Serum holoTC Research Articles

Holo-transcobalamin is not associated with the risk of all-cause mortality in the general population

Background & AimsHolo-Transcobalamin (holo-TC) is the biologically active form of vitamin B12, a vitamin essential in human metabolism. The association between vitamin B12 (total cobalamin) and mortality risk has been controversially reported, whereas the relation between holo-TC and survival is unknown. In a population-based sample (n=862, female share 42.8 %, median age 62.3 years), we related serum holo-TC to the risk of all-cause mortality. MethodsWe measured serum holo-TC by electro-chemiluminescence. Multivariable-adjusted Cox proportional hazards regression models were used to quantify the association between serum holo-TC and all-cause mortality. ResultsOver a median follow-up time of 10.9 years, n=99 individuals died. We did not find significant associations between serum holo-TC and the risk of all-cause mortality (HR: 1.00 [95% CI 0.97–1.03] per 5-point increment in holo-TC), neither in the overall sample, nor in subgroups stratified by sex, diabetes, or hypertension. ConclusionThe biologically active form of vitamin B12, holo-TC, is not related to the risk of all-cause mortality in a moderate-sized sample from the general population.

Associations of Serum Folate and Holotranscobalamin with Cardiometabolic Risk Factors in Rural and Urban Cameroon

A low intake of fruit and vegetables and a high intake of meat are associated with higher cardiometabolic disease risk; however much prior research has relied on subjective methods for dietary assessment and focused on Western populations. We aimed to investigate the association of blood folate as an objective marker of fruit and vegetable intake and holotranscobalamin (holoTC) as a marker of animal-sourced food intake with cardiometabolic risk factors. We conducted a population-based cross-sectional study on 578 adults (mean ± SD age = 38.2 ± 8.6 years; 64% women). The primary outcome was a continuous metabolic syndrome score. The median serum folate was 12.9 (IQR: 8.6–20.5) nmol/L and the mean holoTC was 75 (SD: 34.3) pmol/L. Rural residents demonstrated higher serum folate concentrations (15.9 (9.8–25.9) nmol/L) than urban residents (11.3 (7.9–15.8) nmol/L), but lower holoTC concentrations (rural: 69.8 (32.9) pmol/L; urban: 79.8 (34.9)) pmol/L, p < 0.001 for both comparisons. There was an inverse association between serum folate and metabolic syndrome score by −0.20 in the z-score (95% CI, −0.38 to −0.02) per 10.8 (1 SD) of folate) in a model adjusted for socio-demographic factors, smoking status, alcohol intake, BMI, and physical activity. HoloTC was positively associated with the metabolic syndrome score in unadjusted analysis (0.33 (95% CI, 0.10 to 0.56)) but became non-significant (0.17 (−0.05 to 0.39)) after adjusting for socio-demographic and behavioural characteristics. In conclusion, serum folate and holoTC were associated with the metabolic syndrome score in opposite directions. The positive association between serum holoTC and the metabolic syndrome score was partly dependent on sociodemographic characteristics. These findings suggest that, based on these biomarkers reflecting dietary intakes, public health approaches promoting a higher intake of fruit and vegetables may lower cardiometabolic risk factors in this population.

Clinical Utility of Serum Holotranscobalamin Measurements in Patients with First-Ever Ischemic Stroke

Background Whether holotranscobalamin (holoTC) indicates B12 deficiency more sensitively than total vitamin B12 (B12) is unclear. This study is aimed at determining the impact of serum holoTC level as a risk factor for ischemic stroke and investigating its association with disease severity and short-term outcomes. Methods Serum holoTC, total B12, and homocysteine levels were compared between 130 stroke patients and 138 healthy controls. Biomarker level correlations with disease severity and stroke functional outcomes were investigated. Results holoTC levels were lower and homocysteine levels were higher in stroke patients than in healthy controls (P < 0.05). The holoTC/total B12 ratio and homocysteine level significantly predicted ischemic stroke in the multivariable regression analysis (P < 0.05). Along with hyperhomocysteinemia, patients more often had holoTC than total B12 deficiency (6.2% vs. 3.1%). holoTC levels negatively correlated with homocysteine levels (partial R -0.165, P < 0.05) in stroke patients in multiple linear regression analyses, but not total B12 levels. The holoTC level and holoTC/total B12 ratio, but not homocysteine and total B12 levels, negatively correlated with the National Institute of Health Stroke Scale (partial R, -0.405 and -0.207, respectively, P < 0.01). Conclusions Measurements of serum holoTC levels combined with total B12 and homocysteine levels may provide valuable information for predicting ischemic stroke and its severity and short-term outcomes of ischemic stroke patients.

