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What is the key characteristic of vitamins and how can they be classified?
We cannot synthesize vitamins, they must be sourced from our diet. They can be classified as fat or water soluble.
Discuss the function, RDA, effects and lab test for vitamin A.
Vitamin A is required for vision, reproduction, growth, cell differentiation, and immune function. The RDA is 900ug/d, and effects of deficiency are night blindness, keratomalacia, and impaired mucous secretion. The lab test for vitamin A is HPLC for retinol.
Discuss the function, RDA, effects and lab test for vitamin D.
Vitamin D is required for regulation of calcium and phosphate, bone mineralisation, cell proliferation, and modulation of the immune system. The RDA is 5ug/day and effects of deficiency are impaired bone mineralisation and osteomalacia. Vitamin D can be measured by LC-MS, HPLC or immunoassay.
Discuss the function, RDA, effects and lab test for vitamin E.
Vitamin E is an antioxidant and is required to maintain the nervous system. The RDA is 15mg/d and deficiency causes neurological disorders and haemolytic anaemia. The lab test is HPLC for alpha-tocopherol.
Discuss the function, RDA, effects and lab test for vitamin K.
Vitamin K is the co-enzyme for a vitamin K-dependent carboxylase, involved in blood coagulation and bone metabolism. The RDA is 120ug/day and deficiency causes bleeding, osteoporosis and coronary heart disease. Vitamin K can be measured by LC-MS, HPLC, or functional assays can be done eg. PIVKA-II.
How are fat soluble vitamins absorbed?
Absorbed from the proximal intestine and dependent on bile and pancreatic juice secretion for solubilisation. Any condition causing prolonged intestinal malabsorption of fat will lead to secondary deficiency of fat-soluble vitamins.
Deficient stated lead to the depletion of tissue stores and are indicated by a decrease in circulatory levels long before physiological changes develop.
What happens to vitamin A post-absorption?
Retinol is transported to hepatic stellate cells and is stored as retinyl esters. Mobilisation of stores occurs to counter dietary restriction or malabsorption to maintain circulatory levels and meet metabolic demand for several months.
What happens to vitamin E post-absorption?
90% is stored in adipose tissue, the reserves are released slowly during dietary restriction to buffer vitamin E status. Any reduction in intake is not reflected in circulatory levels for about 1 month.
What happens to vitamin D post-absorption?
Stored in body fat and the liver when present in large quantities. Released gradually into the blood stream. Otherwise converted rapidly to 25(OH)D, which circulates. Dependency on dietary vit D depends on amount synthesized from sunlight, a production partly of function of skin pigmentation and latitude. The wavelength required for conversion of 7-dehydrocholesterol to previtamin D3 is 290 - 315nm which is unable to penetrate glass.
What happens to vitamin K post-absorption?
Stored mainly in the liver - 90% of hepatic stores are menaquinones (vit k2) and are of bacterial origin. The main circulatory form is vitamin K1 (phylloquinone) from plants.
Who is at risk of vitamin K deficiency.
Rare, but potentially fatal syndrome that can lead to bleeding.
Those at the beginning and end of life:
-the anorexic patient (stores depleted <3 days)
-Subclinical deficiency (with respect to functionality of extra-hepatic vitamin k dependent proteins) is common.
Neonates are given vitamin K injection at birth.
Patients with impaired bile excretion, or patients on warfarin are at risk also.
What markers of vitamin K status are there?
Prothrombin time: poor marker - 50% reduction in circulatory levels before there is an increase in PT. Only advanced states of deficiency are detected.
Static markers: circulatory levels of vitamin K1
Functional markers: PIVKA-II (protein induced by vitamin k absence) can detect abnormal prothrombin.
What are the risk factors for deficiency?
Intestinal malabsorption - as a consequence of pancreatic insufficiency and bile salt deficiency.
Liver disease, diarrhoea, frequent antibiotic therapy, bowel restriction, inadequate dietary intake.
Vitamin K antagonist - warfarin
Inborn errors - VKCFD1 (carboxylase) and VKCFD2 (VKOR)
Discuss the function, RDA, effects and lab test for vitamin B1.
Thiamine is a co-enzyme in carbohydrate and branched chain amino acid metabolism. The RDA is 1.2mg/d and deficiency causes beriberi, confusion, cardiac failure, and nerve membrane disorder. Measure thiamine diphosphatase, or erythrocyte transketolase activity.
Discuss the function, RDA, effects and lab test for vitamin B2.
Riboflavin is a coenzyme in numerous redox reactions. The RDA is 1.3mg/d and deficiency doesnt cause severe disease, but impacts B3, B6, and B9, cheilosis and angular stoatitis. Detect riboflavin, FAD and FMN via HPLC.
Discuss the function, RDA, effects and lab test for vitamin B3.
Niacin is required for NAD and NADP which act as acceptor or donor of electrons for redox reactions. The RDA is 16mg/day, and deficiency causes pellagra, dermatitis, diarrhoea and dementia. Only urinary excretion of metabolites (N-methyl-nicotinamide) can be used to detect deficiency.
Discuss the function, RDA, effects and lab test for vitamin B5.
Pantothemic acid is required for formation of coenzyme A, and is involved in fat, carb and protein metabolism. The RDA is 5mg/day and deficiency causes painful peripheral neuropathy. Lab test is for calcium- D-pantothenate.
