Biomaterial

Serum 25-hydroxyvitamin D (25(OH)D) and parathyroid hormone (PTH)


LASAC862 / LAS2B862 / LASAG862 / LAS3B862

Contact: Natasja van Schoor

Background
Vitamin D deficiency is common in older western individuals. Depending on country and used definition the prevalence of vitamin D deficiency ranges from 0 up to 90% (1).
The precursor of active vitamin D, cholecalciferol, is formed in the skin under influence of sunlight or obtained from nutrients, especially fatty fish. Cholecalciferol is hydroxylated in the liver into 25-hydroxyvitamin D3 (25(OH)D). Subsequently, the active metabolite (1,25-hydroxyvitamin D3 (1,25(OH)2D)) is formed in the kidney (2).
Vitamin D is essential in several physiological processes, such as the mineralization of bone (2). In addition, vitamin D deficiency is found to associate with several diseases and conditions, for example diabetes mellitus, low physical performance, chronic obstructive pulmonary disease, and (osteoporotic) fractures (3-6).
Parathyroid hormone stimulates the hydroxylation of 25(OH)D into 1,25(OH)2D. A negative feedback system through calcium and directly through 1,25(OH)2D is responsible for the balance between PTH and vitamin D status (2).

Measurements in LASA

Blood collection
Blood samples for measurement of serum 25(OH)D and parathyroid hormone (PTH) were obtained in 1995/96 (cycle C of first LASA cohort), 2009 (cycle G of first LASA cohort), 2002/03 (cycle 2B of second LASA cohort), 2009 (cycle G of second LASA cohort) and 2012/13 (cycle 3B of the third LASA cohort).

Measurement procedure & variable information
At cycle C and 2B, morning blood samples were drawn. Subjects were only allowed to take tea and toast, but no dairy products. At cycle G and 3B, fasting blood samples were drawn. Subjects were not allowed to take any food or drinks from midnight. The samples were centrifuged and stored at -20˚C until determination. Determination took place in 1997/98 for the samples from cycle C, in 2010/11 for the samples from cycles 2B and G, and in 2016 for cycle 3B. All analyses were performed at the Endocrine Laboratory of the VU University Medical Center.

At cycle C, blood samples were drawn from 1352 respondents. Measurements of 25(OH)D and PTH were available for 1320 individuals. At cycle 2B, blood samples were obtained from 748 individuals. Measurements were available for 739 respondents. At cycle G, blood samples were obtained from 935 respondents. Measurements of 25(OH)D were available for 917 respondents and measurements of PTH were available for 915 respondents. At cycle 3B, blood samples were obtained for 645 respondents. Measurements were available for 639 individuals.

For analyses on serum 25(OH)D, a competitive protein binding assay was used in 1997/98 (Nichols Diagnostics Capistrano, CA, USA) and in 2010/11 a radioimmunoassay was used (Diasorin, Stillwater, Minesota, USA). The Nichols device used serum, whereas the Diasorin device used EDTA for the measurement of 25(OH)D. To compare both methods, cross- calibration was performed by measuring 41 samples from 1995/96 at the Diasorin in 2010. Passing and Bablok regression analyses resulted in the following regression formula: Diasorin=8.0966 + 0.9218*Nichols. The correlation coefficient r=0.8765. In 2016, 25(OH)D was assessed by isotope dilution/online solid-phase extraction liquid chromatography/tandem mass spectrometry (ID-XLC-MS/MS) at the Endocrine Laboratory of the VU University Medical Center (Amsterdam, the Netherlands) (7). 25(OH)D2 and 25(OH)D3 were measured separately. 

Serum PTH was determined by an immunoradiometric assay in 1997/98 (Incstar Corp., Stillwater, MN, USA), and by immunometric assay, Luminescence (Architect, Abbott Laboratories, Diagnostics Division, Abbott Park, Chigaco, Illinois USA) in 2010/11. Both devices used EDTA for the measurement of PTH.

Lower limit of quantitation

Interassay coefficient of variation

Measurement cycle

C

2B & G

3B

C

2B & G

3B

Year of laboratory analyses

1997/98

2010/11                

 2016

1997/98

2010/11

 2016

25(OH)D

10 nmol/l

5 nmol/l

 4 nmol/l

10%

10%

 <8%*

PTH

0.7 pmol/l

0.5 pmol/l

12%

5%

* For concentrations between 25 and 180 nmol/l.

