# Presentation: 146974

## The Assessment of Circulating 25(OH)D and 1,25(OH)2D: Emergence as Clinically Important Diagnostic Tools

- Bruce W. Hollis, Ph.D.
*Departments of Pediatrics, Biochemistry and Molecular Biology Medical University of South Carolina Charleston, SC *

## Vitamin D

- 25(OH)D
- 25(OH)D3-26,23-lactone
- 24,25(OH)2D
- 25,26(OH)2D
- 1,25(OH)2D
- 1,24,25(OH)3D
- 1,25,26(OH)3D

- Vitamin D2 and Vitamin D3 Metabolites that we could measure in a single plasma sample using hplc to separate

## Plasma (3-5 ml) + 3H Metabolites

- Diethylether (2X)

- Methanol: Methylene Chloride (1:3)

- Backwash w/ 0.1m NaPO4 (pH 10.5)

- Sephadex LH-20
- (1:1:9 Chloroform : Methanol : Hexane)

- 25(OH)D

- Sephadex LH-20
- (50:50 Hexane: CHCl3)
- OR
- Lipidex 5000
- (10:90 CHCl3:Hexane)

- hplc on Zorbax Sil
- (4:96 IP :Hexane)

- Quantitate both D2 & D3
- Forms by UV Absorbance

- Dihydroxyvitamin D

- Vitamin D

- Lipidex 5000
- (5:95 CHCl3:Hexane)

- Hplc on Zorbax Sil
- (11:89 IPA: Hexane)

- 24, 25-(OH)2D
- Complex

- 25, 26-(OH)2D3

- 1, 25-(OH)2D

- Quantitate Both  D2 & D3 Forms Simultaneously by Competitive Protein Binding Assay

- Hplc on Zorabx Sil
- (0.25:99.75 IPA:MeCl2)

- Hplc on Zorbax ODS
- (4:96 H2O:MeOH)

- Quantitate both D2 & D3
- By UV Absorbance

- Quantitate by Competitive Protein Binding Assay

- Hplc on Zorbax Sil
- (2:98 IPA:MeCl2

- 25-OHD3-26, 23 Lactone

- 24, 25-(OH)2D2

- 24, 25-(OH)2D3

- 25, 26-(OH)2D2

- Quantitate Individually by competitve protein binding assay

## Circulating 25(OH)D is the best indicator of nutritional vitamin D status.Circulating 1,25(OH)2D confirmatory test for disorders involving the vitamin D endocrine system.

## Indication for 25(OH)D Measurement

- When nutritional deficiency of vitamin D is suspected
- Intestinal malabsorption syndromes
- Limited exposure to the sun
- Limited intakes of oral vitamin D supplements
- Aged, homebound patients
- Skin pigmentation
- EVERYBODY should know their 25(OH)D status

## Indication for 1,25(OH)2D Measurement

- Monitor therapy
- Disease State
- Kidney disease
- Hyperparathyroidism
- Sarcoidosis
- Research

## Early 25(OH)D assays

- J.G. Haddad & K.J. Chyu, Competitive protein-binding radioassay for 25-hydroxycholecalciferol., Journal of Clinical Endocrinology and Metabolism 33  (1971) 992-995.
- R.E. Belsey, H.F. DeLuca & J.T. Potts, Jr., A rapid assay for 25-OH-vitamin D3 without preparative chromatography, J Clin Endocrinol Metab 38 (6) (1974) 1046-1051.

## Various Methods

- Competitive-Protein Binding Assays (CPBA)
- Advantages
- Inexpensive
- Small sample size
_**Co-specific **_**for  25(OH)D****2**** and 25(OH)D****3**
- Disadvantages
- Subject to nonspecific interference
- Requires organic extraction and solvent evaporation
- Requires preparative chromatography
- Is limited to the use of 3H-tracer and thus liquid scintillation counting

## Physician’s View

- A lot of ergocalciferol 50,000 U (Drisdol and Calciferol) is used.
- Don't have easy access to large doses of vitamin D3 at this point is main reason to use vitamin D2.
- Works for me more often than not.

