User:Minihaa/Vitamin B12 status and metabolic traits

1.1 Introduction

Vitamin B12 is an essential water soluble micronutrient, which participates as a cofactor for the synthesis of DNA, fatty acids, and myelin. Vitamin B12 deficiency was previously thought to be limited to populations with a low intake of vitamin B12-rich foods (mainly vegetarians) and older adults, due to their impaired absorption of the vitamin through food. However, alarmingly high prevalence rates of low plasma vitamin B12 status have been recognized to exist in the Indian subcontinent, Mexico, Central and South America, and selected areas in Africa. Symptoms of vitamin B12 deficiency include haematological and neurological impairment. Additionally, observational studies have shown that low vitamin B12 concentrations are accompanied by a wide range of chronic diseases and conditions, including obesity, insulin dysregulation and adverse cardiometabolic outcomes.

Metabolic diseases such as type 2 diabetes and obesity are world-wide health problems, which are now increasingly diagnosed earlier in life. The metabolic diseases are generally caused by the interaction between environmental factors (dietary factors and sedentary lifestyle) and a genetic predisposition to the development of metabolic diseases. Whilst dietary factors are an important contributor to metabolic disorders, this relationship differs across countries, due to the variation in food consumed worldwide. Studies have shown that the intrauterine imbalance of vitamin B12 and folate can affect DNA methylation and ‘programme’ the offspring to develop metabolic disorders later in life providing evidence for interactions between genes and nutrients in the development of metabolic disease.

Many candidate genes have been studied in relation to their potential role in vitamin B12 metabolism, and an association between these genes and vitamin B12 concentrations have been confirmed. To date only two Mendelian Randomization studies (an analytical tool used to measure the causal relations between modifiable risk factors and a clinically relevant outcome, using measured variation in genes of known function) have explored the relationship between a genetically determined decrease in serum vitamin B12 concentrations on body mass index (BMI) and cardiometabolic risk highlighting the need for more studies. Vitamin B12 levels, which are not a homogenous phenotype, are responsive to changes in diet and are dependent on the quality and consumption of animal protein. Therefore, controlling diet is recommended in preventing vitamin B12 deficiency. Given that the genetic make-up varies from individual to individual, it is vital to examine the interactive effects between dietary factors and genetics on vitamin B12 concentrations and metabolic traits, which will ultimately allow us to personalise diet according to each ethnic sub-group [12]. Furthermore, other modifiable factors (e.g. physical activity), which could interact with genetic factors should be taken into account.

The following chapter will (i) explain the nutritional aspects of vitamin B12 (ii) focus on the importance of maintaining adequate vitamin B12 concentrations (iii) describe the symptoms associated with vitamin B12 deficiency (iv) explain the role of genes in influencing circulating vitamin B12 concentrations and (vi) explain the need for a nutrigenetics approach to study the role of genes and diet in the development of vitamin B12 deficiency and metabolic traits.

1.2 Vitamin B12 function

Can basically just be added to the existing text.

Vitamin B12 in the body is crucial for normal erythropoiesis. Both folate and vitamin B12 are required for DNA synthesis, which codes for the production of billions of erythrocytes daily. Deficiency of either folate or vitamin B12 leads to the inhibition of purine and thymidylate synthesis, which impairs DNA synthesis. As a result erythroblast apoptosis and anaemia persists. Additionally, vitamin B12 is an essential co-factor important for cell metabolism, thus a deficiency will have serious clinical consequences. The intracellular conversion of vitamin B12 into two active co-enzymes, methylcobalamin (cytoplasm) and adenosylcobalamin (mitochondria) is essential for the homeostasis of methionine and methylmalonic acid, respectively.

In the cytoplasm, methylcobalamin participates as a co-factor for the enzyme methionine synthase; which converts homocysteine to methionine. This reaction also depends on folate, where the methyl group of methyltetrahydrofolate is transferred to homocysteine, to produce methionine and tetrahydrofolate. A deficiency of vitamin B12 may lead to the increase in homocysteine concentration, which is a known marker of cardiovascular disease (CVD). Furthermore, methionine synthase is important for purine and pyrimidine synthesis.

In mammals, the mitochondrial conversion of methylmalonyl-CoA to succinyl-CoA is catalysed by methylmalonyl-CoA mutase an enzyme which utilizes vitamin B12 (5-deoxy adenosyl cobalamin) as a co-enzyme. Subsequently, succinyl-CoA, which is important for lipid and carbohydrate synthesis, enters the Krebs cycle. A defect in the conversion of methylmalonyl-CoA to succinyl-CoA can cause the build-up of methylmalonyl- CoA which gets converts into methylmalonic acid (MMA), which has detrimental implications on the nervous system. Accumulated MMA is thought to be a myelin destabiliser, where excessive MMA leads to the incorporation of abnormal fatty acids into the myelin sheath.

1.3 Metabolism of vitamin B12

See: Vitamin_B12#Absorption Muss detailliert eingearbeitet werden.

While ingesting food, the salivary and oesophageal glands release transcobalamin-I (TCN1, also known as haptocorrin), which binds strongly to vitamin B12. The function of TCN1 is to protect vitamin B12 from acid degradation in the stomach. Once vitamin B12 reaches the duodenum, proteolytic enzymes from the pancreas release vitamin B12 from TCN1. Vitamin B12 then forms a new complex with intrinsic factor (IF), which is secreted by the gastric epithelium. The vitamin B12-IF complex interacts with the cubam receptor (consisting of cubilin and a receptor-associated protein) present on the apical surface of the distal ileal epithelium, at which the complex enters by endocytosis in the ileum. Upon internalization, IF is degraded in enterocyte lysosomes, releasing the free vitamin B12 to the cytosol in the form of hydroxocobalamin. Next, hydroxocobalamin is either transformed into methylcobalamin in the cytoplasm or to adenosylcobalamin in the mitochondria. Alternatively, vitamin B12 is transported into portal circulation by the ABC drug transport protein (ABCC1), also known as multidrug resistance protein (MRP1). Vitamin B12 then binds to transcobalamin II (TC), which is then secreted into circulation and is transported as holotranscobalamin (holoTC) in serum and is distributed to tissues including the liver by receptor-mediated endocytosis.

In healthy adults, approximately 50-90% of vitamin B12 is stored in the liver as adenosylcobalamin (2000-5000 µg). The remainder of vitamin B12 is stored in muscle, skin and blood plasma. Approximately, 2-5 µg of vitamin B12 is lost daily as a result of cellular metabolism, irrespective of how much vitamin B12 is stored in the body [36]. Vitamin B12 is also excreted into bile (500 µg - 5000 µg) and is reabsorbed across the ileal enterocyte. Very small amounts of vitamin B12 absorption (1%–2% of an oral dose) occur by passive diffusion, and this route of absorption especially important for populations with limited or no intrinsic factor present (e.g., patients with gastric bypass surgery).

1.4 Dietary sources and Bioavailability of vitamin B12

The Wikipedia article is probably much better than the text here....

1.4.1 Bioavailability of vitamin B12

The intake of dietary vitamin B12 cannot be used as a sole measure of nutritional status. It is important to take into consideration how much of the vitamin B12 from the food source can be used systematically through normal body functions. At present, the bioavailability of vitamin B12 is assumed to be between 40-50% for healthy adults with normal gastrointestinal functioning.

Absorption of vitamin B12 are traditionally assessed by measuring faecal extraction of radioactivity, after consuming 100g of a food item labelled with radioactive vitamin B12. In healthy humans, the absorption of vitamin B12 has shown to vary according to the type and quantity of protein consumed within the diet. Studies assessing bioavailability of B12 from different food sources in healthy participants showed that the absorption of vitamin B12 was better in milk (65%) and chicken (61-65%), in comparison to eggs (24-36%).

A further issue to take into consideration, when discussing bioavailability, is that IF- vitamin B12 receptors (present on the distal ileal epithelium), can be saturated and absorb a certain amount of vitamin B12. It is thought that approximately 1.5-2.0 µg of vitamin B12 can be absorbed from a meal, however other studies have reported higher absorption rates (up to 6 µg from a single meal). Bioavailability of vitamin B12 increases as the vitamin B12 content in food increases up to a certain point, and then it decreases if the vitamin B12 content is higher than the absorption capacity of the IF-vitamin B12 receptors.

Consuming processed food, improving hygiene and reheating cooked foods are some factors which reduce the bioavailability of vitamin B12 in foods. Furthermore, the overgrowth of intestinal bacteria (because of poor dietary intake, antibiotics and stress), leads to the competitive uptake of vitamin B12 by bacteria and interferes with the bioavailability of vitamin B12.

Nutritional Aspects of vitamin B12

Vitamin B12 is synthesized from bacteria growing in soil, sewage, water and the intestinal lumen of animals. Vitamin B12 enters animal tissues when animals ingest vitamin B12-producing bacteria present on legumes/roots or produced in the animal’s rumen. Although, micro-organisms in the human colon synthesize vitamin B12, humans cannot absorb it, as the majority of vitamin B12 is absorbed in the small intestine. Consequently, the main sources of biologically active vitamin B12 vitamers are derived from animal products, such as milk, eggs, seafood and poultry. Excellent sources of vitamin B12 include the livers of ruminant animals as well as shellfish, fish and fish roe (Table 1).

