductase (MTHFR) or methionine synthase ( Fig. 1), or defects in transport proteins or enzymes providing vitamin B 12 (B 12 ) to methionine synthase. These so-called remethylation defects lead to increased tHcy, whereas methionine is low or within reference values (11 , 12 ) . Symptoms often develop early in life and include developmental delay, failure to thrive, myelopathy, and sometimes, megaloblastic anemia (11 ) . The effect of therapy with B 12 , folic acid, and betaine is variable (11 ) , but some data indicate that early diagnosis and treatment can reduce complications and delay symptom onset (11 ) . Recently, an acquired cause of impaired remethylation has received increasing attention: many babies have low B 12 concentrations (13 – 15 ) . Such neonatal B 12 deficiency is nearly always attributable to low maternal B 12 status (16 ) . These babies already have low B 12 combined with increased concentrations of tHcy and the specific B 12 marker, methylmalonic acid (MMA; Fig. 1) at birth. They continue to have a low B 12 status during infancy, particularly if the baby is exclusively breastfed (13 , 17 , 18 ) . B 12 deficiency may lead to failure to thrive and developmental delay in infancy and lower cognitive function later in childhood (16 ) . Low B 12 status is usually easily corrected by supplying vitamin B 12 (19 ) . The conventional approach for newborn screening for homocystinuria is detection of increased methionine by the bacterial inhibition assay (3 ) . However, methionine measurement identifies only the more severe and usually pyridoxine-nonresponsive variants of CBS deficiency (3 ) ; it does not detect inborn errors attributable to remethylation defects, nor will it identify babies with B 12 deficiency. In this regard, measurement of tHcy may be a better approach. However, data on tHcy in newborn-screening samples are sparse (20 ) , and a critical evaluation of the use of tHcy measurements as a potential screening tool for identification of babies with homocystinuria or low B 12 status is lacking. In this study, we investigated tHcy and related variables in ϳ 5000 newborn-screening samples. The effect of selected CBS mutations was also examined. Our intention was to identify the various factors determining increased tHcy concentrations in newborn babies and, if possible, to present some recommendations on the use of tHcy screening in newborns. From February to April 1999, approximately 12 000 capillary blood samples were sent to the Rikshospitalet University Hospital in Oslo for routine newborn screening for phenylketonuria and congenital hypothyroidism. From these samples, 4992 samples were randomly selected for the present study. The blood was collected in a gel separator tube, usually 3 – 5 days after birth. The tube was centrifuged locally, and the tube containing both serum and blood cells was then sent to the screening laboratory, where it was kept at 2 – 4 ° C. Aliquots ( ϳ 50 – 100 L) of serum and packed blood cells from each sample were transferred into microtiter plates (52 plates; 96 samples per plate) after the routine screening had been completed, and the samples were then stored at Ϫ 20 ° C until analyses. For 104 babies (the Bergen sample set), we had information about time of blood collection relative to birth. All samples used in the study were unlinked and anonymous. Total sample population. The total number of samples, i.e., microtiter wells, was 4992. However, one serum well was empty, and another six wells contained serum where ...