Vitamin B12 Absorption as Measured by Total B12 and Holo-TC Following A Single Oral Dose of Multivitamin in Healthy Adults

ObjectivesMeasure total B12 and holotranscobalamin (Holo-TC) concentrations in serum to show vitamin B12 absorption following single oral doses of multivitamin (MVI) gummy and tablet. MethodsThis crossover clinical trial involved healthy adults randomized to either gummy or tablet multivitamins (MVI) containing vitamin B12 (60.8 mcg in gummy and 91.8 mcg in tablet) as cyanocobalamin in a single dose in Phase 1 with serial blood samples collected at pre-dose and at 0.5-, 1-, 2-, 4-, 6-, 8-, 9-, 10-, 24-, and 48-hrs after dosing, followed by a 2-week washout period. In Phase 2, participants then crossed over to receive MVI in the form not previously given, with blood draws at the same timepoints. Serum samples were measured for total B12 (n = 19) using an electrochemiluminescence immunoassay (ECLIA) and for Holo-TC (n = 14) using commercial Holo-TC ELISA kit. Measurements from groups of time points were averaged to estimate pre-absorptive (0 to 2 hours) and absorptive (6 to 10 hours) concentrations of total B12 and Holo-TC for each subject. The difference between pre-absorptive and absorptive concentrations for both analytes was calculated to estimate newly absorbed vitamin B12. ResultsMean increases of about 15% in serum Holo-TC in absorptive phase over the pre-absorptive phase were observed for both the gummies and the tablets, which are statistically significant (p < 0.001). These changes were similar to those observed for total B12. There was no significant difference in mean increases between the gummies and the tablets in both total B12 and Holo-TC. These results support the conclusion that the observed increases in total B12 and Holo-TC were the result of intestinal absorption following the single oral dose. ConclusionsOverall, under the conditions of this study, both gummy and tablet multivitamins showed similar absorption of vitamin B12, whether assessed by total B12 or holo-TC measurement. Funding SourcesChurch & Dwight Co. Inc.

Vitamin B-12 Biomarkers Reviewed: Holotranscobalamin Allows Identification of Vitamin B12 Deficiency in Children with Short-Bowel Syndrome

Abstract Objectives Serum total vitamin B12 (tB12) is the most commonly used biomarker to assess B12 status in healthy and patient populations but there is ambiguity as to its performance for early and reliable B12 deficiency diagnosis. Holotranscobalamin (holoTC) is the B12 form taken up by cells and may be a more sensitive B12 indicator. This descriptive, prospective study aimed to compare serum tB12 with serum holoTC concentration in their ability to detect functional B12 deficiency defined as elevated methylmalonic acid (MMA) concentration in a pediatric population at risk of B12 deficiency. Methods A total of 26 infants and children aged 3 mo-15 yrs with a history of ileal resection were recruited from the Children's Intestinal Rehabilitation Program at the British Columbia Children's Hospital, Canada. Between 1 to 7 samples per patient were collected over 2yrs, with complete biomarker data for 41 samples. Clinical, anthropometric and dietary data were also collected. Age-specific reference limits derived from a healthy pediatric reference sample were used as cut-offs to categorize B12 deficiency. Results Of the 41 samples, 29 (71%) had concordant biomarker results all indicating adequate B12 status. Elevated MMA (&amp;gt;398 nmol/L) was found in only 2 samples and those had holoTC concentration indicative of B12 deficiency (&amp;lt;40 pmol/L) but normal tB12 (&amp;gt;209 pmol/L). While holoTC exceeded tB12 in sensitivity for the detection of functional B12 deficiency, holoTC had reduced specificity, 75% compared to 85% for tB12, leading to a reduced positive predictive value (22%). Patients receiving total parenteral nutrition (TPN) had significantly higher tB12 but similar holoTC concentration compared to those not receiving TPN. Conclusions Overall, the results suggest that serum holoTC detects patients with elevated MMA indicative of B12 deficiency, who are not identified by tB12 concentration, in children with short bowel syndrome. It also suggests that the current cut-off for tB12 may be too low. These descriptive findings warrant further evaluation and validation of B12 biomarkers and related cutoffs for adequate, suboptimal and deficient B12 status. Funding Sources Healthy Starts Catalyst Grant from the British Columbia Children's Hospital Research Institute, Vancouver BC, Canada.

