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Chemistry: Cobalamin and Folate - Comment

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1. ATMS B12/Folate PART 2 – CHEMISTRY: COBALAMIN & FOLATE
2. • Vitamin B12 is composed of * Tetrapyrrole ring surrounding Cobalt atom. * 5,6-dimethyl-benzimidazole. * R group. The R group may be either: * Cyanide in Cyanocobalamin (common in supplements, not a physiological form, more stable) * Methyl in methyl cobalamin * 5’- deoxyadenosine in adenosylcobalamin * OH in hydroxocobalamin.(natural form, produced by bacteria, used in supplements and injections) Attached to Cobalt 2
3. Chemistry B12 is the most chemically complex of all vitamins. * Chemical name cobalamin is derived from its central cobalt atom (which is positively charged) (Vitamin B12: Cobalamin 2013) * It’s impressive formula C63H88N14O14PCo reflects the intricate molecular structure (Types of Vitamin B12 2018) * Vitamin B12 is the only metal-containing vitamin. It is also watersoluble and stored in the liver (Vitamin B12: Cobalamin 2013) * For anaerobic bacteria to synthesize B12, cobalt must be in the soil. 3
4. Chemistry B12 is the collective term for a group of cobalt-containing compounds known as corrinoids, which when assembled with 5th and 6th position ligands are known as cobalamins (Krautler 2012) (Vitamin B12: Cobalamin 2013). * The principal cobalamins are: * cyanocobalamin & hydroxocobalamin (Types of Vitamin B12 2018). * The two co-enzyme forms are: * methylcobalamin & 5-deoxyadenosylcobalamin (adenosylcobalamin) (Types of Vitamin B12 2018). 4
5. Chemistry The cobalamins are the only known substances to naturally contain cobalt * Vitamin B12 is often thus treated as synonymous with cobalamin. * It is an odourless, intensely dark-red coloured, crystallized, heat and light sensitive substance (Vitamin B12: Cobalamin 2013). * Cobalamin is almost never found in its chemically pure form as it is usually bound to other molecules. 5
6. Epidemiology of B12 Deficiency Overall prevalence of B12 deficiency is unknown. * Framingham Offspring Study found up to 39% of US adults were at risk of B12 deficiency (serum B12 100 historical documents and 678 surviving letters, that Mrs Lincoln suffered PA in its worst manifestations (Scientist, 2018). * Lincoln died in 1882 at age 63. “In the 19th century there was no treatment, so the disease progressed to its fatal endpoint. * A complete description and cure of pernicious anaemia were discovered in 1926 (Scientist, 2018). 17
18. Marie Curie (D. 1934) 18
19. The Cobalamin Mystery 1920 (approx.) whilst experimentally inducing anaemia in dogs, Whipple discovered that ingesting large amounts of raw liver resolved the condition (Sinclair, 2008). 1926, Minot & Murphy trialled raw liver as tx for pernicious anaemia in 45 adults (120–240 g of liver and 120g of muscle meat/day). * They observed rapid symptomatic improvement and increased red cell count (RCC). Within 4-10 days the formation of new young RBC’s (reticulocyte count) had increased from 1% to approx. 8%, jaundice lessened (less RBC destruction), and Hb concentration and RCC increased (Sinclair, 2008). Minot & Murphy published their results in the Journal of the American Medical Association in 1926 (Sinclair, 2008). * Eating raw liver or drinking liver juice became tx for the previously fatal condition. Whipple, Minot, and Murphy shared the 1934 Nobel Prize in Physiology or Medicine. 19
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21. Raw Liver Smoothie… mmmmm Ingredients 1/4 cup yogurt/kefir/milk Small handful berries 1/2 banana 1 Tbsp frozen raw liver 2 raw egg yolks 1 tsp lemon/lime juice 1 tsp cinnamon Pinch of sea salt Honey/maple syrup Method 1. Blend all ingredients (except egg yolks) until smooth 2. Add egg yolks and pulse to incorporate 3. Enjoy….!!! 21