## UV Quantitation following HPLC (Gold Standard)

- J.A. Eisman, R.M. Shepard & H.F. DeLuca, Determination of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 in human plasma using high pressure liquid chromatography., Analytical Biochemistry 80  (1977) 298-305.
- G. Jones, Assay of vitamins D2 and D3, and 25-hydroxyvitamins D2 and D3 in human plasma by high-performance liquid chromatography., Clinical Chemistry 24  (1978) 287-298.

## UV Quantitation following HPLC (Gold Standard)

- Advantages
- Separate quantitation of 25(OH)D2 and 25(OH)D3
- Very stable and repeatable
- Disadvantages
- Larger sample size required
- Very costly equipment required
- Internal standard [3H]-25(OH)D3, laurophenone
- Need preparative chromatography
- Sometimes assay is subject to interfering UV compounds
- High level of technical expertise required
_**Slower sample turn around time**_

## B.W. Hollis & J.L. Napoli, Improved radioimmunoassay for vitamin D and its use in assessing vitamin D status, Clin Chem 31 (11) (1985) 1815-1819.

B.W. Hollis, J.Q. Kamerud, S.R. Selvaag, J.D. Lorenz & J.L. Napoli, Determination of vitamin D status by radioimmunoassay with an 125I-labeled tracer., Clinical Chemistry 39  (1993) 529-533.

- B.W. Hollis, J.Q. Kamerud, S.R. Selvaag, J.D. Lorenz & J.L. Napoli, Determination of vitamin D status by radioimmunoassay with an 125I-labeled tracer., Clinical Chemistry 39  (1993) 529-533.

- Radioimmunoassay (RIA)

## Radioimmunoassay (RIA)

- Advantages
- Small sample size
- Incorporation of 125I into tracer
- Quantitative extraction so no internal tracer required
- Not subject to nonspecific interference
- Rapid, inexpensive and accurate
- Disadvantages
- Still requires the use of radionuclides
- Some RIAs discriminate between 25(OH)D2 and 25(OH)D3

## LC-MS/MS Quantitation

- Same limitations as HPLC quantitation although sample size is quite small (200 ul)
- Difficult to resolve 3-epi-25(OH)D3
- An inactive metabolite with respect to calcium metabolism

## 25(OH)D3

- HO

- HO

- HO

- HO

- HO

- HO

- 25(OH)D3

- 25(OH)D2

- 3-epi-25(OH)D3

## *RIA doses not detect 3-epi-25(OH)D3

- *RIA doses not detect 3-epi-25(OH)D3

- *Total 3-epi-25(OH)D3 by LC-MS/MS

## Commercial 25(OH)D Assay RIA/EIA Methods

- DiaSorin RIA, 125I-ligand
- 	-ACN extraction, primary and secondary Ab
- 	-Co-specific fo  25(OH)D2 and 25(OH)D3
- IDS RIA, 125I-ligand
- 	-ACN extraction, primary and secondary ab
- 	-discriminates against 25(OH)D2 (0.75)
- IDS EIA
- 	-no extraction
- 	-biotin labeled ligand, avidin labeled HRP
- 	-Co-specific for 25(OH)D2 and 25(OH)D3

## Automated Instrumentation Methods

- DiaSorin Liason, chemilumenescence
- 	-whole serum, w/antibody coated particles
- 	-Recognizes 25(OH)D2 and 25(OH)D3 equally
- 	-180 samples/h
- Nichols Advantage, chemilumenescence
- 	-whole serum, DBP coated particles
- 	-problem recognizing 25(OH)D2
- 	-170 samples/h
- IDS EIA on Grifols TRITURUS