Table 1: Contents of uncooked foods containing a high vitamin B12 content

Beef liver	60-122b
Shellfish1	2-58
Fish2	3.0-8.0
Fish roe3	18

The data has been extracted from a national Food composition data bank (The Danish National Food Institute, 2015). The data should be treated as an estimate, given that the food data base did not disclose how the levels of vitamin B12 were obtained.

¹ Clam, scallop, mussel, shrimp and oyster

² Salmon, trout, mackerel, and tuna

³ Roe from Atlantic cod, lumpfish, and rainbow trout

^aThe adult UK Recommended Nutrient Intake (RNI) for vitamin B12 is 1.5 µg/day

^bThe efficiency of absorption from liver is approximately 11% compared with 50% for other food.

It is believed that individuals following a vegan/vegetarian diet are more susceptible to vitamin B12 deficiency. Any vitamin B12 present on plant-derived products are usually because of bacterial contamination. However, some plants such as dried purple laver (nori), mushroom fruiting bodies fermented soybeans (Tempe), and tea leaves have been found to contain vitamin B12. Most blue-green algae (cyanobacteria) and certain edible shellfish contain vitamin B12 analogues which are inactive in mammals and may inhibit cobalamin-dependent enzymes. As a result, vegetarians and vegans are reliant on vitamin supplements containing vitamin B12, and foods such as breakfast cereals, soy milk and nutritional yeast products which are fortified with vitamin B12.

Methods for the analysis of vitamin B12 in food

See Vitamin_B12#Chemistry

Several methods have been used to determine the vitamin B12 content in foods including microbiological assays, chemiluminescence assays, polarographic, spectrophotometric and high-performance liquid chromatography. The microbiological assay has been the most commonly used assay technique for foods, utilizing certain vitamin B12–requiring microorganisms, such as Lactobacillus delbrueckii subsp.lactis ATCC7830. However, it is no longer the reference method due to the high measurement uncertainty of vitamin B12. Furthermore, this assay requires overnight incubation and may give false results if any inactive vitamin B12 analogues are present in the foods [38]. Currently, radioisotope dilution assay (RIDA) with labelled vitamin B12 and hog IF (pigs) have been used to determine vitamin B12 content in food. Previous reports have suggested that the RIDA method is able to detect higher concentrations of vitamin B12 in foods compared to the microbiological assay method . New techniques employing more specific monoclonal antibodies and specific binding proteins are expected to advance the detection of vitamin B12 in food products.

Recommended dietary intake of vitamin B12

The recommended dietary intake (RDI) of vitamin B12 varies between countries. The European Union recommends 1 µg of vitamin B12, whilst the government of the United Kingdom and United States recommends a daily intake of 1.5 µg and 2.4 µg, respectively. The requirements of vitamin B12 also varies according to age and whether a woman is pregnant or lactating, as shown in Table 2.

Table 2: Recommended Dietary Allowance (RDA)/ Recommended Nutrient Intake (RNI) for vitamin B12

Age or condition	Vitamin B12 requirement (µg/day) in healthy U.S and Canadian populations [45]	Age or condition	Vitamin B12 requirement (µg/day) in a healthy UK population [44]
Pregnant	2.6	Pregnant	1.5
Breast-feeding	2.8	Breast-feeding	2.0
0-6 mo	0.4	0-6 mo	0.3
7-12 mo	0.5	7-12 mo	0.4
1-3 yr	0.9	1-3 yr	0.5
4-8 yr	1.2	4-6 yr	0.8
9-13 yr	1.8	7-10 yr	1.0
14-18 yr	2.4	11-14 yr	1.2
19-50 yr	2.4	15+ yr	1.5
51+ yr	2.4

The RNI of vitamin B12 for healthy British adult men and women is 1.5 µg/day and is based on the estimated average requirement (EAR) of vitamin B12 which is 1.25 µg/day for over 15 year olds (with no different recommendations for pregnant women). Although the daily requirement of vitamin B12 for people over the age of 50 is the same as younger adults (1.5 µg/day), this serves many problems. Individuals over the age of 51 years are at greater risk of vitamin B12 malabsorption, due to inadequate stomach acid and gastritis. As a result, the US institute of Medicine has recommended that individuals over 51 should take vitamin B12 supplements or consume a greater amount of fortified vitamin B12 products. The storage of vitamin B12 in the body is approximately 1000 - 5000 µg, which is relatively high. Therefore, vitamin B12 deficiency may not appear for several years, until stores deplete. However, an inadequate dietary consumption of vitamin B12 is recommended to prevent the onset of vitamin B12 deficiency.

For pregnant women in the UK, the RNI (1.5 µg/day) does not take into consideration the foetal deposition of vitamin B12 (0.10-0.2 µg/day). Furthermore, there is evidence that the maternal absorption of vitamin B12 is more efficient during pregnancy. During lactation, the RNI is further increased to 2.0 µg/day to take account of the approximate secretion of 0.33 µg vitamin B12/day in breast milk.

Vitamin B12 deficiency

Insert as "Vitamin B12 deficiency by country" in the deficiency article.

Symptoms of vitamin B12 deficiency

The clinical manifestations of vitamin B12 deficiency vary in severity and can affect multiple systems in the body. The following section summarizes the current knowledge of the adverse functional effects of vitamin B12 deficiency.

Prevalence of vitamin B12 deficiency from world-wide studies

Transfered to Vitamin_B12_deficiency#Epidemiology

According to the World Health Organization (WHO), vitamin B12 deficiency may be considered a global public health problem affecting millions of individuals. However, the incidence and prevalence of vitamin B12 deficiency worldwide is unknown due to the limited population-based data available (see table below).

Developed countries such as the United States, Germany and the United Kingdom have relatively constant mean vitamin B12 concentrations. The data from the National Health and Nutrition Examination Survey (NHANES) reported the prevalence of serum vitamin B12 concentrations in the United States population between 1999 to 2002. Serum vitamin B12 concentrations of <148 pmol/L was present in < 1% of children and adolescents. In adults aged 20-39 years, concentrations were below this cut-off in ≤3% of individuals. In the elderly (70 years and older), ≈ 6% of persons had a vitamin B12 concentration below the cut-off.

Furthermore, ≈ 14-16% of adults and >20% of elderly individuals showed evidence of marginal vitamin B12 depletion (serum vitamin B12: 148-221 pmol/L). In the United Kingdom, a National Diet and Nutrition Survey (NDNS) was conducted in adults aged between 19 to 64 years in 2000–2001 and in elderly individuals (≥ 65 years) in 1994–95. Six percent of men (n = 632) and 10% of women (n = 667) had low serum vitamin B12 concentrations, defined as <150 pmol/L. In a subgroup of women of reproductive age (19 to 49 years), 11% had low serum B12 concentrations <150 pmol/L (n=476). The prevalence of vitamin B12 deficiency increased substantially in the elderly, where 31% of the elderly had vitamin B12 levels below 130 pmol/L. In the most recent NDNS survey conducted between 2008-2011, serum vitamin B12 was measured in 549 adults. The mean serum vitamin B12 concentration for men (19-64 years) was 308 pmol/L, of which 0.9% of men had low serum B12 concentrations <150 pmol/L. In women aged between 19-64 years, the mean serum vitamin B12 concentration was slightly lower than men (298 pmol/L), with 3.3% having low vitamin B12 concentrations <150 pmol/L. In Germany, a national survey in 1998 was conducted in 1,266 women of childbearing age. Approximately, 14.7% of these women had mean serum vitamin B12 concentrations of <148 pmol/L.

Few studies have reported vitamin B12 status on a national level in non-Western countries. Of these reported studies, vitamin B12 deficiency was prevalent among school- aged children in Venezuela (11.4% ), children aged 1-6 years in Mexico (7.7%), women of reproductive age in Vietnam (11.7%), pregnant women in Venezuela (61.34%) and in the elderly population (>65 years) in New Zealand (12%). Currently, there are no nationally representative surveys for any African or South Asian countries. However, the very few surveys which have investigated vitamin B12 deficiency in these countries have been based on local or district level data. These surveys have reported a high prevalence of vitamin B12 deficiency (<150 pmol/L), among 36% of breastfed and 9% of non-breastfed children (n=2482) in New Delhi and 47% of adults (n=204) in Pune, Maharashtra, India. Furthermore, in Kenya a local district survey in Embu (n=512) revealed that 40% of school- aged children in Kenya had vitamin B12 deficiency.