Laboratory Performance of Serum Holotranscobalamin Assay in the United Kingdom (UK) As Assessed By the UK National External Quality Assessment Scheme for Haematinics

Background Although serum B12 assay is the most commonly used routine test for assessing cobalamin status in the body, it has its limitations (Mackenzie F and Devalia V (2018) Laboratory performance of serum B12 assay in the United Kingdom (UK) as assessed by the UK National External Quality Assessment Scheme for haematinics: implications for clinical interpretation. BLOOD, 132, suppl 1, 2230). Holotranscobalamin assay(HoloTC), also known as 'Active B12' assay, is an alternative test which is increasingly used as a first line test since it is felt to represent the assessment of the functional component of the cobalamin status of the body, and possibly a more relevant assessment clinically. However, its technical performance in terms of reliability and suitability in a routine diagnostic laboratory for clinical assessment has not been published. In the United Kingdom, there are over 30 laboratories performing the assay using five different platforms. The numerical value obtained of any sample can vary considerably between the different methodologies used.. External quality assessment of the assays is organised by UK NEQAS for Haematinics by sending three serum samples every 3 months. An 'all participants' consensus mean is calculated and used as the target value and the results analysed with respect to intra-group variation. The percentage bias from the target value is used to assess performance. Aim We present data on one such assessment to demonstrate the performance of the serum holotranscobalamin assay (Survey number 248, April 2018) and also how it is interpreted by the laboratory for clinical use. Method Three serum samples with a HoloTC target value of approximately 7.98 (low/indeterminate), 35.7 (normal/indeterminate) and 53.2 (normal) pmol/L were sent to participating laboratories for analysis (labelled as 248A, B and C respectively: see figure 1). Laboratories were also asked for an interpretation of their result which would be reported to the requesting clinician, namely from low to high (see figure 2). Results Fig 1 shows an individual laboratory's result in relation to all laboratories using the same technology (shaded histogram) or all methods (open histogram). There is a significant variation with an overall co-efficient of variation of around 10% within all the three different samples. Fig 2 shows the distribution of results in the different methodologies used and how each laboratory interpreted its result. It demonstrates the bias of results obtained by the different methods.The vast majority use the Abbot Architect (AB13) platform, and there is a suggestion of a trend of the results obtained to lie on the lower side than the other platforms used. For sample 248A, all laboratories reported it as 'low' or deficient. For sample 248B, there is a significant variation is reporting the sample as 'low', 'normal' or 'indeterminate' within the same platform used, clearly seen in the Abbot Architecture group. Indeed, the interpretation provided by the laboratory varies even with the same numerical value of the result. Discussion These data demonstrate that serum HoloTC assay has an overall co-efficient of variation around 10%. The numerical value obtained of any sample can vary considerably according to the technology used, and the clinical interpretation provided by the laboratory can be variable and not entirely concordant with the numerical result of the assay used. This is particularly evident around the 30-40 pmol/L range. This may be partly explained by the fact that it is not quite clear what would be regarded as the normal or reference range, which has been previously taken as 40 - 200 pmol/L (according to previous publications). Conclusion The UK NEQAS Haematinics Programme is unique in providing external quality assessment for laboratories using HoloTC assays for determining body cobalamin status in a style that is also unique across EQA/ PT schemes. Laboratories need to assess their performance in analysis of serum HoloTC levels in order to provide appropriate clinical advice. Ideally clinicians should be aware of the limitations of the HoloTC assay as demonstrated in this external quality assessment scheme exercise. Disclosures No relevant conflicts of interest to declare.

Maternal gastric intrinsic factor A68G and paternal cubilin C758T variants increase the risk for neural tube defects in the fetus: A family-triad study from South India

BackgroundNeural tube defects (NTDs) are the leading cause of infant deaths worldwide. Gastric Intrinsic Factor (GIF) and Cubilin (CUBN) genes essential for early fetal development have not been extensively studied in NTDs. In view of this, present study aimed to evaluate association of GIF A68G and CUBN C758T variants with NTD risk using family-based triad approach. MethodsA total of 924 subjects including 124 NTD case-parent trios and 184 healthy control-parent trios diagnosed at Institute of Genetics and Hospital for Genetic Diseases, Hyderabad were considered for the study. Blood samples from study group parents and umbilical cord tissue samples from NTD cases and controls were genotyped for GIF A68G (rs35211634) and CUBN C758T (rs1801222) variants for their association with NTDs. Serum holo-transcobalamin (holo-TC) levels were measured using ELISA. ResultsAssessment of maternal-paternal genotype incompatibility risk for NTD revealed maternal AG genotype in combination with paternal AA genotype of GIF A68G (AGxAA = AG,OR = 2.92,p = .006) and maternal CC genotype with paternal CT genotype combination of CUBN C758T (CCxCT = CT,OR = 3.71,p < .001) significantly increased the risk for NTDs. Allelic gene-gene interactions of GIF A68G and CUBN C758T showed significant association of maternal G-C (OR = 6.34,p < .001) and paternal A-T alleles (OR = 2.61,p = .001) for the occurrence of NTDs. Further, parent-of-origin effects also revealed significant transmission of maternal ‘G’ allele (OR = 3.50,p = .015) and paternal ‘T' allele (OR = 2.57,p = .016) to NTDs. Correlation of serum holo-TC levels with GIF A68G and CUBN C758T genotypes revealed significant association of paternal CT genotype of C758T variant with decreased holo-TC levels compared to controls (p = .013). However, GIF A68G variant showed no significant association with holo-TC levels. ConclusionThe present family-based triad study provides the first report on the risk of NTD susceptibility being potentially influenced by maternal GIF A68G and paternal CUBN C758T variants.