Discuss the function, RDA, effects and lab test for vitamin B6.
Pyridoxine is required for amino acid metabolism, glycogen phosphorylation, and conversion of tryptophan to niacin, or serotonin. Required for hormone function. The RDA is 1.3mg/day and deficiency causes irritability, depression, confusion, mouth ulcers, and inflammation of the tongue. Measure pyridoxal-5-phosphate or urinary 4-pyridoxic acid.
Discuss the function, RDA, effects and lab test for vitamin B9.
Folate is required for one carbon metabolism of nucleic acids and amino acids. The RDA is 400ug/day and deficiency causes megaloblastic anaemia, fatigue, palpitations. Measure RBC folate, serum folate and homocysteine.
Discuss the function, RDA, effects and lab test for vitamin B12.
Cobalamin is a coenzyme for methionine synthase and MMA mutase. The RDA is 2.4ug/day and deficiency causes pernicious anaemia and food bound vitamin B12 malabsorption. Measure serum B12, holotranscobalamin, homocysteine and MMA.
Outline dietary B12 absorption.
Initiated in the stomach by release from food-binding proteins by gastric acid and digestion of food by pepsin. Two proteins compete for free B12 - intrinsic factor (released from gastric parietal cells) and haptocorrins (produced by salivary glands).
Intrinsic factor has a low affinity and haptocorrins have a high affinity for free B12 at an acidic pH.
When they enter the duodenum and an alkaline pH, the haptocorrins are partly digested by proteases secreted by the pancreas which frees up B12 to attach to intrinsic factor. The complex moves to the lower ileum where it binds to specific receptors on the intestinal mucosal enterocytes. It is transported to blood possibly by ATP-dependent carriers.
Outline the reference values for active B12 (holotranscobalamin).
<25pmol/L = suggests vitamin B12 deficiency
25-70pmol/L = vitB12 deficiency cannot be excluded, sample referred for MMA analysis
>70pmol/L = patient unlikely to have B12 deficiency
What is the mechanism behind the two functional markers for vitamin B12 status?
In the cytosol methionine synthase requires methylcobalamin as a cofactor during the remethylation of methionine from homocysteine.
In the mitochondrion methylmalonyl-CoA mutase requires adenosylcobalamin as a cofactor for the conversion of methylmalonic-CoA to succinyl-CoA
Discuss HPLC as a method for vitamin measurement.
HPLC uses a mobile and stationary phase, with high pressure to separate out components of a sample. There are multiple detector types available. The following things must be considered for implimenting this technique:
-type of column
-type of detector
-in-house method? CE marking!
-Bring in commercial kit?
Outline the advantages and disadvantages of HPLC for vitamin measurement.
-relatively low consumable cost
-can be automated
-relatively high equipment cost
-lack of specificity (co-eluting peaks)
Outline LC-MS/MS for measurement of vitamins.
HPLC coupled to mass specs
3MSs - 2 for detection and 1 as collision cell
3 measuring parameters (min) = often very specific
Mass to charge ratio (m/z) - need ionised particles
Different operational modes
Electrospray ionisation - nebuliser gas and high temperature = positive ions
Components: probe, LC column, electrospray ionisation, MS1, collision cell, MS2 and detector.
Things to consider:
-Type of column
-Type of source
-Type of MS (TOF, triple quad)
-Type of mode (MRM, parent ion scan)
-In-house methodology - CE marking!
-Ion suppression and epimers
What are the advantages and disadvantages of LC-MS/MS?
Relatively low consumable cost
can be automated
high equipment cost
Outline immunoassay for measurement of vitamins.
A test that uses antibody and antigen complexes as a means of generating a measurable result.
Competitive vs non-competitive
One-step vs two step
What needs to be considered for immunoassay?
manual or automated?
workload - can your analysers cope?
Cost of reagents
Ability to measure all forms
Is there an assay available?
What are the advantages and disadvantages of an immunoassay?
fully automated process
high equipment cost
lack of specificity
What specific issues are there when analysing vitamins?
sample type, stability and storage
method to use and considerations
how to calibrate
Discuss sample type for vitamin analysis.
Always validate the sample type, stability and storage for your method.
Some labs require samples on dry ice and some protected from light - especially for vitamin A and carotenes.
STEMDRL - vitamin A, E and carotenes highly light sensitive - send on dry ice.
Data for SWBH shows storage conditions and stability do not make much difference.
Check plasma and serum from different tubes behave the same as this can lead to problems - eg ion suppression.
Discuss calibrators for vitamin analysis.
Make in house? - matrix, purity?
Discuss units used in vitamin analysis.
The units used for vitamins across the country are rather variable, as highlighted in the audit of vitamin D measurement in Wales 2012 which showed that 3 labs were using nmol/L, 3 were using ug/L and 1 was using ng/mL. UK now tends to use nmol/L.
What other biochemistry can help in investigation of vitamin status?
Intrinsic factor antibodies in B12 deficiency
FBC in folate/B12 deficiency
PTH, Ca and ALP for vit D deficiency
Bilirubinaemia in hypercarotenaemia
Cholesterol for vitamin E status.
Refeeding and nutritional assessment - Mg, PO
CRP/albumin - acute phase response
Homocysteine and MMA for B12/folate deficiency
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