Descriptives

Mean serum 25(OH)D (standard deviation) (in nmol/L) and PTH (standard deviation) (in pmol/l)

C

2B

G

3B

Serum 25(OH)D

PTH

53.2 (24.0)

56.5 (20.3)§

65.3 (21.8)§

 68.7 (21.9)

3.6(2.1)

6.0 (2.5)

6.3 (2.9)

* See paragraph on outliers
§ values without outliers: 2B: 182.8 nmol/l, G: 624.4 nmol/l and 961 nmol/l
† values without outliers: C: 50.60 pmol/l, 2B: 51.76 pmol/l, G: 51.89 pmol/l

Standardized serum 25(OH)D values
In 2015, serum 25(OHD) values were standardized using the Vitamin D Standardization Program (VDSP) protocol as part of the European ODIN study (“Food-based solutions for optimal vitamin D nutrition and health through the life cycle”) (8, 9). In total, 166 frozen samples from 1995/96, 146 frozen samples from 2002/03 and 159 frozen samples from 2009 were reanalyzed in Ireland by a standardized and certified LC-MS/MS method, which was traceable to the National Institute of Standards and Technology higher-order Reference Measurement Procedure. The resulting serum 25(OHD) values were used to develop master regression equations, which were used to recalibrate the datasets of 1995/96, 2002 and 2009, respectively. This resulted in 1320 standardized serum 25(OH)D values in 1995/96, 739 in 2002/03 and 915 in 2009, which were added to the datasets.  

Deseasonalized serum 25(OH)D values
In one LASA publication, longitudinal changes and seasonal variations of original (unstandardized) serum 25(OH)D values were analyzed (10). Seasonal variations were described using a cosine curve. When 25(OH)D levels are used in (association) analyses, adjustment for season is recommended. A simple method is to add ‘season of blood collection’(winter/summer or winter/spring/summer/autumn) as covariate to the model (e.g. 11, 12). However, a more accurate method may be to use deseasonalized serum 25(OH)D concentrations, which was done in the following LASA publication (13). In this case, deseasonalization of the 25(OH)D concentrations was needed because the intra-individual change between two 25(OH)D measurements (often from different seasons) was calculated. For this deseasonalization, a sinusoidal regression model was fitted:

original 25(OH)D concentration =ß0 + ß1 sin(2πT / 365.25) + ß2 cos(2πT / 365.25)

original 25(OH)D concentration: original 25(OH)D concentration
T: the day of the year of blood draw
ßj(j=0, 1, 2)
: estimated regression coefficients

To obtain individual deseasonalized 25(OH)D concentrations, the residuals (difference between the predicted value and the original value) from this model were saved and added to the annual mean of the model (the intercept: ß0) (14-16). In this way, the seasonal variation of 25(OH)D concentration was adjusted for.

Deseasonalizing can be performed for all participants or for subsamples. In the paper on change in 25(OH)D (13), deseasonalizing was performed separately for each cohort and each measurement cycle (i.e. four times): for C (1995/96) and G (2009) for the first cohort, and for 2b (2002/03) and G (2009) for the second cohort. The reason for this was that the cohorts differ from each other (e.g. in age, generation) and possibly also in the seasonal variation of their 25(OH)D level over the year.

In this file, a syntax for deseasonalizing can be found for cycle C (1995/96, first cohort). This syntax can be adapted for deseasonalizing the 25(OH)D values of 2002/03 (second cohort), 2008/09 (first and second cohort) and 2016 (third cohort).

Remarks:
  • The value of respondent R25750 in 2009 becomes negative (-7.11 nmol/l) after deseasonalizing.
          • When calculating the changes in serum 25(OH)D values over time, some participants had remarkable changes: a decrease from high to very low (R25750) and an increase from normal or high to very high (R51288, R58015 and R17010). These participants might be excluded from the analytical sample in sensitivity analyses.
Outliers
In cycle G, two respondents had unreliable high measured serum 25(OH)D values (624.4 nmol/l and 961 nmol/l), which were replaced by a missing code in the dataset. In cycle 2B, one respondent’s serum 25(OH)D was 182,8 nmol/L (R54047). Although, from a statistical view this value is an outlier, it must be researcher’s own decision whether this value will be handled as missing in the analyses. In addition, in cycle 2B and G, there is one respondent with a PTH level above 50 pmol/l (R41618 at 2B; R25139 at G). These values could be explained by the respondents’ poor renal function. In cycle C, one respondent had a PTH level above 50 pmol/l (R11791). Unfortunately, we had no information on respondent’s renal function. We advise to mark these values as missing in the analyses.

Availability of data per wave

Numbers per wave

25(OH)D

B

C

2B*

G

3B*

All regions

1320

739

916

639

* 2B=baseline second cohort;
   3B=baseline third cohort

PTH

 

B

C


2B*

G



3B*

All regions

 

1320

739

915

 

* 2B=baseline second cohort;
   3B=baseline third cohort

Previous use in LASA
The LASA data has been used to investigate vitamin D deficiency as independent risk factors for osteoporotic fractures. Moreover, the role of vitamin D has been assessed in relation to other indicators of physical functioning, including factors underlying fractures such as bone mineral density, as well as sarcopenia and overall physical performance, confirming that vitamin D status is implicated in much musculoskeletal morbidity in old age, and identifying threshold levels of vitamin D at which intervention is warranted. In addition, standardized serum 25(OH)D values were used in several meta-analyses (e.g. ref 8 by Cashman et al) and several LASA publications (e.g. ref 13). Deseasonalized 25(OH)D values were used in the LASA article on intra-individual 25(OH)D changes over time (ref 13).

