Review
Vitamin D and DBP: The free hormone hypothesis revisited

https://doi.org/10.1016/j.jsbmb.2013.09.012Get rights and content

Highlights

  • Vitamin D metabolites circulate bound to DBP and, to a lesser extent, albumin.

  • 25OHD binds to DBP with high affinity and is thus strongly influenced by DBP.

  • For some cells unbound or ‘free’ 25OHD may be the most bioavailable form of 25OHD.

  • DBP may play a pivotal role in the intracrine synthesis of 1,25(OH)2D by immune cells.

  • Free or bioavailable 25OHD is influenced by DBP concentration and binding affinity.

Abstract

The last five years have witnessed a remarkable renaissance in vitamin D research and a complete re-evaluation of its benefits to human health. Two key factors have catalyzed these changes. First, it now seems likely that localized, tissue-specific, conversion of 25-hydroxyvitamin D (25OHD) to 1,25-dihydroxyvitamin D (1,25(OH)2D) drives many of the newly recognized effects of vitamin D on human health. The second key factor concerns the ongoing discussion as to what constitutes adequate or optimal serum vitamin D (25OHD) status, with the possibility that vitamin D-deficiency is common to communities across the globe. These two concepts appear to be directly linked when low serum concentrations of 25OHD compromise intracrine generation of 1,25(OH)2D within target tissues. But, is this an over-simplification? Pro-hormone 25OHD is a lipophilic molecule that is transported in the circulation bound primarily to vitamin D binding protein (DBP). While the association between 25OHD and DBP is pivotal for renal handling of 25OHD and endocrine synthesis of 1,25(OH)2D, what is the role of DBP for extra-renal synthesis of 1,25(OH)2D? We hypothesize that binding to DBP impairs delivery of 25OHD to the vitamin D-activating enzyme 1α-hydroxylase in some target cells. Specifically, it is unbound, ‘free’ 25OHD that drives many of the non-classical actions of vitamin D. Levels of ‘free’ 25OHD are dependent on the concentration of DBP and alternative serum binding proteins such as albumin, but will also be influenced by variations in DBP binding affinity for specific vitamin D metabolites. The aim of this review will be to discuss the merits of ‘free 25OHD’ as an alternative marker of vitamin D status, particularly in the context of non-classical responses to vitamin D.

This article is part of a Special Issue entitled ‘16th Vitamin D Workshop’.

Introduction

Steroid hormones and sterols such as vitamin D are highly lipophillic, and therefore share a common requirement for serum carrier proteins to ensure effective delivery to target cells. Given the abundance of proteins in serum, some of this transport will be non-specific. Nevertheless, there are many ligand-specific serum carriers of steroid hormones and sterols including corticosteroid-binding globulin (CBG) (glucocorticoids, mineralocorticoids), vitamin A (retinol)-binding protein, vitamin D-binding protein (DBP), sex hormone-binding globulin (SHBG) (estrogens, androgens), and thyroid hormone-binding globulin. Although these proteins have been studied primarily in the context of their impact on serum assays for their respective ligands, it is now clear that they may also fulfill alternative functions. For example, CBG and SHBG not only act as high affinity serum transporters, but are also able to bind to cell membranes in their liganded forms [1], [2], suggesting alternative actions as signal transducers [3], [4], [5]. In a similar fashion, DBP can function as a macrophage-activating factor (MAF) [6] and actin-binder [7], actions independent of its vitamin D metabolite binding functions. Despite the pluripotent properties of steroid binding globulins, many recent studies of these proteins have focused only on their ability to deliver hormone ligands to target cells.

