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FROM LAB PERSPECTIVE

Tjan Sian Hwa

Dec2021
CYP2R1 25(OH)D3 +25(OH)D2

CYP24A1 24,25(OH)2D

CYP27B1

- DBP (85%)
- Albumin, Protein,
Lipoprotein (15%)
- Free ( 0.03%)

J Clin Invest. 2006;116(8):2062-2072

VITAMIN D IN CALCIUM AND PHOSPHATE METABOLISM

Ca and P are regulated by Vit D and PTH

Low vit D

Normal Ca n
P

High normal /
high PTH

High normal
/High ALP

Low 24 hour
urin Ca
NON CALCIUM-BONE METABOLISM FUNCTION OF VIT D:

Many cells have Vitamin D Receptors and also able

converting 25(OH)D to 1.25(OH)2D

• Immunity:
• Increase production of cathelicidin by activated
macrophages
• Cancer :
• Regulates gene that control normal cell
proliferation (p21,p27) , inhibit angioneogenesis
and induced apoptosis
• Diabetes :
• Stimulates insulin production by B cell of
pancreas
• Cardiovascular :
• Down regulate renin production
VITAMIN D DEFICIENCY

5 Holick MF. Rev Endocr Metab Disord 2017;18:153–65.

FACTORS INFLUENCING VITAMIN D STATUS

Synthesis of vitamin D from sunlight Bioavailability of vitamin D Other factors

Exposure to ultraviolet radiation Gastrointestinal malabsorption of vitamin D Kidney disease
• Latitude • Celiac disease • Chronic kidney disease
• Season • Biliary obstruction • Nephrotic syndrome
• Use of sunscreen • Chronic pancreatitis Liver disease
• Clothing • Liver failure • Cholestatic liver disease
Skin • Cystic fibrosis • Parenchymal liver disease
• Pigmentation • Crohn’s disease • Hepatic failure
• Temperature • Gastric bypass
Granulomatous disorders and malignancies
• Scarring, e.g. burns • Bile acid-binding medication (e.g.
• Sarcoidosis, tuberculosis, fungal
• Age colestyramine, colestipol) granulomas, berylliosis
Obesity • Certain tumours (tumour-induced
Enzyme activity osteomalacia)
• 1-α-hydroxylase: serum phosphorus,
parathyroid hormone, genetic mutations
• 25-hydroxylase: concentration of 25(OH)D
• Cytochrome P450 enzymes (CYP24, CYP3A4):
medications (phenobarbital, phenytoin,
carbamazepine, rifampicin, antiretrovirals)

6 Tsiaras WG, Weinstock MA. Acta Derm Venereol 2011;91:115–24.

VITAMIN D TESTING

❑ Not recommended for routine screening

❑ Indicate for:
• Individuals with risk for deficiency of vitamin D
• Individuals with lab or radiographic finding associated with vit D deficiency
• ALP > with Normal Liver Function Tests, High PTH, high/low Ca, low Phosphate
• Treatment monitoring if under or over treatment is suspected
• In Pediatric :
• Sign and symptoms of Rickets,
• Infants of mother with Vit D deficiency
• Exclusively breast fed babies in combination with at least one other risk factor
• Sibling of children with vitamin D deficiency
VITAMIN D TESTING INDICATION
CIRCULATING VITAMIN D
• Vitamin D metabolites:
• 25(OH)D3,
• 25(OH)D2
• 1.25(OH)2D
• 24,25(OH)2D
• 3-epi(OH)D3
• Vitamin D metabolites circulate
• Bound to Vitamin D binding Protein ( 85%),
• Bound Albumin, Protein, Lipoprotein (15%)
• Free ( 0.03%)
• DBP has a higher binding affinity to 25(OH)D3 compared to 25(OH)D2.
• DBP has higher binding affinity to 25(OH)D than 1,25(OH)2D
CIRCULATING VITAMIN D

1. 25(OH)D :
• Consist of 25(OH)D3 + 25(OH) D2
• The predominant circulating form
• Recommended and widely used because it reflects vitamin D supply from cutaneous synthesis
and diet, has a long half-life (2-3 weeks) and is not under tight homeostatic control.
• Decrease in acute inflammation.

https://www.gov.uk/government/groups/scientific-advisory-committee-on-nutrition
2. 1,25 (OH)2D
- Produced in kidney
- Biologically active metabolite of vitamin D,
- Not recommended for monitoring vitamin D status
- Half life 4 hours
- Low concentration (1000 x < 25(OH)D)
- Blood level is tightly regulated by serum levels of PTH, Ca and P
- Not related to vitamin D stores.
25(OH)D deficiency → 1,25-(OH)2D levels still normal

