vitamin D

molecular formula of D2

C28H44O.

what is the role of PTH in vitamin D regulation

maintain normal serum and total body calcium

how does age affect vitamin D?

older people produce less vitamin D due to: -skin aging - kidneys less efficient at converting vit D into calcitriol - tend to spend more time indoors

amount of Vitamin D absorbed is dependent upon

presence of fat in the lumen

vitamin D receptor

present in most tissues and cells within the body.

What are the potential interferences in a patient sample that, if present, may cause erroneous results in immunoassays for vitamin D?

rheumatoid factor, endogenous alkaline phosphatase, fibrin, and proteins capable of binding to alkaline phosphatase.

conversion of vitamin D to calcitriol occurs in other areas besides the kidneys.

skin, prostate, brain, pancreas, adipose tissue, skeletal muscle, heart, colon, monocyte/macrophages and in neoplastic tissues.

mechanism of vitamin D deficiency on calcium

vitamin D deficiency reduces intestinal calcium absorption ==> decreased serum calcium levels ==> triggers release of PTH ==> PTH corrects calcium level by taking calcium from the bone, increasing renal tubular reabsorption, and increasing renal production of 1,25(OH)2D.

decreased in serum ____ levels results in proportionally higher ____

vitamin D, parathyroid hormone (PTH)

vitamin D sources

vitamin D3: fatty fish beef liver cheese egg yolks vitamin D2: some mushrooms

role of vitamin D on the development and maintenance of bones

assist calcium absorption from food in the intestine, and ensure the correct renewal and mineralization of bone tissue.

rickets

-a condition that affects the development of bones in children. -causes soft weak bones, which can become bowed or curved. -common among children 3 months - 18 mos -signs and symptoms: misshaped or deformed skeleton, pain, fragile bones, and poor growth and development. -common cause: extreme vitamin D and/or calcium dificiency

D2 (ergocalciferol)

-absorption and metabolism of calcium and phosphorous via increasing the efficiency of the small intestine to absorb these minerals from the diet. -fat soluble

sample requirements

-fasting specimen is recommended but not required - fresh or frozen serum or plasma collected in red-top or serum-separator tubes as well as green top tubes that contain sodium heparin. -can be stored at < -20ºC, because vitamin D is very stable -Moderately hemolyzed specimens may be used in certain testing methods because red blood cells do not contain Vitamin D. But may interfere with spectrophotometric techniques.

D3 (cholecalciferol)

-helps the body absorb calcium -a steroid hormone produced in the skin when exposed to ultraviolet light (endogenous source) or taken in from dietary sources (exogenous). -The active form has an important role in maintaining blood calcium and phosphorous levels and mineralization of bone. - In the activated form, cholecalciferol binds to vitamin D receptors and modulates gene expression. This leads to an increase in serum calcium concentrations by increasing intestinal absorption of phosphorus and calcium, promoting distal renal tubular reabsorption of calcium and increasing osteoclastic resorption.

liquid chromatography - mass spectrophotometry (LC-MS/MS) measures

-vitamin D2, vitamin D3, and the D3 epimer (one of a pair of stereoisomers) -total vitamin D is calculated

recommended daily intake of vitamin D: Endocrine Society

1 to 18 years old: 600-1,000 IU/day 19 to 70 years old: 1,500-2,000 IU/day 71+ years old: 1,500-2,000 IU/day

recommended daily intake of vitamin D: IOM

1 to 18 yo: 600 IU/day 19 to 70 yo: 600 IU/day 71+ yo: 800 IU/day

Two Vitamin D forms important to humans:

1) vitamin D2, which is made by plants 2) vitamin D3, which is made by human skin when exposed to sunlight.

major circulating form of Vitamin D

25(OH)D3, the precursor of calcitriol (produced in the liver)

molecular formula of D3

C27H44O

absorption issues

GI condition affecting ability to absorb fat. ex: crohn's disease, celiac disease, non-celiac gluten sensitivity, other inflammatory bowel diseases

which form of vitamin D is made endogenously?

Vitamin D3

what happens when the washing step is skipped in an immunoassay test for vitamin D?

Washing steps are necessary to remove unbound material and decrease background, thereby increasing the signal. Insufficient washing will allow high background, which in turn will result in inaccurate increase of results.

25(OH)D3 circulates bound to

a carrier protein (vitamin D binding protein (DBP))

vitamin D metabolism

cholesterol from diet converts to 7-dehydrocholesterol which gets taken up by cells (skin cells) in the blood stream. contact with sunlight converts it to cholecalciferol --> blood stream --> converted to 25-hydroxy vitamin D by liver --> converted to 1,25 dihydroxyvitamin D (active form) in the kidneys

how does serum phosphorus level affect calcitriol synthesis?

high phosphorus = inhibit calcitriol synthesis low phosphorus = stimulate calcitriol syntheis

reference method for vit D analysis

liquid chromatography - mass spectrophotometry (LC-MS/MS)

effect of vitamin D deficiency on bones

osteopenia is acquired first and without treatment will progress to osteoporosis due to decreased calcium absorption.

drugs that raise vitamin D levels

paricalcitol (zemplar) and anti- HIV drug darunavir (Prezista)

immunoassays

separate and quantify vitamin D2 and vitamin D3. But, depending on the specificity of the antibody used in the immunoassay method, some immunoassays measure only one form.

immune system impairment due to vitamin D deficiency

vit D receptor expressed on immune cells (B cells, T cells, antigen presenting cells). vit D binds to intracellular receptors ==> activation of cell signaling cascade or gene transcription ==> modulate immune cell function can also influence natural and adaptive immune response of these cells.

