A metionina sintase redutase (MTRR) catalisa a redução da cobalamina (Cbl) oxidada a metilcobalamina. Em presença de folato, a metionina sintase (MTR) utiliza a metilcobalamina como cofator na metilação da homocisteína (tHcy) a metionina. O objetivo deste estudo foi avaliar os efeitos dos polimorfismos MTR A2756G e MTRR A66G nas concentrações dos metabólitos marcadores de deficiência de Cbl e folato em gestantes e neonatos. Os genótipos dos polimorfismos MTR A2756G e MTRR A66G foram obtidos por PCR-RFLP. O genótipo MTR 2756AA foi relacionado aos maiores valores de tHcy em gestantes e MMA em neonatos. Gestantes com genótipos MTRR 66AG e GG e com menores concentrações de Cbl apresentaram maior risco de apresentar concentrações elevadas de tHcy. Neonatos com genótipos com MTRR 66AG e GG apresentaram menores valores de SAM. Os polimorfismos MTR A2756G e MTRR A66G interferem nas reações dependentes de Cbl e folato em gestantes e neonatos. / Methionine synthase reductase (MTRR) catalyzes the reductive reaction of oxidized cobalamin to methylcobalamin. When folate is present, methionine synthase (MTR) uses methylcobalamin cofactor at homocysteine to methionine methylation process. The aim of this study was to evaluate the effects of MTR A2756G and MTRR A66G polymorphisms on total homocysteine (tHcy), methylmalonic acid (MMA), S-adenosylmethionine (SAM) concentrations and SAM/SAH ratio in Brazilian pregnant women and their newborns. Genotypes of two polymorphisms were determined by PCR-RFLP. MTR 2756AA genotype was associated with higher tHcy and MMA levels in mothers and babies, respectivelly. Lower cobalamin concentrations associated with MTRR 66AG and GG genotypes increased risk to elevated tHcy levels in pregnant women. The SAM levels were lower in neonates with MTRR 66AG e GG genotypes. The polymorphisms MTR A2756G and MTRR A66G could affect cobalamin and folate dependent reactions in pregnant women and newborns.

As vitaminas do complexo B e o ferro estão presentes em farinhas de cereais, alimentos amplamente consumidos pela população brasileira. O teor natural desses compostos em farinhas de cereais pode ser significativo no cálculo de uma dieta bem balanceada e o consumo de produtos industrializados enriquecidos com vitaminas do complexo B e ferro ajuda a contribuir na ingestão diária recomendada desses micronutrientes. Tabelas Mundiais de Composição de Alimentos apresentam dados incompletos das vitaminas do complexo B e do ferro em farinhas de cereais. No Brasil, a adição de ácido fólico e ferro em farinhas de milho e trigo produzidas para fins industriais é obrigatória desde 2002. Deste modo, para a adequação dos teores de rotulagem de produtos enriquecidos com vitaminas e ferro se fazem necessários o desenvolvimento e a validação de metodologias analíticas confiáveis e sensíveis para análise de vitaminas do complexo B e ferro em alimentos que apresentam tais micronutrientes em quantidades baixas, porém significativas, que correspondem aos teores naturalmente presentes. O objetivo do presente trabalho é a avaliação dos teores de sete vitaminas do complexo B (B1, B2, B6, ácido pantotênico, ácido fólico, niacina e biotina) e ferro em cinco farinhas de cereais (aveia, arroz, cevada, milho e trigo) utilizadas como matéria prima de produtos enriquecidos na indústria alimentícia, utilizando métodos validados. / Complex B vitamins and iron are present in some cereal foods, a kind of food largely consumed by Brazilian people. The total of these micronutrients can be significant at the dairy ingestion portion and in the consume of industrialized products enriched with complex B vitamins and iron contribute in the recommended dietary intake of these micronutrients. Table of food composition do not report complete data of complex B vitamins and iron in cereal flours. In Brazil, since 2002 the addition of folic acid and iron in com and wheat flours is compulsory. Therefore, to adapt the label of some products enriched with vitamins and iron, there is necessary the development and validation of analytical methods. These methods must be reliable and with enough sensitivity to analyse complex B vitamins and iron, in low concentration, wich are natural content in food. The purpose of this work is the evaluation, with validated methods, of the content of seven complex B vitamíns (B1, B2, B6, niacin, folic acid, pantothenic acid and biotin) and iron in five kinds of cereal flours (oat, rice, barley, com and wheat). These raw materiais are used in food industry in order to enrich the industrialized products.

