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6 24,25-Dihydroxyvitamin D and 1,24,25-Trihydroxyvitamin D
C. Zierold et al.
present in the inner membrane of the mitochondria of target cells. This enzyme,
also known as CYP24A1, is a cytochrome P-450 mixed function monooxygenase
that requires nicotinamide adenine dinucleotide phosphate oxidase (NADPH), ferrodoxin, and ferrodoxin reductase to introduce molecular oxygen on the carbon 24
of 1,25(OH)2D or 25(OH)D. Both metabolites are substrates for the 24-hydroxylase, but the affinity is tenfold higher for 1,25(OH)2D than 25(OH)D, yet the
25(OH)D circulates at 1,000-fold higher concentrations (ng/mL versus pg/mL).
Recently, human patients with infantile idiopathic hypercalcemia were identified
with mutations in the CYP24A1 gene, and found to have accumulation of serum
1,25(OH)2D, further supporting the role of 24-hydroxylase in the clearance of vitamin D metabolites [1, 2, 6].
A biological role for 24-hydroxylated vitamin D metabolites remains
controversial. While inactivation of the active 1,25(OH)2D makes physiological
sense, the role of inactivation of the not-yet-active 25(OH)D has been questioned.
However, that 24-hydroxylation of 25(OH)D decreases the available substrate for
1α-hydroxylation does afford a viable regulatory step. Animals fed a diet with
24,24-difluoro-25-OH-D3 as their sole source of vitamin D for two generations
had normal growth, reproduction and skeletal mineralization. The difluoro compound cannot be 24-hydroxylated, but behaves as 1,25(OH)2D when
1α-hydroxylated, and the metabolism of vitamin D is not unbalanced, yet
24,25(OH)2D is absent. Furthermore, 24-Hydroxylase-null mice were generated
and show 50 % perinatal lethality. Interestingly the mice that survived past weaning were able to catabolize 1,25(OH)2D by an alternate pathway, and showed normal levels of circulating calcium and phosphate. These surviving homozygous
24-hydroxylase-null mice can be bred, and have offspring with abnormal bone
development, which can however be rescued when CYP24/VDR double null
mutants are generated. This can be explained by the fact that 24-hydroxylase-null
dams have elevated levels of 1,25(OH)2D during gestation which cause the bone
abnormalities in the developing offspring when VDR is present. In the absence of
VDR, 1,25(OH)2D cannot carry out its function even though circulating at elevated levels, and the offspring now have normal bone development in spite of the
absence of 24,25(OH)2D. In light of this, 24,25(OH)2D does not appear to be necessary for normal bone development during development, and is likely the product of the first catabolic step for 25(OH)D, with no known function. This confirms
the early conclusions reached using the 24,24-difluoro-25-OH-D as a sole source
of vitamin D [7, 20].
Regulation of key players in vitamin D metabolism is reciprocal and very tight.
The activating enzyme 1α-hydroxylase, and the catabolic enzyme 24-hydroxylase
are reciprocally regulated by PTH, 1,25(OH)2D, and FGF23. PTH up-regulates
1α-hydroxylase when calcium is needed, while at the same time it downregulates
the 24-hydroxylase. When calcium is normalized, 1,25(OH)2D regulates its own
breakdown by activating the 24-hydroxylase, and decreases its synthesis by downregulating the 1α-hydroxylase. FGF23 is increased when phosphorus levels are
elevated, and shuts down further absorption by down-regulating the 1α-hydroxylase
and up-regulating the 24-hydroxylase .
1 Vitamin D Metabolism in Normal and Chronic Kidney Disease States
Over 33 metabolites of vitamin D have been isolated and identified, and most are
formed only when high doses of vitamin D are administered. The metabolites that
have been isolated under physiological conditions are shown in Fig. 1.5. In addition
to the above mentioned pathways, other important metabolic pathways of vitamin D
that occur at physiologic concentrations are the 23-hydroxylation of 25(OH)D with
subsequent 26-oxidation, and cyclization to form a lactone, and the 26-hydroxylation
of 25(OH)D. Animals maintained on 25(OH)D fluorinated at positions 23, 26, and
27 to prevent hydroxylation at these carbons, were shown to have normal growth,
reproduction, and bone mineralization thus suggesting that the 23- and
26-hydroxylated metabolites do not have important functions in calcium and phosphorus homeostasis, and that these compounds are likely metabolites of another
catabolic pathway that leads to excretion [4, 6].
