biomedicines

Perspective
Physiological Basis for Using Vitamin D to Improve Health
Sunil J. Wimalawansa

Medicine, Endocrinology & Nutrition, Cardio Metabolic Institute, (Former) Rutgers University,
North Brunswick, NJ 08901, USA; [email protected]

Abstract: Vitamin D is essential for life—its sufficiency improves metabolism, hormonal release,
immune functions, and maintaining health. Vitamin D deficiency increases the vulnerability and
severity of type 2 diabetes, metabolic syndrome, cancer, obesity, and infections. The active enzyme
that generates vitamin D [calcitriol: 1,25(OH)2 D], CYP27B1 (1α-hydoxylase), and its receptors (VDRs)
are distributed ubiquitously in cells. Once calcitriol binds with VDRs, the complexes are translocated
to the nucleus and interact with responsive elements, up- or down-regulating the expression of
over 1200 genes and modulating metabolic and physiological functions. Administration of vitamin
D3 or correct metabolites at proper doses and frequency for longer periods would achieve the
intended benefits. While various tissues have different thresholds for 25(OH)D concentrations, levels
above 50 ng/mL are necessary to mitigate conditions such as infections/sepsis, cancer, and reduce
premature deaths. Cholecalciferol (D3 ) (not its metabolites) should be used to correct vitamin D
deficiency and raise serum 25(OH)D to the target concentration. In contrast, calcifediol [25(OH)D]
raises serum 25(OH)D concentrations rapidly and is the agent of choice in emergencies such as
infections, for those who are in ICUs, and for insufficient hepatic 25-hydroxylase (CYP2R1) activity.
In contrast, calcitriol is necessary to maintain serum-ionized calcium concentration in persons with
advanced renal failure and hypoparathyroidism. Calcitriol is, however, ineffective in most other
conditions, including infections, and as vitamin D replacement therapy. Considering the high costs
and higher incidence of adverse effects due to narrow therapeutic margins (ED50), 1α-vitamin D
analogs, such as 1α-(OH)D and 1,25(OH)2 D, should not be used for other conditions. Calcifediol
analogs cost 20 times more than D3 —thus, they are not indicated as a routine vitamin D supplement
for hypovitaminosis D, osteoporosis, or renal failure. Healthcare workers should resist accepting
inappropriate promotions, such as calcifediol for chronic renal failure and calcitriol for osteoporosis
Citation: Wimalawansa, S.J. or infections—there is no physiological rationale for doing so. Maintaining the population’s vitamin
Physiological Basis for Using Vitamin
D sufficiency (above 40 ng/mL) with vitamin D3 supplements and/or daily sun exposure is the most
D to Improve Health. Biomedicines
cost-effective way to reduce chronic diseases and sepsis, overcome viral epidemics and pandemics,
2023, 11, 1542. https://doi.org/
and reduce healthcare costs. Furthermore, vitamin D sufficiency improves overall health (hence
10.3390/biomedicines11061542
reducing absenteeism), reduces the severity of chronic diseases such as metabolic and cardiovascular
Academic Editors: Małgorzata diseases and cancer, decreases all-cause mortality, and minimizes infection-related complications
Żychowska and J˛edrzej Antosiewicz
such as sepsis and COVID-19-related hospitalizations and deaths. Properly using vitamin D is the
Received: 12 April 2023 most cost-effective way to reduce chronic illnesses and healthcare costs: thus, it should be a part of
Revised: 11 May 2023 routine clinical care.
Accepted: 11 May 2023
Published: 26 May 2023 Keywords: 25(OH)D; 1,25(OH)2 D; autocrine and paracrine; endocrine; human; mechanisms;
musculoskeletal; morbidity and mortality; public health

Copyright: © 2023 by the author.

Licensee MDPI, Basel, Switzerland.
1. Introduction
This article is an open access article
distributed under the terms and
From the physiological point of view, humans are expected to generate more than
conditions of the Creative Commons 80% of their vitamin D requirements through ultraviolet sunrays, as intake through diet is
Attribution (CC BY) license (https:// minimal. Significant behavioral changes occurred over the past few decades; more people
creativecommons.org/licenses/by/ are working indoors and avoiding the sun. Consequently, clinically relevant vitamin D
4.0/).

Biomedicines 2023, 11, 1542. https://doi.org/10.3390/biomedicines11061542 https://www.mdpi.com/journal/biomedicines

Biomedicines 2023, 11, 1542 2 of 26

deficiency has become a global pandemic. Vitamin D deficiency increases the global burden
of acute and chronic diseases and healthcare costs.
In those with vitamin D deficiency, increasing 25(OH)D concentrations via D3 sup-
plements or sufficient daily sun exposure reduces the risks and the severity of multiple
disorders, including type 2 diabetes, hypertension, metabolic syndrome, obesity, cancer,
and infections [1–3]. Despite this, there is little consensus from randomized controlled
clinical trials (RCTs) regarding the proper intake and optimum serum 25(OH)D levels for
preventing the mentioned complications. This lack of agreement is primarily due to the
poorly designed studies; many clinical studies piggybacked on evaluating pharmaceu-
ticals, failure to include vitamin D insufficient or deficient subjects in RCTs, infrequent
doses administered, participants are allowed to take over-the-counter supplements (in-
cluding vitamin D) during clinical trials, and the failure to measure circulatory 25(OH)D
concentrations to ensure the target level is achieved.
Adverse effects from vitamin D supplements—hypercalcemic syndrome due to over-
dosing of vitamin D—are extremely rare. When they occur, they are invariably due to
taking mistaken very high (i.e., supra-pharmacological) doses too frequently [4,5]. Vi-
tamin D toxicity should not be diagnosed in isolation—incidental findings of elevated
serum 25(OH)D concentrations should correlate with clinical signs and symptomatology.
Characteristics of the calcitriol-driven hypercalcemic syndrome include serum 25(OH)D
concentration over 150 ng/L (over 375 nmol/L) associated with hypercalcemia (raised
serum ionized calcium), hypercalciuria (urinary calcium, exceeding 400 mg/24 h), and
suppressed parathyroid hormone (PTH) concentration [6]. All three components must be
present to diagnose vitamin D toxicity—an exceedingly rare event in the community.