Table showing worldwide prevalence of vitamin B12 deficiency (serum/plasma B12 < 148 or 150 pmol/L)

Group	Number of studies	Number of participants	Vitamin B12 deficiency (%)
Children (< 1y – 18 years)	14	22,331	12.5
Pregnant women	11	11,381	27.5
Non-pregnant women	16	18,520	16
All adults (Under 60 years)	18	81.438	6
Elderly (60+ years)	25	30,449	19

Data derived from Table 2 available on

Vitamin B12 and metabolic risk in offspring

Vitamin B12 is a critical micronutrient essential for supporting the increasing metabolic demands of the foetus during pregnancy. B12 deficiency in pregnant women is increasingly common and has been shown to be associated with major maternal health implications, including increased obesity, higher body mass index (BMI), insulin resistance, gestational diabetes, and type 2 diabetes (T2D) in later life. A study in a pregnant white non-diabetic population in England, found that for every 1% increase in BMI, there was 0.6% decrease in circulating B12. Furthermore, an animal study in ewes demonstrated that a B12, folate and methionine restricted diet around conception, resulted in offspring with higher adiposity, blood pressure and insulin resistance which could be accounted for altered DNA methylation patterns.

Both vitamin B12 and folate are involved in the one-carbon metabolism cycle. In this cycle, vitamin B12 is a necessary cofactor for methionine synthase, an enzyme involved in the methylation of homocysteine to methionine. DNA methylation is involved in the functioning of genes and is an essential epigenetic control mechanism in mammals. This methylation is dependent on methyl donors such as vitamin B12 from the diet. Vitamin B12 deficiency has the potential to influence methylation patterns in DNA, besides other epigenetic modulators such as micro (RNAs), leading to the altered expression of genes. Consequently, an altered gene expression can possibly mediate impaired foetal growth and the programming of non-communicable diseases.

Vitamin B12 and folate status during pregnancy is associated with the increasing risk of low birth weight, preterm birth, insulin resistance and obesity in the offspring. In addition it has been associated with adverse foetal and neonatal outcomes including neural tube defects (NTDs) and delayed myelination or demyelination. The mother’s B12 status can be important in determining the later health of the child, as shown in the Pune maternal Nutrition Study, conducted in India. In this study mothers with high folate concentrations and low vitamin B12 concentrations, led to babies having a higher adiposity and insulin resistance at age 6. In the same study, over 60% of pregnant women were deficient in vitamin B12 and this was considered to increase the risk of gestational and later diabetes in the mothers. Increased longitudinal cohort studies or randomised controlled trials are required to understand the mechanisms between vitamin B12 and metabolic outcomes, and to potentially offer interventions to improve maternal and offspring health.

Vitamin B12 and cardiometabolic disease outcomes

Multiple studies have explored the association between vitamin B12 and metabolic disease outcomes, such as obesity, insulin resistance and the development of cardiovascular disease. Results from two recent studies have indicated that vitamin B12 deficiency may be associated with obesity during childhood. Pinhas-Hamiel et al., reported that obese children and adolescents (n=164) had significantly lower vitamin B12 concentrations in comparison to normal-weight children (n=228). The report from the Canadian Health Measurement Survey showed that obese children and adolescents aged 6 to 19 years were more likely to have an inadequate vitamin B12 status compared to those with normal weight. In adults, Madan et al., (2006) reported that 13% of patients referred to pre-operative bariatric surgery had vitamin B12 deficiency. On the other hand, Schweiger et al., (2010) only observed vitamin B12 deficiency in 3.4% of patients evaluated in bariatric surgery (n=114). In addition, in a study conducted in post-menopausal women, vitamin B12 concentrations decreased in relation to an increase in BMI. A long-term study where vitamin B12 was supplemented across a period of 10 years, led to lower levels of weight gain in overweight or obese individuals (p < 0.05).

There are several mechanisms which may explain the relationship between obesity and decreased vitamin B12 status. Vitamin B12 is a major dietary methyl donor, involved in the one-carbon cycle of metabolism and a recent genome-wide association (GWA) analysis showed that increased DNA methylation is associated with increased BMI in adults, consequently a deficiency of vitamin B12 may disrupt DNA methylation and increase non- communicable disease risk. Vitamin B12 is also a co-enzyme which converts methylmalonyl- CoA to succinyl-CoA in the one carbon cycle. If this reaction cannot occur, methylmalonyl- CoA levels elevate, inhibiting the rate-limiting enzyme of fatty acid oxidation (CPT1 – carnitine palmitoyl transferase), leading to lipogenesis and insulin resistance. Further to this, reduced vitamin B12 concentrations in the obese population is thought to result from repetitive short-term restrictive diets and increased vitamin B12 requirements secondary to increased growth and body surface area. It has also been hypothesised that low vitamin B12 concentrations in obese individuals are a result of wrong feeding habits, where individuals consume a diet low in micronutrient density. Finally, vitamin B12 is involved in the production of red blood cells, and vitamin B12 deficiency can result in anaemia, which causes fatigue and the lack of motivation to exercise. The investigation into the relationship between cardiovascular disease (CVD) and vitamin B12 has been limited, and there is still controversy as to whether primary intervention with vitamin B12 will lower cardiovascular disease. Deficiency of vitamin B12 can impair the remethylation of homocysteine in the methionine cycle, and result in raised homocysteine levels. There is much evidence linking elevated homocysteine concentrations with an increased risk of cardiovascular disease, and homocysteine lowering treatments have led to improvements in cardiovascular reactivity and coagulation factors. In adults with metabolic syndrome, individuals with low levels of vitamin B12 had higher levels of homocysteine compared to healthy subjects. It is thus possible that vitamin B12 deficiency enhances the risk of developing cardiovascular disease in individuals who are obese. Alternatively, low levels of vitamin B12 may increase the levels of proinflammatory proteins which may induce ischaemic stroke.

It is important to screen vitamin B12 deficiency in obese individuals, due to its importance in energy metabolism, and relationship with homocysteine and its potential to modulate weight gain. More studies are needed to test for the causality of vitamin B12 and obesity using genetic markers. Furthermore, many studies have tested for the association of vitamin B12 with BMI. However, BMI does not accurately measure adiposity, and a high BMI does not necessary indicate that an individual is obese. More studies implementing x-ray absorptiometry, magnetic-resonance imaging computed tomography scans and analysing body fat % may be important for testing the link between obesity-related traits and vitamin B12 concentrations. A few studies have also reported no deficiency of vitamin B12 in obese individuals. Lower vitamin B12 concentrations were observed in overweight Brazilian adolescents compared to normal-weight adolescents, however there was no statistically significant difference between the two groups. Likewise, among Thai adults no statistically significant difference between overweight and obese individuals compared to normal control subjects was detected. In the study by Baltaci et al, approximately 37.7% of overweight and 40.1% of obese Turkish individuals were deficient in vitamin B12. Despite overweight and obese individuals having lower B12 levels in comparison to control non-obese subjects, the difference between the groups were not statistically significant. In a Mendelian randomization study conducted in a Danish cohort, no significant associations were detected between genetically determined decreased serum vitamin B12 concentrations and BMI levels, indicating that there may not be a causal role of low serum vitamin B12 levels in obesity. Finally, a recent literature review conducted over 19 studies, found no evidence of an inverse association between BMI and circulating vitamin B12.

Previous clinical and population-based studies have indicated that vitamin B12 deficiency is prevalent amongst adults with type 2 diabetes. Kaya et al., conducted a study in women with polycystic ovary syndrome, and found that obese women with insulin resistance had lower vitamin B12 concentrations compared to those without insulin resistance. Similarly, in a study conducted in European adolescents, there was an association between high adiposity and higher insulin sensitivity with vitamin B12 concentrations. Individuals with a higher fat mass index and higher insulin sensitivity (high Homeostatic Model Assessment [HOMA] index) had lower plasma vitamin B12 concentrations. Furthermore, a recent study conducted in India reported that mean levels of vitamin B12 decreased with increasing levels of glucose tolerance e.g. individuals with type 2 diabetes had the lowest values of vitamin B12, followed by individuals with pre-diabetes and normal glucose tolerance, respectively. However, B12 levels of middle aged-women with and without metabolic syndrome showed no difference in vitamin B12 levels between those with insulin resistance (IR) and those without. It is believed that malabsorption of vitamin B12 in diabetic patients, is due to individuals taking metformin therapy (an insulin sensitizer used for treating type 2 diabetes). Furthermore, obese individuals with type 2 diabetes are likely to suffer from gastroesophageal reflux disease, and take proton pump inhibitors, which further increased the risk of vitamin B12 deficiency.