This diploma thesis is focused on microdetermination of cobalt by UV-VIS spectrophotometry with organic agents and comparison with atomic absorption spectrometry. In the first part properties, occurance and importance of cobalt in the environment are desribed, complete with as a part of vitamine B12. Attention is also given to cobalt compounds, especially in the oxidative states +II and +III. Methods of preconcentration and preparation cobalt determination in real samples are adduced. In this thesis are described some optical analytical methods with accept on spectrophotometric agents for determination of cobalt. The experimental part of this thesis icludes optimalization of the spectrophotometric determination of cobalt with choosen organic agents 4-(2-pyridylazo)resorcin and 2-(5-Brom-2-pyridylazo)-5-diethylaminophenol and optimalization of atomic absorption spectrometry for cobalt determination. All of real samples of water (surface, underground, mineral and waste) were measured by both 4-(2-pyridylazo)resorcin and 2-(5-Brom-2-pyridylazo)-5-diethylaminophenol via UV-VIS and via atomic absorption spectrometry. Results of determinations were compared. A sample of vitamine B12 was measured too by all techniques.

Clinical diagnosis of vitamin B12/cobalt (Co) deficiency is difficult due to the unspecific nature of the clinical symptoms. The apparent increase in vitamin B12 deficiency in New Zealand in the late 1990’s made it clear that health providers were very reliant on plasma reference ranges to diagnose deficiency. However, the lack of quantitative data of what these reference ranges represent in terms of supply of vitamin B12, has prevented a better understanding of the metabolism of vitamin B12 within sheep. This thesis describes the development of an experimental approach to measure vitamin B12 production and absorption in sheep. The model was then used to investigate whether the type of carbohydrate source affects vitamin B12 production and/or absorption. In the first trial (Chapter 4), an adaptation of the repletion technique of Suttle (1974) for copper was used. Previously vitamin B12 depleted sheep were maintained on a diet of 400 g DM meadow hay and 250 g DM crushed barley and which provided a daily intake of 0.03 mg Co. Sheep were intravenously infused with known quantities of vitamin B12 (0 – 200 nmol hydroxycobalamin/d) to determine whether a relationship exists between plasma vitamin B12 concentration and rate of entry of the vitamin into the bloodstream and to quantify the relationship. Eight ewes fitted with rumen and abomasal cannulae were used in a paired 4x4 Latin square design. Four levels of vitamin B12 were infused into the jugular vein for 4 d followed by recovery period of 17 d and the plasma responses measured during this period. Control animals maintained relatively stable plasma levels while levels in treated animals rose sharply within 4 h after the start of infusion. The responses were proportional to infusion rate with a highly significant difference in concentrations observed between treatments (P<0.001). Appearance of vitamin B12 in the plasma was quantified by the relationship Y(pmol/l) = 238 + 102x0.74 where x = infusion rate(nmol B12/d), R² = 0.945. Plasma levels declined rapidly on cessation of infusion and had returned to original pre-treatment levels by the end of the recovery period. The second trial (Chapter 5) quantified plasma responses in relation to dietary Co supply to the rumen. The same eight rumen and abomasal cannulated ewes were used in a paired 4x4 Latin square with 4 levels of intra-ruminal Co infusion in amounts ranging from 0 – 1 mg Co/d as CoSO₄, infused for 4 d followed by a 19 d recovery period. Plasma vitamin B12 response was measured as were concentration of vitamin B12 in abomasal digesta supernatant and digesta liquid flow, the latter using polyethylene glycol as liquid phase marker. Plasma concentration increased from approximately 300 pmol/l to 750 – 800 pmol/l, peaking at day 5 after commencement of infusions. Control animals infused with water maintained base-line plasma vitamin B12 levels around 250 pmol/l. Digesta liquid flow was estimated at 14.6 l/d. The results from Chapters 4 and 5 allowed estimates of vitamin B12 production and absorption. These results are discussed and efficiency of capture of Co into vitamin B12 from dietary Co intake was estimated to decrease from 8.4% to 0.74% as Co intake increased from 0.03 to 1.11 mg Co/d. Absorption coefficient was estimated from the relationships between abomasal vitamin B12 flow and plasma response. The absorption coefficient was calculated to increase from 7 to 10% as abomasal vitamin B12 flow increased from 108 to 142 nmol/d. The current animal model was subsequently used in the final trial (Chapter 6) to investigate whether the type of carbohydrate supplied in the diet influences vitamin B12 production and absorption. The same eight ewes fitted with rumen and abomasal cannulae were used in a 2x4 cross-over design. Half of the ewes were maintained on the basal diet and crushed barley while, for the remainder, the barley was replaced by 250 g of fructose:sucrose in a 80:20 mix during a 7 d period of adaptation. While on their respective diets, both groups were then infused with 0.3 mg Co/d for 6 d, a level chosen to optimise plasma vitamin B12 response. All ewes were then returned to the basal diet and barley for a 15 d recovery period after which treatments were reversed and the procedure repeated. Rumen pH, valeric, propionic, butyric, succinic, and acetic acid and ammonia concentrations were not significantly affected by diet. Digesta flow was 30% greater on a diet enriched with starch compared to sugar-enriched diet. There was an observed difference in vitamin B12 production. However there was no significant diet x time effect on plasma vitamin B12 concentration so any difference in production was negated by a difference in absorption. Carbohydrate supplied as water soluble carbohydrate or starch does not appear to influence plasma vitamin B12 concentration.