Seminal but not Serum Levels of Holotranscobalamin are Altered in Morbid Obesity and Correlate with Semen Quality: A Pilot Single Centre Study.

Vitamin B12 (cobalamin) is an essential cofactor in the one-carbon metabolism. One-carbon metabolism is a set of complex biochemical reactions, through which methyl groups are utilised or generated, and thus plays a vital role to many cellular functions in humans. Low levels of cobalamin have been associated to metabolic/reproductive pathologies. However, cobalamin status has never been investigated in morbid obesity in relation with the reduced semen quality. We analysed the cross-sectional data of 47-morbidly-obese and 21 lean men at Careggi University Hospital and evaluated total cobalamin (CBL) and holotranscobalamin (the active form of B12; holoTC) levels in serum and semen. Both seminal and serum concentrations of holoTC and CBL were lower in morbidly obese compared to lean men, although the difference did not reach any statistical significance for serum holoTC. Seminal CBL and holoTC were significantly higher than serum levels in both groups. Significant positive correlations were observed between seminal holoTC and total sperm motility (r = 0.394, p = 0.012), sperm concentration (r = 0.401, p = 0.009), total sperm number (r = 0.343, p = 0.028), and negative correlation with semen pH (r = −0.535, p = 0.0001). ROC analysis supported seminal holoTC as the best predictor of sperm number (AUC = 0.769 ± 0.08, p = 0.006). Our findings suggest that seminal rather than serum levels of holoTC may represent a good marker of semen quality in morbidly obese subjects.

Assessing Diagnostic Accuracy of Serum Holotranscobalamin (Active-B12) in Comparison with Other Markers of Vitamin B12 Deficiency.

About 15-40% India is Vitamin B12 deficient (commonly diagnosed by total Vitamin B12) but, as only holoTC (active form) is taken up by body cells, thus measuring holoTC is more reflective of Vitamin B12 status than the former. We aimed to assess diagnostic accuracy of serum holoTC in comparison with total Vitamin B12 and total Homocysteine (HCY) as indicator of serum Vitamin B12 status. 217 human subjects (99 males and 118 females) ranging from 17 to 83years were divided into Vitamin B12 deficient (n = 70), borderline (n = 100) and sufficient groups (n = 47) who were further assessed for markers of Vitamin B12 deficiency-holoTC, HCY, Mean Corpuscular Volume (MCV), Folate, heamoglobin and creatinine. Samples were analysed using Siemens Advia Centaur Xpi. Total Vitamin B12 deficient group had - 84.3% holoTC deficient; 15.7% holoTC sufficient; 72.9% with elevated HCY; 27.1% with normal HCY; 11.4% with megaloblastic anaemia. Borderline group had - 34% holoTC deficient; 28% elevated HCY. A strong positive correlation was found between Total Vitamin B12 and holoTC (r = 0.754, p = <0.001) but strong negative correlation existed between holoTC and HCY (r = - 0.471, p = <0.001). Concordance between Total Vit B12 and HCY (Kappa index = 0.51, p < 0.001); between holoTC and HCY (Kappa index = 0.52, p = <0.001) were statically significant but the latter had a better sensitivity and specificity. Also, statically significant association exists between Total Vitamin B12 and holoTC with HCY (p = <0.001). Therefore, it is ascertained that Active Vitamin B12 assay is a better test and can be considered as an early marker of vitamin B12 deficiency.