C3-Epimer of 25(OH)D
The C3-epimer of 25(OH)D is an isomer of 25(OH)D, having the hydroxyl group
on carbon 3 in the β orientation instead of α. It has been measured in some infant
blood samples, and it can be found at levels similar to or greater than 25(OH)D. Since
its initial detection in pediatric samples, it has also been detected in adults, albeit
less frequently. When present, the C3-epimer can be detected at concentrations
below 10 ng/mL, though in extreme cases levels as high as 30–50 ng/mL have been
found. To date, no physiological role has been attributed to this metabolite, but
increased interest exists as to find why some subjects present with such high circulating amounts while others have none .
24(OH)D2 or 1,24(OH)2D2
Widespread vitamin D deficiency has led to increased use of over-the-counter vitamin D supplements (both vitamin D3 and D2), and prescription strength vitamin D2
supplementation (50,000 IU). Alternate metabolites were observed when either a
single large dose (1,000,000 IU of vitamin D2) or repeated daily doses (1,000–
50,000 IU vitamin D2) were ingested. 24(OH)D2 and 1,24(OH)2D2 were produced
via a pathway that resulted in 24-hydroxylation occurring via a liver 25-hydroxylase,
presumably the CYP27A1 which prefers vitamin D2 as a substrate over vitamin D3.
This alternate pathway has not been observed for vitamin D3 supplements for which
the expected 25(OH)D3 and 1,25(OH)2D3 metabolites were produced even at high
levels of supplementation. 1,24(OH)2D2 was shown to be physiologically active,
and behave similarly to 1,25(OH)2D [21, 22].
C. Zierold et al.
Relation Between Vitamin D, 24(OH)D, and 1,25(OH)2D
Research in the field has over the years consistently and conclusively shown that
1,25(OH)2D is the biologically active, hormonal form of vitamin D. However, the
measurement of inactive 25(OH)D, has commonly been used in studies of disease
association with vitamin D. The conversion of inactive 25(OH)D to active
1,25(OH)2D is a tightly regulated step, and circulating levels of 1,25(OH)2D are not
directly proportional to the circulating 25(OH)D, but are dependent on physiological states, and respective regulatory stimuli. While small increases in 1,25(OH)2D
may result when circulating levels of 25(OH)D increase, due to more substrate
availability, much larger changes of 1,25(OH)2D can result from regulation by the
physiological state, so that for a given serum level of 25(OH)D, levels of 1,25(OH)2D
can vary more than tenfold. Novel methods for a more accurate and precise measurement of 1,25(OH)2D are being developed, and future clinical studies on the
effects of vitamin D should include quantification of not only 25(OH)D, but also
1,25(OH)2D the active effector molecule that binds the VDR, and is responsible for
physiological responses in target cells.
Vitamin D as synthesized in the skin or ingested in the diet is inactive, and must
undergo two successive hydroxylations to form the active metabolite 1,25(OH)2D.
1,25(OH)2D maintains adequate levels of calcium and phosphorus in the blood by
acting on intestine, kidney, and bone, and is responsible for other non-calcemic
functions. 1,25(OH)2D carries out its functions through the vitamin D receptor. The
25(OH)D that is produced in the first bio-activation step is inactive, but its measurement in serum or plasma has been adopted as an indicator of an individual’s vitamin
D status. 25(OH)D levels have also commonly been used in studies of disease association with vitamin D, yet 1,25(OH)2D has dependably been shown to be the biologically active form. The regulation of 1,25(OH)2D is very tight and largely
dependent on the physiological state surrounding calcium and phosphorus homeostasis, and thus not directly correlated to the levels of 25(OH)D. Though
25-hydroxyvitamin D, 1,25(OH)2D, and the 24-hydroxylated metabolites are the
most important and well-studied, other physiological metabolites of vitamin D exist,
and future research may reveal additional physiologically important metabolites.