This study characterizes B vitamin supplement use prior to and during pregnancy, changes in dietary one-carbon nutrient intakes (folate, vitamin B12, vitamin B6, choline, betaine and methionine) and most significant dietary sources. In Canadian women (Toronto, Ontario) supplemental (n=364) and dietary intakes (using a food frequency questionnaire) (n=290) were assessed during early and late pregnancy. Majority reported using a B vitamin-containing supplement prior (60%) to and during early (93%) and late (89%) pregnancy. Median supplemental intakes of folic acid, B12 and B6 were 1000 µg/d, 2.6 µg/d and 1.9 mg/d, respectively. Dietary one-carbon nutrient intakes did not change appreciably between early and late pregnancy. Most significant sources of folate and B6 were fruits and vegetables, of folic acid were cereals and grains and of B12 were dairy and egg products. Overall, this study provides novel information about one-carbon nutrient intakes in pregnancy which are crucial in maternal and child health.

This study characterizes B vitamin supplement use prior to and during pregnancy, changes in dietary one-carbon nutrient intakes (folate, vitamin B12, vitamin B6, choline, betaine and methionine) and most significant dietary sources. In Canadian women (Toronto, Ontario) supplemental (n=364) and dietary intakes (using a food frequency questionnaire) (n=290) were assessed during early and late pregnancy. Majority reported using a B vitamin-containing supplement prior (60%) to and during early (93%) and late (89%) pregnancy. Median supplemental intakes of folic acid, B12 and B6 were 1000 µg/d, 2.6 µg/d and 1.9 mg/d, respectively. Dietary one-carbon nutrient intakes did not change appreciably between early and late pregnancy. Most significant sources of folate and B6 were fruits and vegetables, of folic acid were cereals and grains and of B12 were dairy and egg products. Overall, this study provides novel information about one-carbon nutrient intakes in pregnancy which are crucial in maternal and child health.

Elevated plasma total homocysteine (ptHcy) or hyperhomocysteinemia (hHcy) independently predicts cardiovascular disease in predialysis chronic kidney disease (pCKD). Folate status is one of the known nutritional determinants of ptHcy. In the era of folic acid food fortification, this cross-sectional study aimed to describe in pCKD subjects (n=48): 1) Prevalence of hHcy. 2) Intake and status of nutrients involved in homocysteine metabolism. 3) Determinants of ptHcy. The prevalence of hHcy was 93.8% (95% CI: 81.8 to 98.4). Median (25th, 75th percentile) total folate intake from food and supplements was 389 (282,640) µg DFE/d. No subject was folate deficient (red blood cell < 317 nmol/L). Red blood cell folate (r = - 0.406, p=0.004) and energy-protein undernutrition (r = 0.357, p=0.013) independently predicted ptHcy. To conclude, total folate intake among subjects with pCKD was sufficient to prevent folate deficiency but not able to prevent a high prevalence of hHcy.