22. The Cobalamin Mystery * 1928, chemist Edwin Cohn prepared a liver extract 50-100 times more potent than the natural liver products. The extract was the first workable treatment for the disease. * These events led to discovery of the soluble vitamin B12, from bacterial broths. * 1947 - Shorb & Folkers were given a US$400 grant to develop the so-called "LLD (Lactobacillus lactis) assay" for B12. Dorner, a bacterial strain requiring "LLD factor" for growth was eventually identified as B12 (Scott & Molloy 2012). * Shorb used the LLD assay to rapidly extract the anti-pernicious anaemia factor from liver extracts, and pure B12 was isolated by 1948. * 1956, chemical structure of the molecule was determined by Dorothy Crowfoot Hodgkin (Scott & Molloy 2012). * 1950’s, methods of producing the vitamin in large quantities from bacteria cultures were developed, leading to the modern treatment form. * 1980’s, MIT and Harvard researchers discovered the final piece of the synthesis pathway of B12, the only vitamin synthesised exclusively by microorganisms. 22
23. The Cobalamin Mystery * The biosynthesis of this essential nutrient is intricate, involved and confined to certain members of the prokaryotic world, seemingly never having made the eukaryotic transition (Martens, Barg, Warren, Jah, 2002) * Prokaryote = a unicellular organism that lacks a membrane-bound nucleus, mitochondria, or any other membrane-bound organelle. * Humans require only trace amounts of B12 (approx. 1µg/day), to assist the functions of only two enzymes, methionine synthase and (R)- methylmalonyl-CoA mutase (Martens, Barg, Warren, Jah, 2002). 23
24. Vitamin B12 is produced by soil microbes that live in symbiotic relationships with plant roots (MIT News, 2018). 24
25. 1956 – Dorothy Hodgkin 25
26. “ The Mount Everest of Biosynthetic Problems'. * B12 is pieced together as an elaborate molecular jigsaw involving around 30 individual components. * Unique amongst the vitamins as it is exclusively made by bacteria. * In the early 1990's it was discovered that two pathways exist in nature for the de novo biosynthesis of adenosylcobalamin; the coenzyme form of vitamin B12, to allow its construction - one requiring oxygen and the other not (Moore et al., 2013). 26
27. Anaerobic Pathway The anaerobic (more common) pathway has yet to be fully characterised, due to the instability of its oxygen-sensitive intermediates which are very unstable and rapidly degrade (Moore & Warren 2012) * Bioscientists at the University of Kent have trained a friendly bacterium; Bacillus megaterium to produce all of the components of the anaerobic B12 pathway (Moore & Warren 2012). * This can be used to help persuade bacteria to make the vitamin in larger quantities, thereby contributing to its use in supplements, livestock feed and health and medical research. 27
28. Moore, Simon J., Andrew D. Lawrence, Rebekka Biedendieck, Evelyne Deery, Stefanie Frank, Mark J. Howard, Stephen E. J. Rigby, and Martin J. Warren. “Elucidation of the Anaerobic Pathway for the Corrin Component of Cobalamin (Vitamin B12).” Proceedings of the National Academy of Sciences 110, no. 37 (September 10, 2013): 14906–11. https://doi.org/10.1073/pnas.1308098110. 28
29. Cobalamin - Dietary Sources & Requirements To confirm: Cobalamin is synthesised solely by microorganisms (Linus Pauling Institute, 2014). * Ruminants obtain cobalamin from the foregut, whereas the only source for humans is food of animal origin, (meat, fish, and dairy products). * Vegetables, fruits, and other foods of non-animal origin are free from cobalamin unless contaminated with bacteria. 29
30. Cobalamin - Dietary Sources & Requirements - Summary A normal Western diet contains 5–30μg of cobalamin daily. Adult daily requirements are only approx. 2-2.8μg (Linus Pauling Institute, 2014). Adult daily losses (excreted via urine and faeces) are 1–3μg (~0.1% of body stores). Body stores are approx.2–3mg, sufficient for 3–4 years if supplies are completely cut off. 30