A recent literature review conducted over seven studies, found that there was limited evidence to show that low vitamin B12 status increased the risk of cardiovascular disease and diabetes. Only one study by Weikert et al. reported that low vitamin B12 status increased the risk of cerebral ischaemia. After controlling for homocysteine, the relative risk of cerebral ischaemia reduced by approximately 10%, suggesting that the effects of low vitamin B12 are partially mediated by homocysteine. In two other studies, higher vitamin B12 concentrations were associated with an increased risk of mortality, fatal and non-fatal coronary events. It is important to note that these discrepancies, may be the result of the study population including individuals who were diseased or old. Further to this, both studies did not assess whether individuals were taking vitamin B12 supplements or they did not exclude individuals with liver disease or malignancy, which is important as raised vitamin B12 levels could have been due to a functional deficit. However, the review did not identify any associations between vitamin B12 and cardiovascular disease in the remaining four studies. Currently, no data supports vitamin B12 supplementation on reducing the risk of cardiovascular disease. In a dose-response meta-analysis of five prospective cohort studies, it was reported that the risk of coronary heart disease (CHD) did not change substantially with increasing dietary vitamin B12 intake. Of these five studies, three of the studies stated a non-significant positive association and two of the studies demonstrated an inverse association between vitamin B12 supplementation and coronary heart disease (only one of the studies was significant).

Vitamin B12 and Neural tube defects (NTDs)

Neural tube defects (NTDs), including spina bifida, encephalocele and anencephaly, are debilitating birth defects which result from the failure of neural fold closure during embryonic development. The causes of NTDs are multifactorial, including folate deficiency, genetic and environment factors. The WHO Technical Consultation has concluded that there is moderate evidence for the association between low vitamin B12 status and the increased risk of developing NTDs. Given that vitamin B12 is a co-factor for methionine synthase within the folate cycle. If vitamin B12 supplies are depleted, folate becomes trapped and DNA synthesis and methylation reactions are impaired. DNA synthesis is critical for embryonic development. Further to this, cell-signalling events which control gene-expression are controlled by methylation reactions. As a result, adequate folate and vitamin B12 is needed to help prevent NTDs. Many studies have shown associations between maternal vitamin B12 status and NTD affected pregnancy. Low vitamin B12 concentrations have also been found in the amniotic fluid of NTD affected pregnancy. Additionally, a population- based case-control study (89 women with an NTD and 422 unaffected pregnant controls) in Canada conducted after the fortification of folic acid in flour, found almost a tripling in the risk of NTD, in the presence of low maternal vitamin B12 status (indicated by holoTC). Future studies, using interventions with vitamin B12 supplements or fortification with vitamin B12 is needed to confirm the relationship between vitamin B12 and NTDs.

Vitamin B12 and anaemia

In countries where vitamin B12 deficiency is common, it is generally assumed that there is a greater risk of developing anaemia. However, the overall contribution of vitamin B12 deficiency to the global incidence of anaemia may not be significant, except in elderly individuals and vegetarians. There are relatively few studies which have assessed the impact of haematological measures in response to vitamin B12 supplementation. One study in 184 premature infants, reported that individuals given monthly vitamin B12 injections (100 µg) or taking supplements of vitamin B12 and folic acid (100 µg/day), had higher haemoglobin concentrations after 10-12 weeks, compared to those only taking folic acid or those taking no vitamin B12 injections. In deficient Mexican adult women and pre-schoolers, it was found that vitamin B12 supplementation did not affect any haematologic parameters. Vitamin B12 deficiency is also a major factor leading to megoblastic anaemia, especially in those infants breastfed by strict vegetarian mothers.

Vitamin B12 and Ageing

Vitamin B12 has been associated with disability in the elderly including the development of age-related macular degeneration (AMD) and the risk of frailty. Age-related macular degeneration is the leading cause of severe, irreversible vision loss in older adults. During the advanced stages of age-related macular degeneration, individuals are impaired of carrying out basic activities such as driving, recognising faces and reading. Several risk factors have been linked to age-related macular degeneration, including increasing age, family history, genetics, hypercholesterolemia, hypertension, sunlight exposure and lifestyle (smoking and diet). A few cross-sectional studies have found associations between low vitamin B12 status and age-related macular degeneration cases. It has been shown that daily supplementation of vitamin B12, B6 and folate over a period of seven years can reduce the risk of age-related macular degeneration by 34% in women with increased risk of vascular disease (n=5,204). However, another study failed to find an association between age-related macular degeneration and vitamin B12 status in a sample of 3,828 individuals representative of the non-institutionalized US population.

Frailty is a geriatric condition which is characterized by diminished endurance, strength, and reduced physiological function that increases an individual’s risk of mortality and impairs an individual from fulfilling an independent lifestyle. Frailty is associated with an increased vulnerability to fractures, falls from heights, reduced cognitive function and more frequent hospitalisation. The worldwide prevalence of frailty within the geriatric population is 13.9%, therefore there is an urgent need to eliminate any risk factors associated with frailty. Poor vitamin B status has been shown to be associated with an increased risk of frailty. Two cross sectional studies have reported that deficiencies of vitamin B12 were associated with the length of hospital stay, as observed by serum vitamin B12 concentrations and methylmalonic acid (MMA) concentrations [139, 140]. Furthermore, another study looking at elderly women (n=326), found that certain genetic variants associated with vitamin B12 status (Transcobalamin 2) may contribute to reduced energy metabolism, consequently contributing to frailty. In contrast, a recent study by Dokuzlar et al., found that there was no association between vitamin B12 levels and frailty in the geriatric population (n=335). Given that there are limited studies, which have assessed the relationship between vitamin B12 and frailty status, more longitudinal studies are needed to clarify the relationship.

Vitamin B12 and neurological decline

Severe vitamin B12 deficiency is associated with subacute combined degeneration of the spinal cord, which involves demyelination of the posterior and lateral columns of the spinal cord. Symptoms include memory and cognitive impairment, sensory loss, motor disturbances, loss of posterior column functions and disturbances in proprioception. In advanced stages of vitamin B12 deficiency, cases of psychosis, paranoia and severe depression have been observed, which may lead to permanent disability if left untreated. Studies have shown the rapid reversal of the neurological symptoms of vitamin B12 deficiency, after treatment with high-dose of vitamin B12 supplementation; suggesting the importance of prompt treatment in reversing neurological manifestations.

Vitamin B12 and cognitive decline

Elderly individuals are currently assessed on vitamin B12 status during the screening process for dementia. Studies investigating the association between vitamin B12 concentrations and cognitive status have produced inconclusive results. It has been shown that elevated MMA concentrations are associated with decreased cognitive decline and Alzheimer’s Disease. In addition, low vitamin B12 and folate intakes have shown associations with hyperhomocysteinemia, which is associated with cerebrovascular disease, cognitive decline and an increased risk of dementia in prospective studies.

There are limited intervention studies which have investigated the effect of supplementation of vitamin B12 and cognitive function. A Cochrane review, analysing two studies, found no effect of vitamin B12 supplementation on the cognitive scores of older adults. A recent longitudinal study in elderly individuals, found that individuals had a higher risk of brain volume loss over a 5-year period, if they had lower vitamin B12 and holoTC levels and higher plasma tHcy and MMA levels. More intervention studies are needed to determine the modifiable effects of vitamin B12 supplementation on cognition.

Vitamin B12 and Osteoporosis

There has been growing interest on the effect of low serum vitamin B12 concentrations on bone health. Recent studies have found a connection between elevated plasma tHcy and an increased risk of bone fractures, but is unknown whether this is related to the increased levels of tHcy or to vitamin B12 levels (which are involved in homocysteine metabolism). Results from the third NHANES conducted in the United States, found that individuals had significantly lower bone mass density (BMD) and higher osteoporosis rates with each higher quartile of serum MMA (n= 737 men and 813 women). Given that poor bone mineralization has been found in individuals with pernicious anaemia, and that the content of vitamin B12 within bone cells in culture has shown to affect the functioning of bone forming cells (osteoblasts); it is possible that vitamin B12 deficiency is causally related to poor bone health.

Randomized intervention trials investigating the association of vitamin B12 supplementation and bone health have yielded mixed results. Two studies conducted in osteoporotic risk patients with hyperhomocysteinemia and individuals who had undergone a stroke, found positive effects between supplementation of B vitamins on BMD. However, no improvement in BMD was observed in a group of healthy older people. Further, controlled trials are needed to confirm the impact and mechanisms vitamin B12 deficiency has on bone mineralization.

Causes of B12 deficiency

See Vitamin_B12_deficiency#Causes

The most common reason for vitamin B12 deficiency in spite of eating a diet rich in animal products is poor absorption. It has been long known that vegans, lacto-ovo vegetarians and elderly individuals are at risk of vitamin B12 deficiency. Causes can also relate to having inadequate amounts of IF, gastric atrophy, intestinal disease, gastric surgery, bacterial overgrowth in the small intestine, alcohol consumption, a tapeworm infection, drug-nutrient interactions, as well as some genetic defects [45, 48, 161].

It is well known that strict vegans are at high risk of vitamin B12 deficiency. At present, there are very few studies analysing the association of vitamin B12 deficiency with veganism in large populations. In a group of 131 vegan adults from Germany (aged 20-82 years), individuals who followed a vegan diet for 7.1 years, had a 1.8 increased rate of deficiency compared to those who adhered to a vegan diet for less than 5 years. The study showed that 26% of strict vegans who did not take vitamin B12 supplements, had vitamin B12 deficiency with a cut-off point of 110 pmol/L [162]. Furthermore, in another study looking at 25 vegan adults from California aged 20 to 60 years, showed that 40% of individuals were vitamin B12 deficit; based on either low plasma cobalamin (< 150 pmol/L), macrocytosis, or elevated serum MMA (>376 nmol/L) [163]. Vegans are therefore recommended to take vitamin B12 fortified foods or supplements to meet their recommended daily intake.