PB2273 LABORATORY PERFORMANCE OF SERUM HOLOTRANSCOBALAMIN ASSAY IN THE UNITED KINGDOM (UK) AS ASSESSED BY THE UK NATIONAL EXTERNAL QUALITY ASSESSMENT SCHEME FOR HAEMATINICS

Background:Although serum B12 assay is the most commonly used routine test for assessing cobalamin status in the body, it has its limitations (Mackenzie F and Devalia V (2018) Laboratory performance of serum B12 assay in the United Kingdom (UK) as assessed by the UK National External Quality Assessment Scheme for haematinics: implications for clinical interpretation. BLOOD, 132, suppl 1, 2230). Holotranscobalamin assay is an alternative test which is increasingly used as a first line test since it is felt to represent the assessment of the functional component of the cobalamin status and possibly a more relevant assessment clinically. However, its technical performance in terms of reliability and suitability in a routine diagnostic laboratory for clinical assessment has not been published. In the United Kingdom, there around 31 laboratories performing the assay using five different platforms. The numerical value obtained of any sample can vary considerably between the different methodologies used. It is not quite clear what would be regarded as the normal or reference range, but it has been previously taken as 40 – 200 pmol/L. External quality assessment of the assays is organised by UK NEQAS for Haematinics by sending three serum samples every 3 months. An ‘all participants’ consensus mean is calculated and used as the target value and the results analysed with respect to inter and intra‐group variation. The percentage bias from the target value is used to assess performance.Aims:We present data on one such assessment to demonstrate the performance of the serum holotranscobalamin assay and also how it is interpreted by the laboratory for clinical use.Methods:Three serum samples with a HoloTC target value of approximately 16.89 (low/indeterminate), 44,7 (normal) and 72.4 μg/L (high normal) were sent to participating laboratories for analysis. Laboratories were also asked for an interpretation of their result which would be reported to the requesting clinician, namely from low to high.Results:Fig 1 shows an individual laboratory's result (indicated by the arrow) in relation to all laboratories using the same technology (shaded histogram) or all methods (open histogram). There is a significant variation with an overall co‐efficient of variation of around 11% within all the three different samples. Fig 2 shows the distribution of results in the different methodologies used and how each laboratory interpreted its result. It demonstrates the bias of results obtained by the different methods. The assays using Beckman Coulter Access/Dxl (SF5 and SF6) gave much lower results than other methods. In the group SF5, three laboratories interpreted the result as ‘normal’ and one as ‘indeterminate’. In the groups Abbot Architect (AB13) and Roche Cobas/Modular (BO5), although having higher results than other groups, there are significant laboratories interpreting the result as ‘low’ and also ‘normal’ or ‘indeterminate’.Summary/Conclusion:These data demonstrate that serum HoloTC assay has an overall co‐efficient of variation around 11%. The numerical value obtained of any sample can vary considerably although the clinical interpretation provided by the laboratory does not seem to be markedly discordant. Conclusion: The UK NEQAS Haematinics Programme is unique in providing external quality assessment for laboratories using HoloTC assays for determining body cobalamin status in a style that is also unique across EQA/ PT schemes. As a result, laboratories are made aware of their performance in assessing serum HoloTC level in order to provide appropriate clinical advice.image

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 and Holotranscobalamin Levels in Diabetic Peripheral Neuropathy Patients

ABSTRACT Diabetic neuropathy is the most common and debilitating microvascular complication leading to nontraumatic amputations. Identifying the role of vitamin B12 in the etiology of neuropathy is crucial because simple vitamin B12 replacement may reverse neurologic symptoms if confirmed. Large proportion of vegetarians and patients on metformin have been associated with lowering of vitamin B12 levels. We have undertaken study on 60 vegetarian subjects between the age of 30 and 60 years, 20 healthy controls, 20 type 2 diabetes mellitus patients who were on metformin with peripheral neuropathy (DMWN), and 20 diabetics without peripheral neuropathy (DMWON). Vitamin B12, homocysteine, and folate levels were measured, and holotranscobalamin (holoTC) is also included, to improve the diagnosis of tissue vitamin B12 status, as holoTC is the biologically active fraction and more sensitive index of vitamin B12 status than total vitamin B12 itself. Complete history and clinical evaluation for neuropathy and electroneuromyography were recorded. Results In the DMWN group, vitamin B12 levels were significantly higher than in the DMWON group and controls [1013 (564-1501) pmol/l, 387 (245-731) pmol/l, and 305 (234-472) pmol/l respectively]. Similarly, serum holoTC was also found to be significantly higher in the DMWN [203.8 (129-300) pmol/l] group compared with the DMWON [94.4 (42.2-230.6) pmol/l] group and controls [74 (40-170) pmol/l]. Whereas mean levels of homocysteine and folate did not show any significant difference between groups, significant increase in fasting blood sugar and HbA1c was seen in the DMWN group compared with DMWON group and controls. Duration of diabetes is also significantly more in DMWN group compared with DMWON group. Conclusion We found that vitamin B12 and holoTC levels were high in patients with DMWN and DMWON groups compared with controls. Our study demonstrated that peripheral neuropathy was not associated with vitamin B12 deficiency in diabetics. These findings merit further research on a larger population to investigate into the cause of diabetic neuropathy, the factors involved, and benefit of vitamin B12 supplementation in these patients. Key messages Vitamin B12 status has no association with diabetic peripheral neuropathy. How to cite this article Noorjahan M, Madhavi K, Priscilla C, Jabeen SA. Vitamin B12 and Holotranscobalamin Levels in Diabetic Peripheral Neuropathy Patients. Indian J Med Biochem 2016;20(1):16-20.