1.1. Vitamin D3 (Cholecalciferol)

Vitamin D is an essential micronutrient that is essential for human survival. Humans
are expected to generate most of their vitamin D requirement from exposure to ultraviolet
B rays (UVB) from sunlight. D3 is synthesized in the skin after exposure to UVB rays, while
D2 originates from plants. UVB photons from UVB rays photolyze 7-dehydrocholesterol
in the skin to produce previtamin D3 , which subsequently isomerizes to form vitamin D3 .
The cycle of the generation of vitamin D and its activation steps leading to synthesizing
25(OH)D and 1,25(OH)2 D is illustrated in Figure 1.
Over-exposure to UVB rays can cause erythema and skin damage and must be avoided.
However, excess sun exposure does not overproduce vitamin D or cause hypercalcemia.
Due to an inherent evolutionary feedback mechanism, the synthesis of previtamin D is
inhibited. If any excess D3 is generated, it is photodegraded, preventing vitamin D from
entering circulation and reaching the liver. Free-living hunter-gatherers and lifeguards
in swimming pools and beaches are exposed daily to plenty of sunlight. They main-
tain an average serum 25(OH)D concentration of 46 ng/mL (a range of between 40 and
65 ng/mL) [3,7,8], and do not have excess vitamin D in the circulation.
Concentrations of 25(OH)D between 40 and 65 ng/mL are the physiological concen-
tration in normal circumstances under free-living conditions. However, when there are
extenuating circumstances and stresses, such as infections (endemics/pandemic), comor-
bidities, metabolic or cardiovascular diseases, or cancer, higher serum 25(OH)D levels are
necessary for the precursors of calcitriol [D and 25(OH)D] to enter peripheral target cells
like immune cells to generation of calcitriol within these cells to overcome the situation
(see Sections 1.4 and 4.1, for details).
With the activation of cytochrome P450, the CYP2R1 gene increases the expression of
25-hydroxylase enzyme in the liver, produce 25(OH)D. The latter is further hydroxylated
by the 1α-hydroxylase enzyme, derived by the CYP27B1 gene in the mitochondria in
the endoplasmic reticulum, to form 1,25(OH)2 D (calcitriol) in renal tubules as well as
in peripheral target cells, such as immune cells. Calcitriol production in renal cells is
regulated by PTH and a negative-feedback control by FGF-23 (secreted by osteoblasts) and
Biomedicines 2023, 11, 1542 3 of 26

1,25(OH)2 D
Biomedicines 2023, 11, x FOR PEER REVIEW (Figure 1). However, this negative-feedback control is minimal in peripheral
3 of 27
target cells.

Figure1.1. Schematic
Figure Schematic illustration
illustrationofofgeneration
generationofof
vitamin D and
vitamin its calcium/mineral
D and regulatory
its calcium/mineral func-
regulatory
tions of calcitriol in conjunction with parathyroid hormone (PTH) and fibroblast growth factor-23
functions of calcitriol in conjunction with parathyroid hormone (PTH) and fibroblast growth factor-23
(FGF-23). The figure also depicts sites of activation of vitamin D—25-hydroxylation in the liver and
(FGF-23). The figure also depicts sites of activation of vitamin D—25-hydroxylation in the liver and
1α-hydroxylation in renal tubular cells and peripheral targets cells. 1α-hydroxylation of 25(OH)D
1α-hydroxylation in renal tubular cells and peripheral targets cells. 1α-hydroxylation of 25(OH)D
in proximal renal tubular cells generates the circulatory, hormonal form of calcitriol that controls
in proximal
calcium renal tubular cells generates the circulatory, hormonal form of calcitriol that controls
homeostasis.
calcium homeostasis.
Over-exposure to UVB rays can cause erythema and skin damage and must be
1.2. Fundamental Benefits of Vitamin D
avoided. However, excess sun exposure does not overproduce vitamin D or cause hyper-
Irrespective of the reason for hypovitaminosis D or the condition, vitamin D3 is the
calcemia. Due to an inherent evolutionary feedback mechanism, the synthesis of previta-
choice for replacement therapies to maintain serum 25(OH)D concentration at the desired
min D is inhibited. If any excess D3 is generated, it is photodegraded, preventing vitamin
levels and to maintain a robust immune system [9]. When exposed to high densities of
D from entering circulation and reaching the liver. Free-living hunter-gatherers and life-
bacteria or viral loads, as with SARS-CoV-2, adhering to straightforward public health
guards in swimming pools and beaches are exposed daily to plenty of sunlight. They
measures is crucial to prevent the spread of the infection. No drugs or nutrients are
maintain an average serum 25(OH)D concentration of 46 ng/mL (a range of between 40
available except for traditional vaccines to prevent infections. However, such preventative
and 65 ng/mL) [3,7,8], and do not have excess vitamin D in the circulation.
vaccines are not available against SARS-CoV-2.
Concentrations of 25(OH)D between 40 and 65 ng/mL are the physiological concen-
tration in normal circumstances under free-living conditions. However, when there are
extenuating circumstances and stresses, such as infections (endemics/pandemic), comor-
bidities, metabolic or cardiovascular diseases, or cancer, higher serum 25(OH)D levels are
necessary for the precursors of calcitriol [D and 25(OH)D] to enter peripheral target cells
Biomedicines 2023, 11, 1542 4 of 26

In contrast, maintaining a robust immune system with vitamin D sufficiency, although

it may not prevent infections, substantially reduces the development of symptomatic dis-
ease and complications from any infection. Furthermore, it significantly lowers community
transmissions of infections and outbreaks. In addition to preventing symptomatic dis-
ease, a robust immune system would notably reduce hospitalizations, ICU admissions,
complications, deaths, and healthcare costs.
In 2011, raising the RDA for all age groups by the US Institute of Medicine (IoM) was
a step in the right direction but was grossly inadequate [10]. Moreover, suggestions by the
IoM [10] are public health recommendations for authorities and are not guidance to use
for individuals or diseases. Besides, those suggestions do not apply to persons outside
North America [3,11–13]. However, because of the ambiguity of such narrowly focused
governmental reports, such publications were misinterpreted by organizations and misled
governments and the public outside the USA. Because of uncertainties, they assumed
reports like IoM as recommendations for the public. Consequently, the implementation
of publicized misleadingly minute doses and lower serum 25(OH)D concentrations than
required for better human health have harmed those populations.
Currently, most nutrient RDAs in many countries are outdated. They are insufficient
to raise serum 25(OH)D even beyond 20 ng/mL—a level considered inadequate. Such
is too little to overcome acute or chronic non-skeletal diseases and infections or promote
good health [14,15]. Ironically, the currently recommended vitamin D food fortification
guidelines are also outdated [16,17] and should be increased by three- to four-fold [18].

1.3. Vitamin D Analogs and Their Clinical Uses

There are specific indications for using vitamin D analogs—calcifediol or 1α-hydroxylated
compounds. Synthetic analogs of vitamin D are twenty-fold more expensive than vitamin D3 .
Since the ED50 of these analogs is narrow, unsurprisingly, they have a higher incidence of ad-
verse effects. Consequently, vitamin D analogs are inappropriate to use as longer-term vitamin
D supplements in vitamin D deficiency and osteoporosis [19]. Calcifediol (25-hydroxylated
vitamin D) is only indicated in those with defective 25-hydroxylation, as in hepato-cellular
failure, and when serum 25(OH)D concentrations in emergencies, such as infection [19].
Inefficient 1α-hydroxylation is present in advanced renal failure and rare genetic
disorders, such as pseudo-hypoparathyroidism and hypoparathyroidism. Such individ-
uals require 1α-hydroxylated vitamin D metabolites to maintain serum ionized calcium
concentration in the lower physiological range. These valuable lifesaving agents im-
prove the quality of life in advanced renal failure, hypoparathyroidism, and pseudo-
hypoparathyroidism [19].
Magnesium sufficiency is crucial for many biological activities, including hormone
synthesis and release (e.g., insulin and PTH) and calcitriol with vitamin D (calcitriol) recep-
tor (VDR) interactions [20,21]. The proper biological functions of CYP450 enzymes, vitamin
D, and vitamin D receptors (VDRs) depend on the availability of the correct intracellu-
lar concentrations of magnesium [20,22] and other co-factors. Furthermore, magnesium
adequacy reduces the complications and mortality from post-COVID syndrome [23] and
chronic kidney diseases [24]. Magnesium and vitamin D are consumed during infections.

