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3 Measurement of Vitamin D and Its Metabolites: Analytical and Clinical Considerations

3 Measurement of Vitamin D and Its Metabolites: Analytical and Clinical Considerations

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5 Measurement of Circulating 1,25-Dihydroxyvitamin D


Table 5.1 Analytical characteristics of vitamin D metabolites





Range of








analyzers, HPLC,








analyzers, LCMS/










(vitamin D3/D2)


Free (or





Low concentration. Very lipophilic;

difficult to remove from VDBP; short

half-life; no standardization; no

reference method

Very lipophilic; necessity to displace

from VDBP; standardization

problems; issues observed in some

subgroups of population; can be found

as 25(OH)D2 and 25(OH)D3;

interference with 24,25(OH)2D with

some immune-assays and C3-epimer

with some LCMS/MS

Very low concentration; short half-life;

can be found as 1,25(OH)2D2 and

1,25(OH)2D3; extraction step often

needed prior to determination; lack of

standardization; no reference method

Low concentration; need to have very

good sensitivity to exclude

24-hydroxylase mutation; no

standardization; no reference method

Very low concentration. Need to avoid

interference by 25(OH)D bound to

VDBP or albumin. No reference

method, no standardization. Can be

found as free/bioavailable 25(OH)D2 or

D3. Polymorphism of VDBP. Formula

not extensively validated

Abbreviations: HPLC high performance liquid chromatography, LCMS/MS liquid chromatography

mass spectrometry/mass spectrometry, ELISA enzyme linked immunosorbent assay, RIA radioimmunoassay, VDBP vitamin D–binding protein



The number of 25(OH)D determinations has dramatically increased over the last

10 years. This increasing number of requests has led most of the clinical laboratories

to move from the DiaSorin RIA, the most widely used method in the 1990s and early

1920s, to methods presenting a larger throughput, i. e. automated immunoassays or

liquid chromatographs coupled with two mass spectrometers in tandem (LC-MS/

MS). Anyway, one has to remember that analytical 25(OH)D determination is far

from an easy task. Indeed, several important problems have to be overcome to correctly assess this parameter. Among them, the very high lipophilic nature of the

molecule and its strong association with vitamin D binding protein (VDBP) and, to

a lesser extent, albumin, necessitates a thorough separation step and, for the


E. Cavalier and P. Delanaye

one-phase immunoassays, a good equilibrium between the analyte and the antibodies used in the kits [8]. VDBP can be present at different concentrations according to

some physiological or pathological conditions, like race [9], pregnancy or CKD,

which could influence the kinetic of the liberation of the molecule [10, 11]. Vitamin

D can be found as vitamin D2 or D3 and the assay should measure both 25(OH)D2

and 25(OH)D3 [12]. Different other metabolites of vitamin D, i.e. C3-epimer or

24,25(OH)2-vitamin D can be present in the serum of the patients at different levels,

possibly interfering with either immunoassays or LC-MS/MS methods [13]. Just like

any other immunoassays, vitamin D assays are prone to heterophilic antibodies interference, leading to potential spurious results [14]. Last but not least, the standardisation of the different assays remains a major problem. Hopefully, we have now a

commonly accepted reference method and an ongoing a worldwide standardisation

program, coordinated by the Center of Diseases Control (CDC), the National Institute

of Standards and Technology (NIST) and the University of Ghent [15]. All these

efforts have globally improved the global concordance of different assays for 25(OH)

D determination in the “normal healthy” population, but some issues are still remaining, notably in “special” populations, like pregnant women and African (or Asian)

subjects because of their high and low, respectively, concentrations of VDBP .

CKD and patients treated by hemodialysis (HD) or peritoneal dialysis (PD) are also

particular populations in whom 25(OH)D determination remain problematic. Indeed,

these patients present a serum matrix which is quite different from the general matrix

of the “healthy” population. Uremic toxins, present in the samples of CKD patients,

but not in the calibrators of the assays, can indeed induce a matrix effect. Protein concentrations can also be quite different in patients suffering from nephrotic syndrome

and this can also lead to matrix effects. Proteins are also, to various extents, carbamylated and it remains unknown whether this affects the assays or not. As a result,

most of the immunoassays available on the market tend to underestimate 25(OH) in

HD and PD patients compared to LCMS, even if they are correctly calibrated [16].