Traditionally, vegans were suggested to be the only group at risk of vitamin B12 deficiency, but it is now acknowledged that individuals who consume low animal source foods are also at risk. Lacto-ovo vegetarians and individuals from less-industrialized (where the consumption of meat is rare) have a greater risk of vitamin B12 deficiency compared to individuals who consume an omnivorous diet [48]. Evidence shows that meat contains comparatively more vitamin B12 (1.3 µg/100 kcal cooked meat) than milk (0.6 µg/100 kcal) [48]. In a recent literature review addressing the vitamin B12 deficiency rates amongst vegetarians, it was reported that 32% of young adult vegetarians/ lacto-ovo vegetarians had vitamin B12 deficiency (MMA >271 nmol/L) [164]. As a result, lacto-ovo vegetarians are required to take supplemental vitamin B12 to meet their nutritional needs [50].

Vitamin B12 deficiency is also a common condition among the elderly. Elderly individuals are frequently malnourished, which enhances the risk of vitamin B12 deficiency. Whilst some of these reasons might be the result of underlying ill health, other influences include problems with dentition, depression or anxiety, mobility difficulties (e.g. difficulties with food preparation) and the use of medications which may interfere with appetite or absorption of vitamin B12 [165]. Atrophic gastritis is also a common condition observed in the elderly, which results in the inflammation of the stomach mucous membrane. In atrophic gastritis, there is a reduction or absence in gastric acid secretion which is needed to release vitamin B12 from proteins in food. However, elderly individuals still retain the ability to absorb vitamin B12 in synthetic form (as it is not protein bound), due to sufficient intrinsic factor being secreted [48].

Pernicious anaemia is the final stage of an auto-immune gastritis (Type A atrophic gastritis). In autoimmune gastritis, parietal cells of the corpus and fundus of the stomach are destroyed. These parietal cells are responsible for producing hydrochloric acid and intrinsic factor, which is required for the uptake of vitamin B12 [166]. As there is no therapy at present for auto-immune gastritis, patients are required to take vitamin B12 injections, or large doses of vitamin B12 to prevent the development of megaloblastic anaemia and future neurological complications [50].

Vitamin B12 uptake in the ileum can be reduced by the overgrowth of bacteria or parasites. Intestinal bacteria may have the potential to compete for vitamin B12, convert the vitamin B12 into inactive analogs or impair the absorption of vitamin B12 [48]. At present, Helicobacter pylori infection is one of the most common gastric infections worldwide. H. pylori infection is characterized by gastritis, gastric and duodenal ulcers, achlorhydria and gastric atrophy. Numerous studies have suggested that there may be a causal relationship between H. pylori and food-bound vitamin B12 malabsorption [167]. Furthermore, diseases of the ileum such as Crohn’s Disease, chronic bowel inflammatory disease and gastrointestinal surgery may induce vitamin B12 malabsorption [45].

Vitamin B12 malabsorption is also linked to genetic disorders which regulate the uptake and metabolism of vitamin B12. Vitamin B12 is a cofactor for methionine synthase (MS) and methylmalonyl CoA mutase (MCM). In order to function as a co-factor, the structure of vitamin B12 must be modified [168]. Obstructions in the intracellular processing of vitamin B12 into its co-factor forms; methylcobalamin (MeCbl) for (MS) and adenosylcobalamin (AdoCbl) for MCM or changes in the functional activity of MS or MCM can result in inborn errors of vitamin

B12 utilisation. The genetically inherited blocks can be detrimental for new-borns and children [168]. A number of inborn errors of intracellular vitamin B12 metabolism, designated cblA- cblG, have been determined by biochemical analysis of radioactive metabolites and B12 (complementation analysis). Methylmalonic acidemia (cblA, cblB, cblD variant 2), hyperhomocysteinemia (cblD variant 1, cblE, cblG) or combined methylmalonic acidemia and hyperhomocysteinemia (cblC, classic cblD, cblF) have so far been acknowledged as inborn errors [169]. These disorders and the genes involved in intracellular B12 metabolism are listed in Table 4. Further to this, vitamin B12 levels in the general population are underpinned by molecular mechanisms which are responsible for the absorption, distribution, metabolism and elimination of vitamin B12 [170]. The genetics of vitamin B12 status and genetic variation in different ethnicities within individuals without inborn errors of metabolism will be discussed in detail in chapter 2.

Table 4: Inborn Errors of Cobalamin Transport and Metabolism

Disorder	Gene	Location	Phenotype (Inborn errors)	Function
Intrinsic factor deficiency	GIF	11q13	Intrinsic factor deficiency	Encodes a glycoprotein secreted by parietal cells of the gastric mucosa. The gene encodes the protein that is required for adequate absorption of vitamin B12.
Imerslund– Gräsbeck syndrome (Megaloblastic anaemia 1)	AMN	14q32	Intestinal absorption of dietary cobalamin is impaired (Partial loss of IF binding affinity to cobalamin or the cubam receptor complex)	Involved in the transfer of the cubilin-vitamin B12 complex into the intestinal cell
	CUBN	10p12.1		It encodes the intestinal receptor Cubilin, which is expressed in the renal proximal tubule and intestinal mucosa. Cubilin recognizes the vitaminB12-intrinsic factor complex, and binds to another protein called Amnionless to facilitate the entry of vitamin B12 into the intestinal cells
Transcobalamin deficiency (Transcobalamin II deficiency)	TCN2	22q11.2	Decreased intestinal absorption of B12, uncorrected by intrinsic factor.	It encodes a transport protein called transcobalamin 2 (TC), which binds to vitamin B12 within the enterocyte. The TC-B12 complex enters the portal circulation and makes vitamin B12 available for cellular uptake in target tissues
Haptocorrin deficiency (Transcobalamin I deficiency)	TCN1	11q11–q12	Affects multiple specific granule proteins, and results in low serum B12 levels	It encodes a glycoprotein called Transcobalamin 1, also known as haptocorrin (HC), which binds to vitamin B12. It shields dietary vitamin B12 from the acidic environment of the stomach.
Transcobalamin receptor deficiency	CD320	19p13.2	Loss of a glutamate residue in the extracellular	It encodes the membrane receptor transcobalamin receptor (TCblR), which binds

			domain of the receptor. Decreased receptor‐mediated uptake of TC‐B12 in vitro.	to the transcobalamin-vitamin B12 complex, and mediates the uptake of vitamin B12 into cells
cblA	MMAA	4q31.1–q31.2	Adenosyl- vitaminB12 deficiency in cells	MMAA encodes a protein that may be involved in the translocation of vitamin B12 into the mitochondria. In addition, MMAA could play an important role in the protection and reactivation of Methylmalonyl-coA mutase (MCM) in vitro.
cblB	MMAB	12q24	Adenosyl- vitaminB12 deficiency in cells	Adenosylates cobalamin in an ATP-dependent manner
cblC	MMACHC	1p23.2	The inability to convert cynano- vitaminB12 into biological forms	The MMACHC gene encodes a chaperone protein MMAACHC (cblC protein) which binds to vitamin B12 in the cytoplasm and appears to catalyse the reductive decyanation of cyanocobalamin into cob(II)alamin
cblD	MMADHC	2q23.2	Improper targeting of vitamin B12 to cognate enzymes	This gene leads to the Branching of vitamin B12 within the cell to either the cytosol or the mitochondrion
CblE	MTRR	5p15.3-p15.2	Inactive methionine synthase	This gene is responsible for the reductive methylation of vitamin B12 to generate methylcobalamin from cob(II)alamin
CblF	LMBRD1	6q13	Accumulation of vitamin B12 within lysosome	Potentially helps in the transport of vitamin B12 out of the lysosome
CblG	MTR	1q43	Homocysteine accumulation	Transfers a methyl group from methyltetrahydrofolate to homocysteine to produce methionine

CblJ	ABCD4	14q24.3	Accumulation of cobalamin within lysosome	Transports vitamin B12 from lysosomes to the cytosol
Methylmalonyl CoA mutase deficiency	MUT	6p21	Methylmalonic acid accumulation	The enzyme converts methylmalonylCoA and succinylCoA, reversibly.

Data derived from Table 1 available on https://doi.org/10.1016/bs.afnr.2017.11.005 [1] and from Table 3 available on https://doi.org/10.1186/s12263-018-0591-9

Currently there is concern that the mandatory fortification of folic acid to cereals and grains, may in fact conceal the macrocytic anaemia associated with vitamin B12 deficiency, consequently eliminating an important diagnostic tool [171]. The combination of high folate and low serum vitamin B12 is associated with higher concentrations of methylmalonic acid and homocysteine, contributing to hematologic and neurologic disturbances. The National Health and Nutrition Examination Survey (NHANES) collected on older adults during 1999- 2002 showed that high folate intakes were related to impaired mental functioning and cognitive decline among individuals with a low vitamin B12 status [172]. Considering these findings, there has been interest as to whether vitamin B12 fortification in flour should be implemented. However, as of yet there is not enough data evaluating the bioavailability of the vitamin from fortified flour in specific population groups (such as the elderly with food-bound vitamin B12 malabsorption and others with gastric atrophy) to make a firm decision [121].