3. 3-epi-25(OH)D3
- high in infant less than 1 year
Vitamin D status is assessed by measuring the serum 25(OH)D
METHODS FOR VITAMIN D TESTING
• Immunoassay
• Elisa , Immuno-chemiluminescence assay
• Rapid, High throughput
• Result variation among assays 15-20%
• Liquid chromatography-tandem mass spectrometry (LC-MS/MS)
• Reference method
• Can separately measured vitamin D metabolites → more specific
• Expensive equipment
• Less throughput
• Need expertise
• HPLC, RIA, Competitive Protein Binding assay
CHALLENGES

• Significant differences in the 25(OH)D were observed between assays (15-

20%).
• Causes :
• Traceability
• Cross reaction with other Vitamin D metabolites
• DBP release capability
• Variety of antibody affinity to 25(OH)D2 and 25(OH)D3 used in assays
• Matrix interferences (pregnancy, hemodialysis, ICU)

Standardization and harmonization of 25(OH)D measurements

Br J Clin Pharmacol 2018;84:2194-7

Analytical bias and assessment of vitamin d status

PlosONE5(7):e11555,doi:10.1371/journalpone.0011555
INTERFERENCES
 VITAMIN D STANDARDIZATION PROGRAM (VDSP) REFERENCE
MEASUREMENT SYSTEM COMPONENTS
• In 2010: NIH Office of Dietary Supplements(ODS), Center for Disease Control(CDC) National
Center for Environmental Health(NCEH), National Institute of Standards and Technology (NIST)
and Ghent University, established Vitamin D Standardization Program (VDSP)

• National Institute for Standards and Technology (NIST), Ghent University and
Centers for Disease Control and Prevention (CDC) reference measurement
procedures
• NIST standard reference materials
• Performance standards for accuracy (mean bias <5%) and precision (total CV
<10%)
• CDC Vitamin D Standardization-Certification
• Program Accuracy-based performance testing/external quality assessment
schemes College of American Pathologists’ accuracy-based vitamin D survey
Vitamin D External Quality Assessment Scheme.

Br J Clin Pharmacol (2018) 84 2194–2207 2197

HARMONIZATION OF RESULT

▪ Traceable to reference measurement method

▪ Traceable to standard reference material
▪ Target of 25(OH)D imprecision(CV) of <10% and bias <5%
▪ Participation in accuracy based EQAS
▪ Participation in Vitamin D Standardization-Certification
CROSS-REACTIVITY
Assay A

Assay B

Assay C

Assay D
PREANALYTICAL ASPECTS OF VITAMIN D TESTING

25(OH)D is a relatively stable analyte, can be examined in serum or plasma

• Temperature :
• stable at room temperature and refrigerator for 2 weeks1
• at room temp 7 days decrease 2%3, At 400C decrease 14%3
• Light : Not affected by light
• Centrifuged with or without refrigerated centrifuge1
• Sample type
• Serum → recommended
• Plasma

1. IJSRM Volume 06 Issue 03 March 2018

3. Sys Rev Pharm 2020; 11(4): 695 700
PLASMA VS SERUM
Using LC –MS/MS, PREGNANT
CLIA
REFERENCE VALUES
• 1970 : from healthy adult → 25-137.5 nmol/L (mean+2SD)
• 1998 after supplementation Vit D 50.000 IU weekly for 8 weeks
• there was 35% decrease in PTH in adult with 25(OH)D < 50 nmol/L →
Vitamin D deficiency < 50 nmol/L
• PTH level began to plateau when 25(OH)D were 75- 100 nmol/L
• Post menopause women with 25(OH)D increased from 50 to 75 nmol/L had
65 % increase of intestinal Ca absorption
• → Vitamin D insufficiency 50-75 nmol/L
Br Med Bull 2018;126:57–77.
REFERENCE VALUES
• At latitudes below 37ᵒN, UVB radiation is sufficient for year round vitamin D
synthesis. At higher latitude, vitamin D is not synthesised during the winter
months.
• Lower plasma/serum 25(OH)D concentrations have been observed in people
with dark skin pigmentation compared to those with lighter skin colour.
• 25(OH)D low level is correlated with low level of exercise, obesity, reduced
sunlight exposure

• Variation in reference range criteria

• Variation in analytical performance of assays
 REFERENCE RANGE

IOM1 Endocrine Society2

Interpretation Serum total 25(OH)D
Deficient <12 ng/mL <20 ng/mL
(<30 nmol/L) (<50 nmol/L)
Insufficient 12–20 ng/mL 21–29 ng/mL
(30–50 nmol/L) (50–75 nmol/L)
Sufficient 20–30 ng/mL 30–100 ng/mL
(50–75 nmol/L) (75–250 nmol/L)
No added benefit 30–50 ng/mL
–
(75–125 nmol/L)
Possible harm >50 ng/mL >100 ng/mL
(>125 nmol/L) (>250 nmol/L)