Unfortunately, few manufacturers have taken this issue into consideration, and

assays should be improved. In 2012, Heijboer et al. compared 6 routine immunoassays to a LCMS/MS method and shown that, if the slopes ranged from 0.83 (IDS

iSYS) to 1.09 (Abbott Architect) in healthy individuals, the slopes observed in HD

patients were ranging from 0.50 (Abbott Architect) to 0.82 (DiaSorin RIA). One

year later, Depreter et al. showed that the slopes on Abbott Architect were of 0.39

and 0.50, for Roche Modular 0.92 and 0.92 and for IDS iSYS of 0.61 and 0.80 for

HD and healthy subjects, respectively compared to a LCMS/MS method. They also

concluded that iSYS showed better performance than Modular E170 (Roche) for

the HD patients in the 25(OH)D range 10–40 ng/ml, due to a higher number of

apparently underestimated concentrations by Modular.



As already mentioned, measurement of circulating levels of 1,25(OH)2D should

not be used to evaluate the vitamin D status which is consensually assessed through

the measurement of 25(OH)D [17]. However, it is important for the differential

5 Measurement of Circulating 1,25-Dihydroxyvitamin D


diagnosis of several disorders of calcium/phosphorus metabolism, especially in

case of hypercalcemia, hypercalciuria, and low serum PTH level [18], or in case of

rickets/osteomalacia which persists after vitamin D supplementation [19]. Calcitriol

serum levels are modified in many clinical situations, increased for example during pregnancy or primary hyperparathyroidism (PHPT), and decreased in CKD or


Measurement of 1,25(OH)2D concentration in serum is not an easy task due to its

hydrophobic nature and because it circulates at picomolar levels compared to

25(OH)D which circulates at a 1,000-fold higher concentration. Currently available

1,25(OH)2D assays are either radio-immunoassays that require time-consuming

extraction procedures and long incubation times, semi-automated immunoassays

that also require extraction procedures or LCMS/MS methods (that are not available

in every lab). The sensitivity of the immunoassays is generally not compatible with

the circulating amounts observed in CKD and HD patients. Finally, an interference

between very high 25(OH)D levels (as seen in intoxications) and 1.25 assays occurs,

leading to false positive values [20].

Very recently, a fully automated and rapid method has been launched by DiaSorin

on Liaison XL which does not require an extraction step, needs a low sample volume and has very interesting analytical features, among which a very low limit of

quantification [21]. With the help of this assay, we found low, but often detectable,

1,25(OH)2D in our cohort of HD patients. Also, the levels of 1,25(OH)2D were

significantly increased after 1 year of vitamin D (50.000 IU cholecalciferol/month

for 1 year) supplementation versus placebo [22]. An increase in serum 1,25(OH)2D

after supplementation with 25(OH)D or vitamin D3 in dialysis patients has previously been reported, this is a proof of extra-renal origin of 1-alpha hydroxylase.

Another proof is the detectable concentration of 1,25(OH)2D in serum of anephric

patients [23].



The method of choice for the determination of 24,25(OH)2 levels is the LCMS/

MS. However, one has to be sure that the method used is i) sensitive enough to differentiate a low value from an undetectable value that should be observed in children

suffering from mutations of the CYP24A1 gene ii) specific enough to avoid to consider another metabolic as being 24.25(OH)2D. The quantification of 24.25(OH)2 is

also challenging because of its low serum level and low ionization efficiency [24].

Different authors have described LC-MS/MS methods for simultaneous determination of the major vitamin D metabolites, including 25(OH)D, 1,25(OH)2D and

24,25(OH)2D. The analytical sensitivity of these methods can vary according to the

way the authors have estimated it and it is thus difficult to compare their performances and to know if they are suitable for the clinical purposes. For instance, Berg

et al. have found that the mean 24.25(OH)2D values (± standard deviation) in community-dwelling White and Black Americans were of 3.6 ± 2.0 and 2.1 ± 1.3 ng/mL,

respectively. They present a LOQ of 0.156 ng/mL for their LCMS/MS method,

which is quite low, but which has been obtained on four replicates only. In the same


E. Cavalier and P. Delanaye

vein, de Boer et al. find that 24.25(OH)2D values obtained in 9,596 participants from