Drug-nutrient interactions

There are some drugs which are thought to interfere with the absorption or metabolism of vitamin B12 [50]. These include H2-receptor antagonists, proton pump inhibitors and metformin. Cimetidine is a H2-receptor antagonist which is used to treat peptic ulcers and alleviate heartburn. Cimetidine inhibits the secretion of gastric acid and pepsin and has been reported to inhibit IF secretion [173, 174]. A >1000 mg/day dose may in fact lead to malabsorption of protein-bound vitamin B12 by peptic ulcer patients (n=9 male) and normal subjects (n=4 male) [175], however this malabsorption was shown to be reversible upon discontinuation of cimetidine in another study [174] .

Proton pump inhibitors (PPIs) such as omeprazole and lansoprazole are widely prescribed to treat gastroesophageal reflux disease. It has been suggested that prolonged use of PPIs may influence vitamin B12 status, by inhibiting gastric acid secretion. The effect of omeprazole on vitamin B12 absorption is dose-related, with intakes of 20 mg/day reducing food-bound vitamin B12 absorption by 70%, whilst 40 mg/day reducing absorption by 90% [176]. At present the current literature on the association between PPI usage and vitamin B12 status is mainly based on case-reports or retrospective observational studies, which have produced relatively inconsistent findings [177].

Metformin therapy is used as the first line of therapy for individuals with type 2 diabetes mellitus. Studies have shown that Metformin induces vitamin B12 malabsorption and impaired intrinsic factor secretion in the ileum [178-181]. The mechanism of metformin-related vitamin B12 deficiency is still under debate. Metformin delays glucose absorption in the upper small intestine affecting the motility of the small bowel, which stimulates bacterial overgrowth and consequential vitamin B12 deficiency [178, 179]. Metformin has also been shown to enhance competitive inhibition or inactivation of vitamin B12 absorption, leading to alterations in intrinsic factor (IF) levels and interactions with the cubulin endocytic receptor. Additionally, Metformin inhibits the calcium dependent absorption of the vitamin B12-IF complex at the terminal ileum [181], as a result increasing calcium intake may improve the uptake of vitamin B12 in metformin users [180].

Assessment of Vitamin B12 status

Traditionally, measuring serum cobalamin remains the preferred choice for determining vitamin B12 deficiency. However, using serum vitamin B12 concentrations alone does not confirm the uncertainties of underlying functional and biochemical deficiencies. Nowadays other methods such as measuring plasma methylmalonic acid, serum holotranscobalamin and plasma homocysteine are also used [23].

Vitamin B12 and Holotranscobalamin

The measurement of serum vitamin B12 levels is the most widely used assay to screen vitamin B12 deficiency. However this method is rarely used alone, as it is known to have a poor sensitivity and specificity in detecting vitamin B12 deficiency [182]. Serum B12 assays measures both serum holohaptocorrin (HoloHC) and serum holotranscobalamin (holoTC). HoloHC, represent 70-90% of vitamin B12, but is biologically inert as no cellular receptors exist, except on the liver. On the other hand, HoloTC contains biologically active vitamin B12, which can be taken up by cells, and represents 10-30% of circulating vitamin B12 [183]. Given that the majority of vitamin B12 is bound to HC, results would mask the true deficiency or would falsely infer vitamin deficiency [23]. Vitamin B12 is usually measured using an automated method and a competitive-binding immune chemiluminescence, a low cost test [23]. Depending on the technique used to measure vitamin B12, vitamin B12 deficiency is usually considered when the plasma vitamin B12 concentration is less than 200 pg/mL. However, there is no gold standard value to represent subclinical deficiency of vitamin B12 (Table 5) [184].

Currently, the TC bound to plasma vitamin B12, is more relevant for assessing the functional vitamin B12 status. HoloTC reflects the absorptive capacity of vitamin B12, and any deficiency of TC has been previously associated with neurological and haematological complications [182]. HoloTC is usually measured by immunoassay, and cut-off values for low HoloTC depend on the specific laboratory guidelines [23].

Table 5: Biomarkers of vitamin B12 status

Biomarker (Unit)	Assay Type	Tentative reference intervala*	Tentative cut-off value for vitamin B12 deficiencyb*	Tentative cut- off value for repletion of vitamin B12*
Plasma B12 (pmol/L)	Competitive-binding immune chemiluminescence method/ protein binding assay	200-600	<148	>221
Holotranscobalamin (pmol/L)	Immunological	40-100	<35	>40
Homocysteine (μmol/l)	Immunological, Liquid chromatography– mass spectrometry or gas chromatography mass spectrometry	8-15	>15	<8
Methylmalonic acid (μmol/l)	Liquid chromatography– mass spectrometry or gas chromatography mass spectrometry	0.04-0.37	>0.37	<0.27

^aThe Tentative reference intervals cover approximately 95% of B12 replete individuals. ^bThe tentative cut-off value for vitamin B12 deficiency includes both clinical and subclinical deficiency.*The values indicated in this table are based on previously cited literature. Data derived from Table 1 available on https://doi.org/10.1038/nrdp.2017.40 [2]

Homocysteine and Methylmalonic acid

Homocysteine (Hcy) and methylmalonic acid (MMA) can be used as sensitive biomarkers to detect an underlying vitamin B12 deficiency, even when no apparent sign of clinical vitamin B12 deficiency or low serum vitamin B12 levels are present [182]. There are two B12- dependent enzymatic reactions which use MMA and Hcy as substrates. Vitamin B12 in combination with folic acid is required to convert Hcy to methionine, and vitamin B12 is used to convert MMA to succinyl-CoA [185]. As a result, MMA is a more sensitive indicator of vitamin B12 deficiency compared to Hcy. These two biomarkers can be confounded by both environmental and physiological conditions [161]. Renal failure, heart transplantation, thyroid dysfunction, certain medications, genetic variation in the methylenetetrahydrofolate reductase (MTHFR) gene and high folate and vitamin B6 deficiency can contribute to elevated Hcy concentrations. Furthermore, MMA is elevated in renal impairment and rare inborn errors affecting methylmalonate-CoA mutase activity [50, 186].

Elevated Hcy and MMA concentrations, have been found to be 99.8% sensitive for diagnosing vitamin B12 deficiency [187]. Both Hcy and MMA are usually measured using Liquid chromatography– mass spectrometry or gas chromatography mass spectrometry [188]. According to Carmel (2006), an inadequate vitamin B12 status is described as serum vitamin B12 < 148 pmol/L, or 148–258 pmol/L and MMA > 0.30μmol/L, or tHcy > 13 nmol/L (females) and >15 nmol/L (males) [189]. However, it should be noted that the reference range depends on the individual techniques used to measure Hcy and MMA; as the published estimates for the specificity and sensitivity for diagnosing vitamin B12 deficiency varies extensively (Table 5)[184].

Treatment of vitamin B12 deficiency

Parenteral treatment

In the United Kingdom and other western countries worldwide, most patients with vitamin B12 deficiency are given intramuscular injections of vitamin B12. Intramuscular vitamin B12 exists in two forms: cyanocobalamin or hydroxocobalamin. Hydroxocobalamin is generally used as the first line of treatment, as it is retained in the body for a longer period of time and it can be administered at intervals of up to three months [190]. Approximately 10% (100 µg) of injected hydroxocobalamin is retained in the body after administration of 1000 µg [22].

The standard treatment for patients without neurological symptoms is three injections of intramuscular hydroxocobalamin (1000 µg) three times a week, for a duration of two weeks.

On the other hand, for patients with neurological involvement, injections are given intramuscularly (1000 µg) on alternative days for three weeks or until clear improvement is shown. Individuals with pernicious anaemia are given lifelong treatment. Individuals with severe anaemia and cardiac symptoms are usually treated with transfusion and diuretic agents [22, 23, 191].

Hydroxocobalamin is usually well-tolerated, with serious adverse reactions being rare. However, injections can cause significant amount of pain in thin patients and can be dangerous in anticoagulated patients [190]. Side effects which are rarely observed for hydroxocobalamin include: chills, fever, hot flushes, itching, nausea, dizziness, skin rash and anaphylaxis [23].

Oral treatment

When vitamin B12 deficiency is related to an individual’s diet, a dose of 50-150 µg cyanocobalamin is given between meals [23]. Oral therapy is considered during mild or subclinical vitamin B12 deficiency and when there are no concerns of compliance or abnormalities associated with absorption [192].