Holotranscobalamin is not influenced by decreased renal function in elderly men: the MrOS Sweden study

Subclinical cobalamin deficiency is common in the elderly, but the sensitivity and specificity of serum total cobalamin for this diagnosis is poor. Serum holotranscobalamin (holoTC), a measure of biologically available cobalamin, is considered a better marker for early cobalamin depletion than total cobalamin. However, in elderly populations, health-related reference intervals for holoTC and correlations to renal function are not entirely clear. HoloTC was determined with an automated microparticle enzyme immunoassay (AxSYM®) in 790 elderly non-vitamin-supplemented Swedish men, median age 75.3 years. Renal function was assessed with creatinine, cystatin C and estimated glomerular filtration rate (eGFR calculated from creatinine). Median holoTC was 51.8 pmol/L, the health-related reference interval 19.6-132.3 pmol/L. There was no significant difference in mean holoTC in probands with normal compared to high creatinine (P = 0.80) and cystatin C (P = 0.82). No significant differences between the quartiles of creatinine or cystatin C in mean of log holoTC were seen. HoloTC correlated strongly with total cobalamin (r = 0.69, P < 0.001), weaker with eGFRcreatinine (r = -0.09, P < 0.05) and creatinine (r = 0.09, P < 0.05), the latter correlation was only seen in subjects with creatinine <100 µmol/L. HoloTC correlated negatively with plasma total homocysteine (r = -0.24, P < 0.001), but not with cystatin C and age. Serum holoTC in healthy elderly men shows the same distribution as earlier described for a younger reference population. In this group of elderly subjects, holoTC did not correlate to reduced renal function. Thus, holoTC appears to be a promising tool for evaluating cobalamin status also in elderly populations.

A Vitamin B-12 Supplement of 500 μg/d for Eight Weeks Does Not Normalize Urinary Methylmalonic Acid or Other Biomarkers of Vitamin B-12 Status in Elderly People with Moderately Poor Vitamin B-12 Status

Plasma vitamin B-12 is the most commonly used biomarker of vitamin B-12 status, but the predictive value for low vitamin B-12 status is poor. The urinary methylmalonic acid (uMMA) concentration has potential as a functional biomarker of vitamin B-12 status, but the response to supplemental vitamin B-12 is uncertain. A study was conducted to investigate the responsiveness of uMMA to supplemental vitamin B-12 in comparison with other biomarkers of vitamin B-12 status [plasma vitamin B-12, serum holotranscobalamin (holoTC), plasma MMA] in elderly people with moderately poor vitamin B-12 status. A double-blind, placebo-controlled, randomized 8-wk intervention study was carried out using vitamin B-12 supplements (500 μg/d, 100 μg/d, and 10 μg/d cyanocobalamin) in 100 elderly people with a combined plasma vitamin B-12 <250 pmol/L and uMMA ratio (μmol MMA/mmol creatinine) >1.5. All biomarkers had a dose response to supplemental vitamin B-12. Improvements in plasma vitamin B-12 and serum holoTC were achieved at cobalamin supplements of 10 μg/d, but even 500 μg/d for 8 wk did not normalize plasma vitamin B-12 in 8% and serum holoTC in 12% of people. The response in uMMA was comparable with plasma MMA; 15–25% of people still showed evidence of metabolic deficiency after 500 μg/d cobalamin for 8 wk. There was a differential response in urinary and plasma MMA according to smoking behavior; the response was enhanced in ex-smokers compared with never-smokers. uMMA offers an alternative marker of metabolic vitamin-B12 status, obviating the need for blood sampling.

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.