5 cohort studies and clinical trials range from 0.8 ± 0.5 to 4.1 ± 2.1 ng/mL [25]. Again,

it is difficult to evaluate the performance of the method as the authors only provide a

mean CV of 14.6 % which was obtained on a set of 20 samples measured nine times

on a spanning period of 6 months Kaufman et al. showed that the CV obtained in

14 days on a sample presenting a value around 2.5 ng/mL was 5–7 %, but no limit of

quantification was calculated [26]. If we insist on these analytical details, it is because

there is a growing interest on measurement of 24.25(OH)2D, 25(OH)D and

1,25(OH)2D. Kaufman et al. consider that the ratio of 25-OH-D to 24,25-(OH)2D is

as a pathophysiologically useful ratio as a novel approach for predicting vitamin D

deficiency. The ratio can also be useful for the differential diagnosis between

Idiopathic Infantile Hypercalcemia due to loss-of-function CYP24A1 mutations and

CYP24A1 defects from hypervitaminosis D during vitamin D intoxication [26].

Binkley has recently suggested that the ratio cholecalciferol 24.25(OH)2D could be

used to facilitate “treat-to-target” paradigm and to guide vitamin D supplementation

[7]. In CKD patients, Bosworth et al. have found that the 24,25(OH)2D concentration was strongly associated with glomerular filtration rate (GFR) [27]. In their study,

non-Hispanic black race, diabetes, albuminuria, and lower serum bicarbonate were

also independently and significantly associated with lower 24,25(OH)2D concentrations. The 24,25(OH)2D concentration was more strongly correlated with that of

PTH than was 25(OH)D or 1,25(OH)2D. According to these results, the authors concluded that CKD was thus a state of stagnant vitamin D metabolism characterized by

decreases in both 1,25(OH)2D production and vitamin D catabolism. Indeed, they

suggest that PTH and FGF23 are not the main drivers of vitamin D catabolism in

CKD but that, due to a decrease in renal mass, less delivery of 25(OH)D to proximal

tubular cells, or lower net metabolic capacity of the proximal tubular cells is the

major determinant of renal 24,25(OH)2D production and serum 24,25(OH)2D concentration. Low 24,25(OH)2D may also identify risk of CKD complications as it is

strongly associated with hyperparathyroidism, perhaps because 24,25(OH)2D concentration reflects the extent to which vitamin D metabolism is deranged in

CKD. Finally, the same team also found that low plasma concentrations of 25(OH)D

and 24,25-dihydroxyvitaminD were associated with increased risk of microalbuminuria in type 1 diabetes [28].


Vitamin D–Binding Protein and Free or

Bioavailable Vitamin D

The vitamin D binding protein (VDBP) is the major plasma carrier protein of vitamin D and its metabolites. VDBP also exerts several other important biological

functions, like actin scavenging, fatty acid transport and macrophage activation.

The molecular weight of VDBP is similar to the one of albumin (52–59 kDa). VDBP

is in molar excess (5 × 10−6 M), compared to 25(OH)D (5 × 10−8 M) and has a rapid

turnover rate. This large molar excess may play an important role in protection

5 Measurement of Circulating 1,25-Dihydroxyvitamin D


against vitamin D intoxication. VDBP has also a much shorter plasma half-life

(2.5 days) than 25(OH)D.

An important VDBP polymorphism is observed in humans [29]. Three alleles

Gc1F, Gc1S and Gc2 are the mostly known, but more than 120 other rare variants

have been identified [17]. VDBP has an interesting geographical distribution: whiteskinned individuals have a relatively lower frequency of the Gc1F-allele and a

higher frequency (50–60 %) of the Gc1S-allele. The Gc1F-allele frequency is markedly higher among black Americans and black Africans. The Gc1F- and Gc1S-allele

frequencies display a typical geographical cline from Southeast Asia, through

Europe and the Middle East, down to Africa [30]. The observed variation in the

Gc-allele frequencies in different geographic areas may be correlated with skin pigmentation and intensity of sun light exposure. Pigmented (black) and keratinized

(yellowish) skin types are characterized by a lower rate of UV light penetration and

a higher susceptibility to rickets. The higher frequency of Gc1F in dark skinned

persons may be explained by its greater affinity for and more efficient transport of

vitamin D metabolites.