Context 3
... , 8 ) . In addition to a greatly increased risk of thrombo- embolic events, clinical signs and symptoms include mental retardation, psychiatric disorders, ectopia lentis, and skeletal abnormalities such as osteoporosis and marfanoid stature (3 ) . Approximately 50% of patients respond to pyridoxine with a marked decrease in tHcy. Independent of pyridoxine responsiveness, treatment from infancy with tHcy-lowering agents prevents premature vascular disease and mortality (9 , 10 ) . Homocystinuria may also be caused by severe deficiencies of methylenetetrahydrofolate reductase (MTHFR) or methionine synthase ( Fig. 1), or defects in transport proteins or enzymes providing vitamin B 12 (B 12 ) to methionine synthase. These so-called remethylation defects lead to increased tHcy, whereas methionine is low or within reference values (11 , 12 ) . Symptoms often develop early in life and include developmental delay, failure to thrive, myelopathy, and sometimes, megaloblastic anemia (11 ) . The effect of therapy with B 12 , folic acid, and betaine is variable (11 ) , but some data indicate that early diagnosis and treatment can reduce complications and delay symptom onset (11 ) . Recently, an acquired cause of impaired remethylation has received increasing attention: many babies have low B 12 concentrations (13 – 15 ) . Such neonatal B 12 deficiency is nearly always attributable to low maternal B 12 status (16 ) . These babies already have low B 12 combined with increased concentrations of tHcy and the specific B 12 marker, methylmalonic acid (MMA; Fig. 1) at birth. They continue to have a low B 12 status during infancy, particularly if the baby is exclusively breastfed (13 , 17 , 18 ) . B 12 deficiency may lead to failure to thrive and developmental delay in infancy and lower cognitive function later in childhood (16 ) . Low B 12 status is usually easily corrected by supplying vitamin B 12 (19 ) . The conventional approach for newborn screening for homocystinuria is detection of increased methionine by the bacterial inhibition assay (3 ) . However, methionine measurement identifies only the more severe and usually pyridoxine-nonresponsive variants of CBS deficiency (3 ) ; it does not detect inborn errors attributable to remethylation defects, nor will it identify babies with B 12 deficiency. In this regard, measurement of tHcy may be a better approach. However, data on tHcy in newborn-screening samples are sparse (20 ) , and a critical evaluation of the use of tHcy measurements as a potential screening tool for identification of babies with homocystinuria or low B 12 status is lacking. In this study, we investigated tHcy and related variables in ϳ 5000 newborn-screening samples. The effect of selected CBS mutations was also examined. Our intention was to identify the various factors determining increased tHcy concentrations in newborn babies and, if possible, to present some recommendations on the use of tHcy screening in newborns. From February to April 1999, approximately 12 000 capillary blood samples were sent to the Rikshospitalet University Hospital in Oslo for routine newborn screening for phenylketonuria and congenital hypothyroidism. From these samples, 4992 samples were randomly selected for the present study. The blood was collected in a gel separator tube, usually 3 – 5 days after birth. The tube was centrifuged locally, and the tube containing both serum and blood cells was then sent to the screening laboratory, where it was kept at 2 – 4 ° C. Aliquots ( ϳ 50 – 100 L) of serum and packed blood cells from each sample were transferred into microtiter plates (52 plates; 96 samples per plate) after the routine screening had been completed, and the samples were then stored at Ϫ 20 ° C until analyses. For 104 babies (the Bergen sample set), we had information about time of blood collection relative to birth. All samples used in the study were unlinked and anonymous. Total sample population. The total number of samples, i.e., microtiter wells, was 4992. However, one serum well was empty, and another six wells contained serum where ...
Similar publications
Objectives: To design an algorithm for the differential diagnosis of hyperhomocysteinemia and to apply it to the diagnosis of 27 pediatric patients. Methods: Total homocysteine: HPLC with fluorescence detection of the SBDF derivatives. Amino acids: ion-exchange chromatography. Organic acids: gas chromatography-mass spectrometry. Folate and vitamin...
Citations
... Herein, a negative association between serum 5-MTHF and cystathionine concentrations was observed (Figure 3). A negative correlation between blood total folate and cystathionine concentrations has also been observed in neonates [102] and patients with coronary artery disease [103,104], possibly because Hcy and cystathionine have similar kinetics [104]. This correlation was different than that observed between tHcy and tCys, and the complex transsulfuration pathway needs to be further elucidated. ...
Maintaining optimal one-carbon metabolism (OCM) is essential for health and pregnancy. In this cross-sectional study, folate status was assessed based on 5-methyltetrahydrofolate (5-MTHF) levels, and the association between 5-MTHF and OCM-related metabolites was investigated in 227 female Japanese university students aged 18-25 years. The participants were divided into high and low 5-MTHF groups based on their folate status. Serum samples of the participants were collected while they were fasting, and 18 OCM-related metabolites were measured using stable-isotope dilution liquid chromatography-electrospray tandem mass spectrometry. The association between serum 5-MTHF and OCM-related metabolite concentrations was assessed using Spearman's rank correlation coefficient. Serum 5-MTHF concentrations were negatively correlated with total homocysteine (tHcy) concentrations and positively correlated with S-adenosylmethionine (SAM) and total cysteine (tCys) concentrations. Serum 5-MTHF concentrations demonstrated a stronger negative correlation with tHcy/tCys than with tHcy alone. The negative correlation between betaine and tHcy concentrations was stronger in the low 5-MTHF group than in the high 5-MTHF group. The 5-MTHF status could be linked to Hcy flux into the transsulfuration pathway via SAM. Therefore, the tHcy/tCys ratio may be a more sensitive indicator of the 5-MTHF status than tHcy alone. Furthermore, a low 5-MTHF status can enhance Hcy metabolism via betaine.