Examples of LASA articles on vitamin D:

  • Elstgeest LEM, de Koning EJ, Brouwer IA, van Schoor NM, Penninx BWJH, Visser M. Change in serum 25-hydroxyvitamin D and parallel change in depressive symptoms in Dutch older adults. Eur J Endocrinol. 2018 Jul 27.
  • De Koning EJ, Elstgeest LEM, Comijs HC, Lips P, van Marwijk HW, Beekman AF, Visser M, Penninx BWJH. Vitamin D status and depressive symptoms in older adults: a role for physical functioning? Results from the Longitudinal Aging Study Amsterdam. Am J Geriatr Psych. 2018. doi: 10.1016/j.jagp.2018.03.004. Epub 2018 Mar 12.
  • Van Schoor NM, Heymans MW, Lips P. Vitamin D status in relation to physical performance, falls and fractures in the Longitudinal Aging Study Amsterdam: A reanalysis of previous findings using standardized serum 25-hydroxyvitamin D values. J Steroid Biochem Mol Biol. 2018 Mar;177:255-260.
  • Sohl, E., De Jongh, R.T., Heymans, M.W., Van Schoor , N.M., Lips, P.T.A. Thresholds for Serum 25(OH)D Concentrations With Respect to Different Outcomes. The Journal of Clinical Endocrinology & Metabolism 2015; 100, 6, 2480-2488.
  • Sohl, E., De Jongh, R.T., Swart, K.M.A., Enneman, A.W., van Wijngaarden, J.P., van Dijk, S.C., Ham, A.C., van der Zwaluw, N.L., Brouwer-Brolsma, E.M., Van der Velde, N., De Groot, L.C.P.G.M., te Velde, S.J., Lips, P.T.A., Van Schoor , N.M. (2015). The association between vitamin D status and parameters for bone density and quality is modified by body mass index. Calcified Tissue International 2015; 96, 2, 113-122.
  • Van Schoor NM, Knol DL, Deeg DJH, Peters FPAMN, Heijboer AC, Lips P. Longitudinal changes and seasonal variations in serum 25-hydroxyvitamin D levels in different age groups: results of the Longitudinal Aging Study Amsterdam. Osteoporos Int 2014;25(5):1483-91.
  • Van den Heuvel, E.G.H.M., Van Schoor , N.M., De Jongh, R.T., Visser, M., Lips, P.T.A. Cross-sectional study on different characteristics of physical activity as determinants of vitamin D status; inadequate in half of the population. European Journal of Clinical Nutrition 2013; 67, 360-365.
  • Sohl, E., De Jongh, R.T., Heijboer, A.C., Swart, K.M.A., Brouwer-Brolsma, E.M., Enneman, A.W., De Groot, L.C.P.G.M., Van der Velde, N., Dhonukshe-Rutten, R.A.M., Lips, P.T.A., Van Schoor , N.M. Vitamin D status is associated with physical performance: the results of three independent cohorts. Osteoporos International 2013; 24, 187-196.
  • Kuchuk NO, Pluijm SM, Van Schoor NM, Looman CW, Smit JH, Lips P. Relationships of serum 25-hydroxyvitamin D to bone mineral density and serum parathyroid hormone and markers of bone turnover in older persons. J Clin Endocrinol Metab 2009;94: 1244–50.
  • Van Schoor NM, Visser M, Pluijm SMF, Kuchuk N, Smit JH, Lips P. Vitamin D deficiency as a risk factor for osteoporotic fractures. Bone 2008;42:260–66.
  • Wicherts IS, Van Schoor NM, Boeke AJ et al. Vitamin D status predicts physical performance and its decline in older persons. J Clin Endocrinol Metab 2007;92:2058–65.
  • Snijder MB, van Schoor NM, Pluijm SM, van Dam RM, Visser M, Lips P. Vitamin D status in relation to one-year risk of recurrent falling in older men and women. J Clin Endocrinol Metab. 2006 Aug;91(8):2980-5. Epub 2006 May 9.
  • Visser M, Deeg DJ, Lips P. Low vitamin D and high parathyroid hormone levels as determinants of loss of muscle strength and muscle mass (sarcopenia): the Longitudinal Aging Study Amsterdam. J Clin Endocrinol Metab 2003;88: 5766–72.

References