Regarding affinity, VDBP has a different one for the different metabolites transported: VDBP binds 88 % of serum 25(OH)D with high affinity (Ka = 5 × 108 M−1),

85 % of serum 1,25(OH)2D with a ten-times lower affinity (Ka = 4 × 107 M−1), leaving 0.40 % ‘free’ and the remainder associated with other serum proteins, mainly

albumin [31].

Bioavailable 25OH-D is defined as circulating 25(OH)D not bound to VDBP. The

bioavailable fraction consists of albumin-bound 25(OH)D and the free, defined as

circulating 25(OH)D bound to neither VDBP nor albumin. To be further metabolized or to exert biological activities, 25(OH)D needs to enter target cells. Free

25(OH)D can enter cells passively: this appears to be an ubiquitous mechanism.

According to the “free-hormone hypothesis”, bound fractions of 25OH-D may be

unavailable to enter cells [32]. However, bound 25(OH) (and particularly VDBPbound 25(OH)D) can be sequestered by cubulin on the cell surface before being

internalized by megalin. This megalin cubulin complex of endocytosis is abundant

in the proximal tubular epithelium of the kidney and is also expressed in other cells

such as osteoblasts.

To date, there are no good assays for the measurement of free or bioavailable

vitamin D. Elisa methods have been developed but are lacking sensitivity. LCMS/

MS could be the solution, but it remains a technical challenge. Calculation of the

free fraction could thus be an alternative to lab measurement. Free 25OH-D can be

calculated using specific formula: a simple one was published by Bikle et al. [33]

and a more complex one was adapted from Vermeulen’s work related to estimation

of free testosterone [34]. These formulas are both based on 25(OH)D, albumin and

VDBP concentrations, as well as defined binding proteins’ affinities for 25(OH)

D. Taking the affinity of VDBP for 25(OH)D according to the genotype of the subjects, Powe et al. have shown that, if Black Americans had lower VDBP and lower

25(OH)D than their White counterparts, their bioavailable 25(OH)D was similar

[9]. Accordingly, the low levels of total 25(OH)D frequently observed in Black

Americans does not probably indicate a true vitamin D deficiency and bioavailable


E. Cavalier and P. Delanaye

25(OH)D might be more appropriate than total 25(OH)D to detect vitamin D sufficiency. In a population of young healthy subjects, the same authors also demonstrated that bioavailable 25(OH)D levels was independently associated with bone

mineral density (BMD) in multivariate regression models adjusted for age, sex,

body mass index, and race. In this study, free and bioavailable 25(OH)D were more

strongly correlated with BMD than total 25(OH)D [35]. Bhan et al. have studied 94

HD patients in whom 25(OH)D and 1,25(OH)2D had previously been determined

and they measured VDBP and calculated free and bioavailable vitamin D according

to the formula they published in the New England Journal [9, 36]. They showed

that, as expected, Black patients, compared to Whites, had lower levels of total

25(OH)D, but not bioavailable vitamin D. However, bioavailable, but not total,

25(OH)D and 1,25(OH)2D were each significantly correlated with serum calcium

and, in univariate and multivariate regression analysis, only bioavailable 25(OH)D

was significantly associated with parathyroid hormone levels. They concluded that

bioavailable vitamin D levels were better correlated with measures of mineral

metabolism than total levels in patients on hemodialysis. This study is not free from

criticisms: indeed, the DiaSorin RIA was used, ant not a LCMS/MS method like in

the New England study. This can definitely have an impact in the values observed in

HD patients. Moreover, the formula has never been validated in HD patients. The

serum matrix of HD subjects is very different than the serum matrix on healthy ones

and this could definitely affect the binding of 25(OH)D with albumin and/or

VDBP. Once again, good analytical methods will be needed to confirm these



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Part II

Classical Mineral and Bone Effects

Chapter 6

Vitamin D and Racial Differences in Chronic

Kidney Disease

Orlando M. Gutiérrez

Abstract Compared to Caucasians, African Americans have lower circulating concentrations of 25-hydroxyvitamin D (25(OH)D), the major storage form of vitamin