... Thus, the detection rate of identified B12 deficiency at NBS is low compared to the findings in other Scandinavian studies where 0.31-0.34% of infant populations are clinically diagnosed with symptomatic B12 deficiency [7,8]. Moreover, in a Norwegian NBS study performed in 1999, where NBS specimens were obtained as serum samples, 5% of newborns showed B12 deficiency [32]. A similar study is difficult to reproduce as B12 in pmol/L concentration is challenging to measure using DBS, but the results from 20 years ago are still 100 times higher than those reported in recent NBS studies using algorithms adjusted to detect B12 deficiency [4,20,21]. ...
... If an alternative definition derived from a study of four-day-old infants had been applied instead (tHcy > 11 µmol/L or MMA > 0.6 µmol/L, both corresponding to 97.5th percentile) [34], the same proportion (40%) of newborns would have been categorized as B12-deficient among the false positives reported in our study. According to the definition of Refsum et al. (B12 < 200 pmol/L and tHcy > 10 µmol/L or MMA > 0.40 µmol/L and tHcy >10 µmol/L) [32], approximately the same percentage (24/61, 39%) of our cases were B12-deficient. Bjørke-Monsen and coworkers have recommended tHcy < 6.5 µmol/L as the intervention threshold for vitamin-optimized infants [35]. ...
Background: Untreated vitamin B12 (B12) deficiency may cause delayed development in infants. Several newborn screening (NBS) programs have reported an increased detection rate of B12 deficiency when second-tier dried blood spot (DBS) analyses of total homocysteine (tHcy) and methylmalonic acid (MMA) are included. Methods: This is a retrospective study of newborns reported from NBS during 2012–2021 with confirmed B12 deficiency. DBSs were retrieved from the NBS biobank for second-tier MMA and tHcy analysis. Results: Thirty-one newborns were diagnosed with B12 deficiency out of 552970 screened. Twenty-five were ascertained from sixty-one false positive (FP) cases of methylmalonic acidemia and propionic acidemia (PA), and six infants screened positive for other NBS metabolic diseases with propionylcarnitine (C3) in the normal range. In the original DBS, 7/23 (30%) and 12/23 (52%) of B12-deficient newborns with FP methylmalonic acidemia/PA had MMA and tHcy > 99th percentile. Conclusion: B12 deficiency was a common differential diagnosis of screening positive for methylmalonic and PA. C3 failed to identify a subset of newborns with B12 deficiency. Second-tier MMA and tHcy analyses in the DBS showed suboptimal sensitivity for identifying infants with B12 deficiency. The shortcomings of NBS should be acknowledged when considering B12 deficiency as a primary target of NBS panels.
... 1,2 In 2004, Refsum et al analyzed 4992 newborn screening serum samples in Norway and found that five percent of the newborns had biomarkers suggesting B12 deficiency when applying the criteria serum total homocysteine (tHcy) > 10 mmol/L, B12 < 200 pmol/L or tHcy >10 mmol/L, and serum methylmalonic acid (MMA) > 0.40 mmol/L. 3 We recently demonstrated a 10% prevalence of clinically relevant hyperhomocysteinemia suggestive of B12 deficiency in infants in Norway. 4 tHcy is the preferred functional biochemical marker of infant B12 status, and vitaminoptimized plasma-tHcy is <6.5 mmol/L at 4 months of age. ...
Background
In Norway, 5-10% of neonates and infants have biomarkers suggesting vitamin B12 deficiency from newborn screening tests and unselected clinical screening, respectively.
Aims
The aims were to identify risk factors and describe presenting symptoms and biochemical profiles in infants diagnosed with vitamin B12 deficiency.
Methods
In this case-control study we searched hospital medical records for infants below one year of age born 2011 - 2018, diagnosed with vitamin B12 deficiency. We compared 85 cases with a control group of 252 infants aged 3-7 months. Parents completed questionnaires.