Previous case control studies have suggested that the oral administration of vitamin B12, is equally safe and effective at eliminating vitamin B12 deficiency [193, 194]. Vitamin B12 taken in the oral route, can be absorbed both actively and passively. In passive absorption, the vitamin B12 is absorbed without binding to IF. Approximately 0.5-4% of radioactively labelled oral vitamin B12 can be absorbed by passive diffusion in both healthy and patients with pernacious anemia [190, 195]. On the other hand, in active absorption, vitamin B12 binds to IF in the terminal ileum [190].

It has been noted that patients with IF deficiency can still adequately absorb vitamin B12, provided that they are given high doses of vitamin B12 (1000 µg daily) [190]. A cochrane review of two randomised controlled trials comparing oral with intramuscular administration in 108 participants, found that high oral doses (1000 µg and 2000 µg daily) are as effective as intramuscular injections in responding to neurological and haemotological symptoms [190]. However, due to the limited data available to present the support of oral therapy in individuals with neurological dysfunction, parenteral vitamin B12 is still the prefered method of treatment.

At present, oral vitamin B12 is widely prescribed in Canada and Sweden [190]. However, high doses of oral vitamin B12 are unavailable for prescription under the NHS in the United Kingdom. Given that intramuscular injections require patients to visit a health facility or have a health care visitor to administer an injection, using oral vitamin B12 instead, could potentially save NHS resources and the time of medical staff [190].

Vitamin B12 Toxicity

High serum vitamin B12 is defined as a value above 950 pg/ml; this refers to the upper limit of biological normality [196]. At present, few studies have looked at the toxic effects associated with a high serum vitamin B12 concentration. One study observed that when vitamin B12 was administered at 2 mg (2,000 μg) daily by mouth or 1 mg monthly by intramuscular (IM) injection to treat pernicious anaemia, no toxic effects were identified [197]. It is known that only a certain percentage of vitamin B12 can be absorbed by the body, and any ingested amounts which exceed the absorption capacity of vitamin B12 intrinsic factor receptors are excreted through the urine or faeces. This could partly explain the low toxicity [38]. On the other hand, other studies have noted that high doses of vitamin B12 supplements were associated with a greater risk of CVD in individuals with diabetic nephropathy [198] and a greater risk of autism spectrum disorder in the offspring of pregnant women [199].

It is possible that an increase in plasma vitamin B12 could be a result of a functional deficit. The destruction of hepatocytes in chronic hepatitis, can stimulate the binding of vitamin B12 to haptocorrin (HC) in the plasma, to form holohatocorrin (holoHC- inactive form of vitamin B12) leading to a decline of vitamin B12 attaching to holotranscobalamin (holoTC) II (active form of vitamin B12). As a result, there is an increase in vitamin B12 in the plasma, as vitamin B12 cannot be delivered to the cells [200]. Furthermore, elevated vitamin B12 concentrations could be due to the leakage of vitamin B12 from damaged liver tissue into the plasma [196]. As a result, high vitamin B12 levels are not always beneficial and could underlie a number of underlying pathologies [196].

Nutrigenetics approach

Nutrigenetics is a branch of science that investigates the effect of genetic variants in response to dietary manipulation. The ultimate goal of nutrigenetics is to investigate the molecular and physiological basis of genetic variants associated with health and disease, and how these genotype-phenotype associations can be modified by dietary intake [201]. The field of nutrigenetics is rapidly evolving, with the hope that one day in the future nutritionists will be able to provide personalised dietary recommendations to patients to delay or prevent the onset of disease [202].

Genetic factors and ethnic variation

Vitamin B12 absorption and metabolism involves complex biological pathways containing multiple steps. Genetic variants may alter vitamin B12 tissue status by affecting the proteins involved in vitamin B12 absorption, cellular uptake and intracellular metabolism [143]. In a study using monozygotic and dizygotic twins, the heritability of B12 levels was estimated to be 59%, indicating that the magnitude of genetic influence on vitamin B12 levels are considerable [203]. At present, genetic studies of vitamin B12 status suggest that it is a multifactorial trait (also called complex trait), where several single nucleotide polymorphisms (SNPs) in multiple genes interact with the environment to cause the altered B12 status [204]. The genetics of vitamin B12 status and the genetic variation in different ethnicities are discussed in detail in chapter 2.

A review article from Surendran et al., (2018) which is found in chapter 2 of this thesis, identified 59 vitamin B12-related gene polymorphisms associated with vitamin B12 status, from the following populations: African American, Brazilian, Canadian, Chinese, Danish, English, European ancestry, Icelandic, Indian, Italian, Latino, Northern Irish, Portuguese and residents of the USA [14]. The most compelling evidence has been accumulated for the fucosyltransferase 2 (FUT2) SNP (rs602662), for which homozygosity of the minor G allele has been associated with lower vitamin B12 status. Variants in other B12 metabolic genes, including methylmalonyl CoA mutase (MUT), cubulin (CUBN) and transcobalamin-I (TCN1) have been reported in European populations [205]. Furthermore, an additional four loci, membrane-spanning 4-domains (MS4A3), citrate lyase beta like (CLYBL), fucosyltransferase 6 (FUT6) and 5q32 were constricted to the Chinese population [206]. It has been suggested that ethnic-specific associations are involved in the genetic determination of vitamin B12 concentrations. However, despite recent success in genetic studies, most of the identified genes that could explain variation in vitamin B12 concentrations were from Caucasian populations. As a result, further research utilizing larger sample sizes of non-Caucasian populations is necessary in order to better understand these ethnic-specific associations [14].

Genes alone are not responsible for explaining the variation in vitamin B12 concentrations, as lifestyle factors e.g. dietary factors, also influence vitamin B12 concentrations. Therefore, this is investigated by identifying gene-diet interactions (Nutrigenetics). In my thesis, I aimed to investigate the interaction between dietary factors (modifiable factor) and genetic markers (non-modifiable factors) on vitamin B12 concentrations and metabolic disease trait outcomes.

Rational for studying gene-diet interactions

Many SNPs have been shown to be associated with vitamin B12 status and these SNPs only represent a fraction of the heritability of vitamin B12 status [14]. It is well known that environmental factors, such as diet, can modulate the effects of genes on metabolic traits [12]. However, it is unknown whether dietary factors can interact with genes to impact vitamin B12 status; hence the interaction between genetic and dietary factors must be considered. Findings from gene-diet interactions will contribute to identifying the interactions of genes and diet in the development of vitamin B12 deficiency. Therefore, this knowledge is essential for the primary prevention of vitamin B12 deficiency, and for developing effective dietary strategies for the prevention of vitamin B12 deficiency and its related metabolic outcomes.

Importance of studying gene-diet interactions in different genetic groups

It has been established that genetic studies looking at vitamin B12 status in healthy adults, especially large-scale ones, have been unable to capture the level of diversity which exists worldwide, as they are mainly based on individuals of European ancestry [14]. The under-representation of diverse ethnic groups hampers our full understanding of the genetic architecture of vitamin B12 levels [207]. Furthermore, the limited genetic data on non- Caucasian populations in relation to genetic susceptibility to vitamin B12 deficiency, can also impede our ability to translate genetic research into clinical care, and will exacerbate health inequalities across the current public health policy [207]. Given that vitamin B12 status can also be determined by environmental factors, it is also important to explore gene-diet interactions in different ethnic groups, so that it will be eventually possible to personalise diet according to each ethnic sub-group. It is important to note that, different ethnic groups respond differently to specific dietary interventions [12]. Therefore, using estimates of genetic risk for vitamin B12 deficiency from European-based studies in non-Europeans may result in an inaccurate assessment of risk of vitamin B12 deficiency and could result in an inappropriate environmental intervention (dietary or physical activity) in under-studied populations.

Study designs and their role in identifying gene-diet interactions

Multiple lines of evidence suggest that SNPs may modify gene expression and consequently influence metabolic disease outcomes. Besides, it is well known that interactions may exist between genes and dietary factors to influence metabolic outcomes [12]. Several genes are involved in vitamin B12 metabolism [14] and variants in these genes may modify cardio-metabolic disease outcomes [16, 17]. Beyond the independent gene effects, no studies have evaluated interactions between vitamin B12 gene polymorphisms and macronutrient intake on cardiometabolic disease outcomes. A more detailed understanding of gene- diet interactions is needed to generate information required to develop strategies for diet modification to reduce the incidence of cardiometabolic disease related traits in individuals with specific variants related to vitamin B12 absorption and metabolism. The following section describes the potential study designs which can be employed for gene-diet interactions.

The most commonly used study design is the cross-sectional design. A cross-sectional design is a study design, where disease related-outcomes and exposures in study participants are measured at a single point in time [208]. One of the limitations of a cross-sectional study design is that it is a one-time measurement of exposure and outcome, thus it is difficult to derive causal relationships between risk factors and a disease. Another limitation is that these studies are prone to confounding. Thus, it is important, that confounding factors are adjusted during statistical analysis (within the regression model) [208].