1.4. Consequences of Hypovitaminosis D

Because of the negative genomic control process, severe vitamin D deficiency is
associated with hyperinflammation, oxidative stress, and autoimmunity—an over-reactive
pathological immune response [25]. This could lead to cytokine storms [26,27], causing
acute respiratory distress syndrome (ARDS). When circulating D3 and/or 25(OH)D is
adequate (e.g., over 50 ng/mL), these precursors will diffuse into peripheral target cells,
such as immune cells, in adequate quantities from the circulation, allowing the generation
of sufficient calcitriol intracellularly, which suppress multiple pathological processes. In
addition to genomic effects, these crucial biological processes occur via autocrine and
paracrine signaling mechanisms [28,29]. This reduces the risks of cytokine storms and
Biomedicines 2023, 11, 1542 5 of 26

ARDS and associates severe pulmonary and cardiovascular complications in persons with
COVID-19 [30,31].
Hypovitaminosis impairs intracrine and paracrine signaling, thus weakening the
immune system and increasing vulnerability [29,32]. The pro-inflammatory and oxidative
stress responses associated with cytokine storms in severe viral infections increase the
need for intensive care unit (ICU) admissions and the risk of death. Children with serum
25(OH)D concentrations less than 12 ng/mL (i.e., severe vitamin D deficiency), when
infected with SARS-CoV-2, could develop life-threatening hyper-inflammatory conditions
such as Kawasaki-like disease or multi-system inflammatory syndrome [33–35].
Moreover, the weakened adaptive immunity in hypovitaminosis D reduces the genera-
tion of neutralizing antibodies, impairs the cytotoxic action of immune/killer cells, reduces
the effectiveness of memory cells and macrophages, and causes weaker responses after
(any) vaccination. In those with a more fragile immune system (primarily due to severe
hypovitaminosis D), SARS-CoV-2, infection or immunization could lead to significant
adverse effects, including autoimmune reactions, generalized hyper-inflammation, and
pathological oxidative stresses, which could lead to systemic complications and death.
Consequently, due to the prevailing high incidence of hypovitaminosis among the elderly,
in 2020, COVID-19 became a pandemic, primarily affecting those with severe vitamin D
deficiency [36,37].
In addition, calcitriol-mediated effects in immune cells facilitate the development
of anti-microbial peptides, neutralizing antibodies, stabilizing epithelial and endothelial
cells, and strengthening gap junctions [38]. These broader membrane-stabilization effects
minimize fluid leakage (e.g., preventing pulmonary edema) and viral spread into soft
tissues [3,39–41]. To mitigate the widespread vitamin D deficiency, a personal vitamin D
response index has been suggested (which is not validated) to optimize vitamin D sup-
plementation (or safe sun exposure). However, the associated cost and lack of practicality
dampen its use. A better and more cost-effective option is adhering to broader population
recommendations [42], which leads to population vitamin D sufficiency with minimal
cost [37,43].

1.5. Intracellular Synthesis of Calcitriol and Benefit from Vitamin D

CYP27B1 enzyme is present in all endocrine cells, such as pancreatic islets and parathy-
roid, thyroid, testes, ovary, and placental cells [44], in which calcitriol is synthesized in-
tracellularly; it also modulates hormone release and metabolic functions [45]. In addition,
vitamin D adequacy enhances DNA repair, suppresses malignant cell development [46–48],
and improves the prognosis of malignancies. Proper vitamin D intake is inversely associ-
ated with lung cancer incidence [49]. Sufficient circulatory D and 25(OH)D concentrations
(i.e., above 50 ng/mL) [9] lead to better physiological responses, such as defense against
microbes [50], the prevention of autoimmune diseases such as multiple sclerosis and inflam-
matory bowel disease [51,52], and the synthesis and secretion of hormones [45], controlling
the renin-angiotensin-aldosterone system (RAS) and the FGF23/klotho system [11,53].
Since vitamin D is a nutrient, adequate supplements significantly benefit those with
deficiency [54]. However, giving extra vitamin D to those already sufficient (e.g., serum
25(OH)D concentration above 40 ng/mL) will unlikely provide additional benefits (with
some exceptions discussed below). These exceptions include those with cancer [55,56] and
infection [57], such as septicemia and SARS-CoV-2 [58,59]—these require the maintenance
of serum 25(OH)D concentrations higher than 40 ng/mL.
In addition to the ability to generate 1,25(OH)2 D, the presence of a high density of
VDRs in epithelial cells allows them to optimize barrier functions and other biological activ-
ities. Examples include the lungs [60], breast [61], intestine [62], prostate [63], osteoblasts,
and chondrocytes [64,65]. Unsurprisingly, all these tissues are targets for calcitriol. The
inactivation of VDRs in chondrocytes reduced the expression of FGF23 in osteoblasts. It
increased the expression of 1α-hydroxylase and sodium phosphate cotransporter type Il
in proximal renal cells—another mechanism to increase calcitriol production [64]. These
Biomedicines 2023, 11, 1542 6 of 26

facilitate the modulation of chondrocyte-mediated signaling (e.g., FGF23) in renal tubular

cells, for bone cell formation and osteoid calcification.