immunoglobulin interferences

- interference from heterophile antibodies (human anti -sheep, mouse, rat, or goat antibodies) in the patient sample. -may cause erroneous results -recommended that LC - MS assays be used

2 guidelines of recommended daily intake of Vitamin D

- Institute of Medicine (IOM): in the form of recommended dietary allowances (RDA) or adequate intakes (AI) - Endocrine Society: guidelines for "people who are at risk for deficiency

spectrophotometric interferences

-Hemolyzed samples (concentrations greater than 50 mg/dL), lipemic, or severely icteric samples - may cause falsely elevated results

individuals who are more at risk for vitamin D definciency

-People who live above 37 degrees north or below 37 degrees south of the equator, because the skin makes very little vitamin D from the sun in these areas -people who are homebound

procedure for most FDA approved methods of immunoassays

-Step One: 25-hydroxy vitamin D is dissociated from its binding protein and binds to the specific solid phase antibody. -Step Two: Vitamin D-isoluminol tracer is added and unbound material is removed with a wash cycle. -Step Three: The reagents are added to initiate the chemiluminescent reaction. -Step Four: The light signal is detected by a photomultiplier as relative light units; this measurement is inversely proportional to the concentration of 25-hydroxy vitamin D.

vitamin D's role in the growth and development of children

-build strong teeth and bones -strengthen the immune system -improve cardiovascular health

HPLC

-can quantitate 25-hydroxy vitamin D2 and D3.

Effect of Renal disease on vitamin D

-can't convert 24-hydroxylase or 1-alpha-hydroxylase to 24,25-(OH)2D3 or to calcitriol -elevated phosphorus due lack of excretion

Calcitriol (1,25-dihydroxyvitamin D)

-carries out major functions of vitamin D in the body. - increases calcium blood levels by increasing calcium uptake in the GI tract, increasing reabsorption by the kidneys, and increasing the release of calcium into blood from bones.

Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS)

-combines the physical separation capabilities of liquid chromatography with the mass analysis capabilities of mass spectrophotometry. - sensitive = lower limits of detection -recognize analytes at 2 physical properties: precursor and product ion mass

medications

-drugs that increase the enzymes that catabolize vitamin D3 and vitamin D2 into inactive forms have the potential to decrease vitamin D levels. - or drugs that interfere with the digestive enzymes -Antibiotics: Rifampin (rifampicin) and isoniazid, commonly used to treat TB. Vitamin D levels can sometimes fall after as little as two weeks exposure to these drugs. -Anti-seizure drugs - Phenobarbital, carbamazepine, phenytoin -Anti-cancer drugs - Taxol and related compounds -Anti-fungal agents - Clotrimazole and ketoconazole -Anti-HIV drugs - Refavirenz (Sustiva, Stocrin, and in Atripla) and AZT (Retrovir, zidovudine and in Combivir and Trizivir) may reduce vitamin D levels in some people. -Herbs - St. John's wort or its extracts (hypericin, hyperforin) -Anti-inflammatory drugs: Corticosteroids

where do the products of vitamin D metabolism go

-excreted through the bile into the feces -small amount eliminated through the urine due to renal reuptake of vitamin D metabolites bound to DBP (vitamin D binding protein).

risk factors associated with vitamin D deficiency in children

-higher child age -more television watching -less playing outside -less biking to school -lower maternal age -lower household income -multiparity -higher maternal BMI

paricalcitol (zemplar)

-is the active form of vitamin D used to prevent and treat secondary hyperparathyroidism in patients with chronic kidney disease and on dialysis. -Vitamin D levels should not be tested in patients who have received Paricalcitol within 24 hours of obtaining the sample.

major role of vitamin D

-maintain normal blood levels of calcium and phosphorus

2 counter-acting hormones regulating renal synthesis of calcitriol

-up-regulation via parathyroid hormone (PTH) -down-regulation via fibroblast-like growth factor-23 (FGF23)

Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) principle

-uses two mass filters arranged sequentially with a collision cell between them. The filters can be used in static or scanning mode to select a particular mass-to-charge ratio or range. In the middle collision cell, the precursor ions collide with gas molecules and are fragmented into smaller ions referred to as product ions. The resulting product ions are then separated and detected in a second stage of mass spectrometry.

forms of vitamin D

D2 (ergocalciferol) D3 (cholecalciferol) Calcidiol (25-hydroxy vitamin D) Calcitriol (1,25-dihydroxyvitamin D)

T/F vitamin D is absorbed with other dietary fats

True

T/F vitamin D is fat soluble

True

body's requirements of vitamin D are mainly achieved by which UV induced photosynthesis in the skin?

UVB

enzymatic hydroxylation of vitamin D (D2 and D3)

biological activation of vitamin D first rxn: -occurs in liver - mediated by the enzyme 25-hydroxylase which forms 25-hydroxyvitamin D3 [25(OH)D3 2nd rxn: -takes place in the kidney -mediated by 1α-hydroxylase, which converts 25(OH)D3 to the biologically active hormone, calcitriol. -Once transformed, it binds to the vitamin D receptor

secondary hyperparathyroidism

caused by chronic vitamin D deficiency

most common cause of vitamin D deficiency

decreased intake (vegan diet)

the diagnostic difference between osteopenia and osteoporosis

degree of decline of bone mineral density

Calcidiol (25-hydroxy vitamin D)

inactive form of vitamin D. calcidiol converted to calcitriol (the active form) in the kidneys