Homocysteine and holotranscobalamin and the risk of Alzheimer disease

To examine the relation between serum levels of homocysteine (tHcy) and holotranscobalamin (holoTC), the active fraction of vitamin B12, and risk of incident Alzheimer disease (AD) in a sample of Finnish community-dwelling elderly. A dementia-free sample of 271 subjects aged 65-79 years derived from the Cardiovascular Risk Factors, Aging, and Dementia (CAIDE) study was followed up for 7 years to detect incident AD. The association between serum tHcy and holoTC with AD was analyzed with multiple logistic regression after adjusting for several potential confounders, including common vascular risk factors. The odds ratios (ORs) (95% confidence interval [CI]) for AD were 1.16 (1.04-1.31) per increase of 1 μmol/L of tHcy at baseline and 0.980 (0.965-0.995) for each increase of 1 pmol/L baseline holoTC. Adjustment for several potential confounders including age, sex, education, APOE ε4 allele, body mass index, Mini-Mental State Examination, smoking, stroke, and blood pressure did not alter the associations: ORs (95% CI) for AD became 1.19 (1.01-1.39) for tHcy and 0.977 (0.958-0.997) for holoTC. Adjusting for holoTC attenuated the tHcy-AD link (OR changed from 1.16 to 1.10, 95% CI 0.96-1.25). The holoTC-AD relationship was less influenced by controlling for tHcy (OR changed from 0.980 to 0.984, 95% CI 0.968-1.000). Addition of folate did not change any of the results. This study suggests that both tHcy and holoTC may be involved in the development of AD. The tHcy-AD link may be partly explained by serum holoTC. The role of holoTC in AD should be further investigated.

Nutritional and oxidative status in occupational obese subjects

Nutritional status, referred to meat and vegetable food consumption, is related to folate and B12 vitamin levels; hyperhomocysteinemia (HHcy) is due to folate and B12 vitamin–methionine metabolism imbalance, which can lead to oxidative stress, OS (imbalance between reactive oxygen species, ROS, and total antioxidant capacity, TAC). Possible early vitamin B12 (B12) deficiency can be highlighted by holotranscobalamin (HoloTC, bioactive cobalamin fraction) assay. Erythrocyte folate is a biomarker of 2- to 3-month folate storage. To evaluate nutritional status and OS we conducted an observational study on 118 occupational obese subjects (34M/84F, aged 16–69, median 46.5 years; BMI 26.5–54.1, median 33.3 kg/m2) without previous cardiovascular disease. Serum TAC and ROS (spectrophotometry, Diacron International, Italy), serum B12 and HoloTC, serum and erythrocyte folate, plasma Hcy (immunoenzymatic, AxSYM, Abbott Diagnostics, USA), lipid panel and inflammatory parameters by routine methods. All subjects showed adequate serum and erythrocyte folate levels, but HoloTC values revealed cobalamin deficiency in 30% of cases not congruent with B12 concentrations, low only in 10%. 43% of cases showed mild HHcy (>10.5 μmol/L; median 12.44 μmol/L, IQR 11.2–16.2). OS was found by normal mean TAC values (370.5 micromolHClO/mL, 340–405; n.v. > 350) but increased mean ROS concentrations (386 CarrU, 337–434; n.v. 250–300) in 93% of subjects. Normal folate and B12 vitamin levels revealed a good nutritional status in our occupational obese subjects. The presence of Oxidative Stress, due to imbalance between ROS and TAC and mild hyperhomocysteinemia may heighten the obesity-related cardiovascular risk.

Comparison between Serum Holotranscobalamin and Total Vitamin B12 as Indicators of Vitamin B12 Status

This study aims to assess the usefulness of serum holotranscobalamin (holoTC), the fraction of vitamin B12 that is available for tissue uptake, compared with total vitamin B12 in patients investigated for vitamin B12 disorders. Serum samples were randomly selected from 76 patients (48 females, 28 males; age range 12-69 years) referred to the Clinical Biochemistry Laboratory, Royal Hospital for the assessment of vitamin B12 status. For each patient, serum total vitamin B12 level was determined by chemiluminescent microparticle immunoassay on Architect 2000 analyzer and holoTC (active vitamin B12) level was determined by microparticle enzyme immunoassay on Axsym analyzer (both from Abbott, USA). Comparison of the data was conducted to reflect the mean, standard deviation (SD) and correlation coefficient between the two groups. The mean (SD) for serum holoTC and total vitamin B12 were 46.5(32.2) pmol/L and 316.3(165.6) pmol/L respectively. There was a significant correlation between holoTC and total vitamin B12 (r= 0.765, P< 0.001) and the regression equation was expressed as; y = a + bx (i.e: holoTC = 1.5 + 0.14 total vitamin B12). Also, the results were assessed for any misclassification when comparing holoTC and the total vitamin B12 in terms of whether each or both values agree or disagree for classifying the patients as having normal or abnormal (low or high) results, based on the cut-off thresholds of the kit's quoted reference range for holo TC of 9 - 123 pmol/L and for total vitamin B12 of 140-600 pmol/L. Accordingly, in 69 (90.8%) samples, there was a parallel agreement/ classification of results, both being normal or abnormal. In 61 (80.4%) patients, both results were normal, whereas in 4 (5.2%) patients, both results were high, and in 4 (5.2%) patients both results were low. However, in 7 (9.8%) cases, there was disagreement/ misclassification of results; 6 (7.8%) patients, holoTC was normal while total vitamin B12 was low, and in 1 (1.4%) patient, holoTC was normal while total vitamin B12 was high. It can be recommended that holoTC and total vitamin B12, alone and in combination, have almost equal diagnostic efficiency in screening/diagnosing vitamin B12 deficiency for the majority of patients. In very few patients, holoTC appeared to be a better reflector of vitamin B12 status. Further comparison studies based on a gold standard method for classifying vitamin B12 status are worth considering.