1.6. Why This Study Is Important and Necessary

Due to the lack of agreement between different medical and scientific societies and
the negative contribution from the IoM, there is apathy and confusion about the necessary
oral doses and the optimum serum 25(OH)D concentrations. In addition, some healthcare
workers are unfamiliar with the proper circumstances to use vitamin D metabolites and
their benefits vs. risks. Despite the crucial biological functions of vitamin D, textbooks
in immunology do not even discuss the role of vitamin D (or nutrients) on the immune
system. Missing such fundamental education impedes advancing knowledge and practice
to benefit patients, even among specialists such as immunologists.
These ambiguities have led to recommendations for using sub-optimal doses by
scientific societies, erroneous advice provided to government bureaucrats by nutrition-
related committees like IoM and the USPTO in the USA, the scientific advisory committee
(SCAN) and National Institute for Health and Care Excellence (NICE) in the UK, dampen
the creation of updated vitamin D guidelines. In addition, the use of high doses of vitamin
D intermittently—infrequent intervals of more than two weeks intervals instead of using
daily or once a week D3 , inappropriately prescribing expensive synthetic vitamin D analogs,
and grossly exaggerating rare adverse effects of vitamin D leads to fear-mongering of using
this safe nutrient are additional examples. These errors in clinical practice recommendations
and myths prevent the betterment of the population. This study aims to overcome some of
the errors and myths mentioned above.
The recommending of sub-optimal (standard–outdated) vitamin D doses to everyone,
ignoring their body weight (and obesity and other variables affecting their serum 25(OH)D
concentrations), or not using serum 25(OH)D-based calculation of the proper doses for
individual persons [9,20], has led to using pediatric doses of vitamin D in adults with
no benefit to recipients. This manuscript also illustrates examples and circumstances
where the efficacy, clinical requirement to raise serum 25(OH)D rapidity (as in emergencies
such as sepsis, SARS-CoV-2-infection, at ICU set-up, etc.), and the target serum 25(OH)D
concentration necessary to overcome the underlying illness such as infection or cancer
needs (see Section 3.4).
The content of the manuscript is primarily based on published evidence. Over four
decades of practical and clinical experience and research by the author on vitamin D and
over fifty peer-reviewed publications related to vitamin D enrich his perspective. Data were
gathered from literature searches on scientific databases, including PubMed and the Library
of Congress (Medicine). The author personally carried out all searches, accumulating and
synthesizing data, and generating figures, and created the list of pertinent references. The
author has no conflicts of interest—he did not receive pharmaceutical, writing, or external
assistance or input in developing this manuscript.

2. Vitamin D Generation—Genomic and Non-Genomic Actions

The primary hormonal function of calcitriol is maintaining calcium homeostasis and
the conservation of calcium and minerals. Humans have developed multiple evolutionary
mechanisms using calcitriol that enhance intestinal calcium absorption, calcium conser-
vation in renal tubules, and dynamic interaction on skeletal calcification and resorption
(Figure 1). In conjunction with parathyroid hormone (PTH), calcitriol profoundly affects
renal tubular calcium reabsorption [66]. Calcitriol has a broader regulatory function and
maintains tight calcium-phosphate homeostasis through VDR–genomic interactions and
non-genomic membrane effects. Collectively, these actions prevent fluctuations of serum-
ionized calcium, keeping a narrow range. These effects are summarized in Figure 1.
Biomedicines 2023, 11, 1542 7 of 26

2.1. Genomic Actions of Calcitriol

Calcitriol is an essential regulator of calcium-phosphate homeostasis and participates
in tightly regulated VDR genomic activity. In conjunction with PTH, the hormonal calcitriol
promotes osteoid calcification and maintains skeletal integrity, thus effectively preventing
rickets and osteomalacia [67]. The endocrine action of calcitriol facilitates musculoskeletal
functions, balance and coordination, and improves reflexes via nerve conduction—overall
improvement in muscular functions [68]. Genomic actions of calcitriol are slow to initiate
but are associated with biological outcomes over a longer duration.
When the concentration of 1,25(OH)2 D increases above the physiologic threshold,
it increases the expression of CYP24A1, 24-hydroxylase enzyme, and the production of
FGF-23. This feedback system also inhibits PTH production, thus reducing the generation
of 1,25(OH)2 D. It simultaneously increases the expression of CYP24A1, increasing the
catabolism of 25(OH)D and 1,25(OH)2 D into their respective inactive 24-hydroxylase
metabolites: 24,25(OH)D and 1,24,25(OH)2 D [44]. The 24-hydroxylation-mediated catabolic
products of calcifediol and calcitriol (Figure 2) have no known biological actions. In
contrast, low circulatory calcitriol suppresses CYP24A1 and stimulates the CYP27B1 gene
to produce the hormonal form of calcitriol from the kidney. Calcitriol and its inactive
Biomedicines 2023, 11, x FOR PEER REVIEW 8 of 27
catabolic derivatives are illustrated in Figure 2.

Figure2.2.Common
Figure Commoncatabolic
catabolicproducts
productsofof1,25(OH)D
1,25(OH)D (calcitriol).The
2 2(calcitriol). Theinactive
inactivemetabolic
metabolicproducts
productsofof
calcitriol and its excretory products, calcitroic acid, are illustrated.
calcitriol and its excretory products, calcitroic acid, are illustrated.

Aswith
As with some
some other
other micronutrients,
micronutrients, vitamin
vitaminDDrepresents
representsanother example
another example with po-
with
tential use
potential useasasa personalized
a personalized or targeted
or targetedapproach
approach to disease prevention,
to disease minimizing
prevention, com-
minimizing
plications from
complications critical
from illnesses,
critical including
illnesses, infection.
including However,
infection. However,the target circulator
the target levels
circulator
of individuals
levels of individualsvaryvary
(depending
(dependingon the target
on the tissue,
target disease,
tissue, disease,andandunderlying
underlyingvitamin
vitaminD
status) for achieving therapeutic goals [11,53] (see Section 3.6 for
D status) for achieving therapeutic goals [11,53] (see Section 3.6 for details). Vitamin details). Vitamin D-re-
D-
lated metabolomics, transcriptomics, and epigenetics studies likely
related metabolomics, transcriptomics, and epigenetics studies likely provide paths for provide paths for bet-
ter critical
better outcomes
critical outcomes [69,70].
[69,70].
Themost
The most biologically
biologically active
active formform of vitamin
of vitamin D, 1,25(OH)
D, 1,25(OH) 2D, regulates
2 D, regulates over
over 1200 1200
genes
genes the
within within
human the human
genome, genome,
and gene and gene polymorphisms
polymorphisms and epigenetics
and epigenetics would would
furtherfurther
influ-
ence its mechanism
influence its mechanismof action [71–73].
of action Meanwhile,
[71–73]. the up-regulation
Meanwhile, the up-regulationof gene transcription
of gene transcrip-
tion leads to the release (stimulation) of anti-inflammatory and antioxidant cytokines, and
the down-regulation (suppression) of inflammatory cytokines leads to a cascade of bene-
ficial effects [28,50,74–76].
Biomedicines 2023, 11, 1542 8 of 26

leads to the release (stimulation) of anti-inflammatory and antioxidant cytokines, and the
down-regulation (suppression) of inflammatory cytokines leads to a cascade of beneficial
effects [28,50,74–76].

2.2. Non-Genomic Actions of Calcitriol

In addition to the presence of VDRs in the cytosolic compartment, VDRs are also
located in cell membranes [77]. The internalization of vitamin D and its D-binding protein
(VDBP) complex is another example of rapid membrane responses [78]. This internaliza-
tion occurs in specific cells, such as renal tubular, fat, and muscle cells, enabling active
transportation. This transpires via the megalin with its co-receptor, cubilin [79]. In addition,
vitamin D influences mitochondrial functions directly and indirectly, including fusion,
energy production, modulation of mitochondrial membrane potential, regulation of ion
channels, and apoptosis [78].
In contrast to genomic actions, non-genomic responses to vitamin D are rapid but last
for a shorter duration [78]. Calcitriol triggers rapid calcium influx through epithelial cells
(membranes) and uptake by various cells, such as gastrointestinal, renal tubular, osteoclast,
and osteoclast cells. Some of these actions occur via its membrane vitamin D receptor
and the protein disulfide-isomerase A3 (PDIA3), responsible for some rapid non-genomic
responses [78]. Calcitriol also stimulates the release of second massagers via membrane
receptors, which modulate several intracellular processes. The latter include the control
of cell cycle, proliferation, and immune responses through wingless (WNT) [80], sonic
hedgehog (SSH), STAT1-3, or NF-kappaB pathways [81]. These calcitriol-mediated rapid
actions facilitate calcium spikes that modulate signaling and multiple other physiological
pathways [82].
Despite some advances, the exact mechanisms of non-genomic responses to vitamin D
are not fully understood. Vitamin D also, directly and indirectly, influences mitochondrial
function, including fusion-fission, energy production, mitochondrial membrane potential,
the activity of ion channels, and apoptosis. However, the mechanisms of the non-genomic
responses to vitamin D are still not fully understood [78].