31. Natural Sources of B12 * Eat animal products, particularly liver & meat * Eat insects/worms * Eat vegetables with soil still attached Or more adventurously….. * Eat contents of ruminant stomachs and intestines * Eat fermented and part-digested moss from reindeer gut (Inuits evenused to eat the feaces) * Eat molluscs * Eat poop 31
32. Rabbit Food! * Faeces are a rich source of vitamin B12 and many species, including dogs, cats, and rabbits eat faeces. * Species within the Lagomorpha (rabbits, hares, and pikas) produce two types of feacal pellets: hard ones, and soft ones called cecotropes. * Animals in these species re-ingest their own cecotropes, which consist of chewed plant material that was metabolised by bacteria in the cecum, a chamber between the small and large intestines. * Cecotropes contain digestible carbohydrates and B vitamins synthesised by the resident bacteria (Intestinal Bacteria as a Vitamin B12 Source 2018). 32
33. Insects, a Potential B12 Food Source * Insects containing B12 include:House crickets (Acheta domesticus) (5.4μg/100g in adults and 8.7μg/100g in nymphs)(Anankware, Fening, Osekre, Obeng-Ofori, 2015). * Mealworm larvae (Tenebrio molitor) 0.47 μg per 100 g (Anankware, Fening, Osekre, Obeng-Ofori, 2015). More research is needed to identify edible insects rich in B vitamins, and the breakdown and utilisation from human consumption of insects. 33
34. Microbial corrinoid metabolism in the gut * In ruminants, gut microbes provide a direct source of cobalamin (direct effect)(Degnan, Taga, Goodman 2014). * In insects, corrinoids are essential cofactors for obligate symbionts that provide key nutrients to the host (indirect effect) (Degnan, Taga, Goodman 2014). * Competition and exchange of corrinoids likely shape gut microbiota composition and expressed functions in humans and other animals (microbiome remodeling) (Degnan, Taga, Goodman 2014). 34
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36. Cobalt Deficiency in Stock * Presence of cobalt in soils markedly improves the health of grazing animals (Huwait, Kumosani, Moselhy, Mosaoa & Yaghmoor 2015). An uptake of 0.20 mg/kg a day is recommended because they have no other source of vitamin B12. * In the early 20th century during the development of farming on the North Island Volcanic Plateau of New Zealand, cattle suffered from what was termed "bush sickness". It was discovered that the volcanic soils lacked the cobalt salts essential for the cattle food chain. * The "coast disease" of sheep in the Ninety Mile Desert of the Southeast of South Australia in the 1930s was found to originate in nutritional deficiencies of trace elements cobalt and copper (AgricWA, 2018). 36
37. Capping it off! 37
38. Summary * B12 is only produced by some prokaryotes (certain bacteria and archaea) (Martens, Barg, Warren, Jah, 2002). * B12 is synthesised by some gut bacteria in the human colon, however cannot be absorbed as the colon is too far from the small intestine, where B12 absorption occurs. * For gut bacteria of ruminants to produce B12, the animal must consume sufficient amounts of cobalt (Huwait, Kumosani, Moselhy, Mosaoa & Yaghmoor 2015). * Grazing animals pick up B12 and bacteria that produce it from the soil at the roots of the plants they eat (Huwait, Kumosani, Moselhy, Mosaoa & Yaghmoor 2015). 38
39. Methylation Pathways - Methionine Synthase * Methylcobalamin is required for the function of the folate-dependent enzyme methionine synthase (Linus Pauling Institute, 2014). * This enzyme is required for the synthesis of the amino acid methionine from homocysteine (Linus Pauling Institute, 2014). * Methionine is required for the synthesis of S-adenosylmethionine (SAMe), a methyl group donor used in many biological methylation reactions, including the methylation of a number of sites within DNA and RNA (Linus Pauling Institute, 2014). * Inadequate function of methionine synthase can lead to an accumulation of Hcy, which has been associated with increased risk of cardiovascular disease (Linus Pauling Institute, 2014). 39