Results
Of the 85 cases with vitamin B12 deficiency 80% presented within the first two months of life with spells (37%) of apneas, motor seizures or absences. Tremor (29%) and irritability (18%) were the most common findings at the first examination. Serum total homocysteine ≥10 μmol/L was found in 77% of cases compared to 28% of controls (p<0.001). None of the mothers were vegetarians, but 25% reported a previous history of vitamin B12 deficiency and 7% had celiac disease. The dose of nitrous oxide given during labor was significantly associated with infant serum total homocysteine level at diagnosis (r=0.37, 95% CI 0.16-0.55, p<0.001) for cases, but not for controls.
Conclusion
Spells, tremor, and irritability are common findings in early infant vitamin B12 deficiency. Nitrous oxide given during labor is proposed as a contributing risk factor to the development of early infant vitamin B12 deficiency.
... The infants included in the present study were also participants in a larger, hypothesis generating, cross-sectional study for the purpose of investigating B12 status in a cohort of 252 healthy infants. 11 B12 deficiency was suggested in 9.5% of infants participating in the present study, in accordance with other prevalence studies, 22 and was associated with tremor or excessive sleep. 11 As the latter symptoms are common in healthy infants and in particular as no associations between HINE scores and markers of B12 status could be demonstrated, no infants were excluded from the present study. ...
Objective:
To provide a valid, continuous reference interval, including a 10th percentile cut-off, for Hammersmith Infant Neurological Examination (HINE) scores based on 3-7 months old term infants with weight appropriate for gestational age.
Study design:
In a prospective study we examined 168 Norwegian infants at one timepoint with HINE at 3-7 months of age. In 134 of these infants Ages and Stages Questionnaire was completed by their parents at two years of age to ensure typical motor development. We calculated a reference interval for HINE scores with the 10th percentile as cut-off for age-dependent optimal scores.
Results:
The best fitting mean model for HINE total score was 78.1358+9659.231*1/age2-5104.174*LN(age)/age2, which explained 49.8% of the variance. The HINE total score 10th percentile cut-off corresponded to 52.1 points at age 12 weeks, 55.6 points at 16 weeks, 59.0 points at 20 weeks, 61.8 points at 24 weeks and 63.8 points at 28 weeks. We found an excellent intra-class correlation coefficient of 0.953 (0.931-0.968) between two examiners. The infants had a typical motor development at two years follow-up.
Conclusion:
We have presented a valid, continuous reference interval and a 10th percentile cut-off for HINE scores for infants aged 3 to 7 months.
... Bjørke-Monsen et al. showed that B12 optimized infants had a tHcy<8 µmol/L at 4 months of age 16 . Consequently, we propose that the clinically relevant HHcy represents clinically relevant B12 deficiency and that our study adds a clinical aspect to other prevalence studies defining B12 deficiency from biochemical test results only 16,18,19,30 . On the other hand, 89 of 114 (89%) infants with HHcy did not present any of the associated symptoms, why it is challenging to decide whom and when to replenish. ...
... In a prevalence study of newborns with biochemical B12 deficiency in Norway, Refsum et al. analyzed 4992 serum samples from the Norwegian newborn screening program and estimated a 5% prevalence of B12 deficiency at birth using the combination of S-tHcy>10 pmol/L and S-B12<200 pmol/L as cut-off values 30 . These are rather strict biochemical criteria for newborn B12 deficiency and may underestimate the true prevalence. ...
Background
Previous studies have demonstrated a high prevalence of biochemical vitamin B12 deficiency in infants in Norway. Increased total homocysteine (tHcy) is the most important marker of B12 deficiency in infants. There is a need to evaluate its clinical relevance.
Aims
To investigate the prevalence of hyperhomocysteinemia (S-tHcy > 8 μmol/L) suggestive of suboptimal B12 status and the prevalence of clinically relevant hyperhomocysteinemia in presumed healthy infants in Norway. Further, to evaluate risk factors, presence of symptoms and psychomotor development in these children.
Methods
In a prospective study we clinically examined 252 infants aged 3–7 months using standardized neurological and psychomotor tests prior to analyzing biochemical B12 deficiency markers in 250 infants.