3. Clinical and Randomized Control Studies (RCTs)

Well-designed and statistically powered RCTs that used proper doses of vitamin D
as the primary interventional agent have all reported favorable clinical outcomes. These
positive clinical outcomes are not restricted to the musculoskeletal system—rickets and osteo-
malacia [83], but also apply to other body systems [81,82], disorders, and diseases [3,84–86].
The main issue with the clinical trials performed to date is that a few have been explicitly
designed to test the hypotheses that hypovitaminosis is a causative factor and have spe-
cific vitamin D-related endpoints. In addition, the designs of many clinical studies lack
the targeting required to achieve pre-specified 25(OH)D concentrations. In contrast, all
well-designed studies with sufficient subjects (statistical power) in persons with hypovita-
minosis D using the proper doses for an adequate duration have reported favorable clinical
outcomes [87–89].

3.1. Contributions from Recent Clinical Studies

Many studies have confirmed the association between vitamin D intake and the reduction
of viral respiratory infections [90,91], including in persons with COVID-19 [92–95]. Further,
more than 120 peer-reviewed clinical studies used vitamin D as the primary intervention
to investigate COVID-19. Clinical outcomes from these studies [96] were published on
the following URLs (https://c19early.org/d, accessed on 27 March 2023) [96]. This site
provides all negative and positive published studies on vitamin D and COVID-19 [96].
This data have been made available with unrestricted access, making this dataset more
valuable than all RCTs, meta-analyses, and systematic reviews, especially considering some
meta-analyses are poorly designed, biased, and address a few cherry-picked RCTs.
Biomedicines 2023, 11, 1542 9 of 26

Although not essential to fulfilling whole criteria, vitamin D fulfills all of Hill’s causa-
tion criteria [97]: hypovitaminosis D significantly increases vulnerability, causing complica-
tions and deaths from COVID-19. Vitamin D- and COVID-19-related data published in over
390,000 subjects should have been examined in 2020 using a Big Data meta-analysis. That
would have provided additional validation with a larger effect size, with higher statistical
power to make firm conclusions.

3.2. Reasons for Failure of Recent Vitamin D Randomized Controlled Clinical Studies
During the last few years, a few negative clinical studies were published related to
vitamin D. The failures of these highly publicized prominent RCTs have been amplified by
dozens of duplicate publications by the same and other authors using the same database;
the VITAL study is one such example [98]. Failed clinical outcomes are directly related to
poor study designs. These include unfamiliarity with the biology and physiology of vitamin
D and/or how vitamin D benefits the body system (e.g., mechanisms of vitamin D on
disease prevention and stimulating the immune system). In some cases, study investigators’
lack of understanding of the biology or conflicts of interest has led to designing studies to
have failed outcomes.
It is important to note that there is no rationale in expecting studies to demonstrate the
beneficial effects of vitamin D supplementation/treatment in persons who are not deficient.
Despite these, studies such as VITAL and others recruited subjects who were not vitamin
D deficient. Whether such flawed study designs are used mistakenly, due to ignorance
of the biology and physiology of vitamin D, or deliberately downplay the importance
of this unpatented, natural nutrient is unclear. Table 1 characterizes components of a
well-designed nutrient/vitamin D clinical study/RCT.

Table 1. Key nutrient clinical study recruitment criteria: Clinical studies should be conducted to test
the hypothesis that intervention (e.g., vitamin D supplements) benefit recipient subjects.

The Goal Action Needed

Measurement of baseline serum 25(OH)D concentration. To recruit
Recruit only vitamin D-deficient subjects subjects with 25(OH)D serum levels less than 20 ng/mL (50 nmol/L) for
clinical studies and RCTs
Based on statistical Power calculation (on the effects size and the
Sufficient sample size
standard error of the mean)
Avoid administration of vitamin D at a frequency or less than once in
Sufficient doses and the right frequency of
two weeks
administration (e.g., daily or weekly—not monthly
Use the appropriate vitamin D dose to raise serum 25(OH)D
or semi-annually)
concentration sufficient to achieve the intended outcome
To optimally function, supplemented nutrients interact and act
synergistically with other nutrients. In the case of vitamin D (or calcium),
Assure the sufficiency of co-nutrients and co-factors
ensure the availability of co-factors and supporting elements, such as
magnesium, vitamin K2 , etc.
Assure the desired blood concentration is achieved In longer-duration trials, measurement of serum 25(OH)D concentrations
(e.g., 25(OH)D concentration) after initiation of the intervention (e.g., approximately in four months)
Shorter trials—Acute (e.g., infections) that last a few weeks vs. longer
Sufficient duration of the study trials—Chronic diseases such as metabolic disorders, cancer, and
osteoporosis., requiring several years of follow-up
Keep the study clean Avoid nutrients, piggyback on pharmaceutical trials
Use the simpler (uncomplicated) protocol with minimum study groups
necessary to test the hypothesis. This improves statistical power, makes
Keep the study simple
more straightforward interpretations and conclusions
become meaningful
Biomedicines 2023, 11, 1542 10 of 26

Table 1. Cont.

The Goal Action Needed

Clinical study protocol needs to test a hypothesis based on a clinically
meaningful increase of the indexed nutrient in circulation
Clinical and statistical meaningfulness
[i.e., 25(OH)D]—achieving and maintaining the blood levels above the
minimum target
Balanced randomizations Minimize confounders
Supplementation should bring serum 25(OH)D levels to a sufficiency
Target serum 25(OH)D concentration (the
level (at least above 40 ng/mL; 100 nmol/L) (in the case of infections
therapeutic window)
above 50 ng/mL)—the target goal of the clinical study
Maintain circulatory 25(OH)D concentrations above the target level
Maintain a sustained effect
during the entire study period in the interventional group [99]
The protocol should define hard primary endpoint(s)—e.g.,
Have firm–hard endpoints complications such as reduced fractures, number needed to treat (NNT)
to save one life, hospitalizations, ICU admissions, or deaths.
Over-the-counter nutrients, supplements, and
vitamins should not be taken during clinical Avoid or minimize interference with statistical power
studies [98,100,101]
Correlation should be made with serum 25(OH)D concentrations
Statistical analyses achieved (active vs. control group) after supplements [or at least, the
changes (∆) from the baseline], but not with the administered dose

Disregarding vitamin D as a nutrient and treating it as a pharmaceutical agent is

another major fallacy of several recent RCTs. These fundamental errors, unfortunately,
exist in recent larger, sponsored clinical studies, including the VITAL study [98,100]. RCTs
that were designed correctly and administered age- and condition-appropriate vitamin D
doses consistently improve the intended clinical outcomes. Examples include insulin sensitiv-
ity [42], facilitating weight loss [102], reducing cardiovascular risks [47,103], improving renal
functions [104], and skeletal disorders [105].