40. Methylation Pathway - Methylmalonyl-CoA Mutase Methyl or Adenosylcobalamin are required by the enzyme that catalyses the conversion of Methylmalonyl-CoA to Succinyl CoA (BMJ, 2018) * Succinyl CoA then enters the citric acid cycle (BMJ, 2018). Succinyl CoA functions include: * an important role in the production of energy from lipids and proteins (BMJ, 2018). * an essential role in the synthesis of Hb (oxygen-carrying component of RBCs) (BMJ, 2018). 40
41. Genetic Defect 41
42. Transsulfuration/Methylation/Re-methylation Pathway 42 Higdon. 2003
43. Transsulfuration Pathway – Hello B6☺ ▶ The transsulfuration pathway is the major route for the metabolism of the sulphur-containing amino acids. 43 Higdon. 2003
44. Absorption of Vitamin B12 * Passive (Linus Pauling Institute, 2014) * via buccal duodenal & ileal mucosa * rapid but inefficient with under 1% absorbed by this process * Active (Linus Pauling Institute, 2014) * occurs through the ileum * mediated by gastric intrinsic factor (gene at chromosome 11q13) (IF) 46
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46. Proteins associated with vitamin B12 metabolism * R protein (aka Haptocorrin or TCN I ) * Intrinsic factors * Cubilin receptors * Transcobalamin II * Cell surface receptors for TCNII-B12 complex * Enzymes involved in formation of- adenosyl and methyl cobalamin forms (Neale 1990). 51
47. Forms of B12 by Structure 52
48. Cyanocobalamin (CNCbl) Synthetically manufactured form of vitamin B12 (Paul & Brady 2017). * It is the most stable form of B12, due to the presence of a cyanide molecule (Paul & Brady 2017). * While the amount of cyanide is not dangerous, it does require the body to expend energy to convert and remove it. * CNCbl is a stable and inexpensive synthetic form commonly used for food fortification and oral supplements (Paul & Brady 2017). 53
49. Methylcobalamin (MeCbl) This is the most active form in the human body (Paul & Brady 2017). Converts homocysteine to methionine, which helps protect the cardiovascular system. * Offers overall protection to the nervous system. * Can also cross the blood-brain barrier (without assistance) to protect brain cells. * It contributes essential methyl groups needed for detoxification and to start the body’s biochemical reactions (Paul & Brady 2017). * In mammalian cells, MeCbl is a cofactor for the cytosolic methionine synthase (Paul & Brady 2017). 54
50. Hydroxocobalamin An abundant and physiologically relevant intermediate form (Paul & Brady 2017). * Bacteria naturally create this form of vitamin B12, making it the main type found in most foods. * It easily converts into methylcobalamin in the body (Paul & Brady 2017). * Hydroxocobalamin is commonly used via injection as a treatment for B12 deficiency as well as a treatment for cyanide poisoning (Thakkar, Billa 2015; Zhang, Ning 2008). 55
51. Adenosylcobalamin (AdoCbl) * AdoCbl is a cofactor for the mitochondrial methylmalonyl-CoA (MM- CoA) mutase (Thakkar, Billa 2015, Paul & Brady 2017). * The energy formation that occurs during the Citric Acid cycle requires this form of B12 (Thakkar, Billa 2015). * Although naturally occurring, it is the least stable of the four types of B12 outside the human body and does not translate well into a tablet-based supplement (Thakkar, Billa 2015, Paul & Brady 2017). * Harder to find in supplement form. 56
52. Supplementation * When supplemented, CNCbl needs to be converted into MeCbl and AdoCbl in order to exert its anticipated biological effect on the cell (Thakkar, Billa 2015, Paul & Brady 2017). * The concept of replacing CNCbl/HOCbl with the coenzyme forms as ready-to-use sources of the cofactors has emerged. * Supplementation of MeCbl and AdoCbl is postulated to be preferable to HOCbl and especially CNCbl/, depending on factors including genetics, environment, age and GIT function (Thakkar, Billa 2015, Paul & Brady 2017) 57
53. Example Dosages Found in Common Supplements * Sublingual Cyanocobalamin Spray: 500ug per spray * Sublingual Cyanocobalamin Tablet: 1000ug per tablet * Chewable Hydroxocobalamin Tablet: 2000ug per tablet * Liposomal Hydroxocobalamin Liquid: 500ug per 0.5ml dose * Liposomal Methylcobalamin: 200ug per Spray * Sublingual Methylcobalamin tablet: 5000ug (HIGH DOSE!!) 58