The mechanisms by which ligands are released from binding globulins and acquired by target cells are crucial to steroid hormone signaling pathways. This is particularly important for vitamin D where there is increasing evidence for extra-renal, intracrine, conversion of pro-hormone 25-hydroxyvitamin D (25OHD) to active 1,25-dihydroxyvitamin D (1,25(OH)2D) [8], [9], [10]. In this setting, the impact of vitamin D will be very much dependent on tissue-specific expression of the vitamin D-activating enzyme 25-hydroxyvitamin D-1α-hydroxylase (1α-hydroxylase) and the nuclear receptor for 1,25(OH)2D, the vitamin D receptor (VDR). Another crucial factor influencing this mechanism will be the availability of substrate 25OHD for activation by 1α-hydroxylase. As serum concentrations of 25OHD are usually considered to be the principal marker of vitamin D ‘status’ for any given individual, the intracrine model has been proposed as a potential explanation for studies linking vitamin D-deficiency with various human health parameters [11]. However, in proposing a model for vitamin D function centered on serum 25OHD (rather than the endocrine 1,25(OH)2D model), it is important to recognize that 25OHD circulates bound to its cognate binding globulin, DBP [12]. Furthermore, because 25OHD is bound by DBP with much higher affinity than 1,25(OH)2D, it is likely that DBP will have a much greater impact on 25OHD-mediated intracrine responses. In this way, DBP and its interaction with 25OHD may be an important consideration in our interpretation of the physiological impact of vitamin D and this will be discussed in greater detail in the following review.

Section snippets

DBP, megalin and the free hormone hypothesis

Although 99.9% of 25OHD circulates bound to DBP or other serum proteins, the general assumption for lipid soluble molecules such as vitamin D is that biological activity involves unbound or free fractions even though this component in serum is very small [13], [14]. Indeed the ‘free-hormone hypothesis’ has been proposed as a universal mechanism for cellular uptake of steroid hormones [15], [16], largely because these molecules are highly lipophillic and therefore have the potential to rapidly

Vitamin D responses in DBP knockout mice

In common with megalin knockout animals, analysis of mice with ablation of the gene for DBP has provided further insight into the role of this protein in vitamin D physiology. DBP knockout mice are healthy and fertile despite having lower circulating levels of 25OHD and 1,25(OH)2D [28]. In the absence of DBP, vitamin D metabolites are more likely to bind to albumin, which has a lower affinity for both 25OHD and 1,25(OH)2D relative to DBP and will therefore be less effective in preventing

Extra-renal 1α-hydroxylase, the free hormone hypothesis revisited

The recent upsurge of interest in vitamin D and human health has stemmed from two key developments in the vitamin D field. The first is the ongoing debate as to what constitutes optimal vitamin D status and how to achieve this safely through normal sunlight exposure and/or dietary supplementation. The second concerns the more widespread acceptance of extra-renal synthesis of 1,25(OH)2D as a mechanism for mediating potential beneficial effects of enhanced serum 25OHD levels. As outlined above,

Bound, free and bioavailable 25OHD in the circulation

In serum, the vast majority of vitamin D metabolites bind preferentially to DBP, but they are also known to associate with serum albumin. The affinity of 25OHD (Ka = 6 × 105 M−1) and 1,25(OH)2D (Ka = 5.4 × 104 M−1) for albumin is substantially lower than that observed with 25OHD (Ka = 7 × 108 M−1) and 1,25(OH)2D (Ka = 4 × 107 M−1) for DBP [13], [36]. However, because of the relative abundance of albumin in serum (650 μM) compared to DBP (5 μM) the potential remains for some vitamin D metabolites to be transported in

Future studies on DBP and free vitamin D

Further studies of free and bioavailable 25OHD in humans are required, notably in the setting of vitamin D supplementation trials and under conditions of enhanced or suppressed expression of DBP. Recent publications have shown a correlation between serum concentrations of 25OHD and DBP [51], [52]. Also, recent reports have described changes in total serum concentrations of 25OHD, 1,25(OH)2D and DBP in subjects following supplementation with either cholecalciferol (vitamin D3) or ergocalciferol

Conclusions

In the last five years there has been revived interest in the role of DBP in vitamin D and human health, with the target levels of serum 25OHD required for vitamin D sufficiency being subject to much scrutiny. Based on recent studies, it is possible that free or bioavailable 25OHD will provide a more meaningful marker of vitamin D function. For example, an individual with low serum 25OHD according to current parameters (less than 50 nM 25OHD), might nevertheless have adequate levels of free

Conflict of interest

The authors have no disclosures to declare.

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