3.3. Examples of Larger Vitamin D Interventional RCTs with Significant Study Design Errors
In many recent clinical studies, including the VITAL study (2000 IU/day; ∆ ~ 10 ng for
5.3 years) [106,107], vitamin D assessment (ViDA) study (∆ ~ 28 ng: 200,000 IU, monthly
doses) [108], D2d study (cancer and pre-diabetes) [109], vitamin D to improve outcomes
by leveraging early treatment (VIOLET) RCT (∆ ~ 35 ng: a single dose of 40,000 IU in
critically ill patients) [110], and vitamin D on all-cause mortality in heart failure (EVITA)
study (∆ ~ 16 ng: 4000 IU/day) [111], all had significant study design errors. In several of
these, as with VITAL [107,112], the control groups were allowed to take over-the-counter
supplements [100], including vitamin D [98,100,101]. This reduced the effect size between
the active and placebo (or the control) groups, losing statistical power led to falsely non-
conclusive outcomes.
In addition, RCTs conducted for a too-short duration and the administration of too-
low and/or too-infrequent doses of vitamin D make the outcomes from such studies not
only negative but also unreliable {Grant, 2018 #11252}. Consequently, irrespective of the
study size, the cost, credibility, or where it was conducted (e.g., university name), clinical
outcomes from poorly designed studies (see Table 1) are unreliable and should not be
generalized or used for policy decision-making. RCTs that fail to adhere strictly to the
nutrient-related essential criteria and research principles mentioned in Table 1 will fail to
generate useful or meaningful data. Detailed guidelines for conducting nutrient clinical
trials and systematic reviews have been reported [41,113,114].
Biomedicines 2023, 11, 1542 11 of 26

3.4. Contrasts between Negative and Positive RCTs

The main reason for RCTs’ outcome failures in critically ill subjects, as in ICUs, is that
raising circulating concentrations of 25(OH)D in seriously ill patients would take more
than a week—another example of a lack of insight into basic biology while designing RCTs
leading to poor outcomes. In seriously ill patients, even very high doses of vitamin D,
such as those over 400,000 IU, administering as a single [110] or intermittent doses will
have no beneficial effects. Therefore, favorable outcomes cannot be expected in RCTs with
such major study design flaws. In contrast, a systematic review analysis of six vitamin
D intervention trials in acute SARS-CoV-2 infection reported a significant relative risk
reduction (RRR) of 0.60 (95% CI 0.40 to 0.92, p = 0.02), and the rates of RT-CR positivity
reduced significantly in the intervention group, with a RRR of 0.46 (95% CI 0.24 to 0.89,
p = 0.02) [115].
As described in this article and others recently [9,20], these significant study design
errors were due to the lack of understanding of the biology of vitamin D and the mech-
anisms of calcitriol modulating body systems. In the case of subjects in the ICU, failed
outcomes are predictable, with any dose of vitamin D, even in those with severe vitamin D
deficiency. These outcome failures could have been eliminated by administering the right
type of vitamin D—calcifediol instead of vitamin D (see Section 4.6 for rationale and dose
recommendation) [92,93,95]. Hence the importance of having a deeper understanding of
the biology of different vitamin D metabolites.
RCTs with study design errors fail to demonstrate benefits (i.e., no significant differ-
ences of primary endpoints from placebo) in diabetes, cancer, osteoporosis, cardiovascular
endpoints, mortality, etc. However, subgroup outcome analyses of most of these studies
using serum 25(OH)D concentrations as a denominator demonstrated beneficial positive
endpoints. This reflects the lack of insights and weaknesses of these RCTs. In parallel,
some meta-analyses reported beneficial clinical outcomes when used multiple failed RCTs,
despite their failure individually. This suggests that some of these individual RCTs did
not have the statistical power to differentiate outcomes in active vs. control groups. Such
examples include reductions in fractures and/or falls, early recovery from respiratory tract
infections, reduced severity of cancer, and low death and all-cause mortality rates [100].

3.5. What to Expect from RCTs

Before accepting the results and conclusion of any nutrient RCT, one must carefully
evaluate (A) the study design, (B) protocol, (C) adherence to the stipulated (pre-defined)
procedures, (D) statistical methods used, and (E) conflicts of interest and sponsorships.
Several currently ongoing large RCTs with significant study design errors are not exceptions.
These studies are unlikely to generate meaningful or valuable data. RCTs not designed to
test a primary hypothesis, those lacking robust clinical study protocols and/or statistical
power to make meaningful conclusions, would not fill the gaps in the knowledge.
Apart from 4 RCTs related to vitamin D and COVID-19 with poor study designs,
over 120 other clinical studies, including 41 RCTs [116,117], provided a larger effect size
and strong associations between vitamin D and clinical outcomes of COVID-19. These
studies provided overwhelming evidence that vitamin D deficiency fulfills Bradford Hill’s
criteria for causality—“hypovitaminosis D significantly increases vulnerability causing
complications and deaths from COVID-19” [9,20,118,119]. Other studies with robust pro-
tocols reported statistically significant clinical outcomes, such as with multiple sclero-
sis [9,20,118,119], periodontal disease [9,20,118,119], and cancer [Munoz, 2022], especially
against important cancer types [120].

3.6. Systems and Different Tissues May Need Different Serum Concentrations of 25(OH)D
It is noteworthy that the circulatory 25(OH)D concentrations required to overcome
disease conditions, such as drug-resistant migraine/cluster headaches, psoriasis, asthma,
etc., and during infectious pandemics and endemic, are higher than the generally re-
comended concentrations. Published data over the past two decades suggest the possibility
Biomedicines 2023, 11, 1542 12 of 26

of tissues/body
Biomedicines 2023, 11, x FOR PEER REVIEW systems related to different circulatory levels of 25(OH)D for 13 optimal
of 27
function [3]. A summary of conclusions from many clinical studies is illustrated in Figure 3.