54. Forms of Cobalamin 59
55. B12 Forms and absorption 60
56. Vitamin B12 Lab Values & Deficiency Signs * While severe deficiency causes permanent neurological damage, earlier manifestations are generally subtle or asymptomatic * Vitamin B12 deficiency is a common condition that can manifest with Neurologic signs & sx * Psychiatric signs & sx * Hematologic disorders (BMJ Best Practice2018) The lab guidelines for B12 deficiency can be vague. In Australia : 258 pmol/L indicates that deficiency is unlikely?? 61
57. Screening Factors * Paraesthesias * Vegan/vegetarian * Medication use * Positive Romberg test - Ataxia * Decreased vibration sense * Pallor, Fatigue B12 is required to make the protective coating (myelin sheath) surrounding nerves, so inadequate B12 can expose nerves to damage (Shipton & Thachil 2015). 62 * Petechiae * Glossitis * Angular cheilitis * Cognitive impairment * Swollen tongue, bleeding gums * decreased appetite.
58. Screening Factors Vegans * Vegans not supplementing with B12 are potentially at an increased risk of vitamin B12 deficiency. * Up to 88% of vegans were found to have evidence of vitamin B12 deficiency in one study. Age >65 years * Prevalence of B12 deficiency increases with advancing age (Clarke et al. 2004). * 12% to 15% of people aged >65 years have biochemical evidence of vitamin B12 deficiency * MOA = Poorer GI absorption, and higher prevalence of atrophic gastritis 63
59. Risk factors (Medications) H2 receptor antagonist or proton-pump inhibitors (PPIs) * Vitamin B12 bound to food must be freed by peptic acid secreted from the stomach. * Taking H2 receptor antagonists or PPIs may increase risk of deficiency * >2 years' use of PPI or H2 receptor antagonists increase the risk of B12 deficiency (Lam 2013) Metformin * Chronic metformin use may cause low serum vitamin B12 levels and place patients at risk of vitamin B12 deficiency (Gupta, Jain, & Rohatgi 2018). * Patients using metformin for 4.3 years have an increased risk of vitamin B12 deficiency. 64
60. Helicobacter pylori infection * Studies reveal a potential association between H pylori infection and vitamin B12 deficiency. * Unclear whether it is the organism or atrophic gastritis that causes vitamin B12 deficiency (Serin et al. 2002, Avcu et al. 2001). Risk Factors (Health Conditions) 65
61. Terminal ileum disease * The majority of patients with terminal ileum resection were found to have evidence of vitamin B12 deficiency in an early study (Skidmore 1989). Crohns Disease * 50% of patients with Crohns disease who have had >20 cm of terminal ileum removed have evidence of vitamin B12 deficiency. Gastric Surgery * Gastric surgery causes inadequate B12 absorption via decreased IF (Kapadia 1995, Rhode 1995, Sumner 1996). Risk Factors (Health Conditions) 66
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63. Diabetes * Neuropathy in patients with diabetes is common (Gupta 2018, Wang 2017). * This means that concomitant vitamin B12 deficiency may be easily overlooked (Tavares 2017, Pflipsen 2009, Wang 2017, Metaxis 2018). * B12 status should therefore be part of diabetic health screening. 68
64. Pregnancy * B12 deficiency is common during pregnancy as levels of vitamin B12 decrease from the first to the third trimester (Rogne et al. 2017, Lai 2017, Dayaldasani 2014). 69
65. Investigations: Full Blood Count (FBC/CBC/FBE) Provides important information about the types, populations, and health of blood cells. * Aids diagnosis and assessment of anaemia, nutritional deficiencies, blood disorders, infection, and many other disorders. An FBC consists of: * RBC (including red cell indices) * WBC (including white cell indices) * Haemoglobin (Hb) * Haematocrit (Hct) * Platelets 70