1. Institute of Medicine (US). Dietary Reference Intakes for Calcium and Vitamin D.
Washington, DC: National Academies Press 2011;
28 2. Holick MF et al. J Clin Endocrin Metab 2011;96:1911–30.
VITAMIN D SUPPLEMENTATION

• Vitamin D supplementation is recommended in those who are

deficient
• Post-supplementation serum levels of 25(OH)D vary substantially

between individuals, highlighting the importance of ongoing testing

• Intoxication is rare and generally occurs after inappropriate

supplementation
Vitamin D intakes recommended by the
IOM1 and the Endocrine Society

aUL indicates level above which there is risk of adverse events. The UL is not intended as a target intake.
bReflectsAI reference value rather than RDA. RDAs have not been established for infants.
cMother’s requirement 4000–6000 (mother’s intake for infant’s requirement if infant not receiving 400

IU/d).

Adapted from Holick MF. Rev Endocr Metab Disord 2017;18:153–65. 1. Institute of Medicine (US). Dietary Reference Intakes for Calcium and Vitamin D.
AI, adequate intake; IOM, institute of medicine; RDA, recommended dietary allowance; Washington, DC: National Academies Press 2011;
UL, upper limit 30 2. Holick MF et al. J Clin Endocrin Metab 2011;96:1911–30.
WHEN TO MONITOR
• Response to vitamin D supplementation varies among individual, depend on
• Basal vitamin D level
• BMI or body fat percentage
• Ca intake
• Genetic
• Oestrogen supplementation
• Fat diet
• Disease and medication
• Season
• Monitoring 3 months after treatment or change of doses
• Half life 25(OH)D 2-3 weeks → steady state 4-5 times T1/2
• Monitoring for individual with risk factors for vitamin D deficiency
(pregnancy, minimal exposure to sunlight, elderly, CKD )
• Monitoring preferably at end of winter
• Monitoring should be done at the same lab and same method
VITAMIN D DEFICIENCY IN CHRONIC KIDNEY DISEASE
Vit D deficiency is prevalent among Chronic Kidney Disease
• Potential causes : proteinuria, reduced skin synthesis of vitamin D, peritoneal dialysis,
diabetes mellitus and reduced 25(OH)D receptors
• reduced 1,25(OH)2D
KDIGO CKD-MBD recommendations:

• In patients with CKD G3a–G5D: 25(OH)D (calcidiol) levels might be measured, and repeated testing
determined by baseline values and therapeutic interventions (2C).
• In patients with CKD G1T–G5T, we suggest that 25(OH)D (calcidiol) levels might be measured, and
repeated testing determined by baseline values and interventions (2C).
• In patients in the first 12 months after kidney transplant with an eGFR> 30 ml/min/1.73 m2 and low
BMD: suggest that treatment with vitamin D, calcitriol/alfacalcidol, and/or antiresorptive agents be
considered (2D). We suggest that treatment choices be influenced by the presence of CKD-MBD, as
indicated by abnormal levels of calcium, phosphate, PTH, alkaline phosphatases, and 25(OH)D (2C).

Kidney International Supplements (2017) 7, 1–59

VITAMIN D AND COVID 19

Forest plots of the association

between serum 25(OHD) levels
<20 ng/ml and COVID-19
outcomes.

A. COVID-19 mortality.
B. ICU admission.
C. Invasive mechanical ventilation
requirement.
D. Non-invasive ventilation
requirement.
E. SARS-CoV-2 positivity status.

Front Public Health. 2021; 9: 624559

TAKE HOME MESSAGE
• Vit D deficiency is a global problem
• Measurement of Vitamin D testing is not recommended for routine screening, but is
recommended in individuals at risk of vitamin D deficiency
• Measuring Total 25(OH)D is recommended
• Method for vitamin D testing are LC-MS/MS, Immunoassay, Protein Binding and RIA.
Immunoassay is the most common used.
• Traceability, precision, cross-reactivity to other vitamin D metabolites and affinity to DBP cause
variation of vitamin D result among assays.
• Vitamin D analytical performance criteria : traceable to reference method and standard with bias
< 5% and precision < 10% , enroll in EQAS and Vitamin D Certification program
• Recommended monitoring interval for vitamin D supplementation is 3 months using same assay
• Standardization and verification of reference range is needed.
• Vitamin D is a stable analyte and the recommended specimen is serum.