## DiaSorin Liaison

- 180 determinations/h

- Nichols Advantage
- 170 determinations/h

## DiaSorin Liaison 

|   | current kit | TOTAL kit |
| --- | --- | --- |
| analytical sensitivity | 2.1 ng/mL | 0.57 ng/mL |
| functional sensitivity | ~5 ng/mL | ~3 ng/mL |

## precision

|   | current kit |  |  | TOTAL kit |  |  |
| --- | --- | --- | --- | --- | --- | --- |
|   | Mean
(ng/mL) | Intra-assay CV | Inter-assay CV | Mean
(ng/mL) | Intra-assay CV | Inter-assay CV |
| KC1 | 16.6 | 12% | 14% | 12.5 | 8% | 8% |
| KC2 | 56.2 | 10% | 11% | 45.0 | 4% | 5% |
| PP1 | 11.0 | 11% | 14% | 13.1 | 3% | 6% |
| PP3 | 35.0 | 7% | 12% | 36.3 | 4% | 5% |
| PP4 | 15.8 | 9% | 11% | 16.0 | 3% | 5% |
| PP5 | 61.8 | 4% | 9% | 72.8 | 3% | 5% |
| PP9 | 53.6 | 6% | 8% | 57.4 | 6% | 8% |
| PP10 | 73.5 | 5% | 8% | 89.6 | 3% | 5% |

## Direct Methods

- HPLC, [3H]-internal standard or laurophenone
- 	-extraction
- 	-pre-purification
- 	-HPLC/UV quantification
- LC-MS/MS, deuterated internal standard
- 	-ACN extraction
- 	-HPLC
- 	-mass detection

## Vitamin D External Quality Assessment Scheme (DEQAS)

- Currently 322 worldwide participants in this QC survey
- 5 samples sent quarterly for 25(OH)D and /or 1,25(OH)2D quantitation

## 10

- 20

- 30

- 40

- 50

- 60

- 70

- N=53

- N=16

- N=16

- N=21

- N=9

- N=4

- N=21

- DEQAS

- 0

- DiaSorin RIA

- DiaSorin Liason

- IDS RIA

- IDS OCTEIA

- Nichols Advantage

- HPLC

- LC-MS

- ng/ml

- 39.2

- 36.8

- 36.4

- 47.3

- 58.9

- 42.6

- 38.2

## DEQAS July 2005 

- DEQAS July 2005
- Ability to measure 50.9 nmol/L of added 25(OH)D2 or 25(OH)D3

- 0

- DiaSorin RIA

- DiaSorin Liason

- IDS RIA

- IDS OCTEIA

- Nichols Advantage

- HPLC

- LC-MS

- % Recovered

- 20

- 40

- 60

- 80

- 100

- 120

- 25(OH)D3

- 25(OH)D2

- N=53

- N=16

- N=16

- N=21

- N=9

- N=4

- N=21

- 82.1

- 83.3

- 81.4

- 88.6

- 54.2

- 29.1

- 78.8

- 56.4

- 46.3

- 43.2

- 112.2

- 97.1

- 111.5

- 118.1

## Mean deviation  (% bias) of the method mean from the target value (ALTM), over the period January 2000 to January 2004 unless stated otherwise. 

**M**

**E**

**A**

**N**

** **

**%**

** **

**B**

**I**

**A**

**S**

- -

- 1

- 0

- 0

- 1

- 0

- 2

- 0

- 3

- 0

- 4

- 0

**% BIAS FROM ALTM**

- Mean deviation  (% bias) of the method mean from the target value (ALTM), over the period January 2000 to January 2004 unless stated otherwise.