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Epidemiology of Vitamin D Deficiency
in Chronic Kidney Disease
Marie Metzger and Bénédicte Stengel
Abstract Vitamin D deficiency is common in both the general population and
CKD patients. Currently defined as a circulating 25-dihydroxyvitamin D (25(OH)
D) level below 20 ng/mL (50 nmol/L), it is a major risk factor for bone and mineral
disorders and has been related to increased risk of non-skeletal health outcomes
including mortality, diabetes, and cardiovascular disease. A greater prevalence of
this deficiency is expected in patients with CKD because they are older and more
likely to have dark skin, obesity, and associated comorbidities such as diabetes and
hypertension. In studies of clinical populations, the mean circulating 25(OH)D levels ranged from 18 to 29 ng/mL for patients with non-end-stage CKD and from 12
to 32 ng/mL for those on dialysis. Large population-based and clinical studies, however, describe inconsistent findings about the association between kidney function
and vitamin D level. While some studies report significant, positive, and independents associations between glomerular filtration rate and circulating 25(OH)D values, others show low levels only in advanced CKD stages. Still others show no or
even an inverse association, with paradoxically higher levels of 25(OH)D in individuals with moderate CKD than in those without CKD. Similarly, it remains
unclear whether these discordant relations are direct and causal, or indirect because
of confounders. Only a few studies have examined the relations between proteinuria
or albuminuria and circulating 25(OH)D levels; they generally report significant
negative associations. Potential mechanisms supporting a causal relation between
kidney function and damage and vitamin D are discussed at the end of this chapter.
Keywords Chronic kidney disease • Dialysis • Transplantation • Vitamin D deficiency • Recommendations • Epidemiology • Risk factors • Prevalence • Glomerular
filtration rate • Albuminuria • Proteinuria
M. Metzger, MD (*)
Inserm UMR 1018, Centre de Recherches en Epidémiologie et Santé des Populations (CESP),
B. Stengel, MD, PhD
Center for Research in Epidemiology and Population Health (CESP),
Renal and Cardiovascular Epidemiology Team, University Paris-Saclay,
© Springer International Publishing Switzerland 2016
P.A. Ura Torres et al. (eds.), Vitamin D in Chronic Kidney Disease,
M. Metzger and B. Stengel
Vitamin D deficiency is a global issue affecting nearly a billion people across the
globe and across the life span, from childhood to advanced age [1, 2]. According to
a 2014 systematic review of vitamin D status in populations worldwide in 2014,
37 % of the 195 studies included from 44 countries reported mean circulating
25-hydroxy vitamin D levels below 50 nmol/mL (20 ng/mL), the most common
threshold for defining deficiency .
Vitamin D is involved in bone and mineral metabolism; its key role in bone
health is consistent with a causal relation . Risks associated with vitamin D deficiency include rickets in children and adolescents  and osteomalacia and osteoporosis, which can lead to fractures, in adults and the elderly . Since 2000, many
observational studies, reviewed by Theodoratou et al. , have also documented
vitamin D deficiency as a potentially independent risk factor for non-skeletal outcomes including overall mortality, cancer, cardiovascular disease, autoimmune disease, and several other outcomes. In addition, a recent Cochrane meta-analysis of 56
randomized clinical trials (RCTs) showed that vitamin D supplementation slightly
but significantly reduces mortality, especially among the elderly . Several other
RCTs on the effects of vitamin D are ongoing . Whether or not vitamin D is causally related to extraskeletal complications, however, is currently debated. Some
authors argue that the associations observed may have resulted from confounding or
reverse causation . Rather than being a risk factor for these outcomes, vitamin
D deficiency may merely be a marker of poor health status associated with malnutrition and sedentary lifestyle.
Vitamin D deficiency is also common in chronic kidney disease (CKD) , and
its role in the development of secondary hyperparathyroidism (SHPT) and CKDrelated bone and mineral disorders (MBD) is well established (see section II of this
book). Supplementation with calciferol, as well as with calcitriol or its analogs, has
proved to be effective in reducing SHPT in patients with CKD, on dialysis or not
[12–14] (but see ). Observational studies have also shown associations between
vitamin D deficiency and other CKD complications, including anemia , insulin
resistance , and inflammation . Moreover, vitamin D deficiency has been
associated with higher risks of overall and cardiovascular mortality in patients with
end-stage [19, 20] and non-end-stage CKD , as well as with faster decline in the
glomerular filtration rate (GFR) and earlier progression to end-stage renal disease
This chapter begins with a review of the methods used to assess vitamin D status
and a description of the definitions of deficiency used in studies in the general
population and among patients with CKD. A brief summary of the main findings
about the epidemiology and risk factors of vitamin D deficiency worldwide follows.