Elevated plasma total homocysteine (ptHcy) or hyperhomocysteinemia (hHcy) independently predicts cardiovascular disease in predialysis chronic kidney disease (pCKD). Folate status is one of the known nutritional determinants of ptHcy. In the era of folic acid food fortification, this cross-sectional study aimed to describe in pCKD subjects (n=48): 1) Prevalence of hHcy. 2) Intake and status of nutrients involved in homocysteine metabolism. 3) Determinants of ptHcy. The prevalence of hHcy was 93.8% (95% CI: 81.8 to 98.4). Median (25th, 75th percentile) total folate intake from food and supplements was 389 (282,640) µg DFE/d. No subject was folate deficient (red blood cell < 317 nmol/L). Red blood cell folate (r = - 0.406, p=0.004) and energy-protein undernutrition (r = 0.357, p=0.013) independently predicted ptHcy. To conclude, total folate intake among subjects with pCKD was sufficient to prevent folate deficiency but not able to prevent a high prevalence of hHcy.

This study aims to analyze the effect of an improvised user-generated health awareness campaign which was based on a personal narrative and first-hand experience with the B12 deficiency symptoms in babies and toddlers. The campaign was conducted in 2015 with a follow-up video in 2016 and shared through social media outlets, informing about the topic and empowering individuals to take responsibility for their own or their child’s health by providing information that could serve as a guideline for early diagnosis and intervention and by presenting an outlook of how people with similar issues manage the condition.The impact of the campaign is being qualitatively and quantitatively accessed by interviews with medical professionals and respectively survey data from a national survey and statistics from the YouTube console. In consideration has been taken the trust the respondents have in the medical service and the usual access to health information both online and offline The quantitative data were collected using a national online survey in which 1185 individuals took part. It aimed to additionally identify the general public attitude towards medical service in and the awareness about the vitamin B12. Four people participated in the interviews, divided into two groups - parents of children, who had symptoms similar to those, shown in the videos; and health practitioners who have seen the videos and comment on its qualities as a self-diagnosing material as well as the effect that attention to the issue created on their medical practices. The results confirmed that the personal narrative of a campaign could help to create identification and thus be more persuasive and with further increased sharing potential of the message through social media. The concrete campaign reached cumulatively over 167.000 people through YouTube, which is around 2.4% of the population of Bulgaria and possibly creating a lasting impact on the public attitude towards vitamin/mineral and other deficiencies. We found out that social media and YouTube could serve as an impactful medium for disseminating health-related information online when accurate and persuasive information is being used. When addressing a wide audience with little or no prior knowledge of the subject the personal narrative or testimonial could create more impact than a neutral fact-providing material.

Background: Rare plants that contain corrinoid compounds mostly comprise cobalamin analogues, which may compete with cobalamin (vitamin B12 (B12)) metabolism. We examined the presence of B12 in a cultivated strain of an aquatic plant: Wolffia globosa (Mankai), and predicted functional pathways using gut-bioreactor, and the effects of long-term Mankai consumption as a partial meat substitute, on serum B12 concentrations. Methods: We used microbiological assay, liquid-chromatography/electrospray-ionization-tandem-mass-spectrometry (LC-MS/MS), and anoxic bioreactors for the B12 experiments. We explored the effect of a green Mediterranean/low-meat diet, containing 100 g of frozen Mankai shake/day, on serum B12 levels during the 18-month DIRECT-PLUS (ID:NCT03020186) weight-loss trial, compared with control and Mediterranean diet groups. Results: The B12 content of Mankai was consistent at different seasons (p = 0.76). Several cobalamin congeners (Hydroxocobalamin(OH-B12); 5-deoxyadenosylcobalamin(Ado-B12); methylcobalamin(Me-B12); cyanocobalamin(CN-B12)) were identified in Mankai extracts, whereas no pseudo B12 was detected. A higher abundance of 16S-rRNA gene amplicon sequences associated with a genome containing a KEGG ortholog involved in microbial B12 metabolism were observed, compared with control bioreactors that lacked Mankai. Following the DIRECT-PLUS intervention (n = 294 participants; retention-rate = 89%; baseline B12 = 420.5 ± 187.8 pg/mL), serum B12 increased by 5.2% in control, 9.9% in Mediterranean, and 15.4% in Mankai-containing green Mediterranean/low-meat diets (p = 0.025 between extreme groups). Conclusions: Mankai plant contains bioactive B12 compounds and could serve as a B12 plant-based food source.