A case-control study determines whether an exposure is associated with an outcome of interest (e.g. disease). In simple terms, a case-control design, is a study which compares a group of individuals who have a disease or an outcome of interest (cases), with patients who are free

from a disease/outcome at a given point of time [209]. The study design is very similar to a cross-sectional study design, and both studies share many strengths. The main strengths of observational studies are that they can be used to generate a hypothesis, or they may be the only study design which is feasible or ethically viable to be carried out. Furthermore, observational studies are quick, easy and relatively inexpensive. They have the potential for large numbers of samples to be collected [210]. Under a nutrigenetics perspective, observational studies (cross sectional and case control) can impose substantial limitations. Firstly, phenotypes can vary across different time periods. For example, when testing gene-diet interactions, TAG concentrations vary upon the time of collection [211], thus only collecting fasting blood samples may be a limitation. Further to this, observational studies lack replication, making it difficult to conclude whether the findings are due to chance. Observational studies rely on FFQs, which are self-reported by participants and this can introduce bias. Ultimately, cross-sectional studies are beneficial as they are able to identify genetic variants which may be associated with diseases, and these variants are less likely to be affected by confounding variables [212].

The next study design is a randomized clinical trial (RCT). RCTs are part of an experimental study design, where volunteers are randomly assigned to receive an experimental treatment (intervention) or a control treatment (where they receive the current standard treatment: this could be no treatment, a placebo or the best existing treatment currently available) [213]. As a result, any observed changes in the outcome e.g. vitamin B12 levels, is a result of the intervention treatment. The main advantages of an experimental trial are that both participants and trialists are unaware of whether the participant is receiving the treatment or control diet, until the study is completed. Although randomised control trials are powerful tools, studies are often limited by the sample size. It is difficult to have large sample sizes as it is not cost effective, furthermore participants may drop out or have poor compliance with the treatment [214].

Alternatively, another type of experimental study is the ‘cross over’ study design. In a cross over study design, half of the study samples are randomly assigned to a control diet for a certain period of time, they then undergo a wash out period, and they then switch to the experimental dietary intervention. The other half of the study sample, start off the experimental dietary intervention, undergo a wash out period, and then switch to the control diet [215]. In this type of study, the groups exchange their respective arms at a specific point of the experiment. The advantages of following cross-over studies are that they verify the findings of the first phase of the study, by reproducing it in the second phase, consequently reinforcing the conclusion of the study. Furthermore, intervention studies minimize the effect of confounding factors. However, one of the main concerns of dietary intervention studies is the need for a washout period between studies and that the trials often have a small sample size, which may reduce the power of detecting gene-diet interaction effect sizes [212, 216].

The postprandial study design (sequential meal design) is a type of experimental design. In this protocol two test meals are given to participants at different time intervals. The purpose of the following test design is to determine how chronic dietary fat or cereal/non-digestible carbohydrate supplements manipulate the lipaemic response. Secondly, this test design is used to determine the acute impact of specific fatty acids on the first meal on the postprandial lipaemic response of the second meal. As a result, looking at the changes in biochemical variables during the postprandial state highlights the importance of using the postprandial design in investigating gene-diet interactions [217]. An overview of the different study designs employed in gene-diet interactions is shown in Table 6.

Table 6: Types of studies used to perform gene-diet interactions

Type of Study design	Overview	Strengths	Disadvantages
Cross- sectional	Disease related-outcomes and exposures in study participants are measured at a single point in time.	Can be used to generate a hypothesis. Quick and Easy Relatively inexpensive. Potential for a large sample to be collected Provides estimates of prevalence of all factors measured	It is not possible to say whether the exposure or the outcome is the cause, and which is the effect. Results are prone to confounding, so it important that confounding factors are adjusted during statistical analysis (within the regression model) Phenotypes can vary across different time periods, thus only collecting fasting blood samples may be a limitation. Using an FFQ, measures the current diet in a group of individuals. The current diet may be altered by the presence of a disease. The reliance of FFQs, which are self-reported by participants, can introduce Bias.
Case- control	A study which compares a group of individuals who have a disease or an outcome of interest (cases), with patients who are free from a disease/outcome at a given point of time	Can be used to generate a hypothesis. Can study several exposure factors simultaneously Quick Easy	Phenotypes can vary across different time periods, thus only collecting fasting blood samples may be a limitation. Selection bias Not useful for rare exposures

		Relatively inexpensive. Potential for a large sample to be collected	The reliance of food frequency questionnaires, which are self-reported by participants, can introduce Bias. Incidence rate cannot be computed
Randomize d clinical trials	Volunteers are randomly assigned to receive an experimental treatment (intervention) or a control treatment	In blinded study designs, both participants and trialists are unaware of whether the participant is receiving the treatment or control diet, until the study is completed. However, often in nutrition studies it is difficult to blind interventions. Ability to detect causal relationships	Studies are usually limited by sample size. It is not cost effective to have a large sample size. Participants may have poor compliance with the treatment.
Cross-over	Half of the study samples are randomly assigned to a control diet for a certain period of time, they then undergo a wash out period, and they then switch to the experimental dietary intervention. The other half of the study sample, start off the experimental dietary intervention, undergo a wash out period, and then switch to the control die	Verification of the findings of the first phase of the study can be conducted, by reproducing it in the second phase, consequently reinforcing the conclusion of the study. This study has minimal effect from confounding factors	The small sample size, which may reduce the power for detecting gene-diet interaction effect sizes.
Postprandia l	In this protocol two test meals are given to participants at different time intervals. The purpose of the following test design is to determine how chronic dietary fat or cereal/non-	The frequency of blood sampling, with on average 10–13 blood samples taken during each postprandial assessment	Determination of the postprandial response is complex Lack of standardisation of methodologies, test meal size and composition, between different studies and research groups

digestible carbohydrate supplements manipulate the lipaemic response. Secondly, this test design is used to determine the acute impact of specific fatty acids on the first meal on the postprandial lipaemic response of the second meal.

Small subject numbers

From Nutrigenetics to Personalised nutrition

It is becoming increasingly evident that genes and nutrients interact and influence an individual’s risk of developing metabolic disease related traits [11]. Approximately over 1000 genes have been shown to be associated with human diseases, [218]; however, many of these genes will not increase the risk of developing a disease without exposure to certain dietary compounds [219]. Given that 80% of chronic diseases can be prevented by lifestyle and dietary modifications [220], it is important that dietary prevention strategies and dietary guidelines are revised. It is now possible to individualise diets using dietary, phenotype and genotypic data [221]. Greater attention is now being placed in switching dietary interventions from being population-based to being ‘personalised’ according to an individual’s genotype. The concept of personalised nutrition is continually changing as research is developing in the field. Grimaldi et al, describes it as an approach that ‘uses information on individual characteristics to develop targeted nutritional advice, products, or services’ [222]. The importance of personalised nutrition was shown in a retrospective study, which found that participants who were truly matched to a diet based on their genotype, had a twofold to threefold greater reduction in body weight during a 12-month period, compared to individuals falsely matched to a diet [223].

At present, personalised nutrition is in its infancy. The success of personalised dietary advice relies on its ability to drive dietary change and attract consumer interest [221]. Although nutrient-gene interactions are a promising field of research, the molecular and pathophysiological mechanisms underlying these interactions is unclear. It is important that functional studies are carried out to clarify the biological significance and potential clinical applications of gene-diet interactions [12]. Furthermore, it has been shown that gut microbiota could interact with gene-diet interactions, to modify the risk of developing metabolic diseases [224]. As a result, future studies should profile individuals for metabolites, so that personalised dietary advice can be based on an individual’s metabotype [12]. It is essential that before personalised nutrition is introduced; larger, well-powered studies should be conducted in a range of ethnic groups. Furthermore, other modifiable factors (e.g. physical activity), which could interact with genetic factors should be taken into account.

Conclusions

The findings from these studies indicate that diet modifications, which attempts to optimize vitamin B12 concentrations and other lipid traits, must consider genetic factors. Gene- diet interaction studies are important for clarifying the relationship between nutrients, genetic variants and vitamin B12 status. Although nutrigenetics research is developing and garnering public health interest, consistent challenges have emerged surrounding the nature of nutrigenetics research. Several unregulated websites offering tests and dietary advice are available, with limited scientific evidence [225]. It is believed that there are no defined standards of how to conduct nutrigenetics studies. Additionally, the majority of nutrigenetics studies, have been published as secondary analyses to studies, the purpose of which was not to study gene-diet interactions [226]. Future studies will require an appropriate study design and a well-powered sample size. Furthermore, certain genetic variants may contribute to interindividual variability during postprandial states [211]; therefore, gene-diet interactions studies must examine both fasting and postprandial states.

In summary, there is a need to increase the number of nutrigenetics studies to establish the link between SNPs, dietary factors and health outcomes. It is also important to identify how gene-diet interactions influence vitamin B12 metabolic and lipid metabolism pathways at the molecular level, in order to determine the mechanism of action. Once this has been determined and validated in various ethnic groups, personalised dietary advice can be enforced to prevent diet-related diseases.

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