Results
Twenty-five of 250 (10%) infants had hyperhomocysteinemia combined with clinically relevant symptoms suggestive of B12 deficiency. Hyperhomocysteinemia was associated with tremor, excessive sleep, and sub-normal scores in the fine motor section of the Ages and Stages Questionnaire. One-hundred and fourteen of 250 (46%) infants had hyperhomocysteinemia. Multiple regression analysis showed months of infant formula use as the strongest negative predictor for hyperhomocysteinemia.
Conclusion
We have demonstrated associations between symptoms suggestive of infant B12 deficiency and increased levels of tHcy in presumed healthy infants The combination of hyperhomocysteinemia and associated relevant symptoms suggestive of B12 deficiency was a common finding, albeit most infants with hyperhomocysteinemia did not show symptoms.
... SAAs were assayed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using a Prominence LC-20AD XR binary pump (Shimadzu, Kyoto, Japan) coupled to a QTRAP 5500 hybrid triple quadropole mass spectrometer (AB Sciex, Framingham, MA, US). Plasma tCys, total homocysteine (tHcy), and total glutathione (tGSH) were analysed using a modification of a previously described method (Refsum et al. 2004), whereas taurine was extracted and assayed separately as described (Elshorbagy et al. 2017). SAM, SAH, rCys, rGSH, cystine, as well as the free (nonprotein bound) fractions of cysteine, homocysteine and glutathione were extracted from PCA-treated plasma using the same protocol and conditions as for the tCys assay (Refsum et al. 2004), adjusted for the dilution of the samples. ...
... Plasma tCys, total homocysteine (tHcy), and total glutathione (tGSH) were analysed using a modification of a previously described method (Refsum et al. 2004), whereas taurine was extracted and assayed separately as described (Elshorbagy et al. 2017). SAM, SAH, rCys, rGSH, cystine, as well as the free (nonprotein bound) fractions of cysteine, homocysteine and glutathione were extracted from PCA-treated plasma using the same protocol and conditions as for the tCys assay (Refsum et al. 2004), adjusted for the dilution of the samples. Quantitation of all analytes was based on comparison with standard curves corrected for presence of isotopically labelled internal standards using a 1/x weighting. ...
Plasma cysteine is associated with human obesity, but it is unknown whether this is mediated by reduced, disulfide (cystine and mixed-disulfides) or protein-bound (bCys) fractions. We investigated which cysteine fractions are associated with adiposity in vivo and if a relevant fraction influences human adipogenesis in vitro. In the current study, plasma cysteine fractions were correlated with body fat mass in 35 adults. Strong positive correlations with fat mass were observed for cystine and mixed disulfides ( r ≥ 0.61, P < 0.001), but not the quantitatively major form, bCys. Primary human preadipocytes were differentiated in media containing cystine concentrations varying from 10–50 μM, a range similar to that in plasma. Increasing extracellular cystine (10–50 μM) enhanced mRNA expression of PPARG2 (to sixfold ) , PPARG1 , PLIN1 , SCD1 and CDO1 ( P = 0.042– < 0.001). Adipocyte lipid accumulation and lipid-droplet size showed dose-dependent increases from lowest to highest cystine concentrations ( P < 0.001), and the malonedialdehyde/total antioxidant capacity increased, suggesting increased oxidative stress. In conclusion, increased cystine concentrations, within the physiological range, are positively associated with both fat mass in healthy adults and human adipogenic differentiation in vitro. The potential role of cystine as a modifiable factor regulating human adipocyte turnover and metabolism deserves further study.
... Homocystinuria is an inherited metabolic disorder resulting from a defect in the methionine (Met) metabolism usually characterized by an aberrant buildup of total homocysteine (tHCY) and its cognates such as HCY, HCY-cysteine complex, and others in blood and urine of the affected neonate. Clinical presentation for homocystinuria is often accompanied by symptoms such as mental retardation, thromboembolism, psychiatric disorders, ectopia lentis, and skeletal abnormalities [8][9][10]. However, the re-methylation disorder is often characterized by diverse clinical presentations such as developmental delay, seizure, hypotonia, microcephaly, feeding difficulty, neurological deterioration, ABSTRACT Background: Tandem mass spectrometry (TMS) -based screening for homocystinuria in the routine newborn screening (NBS) primarily relies on the elevated levels of methionine (Met) in the dried blood spot (DBS) of affected neonates. ...