Figure3.3.Illustrates
Figure Illustratescalculated
calculated serum
serum 25(OH)D
25(OH)D concentrations
concentrationsneeded
neededtotoovercome
overcomedifferent
differentgroups
groups
of conditions and disorders and the reported average (percentage) improvements/responses in pri-
of conditions and disorders and the reported average (percentage) improvements/responses in
mary clinical outcome. The figure summarizes cumulated data from many outcome-based vitamin
primary clinical outcome. The figure summarizes cumulated data from many outcome-based vitamin
D-related clinical studies.
D-related clinical studies.
As described above, higher circulatory 25(OH)D and D3 concentrations are crucial
As described above, higher circulatory 25(OH)D and D concentrations are crucial
for maintaining a robust immune system to impede viruses 3such as SARS-CoV-2 [59].
for maintaining a robust immune system to impede viruses such as SARS-CoV-2 [59].
Nevertheless, prevention efforts should be directed at the “entire person,” not at a given
Nevertheless, prevention efforts should be directed at the “entire person,” not at a given
tissue or a system. Furthermore, conditions and diseases (e.g., infections) can manifest
tissue or a system. Furthermore, conditions and diseases (e.g., infections) can manifest
quickly, precluding the opportunity to optimize serum 25(OH)D concentrations. There-
quickly, precluding the opportunity to optimize serum 25(OH)D concentrations. Therefore,
fore, the prevailing data strongly suggest that maintaining serum 25(OH)D concentrations
the prevailing data strongly suggest that maintaining serum 25(OH)D concentrations above
above 50 ng/mL (125 nmol/L) as the best option for the broadest protection [9,20].
50 ng/mL (125 nmol/L) as the best option for the broadest protection [9,20].
4. Pharmacodynamics and the Underlying Mechanisms of Calcitriol
4. Pharmacodynamics and the Underlying Mechanisms of Calcitriol
Calcitriol acts via multiple mechanisms, benefiting humans. It activates the innate
Calcitriol acts via multiple mechanisms, benefiting humans. It activates the innate and
and adaptive immune systems and has broader genomic and non-genomic actions. Sev-
adaptive immune systems and has broader genomic and non-genomic actions. Several
eral key biological and physiological functions of calcitriol, including autocrine and para-
key biological and physiological functions of calcitriol, including autocrine and paracrine
crine signaling mechanisms, are mediated from intracellularly synthesized 1,25(OH)2D
signaling mechanisms, are mediated from intracellularly synthesized 1,25(OH)2 D (calcitriol)
(calcitriol) in peripheral target cells [121], and not the circulating hormonal form of calcit-
in peripheral target cells [121], and not the circulating hormonal form of calcitriol, of which
riol, of which the concentrations are far too little to enter peripheral target cells. This lo-
the concentrations are far too little to enter peripheral target cells. This locally generated
cally generated calcitriol in these peripheral target cells (e.g., immune cells), such as mac-
calcitriol in these peripheral target cells (e.g., immune cells), such as macrophages, T- and
rophages, T- and B-lymphocytes, and dendritic cells, provide essential signaling for bio-
B-lymphocytes, and dendritic cells, provide essential signaling for biological functions,
logical functions, such as autocrine and paracrine signaling mechanisms of vitamin D and
such as autocrine and paracrine signaling mechanisms of vitamin D and prevention of
prevention of autoimmunity [121] (see next section).
autoimmunity [121] (see next section).
4.1. Autocrine and Paracrine Signaling
4.1. Autocrine and Paracrine Signaling
In addition to the direct effects of calcitriol on the genome, it also exerts indirect ef-
In addition to the direct effects of calcitriol on the genome, it also exerts indirect effects
fects via autocrine and paracrine signaling [20]. Examples of these include the impact of
via autocrine and paracrine signaling [20]. Examples of these include the impact of cal-
calcitriol switching T helper cell 1 (Th1) to T helper cell 2 (Th2) and Th17 to Treg cells,
citriol switching T helper cell 1 (Th1) to T helper cell 2 (Th2) and Th17 to Treg cells, which
which transforms pro-inflammatory status to anti-inflammatory status [28,29]. When the
transforms pro-inflammatory status to anti-inflammatory status [28,29]. When the intracel-
intracellular calcitriol concentration is low, statutes of Th1 and Th17 cells remain inflam-
matory, contributing to cytokine storms and the development of ARDS following viral
Biomedicines 2023, 11, 1542 13 of 26

lular calcitriol concentration is low, statutes of Th1 and Th17 cells remain inflammatory,
contributing to cytokine storms and the development of ARDS following viral infections in
vulnerable people [122,123]. In addition, intracellular calcitriol in immune cells, directly
and indirectly, enhances the expression of anti-microbial peptides and antibody synthesis.
While the exact mechanism of stimulation of some of the above pathways is un-
clear, it is known to involve transcription factors C/EBPβ and inhibit the orphan receptor
NR4A2 [124]. The regulation of the CYP27B1 gene (1α-hydroxylase enzyme) by a transcrip-
tional factor promoter, NR4A2, is inhibited by C/EBP-beta. Furthermore, over-expression
of C/EBP-beta decreases NR4A2 and CYP27B1 mRNA levels [124]. In contrast, FGF-23
counteracts the 1α-hydroxylase enzyme through FGF receptors in the presence of the
co-receptor (an aging-related factor), Klotho [11]. At the same time, the ablation of Klotho
leads to the over-expression of the FGF23 phenotype, consistent with Klotho deficiency [11].
This signaling also activates the mitogen-activated protein kinase (MAPK) cascade, but its
role in CYP27B1 expression remains unclear [125].
Despite the above, there is little evidence from RCTs regarding the optimum serum
25(OH)D levels for preventing various disease-related complications. This confusion
derives from the non-standardized clinical studies using different serum 25(OH)D con-
centrations, correlated with minimum effective concentrations, and poorly designed and
conducted RCTs [100] (see Section 3). The fundamental flaw is that investigators failed to
regard vitamin D as a nutrient: thus, designing RCTs considering vitamin D as a synthetic
pharmaceutical agent. Clinical outcome failures are common, as the pharmacokinetics of
these two sets of agents are very different (Figure 3). Furthermore, they failed to adhere to
standard published guidelines with nutrient clinical studies (Table 1) [113].

4.2. Optimum Concentrations of Circulating 25(OH)D Concentrations

As illustrated above (Figure 3), there are tissue-specific serum concentrations of
25(OH)D to elicit biological effects. The high affinity of vitamin D and 25(OH)D to the
VDBP provides the means for transporting and enhancing the half-life of 25(OH)D in circu-
lation. In addition, the specific evolutionary mechanism of the megalin–cubilin-mediated
membrane-driven internalization of vitamin D and 25(OH)D molecular complexes provides
the energy-dependent entry of these compounds into renal cells and vitamin D and other
nutrient storage tissues [126,127]. This entry facilitates the synthesis of the hormonal form
of calcitriol in renal tubular cells and storage mechanisms for D3 and 25(OH)D in muscle
and fat cells. In contrast to pharmaceuticals, these mechanisms allow 25(OH)D to have a
naturally longer circulatory half-life [128,129]. Administration of a nutrient such as vitamin
D in a deficient person, the change in nutrient status (∆) (see Section 3.3), would produce a
measurable, statistically significant clinical outcome. However, that is present only in those
with nutrient-deficient status.
In contrast to micronutrients like vitamins, iron, iodine, etc., pharmaceutical agents
neither have a specific internalization process nor a physiologic storage mechanism—a
significant difference. Consequently, pharmaceutical agents must be administered once
or multiple times daily to maintain the necessary serum concentrations to achieve the
intended benefits [130,131]. To overcome this, pharmaceutical agents have been modified
to administered as precursor, slow-released compounds, or converted into longer-acting
formulas (e.g., depot format), thus enabling them to prolong their half-lives and actions.
Consequently, the dose-response curves for the mentioned two categories differ vastly.
Therefore, one cannot evaluate these two categories using the same principle, protocols,
or study designs. Figure 4 illustrates the fundamental differences between (Figure 4A)
circulatory concentrations of a nutrient and (Figure 4B) a pharmaceutical agent.
Biomedicines 2023,
Biomedicines 11,11,
2023, 1542
x FOR PEER REVIEW 14
15ofof2627