D, leading to the widespread assumption that blacks are at higher risk of vitamin D

deficiency. Since low 25(OH)D is associated with adverse cardiovascular and kidney outcomes, this has supported the notion that low 25(OH)D concentrations partly

underlie racial disparities in health outcomes, including faster progression of

chronic kidney disease (CKD) in blacks vs. whites. However, the finding that blacks

maintain better indices of musculoskeletal health than whites throughout their lifespan despite having lower circulating 25(OH)D concentrations suggests that the relationship between vitamin D deficiency and racial health disparities may not be so

straight forward. This has been further underscored by epidemiologic studies showing major racial heterogeneity in the association of 25(OH)D with cardiovascular

outcomes. When coupled with emerging data showing genetically determined differences in the bioavailability of vitamin D by race, these data suggest that there are

important differences in vitamin D metabolism by race which need to inform and

perhaps revise our current understanding of the role of vitamin D in racial disparities in CKD outcomes.

Keywords Race and ethnic differences • Chronic kidney disease • Bone mineral

density • Vitamin D



Vitamin D is an essential hormone involved in the regulation of numerous physiological systems. While the primary actions of vitamin D involve calcium homeostasis, vitamin D receptors (VDRs) are present in many tissues not explicitly involved

O.M. Gutiérrez

Division of Nephrology, Department of Medicine, School of Medicine; Department of

Epidemiology, School of Public Health, University of Alabama, Birmingham, AL, USA

e-mail: ogutierr@uab.edu

© Springer International Publishing Switzerland 2016

P.A. Ureña Torres et al. (eds.), Vitamin D in Chronic Kidney Disease,

DOI 10.1007/978-3-319-32507-1_6



O.M. Gutiérrez

in calcium regulation, including cardiac, vascular smooth muscle, endothelial, juxtaglomerular, and immune cells [1]. These data provide biological plausibility for a

link between vitamin D deficiency and cardiovascular disease (CVD). Consistent

with this, low 25-hydroxyvitamin D (25(OH)D) concentrations have been associated with hypertension, insulin resistance, endothelial dysfunction, and higher risk

of CVD events, chronic kidney disease (CKD) and death [2–34]. Since vitamin D

deficiency is common in the general population, this has fueled interest for treating

vitamin D deficiency as a novel approach to reducing the risk of CVD and CKD.

Enthusiasm for diagnosing and treating vitamin D deficiency has been particularly high in efforts to address racial disparities in CVD and CKD outcomes. This is

because the prevalence of 25(OH)D deficiency is substantially higher in individuals

of black race than individuals of white race [35, 36]. On the basis of these findings,

black individuals have long been assumed to be at increased risk of vitamin D deficiency [1]. When coupled with epidemiologic data linking low 25(OH)D with

adverse outcomes, these data support the notion that vitamin D deficiency should be

a prime target of therapy for improving health disparities, such as the excess risk of

CKD and end-stage renal disease (ESRD) among black individuals.

However, inconsistencies in the relationship between vitamin D and health outcomes in black individuals have raised doubts about these conclusions. Despite having lower circulating 25(OH)D concentrations and lower calcium intake than white

individuals, black individuals have markedly lower rates of osteoporosis and skeletal fractures than age- and gender-matched white individuals [37–41]. Similarly,

despite a wealth of data showing positive associations of serum 25(OH)D with

muscle mass and strength [42–46], black individuals have better indices of muscle

health than whites across the life span [47]. Additionally, associations of 25(OH)D

and CVD outcomes have been inconsistent in cohorts with large numbers of black

participants, with a number of studies showing no association of lower 25(OH)D

with CVD risk in blacks whereas a strong association was observed in whites [4, 14,

31, 32, 48]. Why this heterogeneity exists is unclear. However, advances in the

understanding of racial differences in vitamin D metabolism have revealed important new insights that may require the development of more refined approaches to

the assessment and management of vitamin D status in racially-diverse populations.

This is particularly the case in individuals with CKD [49], who are prone to the

development of vitamin deficiency, and for whom racial differences in vitamin D

metabolism may most strongly inform disparities in outcomes.


The Case for Vitamin D as a Factor Underlying Racial

Disparities in CKD

As mentioned above, the much higher prevalence of 25(OH)D deficiency in blacks

than whites has led to the hypothesis that low 25(OH)D concentrations partly underlie racial disparities in a variety of health outcomes, including diabetes, heart disease, CKD progression and cancer [35, 36]. In support of this, studies have shown

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3 Measurement of Vitamin D and Its Metabolites: Analytical and Clinical Considerations

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