Tandem mass spectrometry (TMS) -based screening for homocystinuria in the routine newborn screening (NBS) primarily relies on the elevated levels of methionine (Met) in the dried blood spot (DBS) of affected neonates. However, due to the lack of required specificity of Met as a marker for homocystinuria, an additional re-sampling and follow-up analytical testing for a definitive diagnosis, emphasis us to develop second-tier testing for a more specific marker, that is homocysteine (HCY) in the same residual blood spot without any new sample. Objective: The objective of the study was to develop a novel approach for the second-tier estimation of total HCY in residual blood spot using TMS. Materials and Methods: Retrospective analysis of DBS from all neonates with deranged levels of Met and Met to phenylalanine in primary NBS was included in the study. The study duration was for a period of 24 months (November 2015-October 2017). Heel prick samples were collected within 24–72 h of birth and after the first feed. Further, the samples with abnormal Met was analyzed for total HCY by isotopic dilution approach using multiple reaction monitoring modes for mass transition m/z 136?90 for HCY, m/z 140?94 for d4-homocysteine, and m/z 269.2?134.4 for d8-HCY. Results: The newly developed methodology demonstrated a sensitive method for the quantification of total HCY over a linear range of 2 ?mol/L–100 ?mol/L. Assay validation parameters such as intraday and interday assay precision, accuracy, and recovery were also within an acceptable range. Conclusion: In the present study, an in-house tandem mass spectrometric-based assay for the quantification of total HCY was developed which can be employed as an important strategy for the differential diagnosis of homocystinuria in neonates.
... Vitamin B12, also known as cobalamin, acts as a coenzyme of methionine synthase which catalyzes the conversion of homocysteine to methionine (7). Vitamin B12 deficiency leads to hyperhomocysteinemia characterized by high levels of homocysteine (8). ...
Purpose: Inflammatory bowel disease (IBD) refers to a spectrum of autoimmune diseases, which result in chronic intestinal inflammation. Previous findings suggest a role for diet, nutrition and dysbiosis of the gut microbiota in both the development and progression of the condition. Vitamin B12 is a key cofactor of methionine synthase and is produced solely by microbes. Previous work links increased levels of homocysteine, a substrate of methionine synthase, MetH, to IBD indicating a potential role for vitamin B12 deficiency in intestinal injury and inflammation. This study assessed the role of vitamin B12 in shaping the gut microbiota and determining responses to intestinal injury using a reproducible murine model of colitis.
Methods: The effects of vitamin B12 supplementation and deficiency were assessed in vivo; 3-week-old post-weanling C57Bl/6 mice were divided into three dietary treatment groups: (1) sufficient vitamin B12 (50 mg/Kg), (2) deficient vitamin B12 (0 mg/Kg) and (3) supplemented vitamin B12 (200 mg/Kg) for a period of 4 weeks. Intestinal injury was induced with 2% dextran sodium sulphate (DSS) via drinking water for 5 days. The impact of varying levels of dietary vitamin B12 on gut microbiota composition was assessed using 16S rRNA gene sequencing from fecal samples collected at day 0 and day 28 of the dietary intervention, and 7 days following induction of colitis on day 38, when blood and colonic tissues were also collected.
Results: No significant alterations were found in the gut microbiota composition of disease-free animals in response to dietary interventions. By contrast, after DSS-induced colitis, >30 genera were significantly altered in vitamin B12 deficient mice. Altered B12 levels produced no significant effect on composite disease-activity scores; however, administration of a B12 deficient diet resulted in reduced DSS-induced epithelial tissue damage.
Conclusions: Vitamin B12 supplementation does not alter the gut microbiota composition under healthy conditions, but does contribute to differential microbial responses and intestinal dysbiosis following the induction of experimental colitis.
... The concentration of amino acid contained in blood specimens can be quantified by the tandem mass method using LC-MS [8,9]. HPLC and LC-MS /MS are also used for microdetermination due to high accuracy. ...
... In newborn screenings, blood specimens are quantified by the tandem mass method using LC-MS [8,9]. HPLC and LC-MS/MS are also used for microdetermination due to a high degree of accuracy. ...