We then report the prevalence of vitamin deficiency in patients with non-end-stage
CKD, those on dialysis, and those living with a kidney transplant. Finally, we discuss
whether or not CKD, defined by either increased albuminuria or decreased GFR, is
associated with vitamin D deficiency, independently of other established risk factors.
Epidemiology of Vitamin D Deficiency in Chronic Kidney Disease
Assessment of Vitamin D Status and Definition
of Vitamin D Deficiency
Vitamin D status is best assessed by the level of its major circulating form,
25-hydroxyvitamin D (25(OH)D), also called calcidiol [4, 26]. Calcitriol or 1,25
dihydroxyvitamin D (1,25(OH)2D3), the active hormonal form of vitamin D, has a
very short half-life and its concentration is tightly regulated by PTH, calcium, and
phosphate levels (see Chap. 1) and is thus a poor indicator of vitamin D status .
Defining normal vitamin D values and comparing mean values or percentages of
abnormal values between studies require a standard definition and a reference assay,
neither of which currently exists. Normal or reference values for a given biological
parameter are usually determined from a large sample of healthy volunteers.
However, the production of 25(OH)D depends on sun exposure to UVB and liver 25
hydroxylation, as well as on endogenous factors such as age and skin color (see
Chap. 6); because its levels therefore vary strongly according to latitude and season,
there is no consensus about this approach. This limitation necessitates caution in the
interpretation of variations in the vitamin D estimates between studies, especially in
light of the lack of standardized assays for it.
Since 2010, various national or international medical societies and expert groups
have published different guidelines for the evaluation, treatment, and prevention of
vitamin D deficiency. Experts establishing recommendations for a level sufficient to
prevent adverse outcomes have considered primarily its bone and mineral action [6,
27, 28]. Some are based on the relation between the circulating levels of 25(OH)D
that reduce PTH level or increase intestinal calcium absorption. Others use findings
from RCTs investigating the vitamin D threshold necessary to prevent hip and nonvertebral fractures . The major definitions currently proposed for vitamin D
deficiency or insufficiency are summarized in Table 2.1.
The threshold of 10 ng/mL (25 nmol/L) for 25(OH)D has long been used to
define low vitamin D values with clinical effects, such as osteomalacia [30, 31].
Most recent guidelines, however, have proposed serum 25(OH)D levels below
20 ng/mL (50 nmol/L) to define vitamin D deficiency in the general population.
Debate nonetheless continues about the definition of sufficiency, particularly
because of the potentially pleiotropic effects of vitamin D. The Institute of Medicine
(IOM) has concluded that values above 20 ng/mL are adequate for all ages and both
genders . The Endocrine Society, on the other hand, considers that optimum
values should exceed 30 ng/mL and suggested defining deficiency as 25(OH)D
below 20 ng/mL and insufficiency by values between 20 and 30 ng/mL (75 nmol/L)
. The discrepancies between guidelines for the minimum level of 25(OH)D
required to be in “good health” and the need for supplementation reflects the low
level of evidence for the extraskeletal effects of vitamin D . Further RCTs are
needed  to justify raising the vitamin D threshold, in view of the major economic
and public health consequences of such an action: the levels of supplementation
and/or sun exposure needed to achieve a circulating 25(OH)D concentration ≥30 ng/
mL are far greater than those to exceed 20 ng/mL ).
Adult general population (with
exception of pregnant and
Patients at risk for deficiency
Adult general population
Adult patients with or at risk
of bone disease (excluding
those with CKD stage 4–5)
NR not reported
Adequate level in at least 97.5 % of the population of “normal healthy individuals”
Low vitamin D status
NICE – UK 
KDIGO [33, 34]
US National Academies:
Institute of Medicine
Clinical practice guidelines 
French osteoporosis research
National Osteoporosis Society –
Canadian Medical Association
Osteoporosis Canada 
Definitions of Vitamin D status
Table 2.1 Definitions of vitamin D status based on circulating 25(OH)D levels (in ng/ml) according to scientific societies, healthcare and medical institutions
M. Metzger and B. Stengel