- Accuracy of 25OHD methods used by DEQAS participants

- DiaSorin RIA

- IDS RIA

- IDS OCTEIA
- (10/02-1/04)

- CPBA

- HPLC

- Nichols Advantage
- (7/01-1/04)

## Head to Head comparisons

## Comparison of hplc vrs. LC-MS/MS 

- Comparison of hplc vrs. LC-MS/MS
- Lensmeyer et al.,  Clin Chem 52:2006

## Method Comparison - 25(OH)D2 negative samples 

- Method Comparison - 25(OH)D2 negative samples

- data from Dr. Ravinder Singh, Mayo Clinic

## CORRELATION OF SAMPLEScontaining significant amount of 25(OH)D2

- data from Dr. Ravinder Singh, Mayo Clinic

## LC-MS is clearly a superior analytical technique by which to assess circulating 25(OH)D

- The data clearly do not support this statement !
- DEQAS results demonstrate that DiaSorin Liaison and LC-MS are clinically equivalent IF LC-MS is properly performed.
- Further, a recent publication by Binkley et. al. (Clin Chem 2006) states that separate determination and reporting of 25(OH)D2 and 25(OH)D3 does nothing more than confuse the physician.
- It is TOTAL 25(OH)D that is important !

## data from Dr. Ravinder Singh, Mayo Clinic Clin Chem 2005: 51 (Suppl 6):A114-5

- data from Dr. Ravinder Singh, Mayo Clinic Clin Chem 2005: 51 (Suppl 6):A114-5

## DEQAS: Comparison of Low, Medium and High Standards                (n)             25(OH)D (nmol)       % CV

- Liaison		29		19.4		29.8
- LC-MS		13		18.5		32.3
- Liaison		37		34.1		18.9
- LC-MS		14		38.1		28.5
- Liaison		38		96.6		16.1
- LC-MS		14		104.7		21.9

## DEQAS Linearity Study (1/07)25(OH)D nmol              1        2       3        4       5

- DS RIA  19.4  34.1  52.5  70.1  90.9
-                         (37.3)   (55.2)  (73.1)
- DS LS    21.3  39.6  57.4  75.3  91.0
-                         (38.8)  (56.2)  (73.6)
- LC-MS   18.5  38.1  63.4  80.8  104.7
-                         (40.1)  (61.6)  (83.2)
- ALL HAVE EXCELLENT LINEARITY

## Slide 35

## Slide 36

## Who Is Right?                    25(OH)D2    25(OH)D3    25(OH)D

- LabCorp DS-LI                              15.6
- Quest LC-MS-MS  <4        45.0       45.0
- Hollis Lab DS-RIA                          21.3
- Mayo Labs LC-MS <1        20.9       20.9
- All values in ng/ml

## Picking an assay platform-recognize limitations

- Assay recognizes vitamin D2 and vitamin D3 metabolites _equally_. Sigma sells standards!!  _**Be sure amounts are correct and compounds are pure.**_
- HPLC or LC-MS/MS
- Low throughput
- Cleanup steps are crucial
- Sample volume can be an issue
- More expertise required
- RIA or automated platform
- Higher throughput, small sample volume
- Can be more variable
- Prefer methods that have been validated against gold standard methods and accuracy is continuously being evaluated in external quality assessment schemes

## 1,25(OH)2D

- RIA
- 	DiaSorin--ACN, C18OH column
- 	IDS--Solid phase extraction using Mab
- RRA
- 	Immunodiagnostiks--Two column extraction
- ELISA
- 	IDS--Immunoextraction
- 	Immunodiagnostiks--Two column extraction

## No need to develop an in-house 1,25(OH)2D assay.
Commercially available kits work fine
Each laboratory should have own internal control sample
Pool of serum or plasma run in each assay to evaluate intra and inter assay variability
Need for Standard Reference Standard (NIST)

- Commercially available kits work fine
- Each laboratory should have own internal control sample
- Pool of serum or plasma run in each assay to evaluate intra and inter assay variability
- Need for Standard Reference Standard (NIST)

- Other Issues

## Conclusion

- There are several valid methods by which to determine circulating 25(OH)D and/or 1,25(OH)2D.
- BE SURE THAT WHOMEVER PERFORMS THEM KNOWS WHAT THEY ARE DOING, HAS VALIDATED THE METHOD AND PARTICIPATES IN DEQAS !

- THANK YOU