Vitamin B12 intake above current recommendations may be needed to ensure optimal vitamin B12 status in sub‐groups of healthy adults

The study objectives were to assess vitamin B12 (B12) intake and status of non‐supplement consuming healthy young (18–49 y) adults (n=299) and to characterize dietary patterns most likely associated with normal B12 status. Vitamin B12 status was assessed by plasma B12, serum MMA, and serum holo‐TC. Dietary intake was estimated using a modified Dietary History Questionnaire and reduced rank regression was used to identify dietary patterns predicting B12 status. Vitamin B12 status was abnormal for at least one biomarker (B12 &lt; 148 pmol/L, MMA &gt; 270 nmol/L, holo‐TC &lt; 35 pmol/L) in 30% of individuals (23% meat consumers and 42% non‐meat consumers). Dietary patterns predicting variance in B12 biomarkers consisted primarily of fortified foods (eg RTE breakfast cereals and vegetarian meat substitutes), and fish, which were positively associated with vitamin B12 status. The median B12 intake associated with high vs normal MMA and low vs normal holo‐TC was 2.9 vs 4.7 g/d and 3.0 vs 4.8 g/d, respectively. The median B12 intake associated with the cut‐off for normal B12 status ranged from 3.2 to 4.1 μg/d for all biomarkers. Based on regression analysis, these data suggest that vitamin B12 requirement of healthy young adults may currently be underestimated. Individuals who wish to reduce meat consumption somewhat, may enhance B12 intake by including fish and fortified foods in their diet.

Measuring holotranscobalamin II, an early indicator of negative vitamin B12 balance, by radioimmunoassay in patients with ischemic cerebrovascular disease

Circulating homocysteine is a risk factor of cardiovascular and cerebrovascular events. Hyperhomocysteinemia may be an early indicator for vitamin B12 disorders because cobalamin is a cofactor in the remethylation process of homocysteine. Serum holotranscobalamin (holoTC II) becomes decreased before the development of metabolic dysfunction. In this study, we assessed circulating holoTC II to estimate the diagnosis of vitamin B12 deficiency in the first ischemic cerebrovascular attack. We also compared the efficacy of the measurement of plasma holoTC II with the other standard biochemical and hematological markers used to reach the diagnosis of cobalamin deficiency. Forty-five patients (age 71 years (range 35-90), 16 men/29 women) within the first ischemic cerebrovascular event were included in this prospective study. All the enrolled patients have been administered vitamin B12 1 mg intramuscular injection once a day for 10 days. At the baseline and on the tenth day of treatment, plasma levels of holoTC II and the proper biochemical and hematological markers in diagnosing cobalamin deficiency were measured. After admission, anemia and diminished serum vitamin B12 levels were determined to be only 20% (9/45) and 44% (20/45), respectively; 78% (35/45) of the patients had low serum holoTC II (<37 pmol/l). Serum homocysteine was higher in patients (49% of them) who had previously suffered a stroke. Thrombocytopenia, hypersegmentated neutrophils, and indirect hyperbilirubinemia were observed in 20% of the patients. Leukopenia and macrocytosis were not evident in any of them. In 18 of 27 patients (67%) that had low holoTC II levels after joining the study and who remained in the study until the end of cobalamin treatment, serum holoTC II levels returned to normal values. Cobalamin deficiency should be considered in patients with cerebrovascular diseases, even if anemia, elevated mean cell volume, depression of the serum cobalamin, or other classic hematological and/or biochemical abnormalities are lacking. Furthermore, measurement of serum holoTC II looks promising as a first-line of tests for diagnosing early vitamin B12 deficiency.