66. Microscopy A peripheral blood smear (a way of looking at blood cells under the microscope) may also provide useful information. In a normal peripheral blood smear, RBC appear regular & round with a pale centre. Variations in the size or shape of these cells, otherwise known as anisocytosis, may suggest a blood disorder. 71
67. Primary Pathological Investigations FBC – (Screening tool not diagnostic) * To determine baseline RDW (not always included in an FBC) as well as MCV, Hb and Hct. * Many individuals with documented vitamin B12 deficiency do not have macrocytosis or anaemia (Chui et al. 2001). * Not useful for diagnosing early vitamin B12 deficiency (except increased RDW) * Not useful to rule out vitamin B12 deficiency. * B12 deficiency may cause: * Increased RDW, increased MCV and increased Hct, depending on the stage of deficiency. 72
68. Serum Vitamin B12 Optimal serum vitamin B12 levels for hematologic and neurologic function are still undetermined. 258 pmol/L indicates that deficiency is unlikely???? 73
69. Primary testing Methylmalonic Acid (MMA) * Produced in very small amounts, used in metabolism and energy production (Methylmalonic Acid 2018). * In one step of metabolism, B12 promotes the conversion of methylmalonyl CoA to succinyl CoA (Methylmalonic Acid 2018). * If there is not enough B12 available, then the MMA concentration begins to rise, resulting in an increase of MMA in the blood and urine. * Elevated urinary MMA is a sensitive and early indicator of B12 deficiency (Sun et al. 2014). 74
70. MMA * Test * Potential differential diagnosis = renal disease, in which increased MMA levels also occur. * Increased MMA may be misleading and requires follow-up to determine whether MMA normalises with adequate treatment (Oh 2018). * Result * Increased (lab-specific) 75
71. Homocysteine (Hcy) Test Not as specific as MMA for vitamin B12 deficiency (Vashi, Edwin, Popiel, Lammersfeld & Gupta 2016). * Differential diagnosis = folate deficiency and hypothyroidism, in which Hcy is also increased. * More readily available than MMA * Result * Increased (lab-specific) 76
72. Intrinsic Factor (IF) antibody * Once vitamin B12 deficiency is confirmed, IF antibody can determine whether pernicious anaemia is the cause. * Only 50% sensitive, but highly specific for pernicious anaemia. * Result * Positive if pernicious anaemia is the cause 77
73. Antiparietal Cell (APC) antibody * Parietal cell antibodies are proteins produced by the immune system that mistakenly target a type of specialised cells that line the stomach wall. This test detects these antibodies in the blood to help diagnose pernicious anaemia. So….. Wrap your head around this……. 78
74. If both parietal cell antibody and intrinsic factor antibody are positive = Immunological evidence of Pernicious Anaemia. If both parietal cell antibody and intrinsic factor antibody are negative = No immunological evidence of Pernicious Anaemia. If parietal cell antibody is positive but intrinsic factor antibody is negative = Gastric Parietal cell antibody is associated with > 90% of patients with Autoimmune Gastritis, the end result of which may be Pernicious Anaemia. In 20-30% of patients, relatives of patients with pernicious anaemia, autoimmune thyroiditis a small percentage of healthy persons may be positive and run an increased long term risk of pernicious anaemia. ○ A negative Intrinsic Factor antibody result does not exclude the diagnosis of Pernicious anaemia, as only 60% of patients with pernicious anaemia will have this antibody. 79
75. Intrinsic Factor Antibody Two types: * Type 1(blocking) antibody prevents the attachment of vitamin B12 to intrinsic factor: present in 50-60% of patients with pernicious anaemia (RCPA 2018). * Type 2 (precipitating) antibody prevents attachment of the vitamin B12-intrinsic factor complex to ileal receptors: present in 30% of patients with pernicious anaemia (RCPA 2018).
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Posted by :  peter88 Post date :  2019-11-06 16:48
Category :  Health & Medicine Views :  35

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