For many years, clinical studies have suggested that blood levels of l-methionine and L-homocysteine correlate with health status or homocystinuria/hypermethioninemia. l-Methionine in a solution containing 0%, 10%, or 20% human serum was detected in 10–200 µM using l-methionine decarboxylase (MetDC). Spike and recovery tests showed that the enzymatic assay could accurately and reproducibly determine the increases in l-methionine in serum samples. These results suggest that our enzymatic method using MetDC is useful for primary screening of hypermethioninemia or homocystinuria based on serum l-methionine concentration. Additionally, we confirmed that l-methionine (100 nmol) in solution was degraded to less than the detection limit by incubation at 37ºC for 10 min using 2 U of MetDC. Therefore, l-homocysteine in serum samples can be detected with equivalent sensitivity using l-methionine γ-lyase (MGL), in solutions that either did not contain l-methionine or contained l-methionine preincubated with MetDC.
Abbreviations: DTT: dithiothreitol; IPTG: isopropyl-β-d-thiogalactopyranoside; KPB: potassium phosphate buffer; MBTH: 3-methyl-2-benzothiazolinonehydrazone; mdc: the gene coding l-methionine decarboxylase; MetDC: l-methionine decarboxylase; mgl: the gene coding l-methionine γ-lyase; MGL: l-methionine γ-lyase; PLP: pyridoxal 5ʹ-phosphate
... Plasma MMA concentration thus serves as a functional biomarker of B12 status and reflects intracellular B12 deficiency [16]. Compared to serum total B12, MMA is also a sensitive indicator of B12 deficiency: circulating MMA concentrations are increased in cases of acquired B-12 deficiency due to dietary restrictions or malabsorption [17,18]. In individuals with inherited disorders involving MMA metabolism, blood MMA ranges from only mildly to markedly elevated (2 to 1000-fold) concentrations. ...
... Methylmalonic acid concentrations are strongly inversely correlated with B-12 status in newborns [18,32]. The cutoff for elevated DBS MMA concentrations can be computed as the mean upper limit of the reference interval. ...
... The patient with propionyl-CoAcarboxylase deficiency had DBS MMA concentrations below the upper limit of the reference interval as expected for this inborn error of metabolism (21). Additionally, our results confirm that newborns have higher circulating MMA concentrations compared to adults [8,18,32]. DBS MMA concentrations of these 160 healthy newborns were approximately 6-times higher than DBS MMA concentrations of healthy, young adult women (2.93 pmol per 8-mm DBS; n = 96) as previously reported [23,33]. Others also reported elevated plasma MMA concentrations in 4-day old newborns [32] and in cord blood samples [34,35] when comparing to adult derived plasma MMA cutoffs. ...
Background
Maternal vitamin B12 (B-12) adequacy is important for maternal health and optimal fetal growth. However, pregnancy-specific cut-offs for B-12 biomarkers are lacking.
Methods
Reference intervals for serum total B-12, holotranscobalamin (holoTC) and methylmalonic acid (MMA) concentrations were calculated following CLSI EP28-A3c guidelines in 723 pregnant women of European (50%) and South Asian (50%) ethnicity, residing in British Columbia, Canada, at median (range) 11.4 (8.3–13.9) and 16.1 (14.9–20.9) weeks of gestation. Change point analyses described relationships between log serum MMA concentration with serum total B-12 and holoTC concentrations, assuming linear-linear relationships.
Results
The central 95% reference interval limits indicated that serum total B-12 <89.9 and <84.0 pmol/L, holoTC <29.5 and <26.0 pmol/L and MMA >371 and >374 nmol/L, in the first and second trimesters, respectively, may indicate B-12 deficiency in pregnant women. The lower limits of total B-12 and holoTC and the upper limits of MMA significantly differed by ethnicity in both trimesters. According to the change point analysis, total B-12 <186 and <180 pmol/L and holoTC <62.2 and <67.5 pmol/L in the first and second trimesters, respectively, suggested an increased probability of impaired intracellular B-12 status, with no difference between ethnicities.
Conclusions
We present novel reference limits and change points for B-12 biomarkers, which may be employed to identify possible B-12 deficiency in women during early and mid-pregnancy. Future research is needed to validate these cut-offs and determine the predictors and functional outcomes associated with impaired B-12 status in ethnically diverse populations.