Figure4.4.Illustrates
Figure Illustratesthe
thedose-response
dose-responseandandpharmacodynamic
pharmacodynamicdifferences
differencesbetween
betweennutrients
nutrientsand
and
pharmaceutical agents (inverted curves with nutrients vs. sigmoidal-shaped pharmaceutical re-
pharmaceutical agents (inverted curves with nutrients vs. sigmoidal-shaped pharmaceutical response
sponse curves). (A). An example using vitamin D dose responses. When it reached its sufficiency
curves). (A). An example using vitamin D dose responses. When it reached its sufficiency for a given
for a given tissue/system, providing more would not have additional physiological benefits. The
tissue/system,
response range providing more
is narrow, would
about halfnot
thehave
orderadditional physiological
of magnitude. (B). The benefits.
responseThe response
range range
expands with
ispharmaceutical
narrow, about half the over
agents orderan
oforder
magnitude. (B). Theand
of magnitude, response range expands
the response curve iswith pharmaceutical
shallow.
agents over an order of magnitude, and the response curve is shallow.
As illustrated above, many recent larger vitamin D RCTs have been poorly designed
As illustrated above, many recent larger vitamin D RCTs have been poorly designed
for bizarre reasons. Some were piggybacked on industry-sponsored studies as a shortcut
for bizarre reasons. Some were piggybacked on industry-sponsored studies as a shortcut
to generating data without additional expenses. Their primary endpoints are focused on
to generating data without additional expenses. Their primary endpoints are focused on
pharmaceutical agents, not nutrients. In such studies, vitamin D-related outcomes are in-
pharmaceutical agents, not nutrients. In such studies, vitamin D-related outcomes are
cidental or secondary; thus, they cannot be relied upon for drug approvals, guidelines, or
incidental or secondary; thus, they cannot be relied upon for drug approvals, guidelines, or
policy decision-making. In addition, the doses, frequency of administration, and duration
policy decision-making. In addition, the doses, frequency of administration, and duration
of studies were inappropriate in many vitamin D RCTs. As a result, their endpoints and
of studies were inappropriate in many vitamin D RCTs. As a result, their endpoints and
conclusions were unreliable.
conclusions were unreliable.

4.3. Importance of Raising Population 25(OH)D Concentrations to Reduce Morbidities

The guidelines of the US Endocrine Society [the minimum adequate serum 25(OH)D
of 30 ng/mL (75 nmol/L)] were for individual patients, not for disease entities or special
Biomedicines 2023, 11, 1542 15 of 26

or vulnerable groups [16]. In the absence of adequate exposure to sunlight, to raise and
maintain a blood level of 25(OH)D above 30 ng/mL, individuals require a daily minimal
oral intake of 2000 to 4000 IU (50 to 100 µg) of vitamin D3 , with a longer-term safe upper
limit of 10,000 IU [116,132–135]. Different scientific organizations have recommended
varied serum 25(OH)D concentrations. However, the minimum level to be maintained to
overcome infections, cancer, autoimmunity, and heart disease and for robust immunity is
50 ng/mL [9,20,50,57,136].
The natural vertebrate-form of vitamin D3 (the parental vitamin D) is the desirable
supplementation, preferably obtained via routine safe skin exposure to ultraviolet sun rays
and/or daily or weekly supplementation or targeted food fortification programs [14]. These
mentioned modes can provide sufficient amounts of vitamin D to maintain the population’s
vitamin D sufficiency [i.e., maintaining the mean serum 25(OH)D concentrations above
40 ng/mL) [43,137], which would enhance the population’s immunity and significantly
reduce illnesses and absenteeism, thus increasing productivity. A robust immunity in the
population can inherently curtail the spread of pathogenic microbial infections, partic-
ularly viral epidemics, and pandemics such as SARS-CoV-2, and reduce the associated
hospitalization and deaths from infections [59,138].

4.4. Factors That Modify the Functions of CYPP450 Enzymes and VDRs
The quantity and the efficiency of generating vitamin D in the skin and the subsequent
two hydroxylation steps are reduced by several factors. These include the location of
residence (i.e., latitude) where no UVB rays reach during winters (i.e., seasonality), air
pollution that blocks UVB rays, sun-avoidance behavior such as excessive clothing, using
umbrellas, and UV blockers, higher melanin skin pigmentation, scarring or aging of the
skin, etc.
While VDR abnormalities such as pleomorphism are not uncommon [139], genetic vari-
ants of CYP450 vitamin D-related enzyme abnormality are rare. For the proper functioning
of CYPP450 enzymes, it is crucial to have adequate intracellular concentrations of not only
calcitriol but also magnesium [140,141]. In addition, diseases, especially infections, increase
the consumption of vitamin D, thus requiring maintaining a higher intake. Furthermore,
acute and chronic inflammation increases reactive oxygen species (i.e., generating oxidative
stress) that reduce mitochondrial functions [142], which further aggravates oxidative stress
and cytochrome 25- and 1α-hydroxylase activity and impairs recovery [143].
The continual availability of sufficient D3 in conjunction with the pleiotropic distribu-
tion of VDRs, co-factors, and the associated signaling systems enables a healthy life [43,144].
The vitamin D control system—CYP450 enzymes that activate and degrade vitamin D and
metabolites, PTH and FGF23—associated feedback mechanisms, and VDRs have evolved
over millions of years [145]. This complex system has been fine-tuned through vertebrate
evolution, resulting in genetic variants and nucleotide polymorphisms in modern hu-
mans [146,147]. Calcitriol–VDR interactions and the resultant gene expressions related to
target metabolic genes and subsequent biological activities. These are further modulated
by epigenetic variations [72,147] and switching from traditional lifestyles to modern [148].

4.5. The Importance of Prescribing the Proper Type and Doses of Vitamin D
Since vitamin D is a nutrient, the benefits from supplements are evident only in those
with absolute or relative deficiencies. Therefore, while broader guidance on vitamin D
supplementation is generally helpful, individual recommendations must be based on their
vitamin D status, body weight, and requirement [9,20]. Therefore, as with other medical
disorders, healthcare workers should investigate patients’ history, background, habits, and
needs before recommending or prescribing vitamin D supplements. Along with lifestyle
changes, this would allow them to advise individuals to take the correct dose, eliminate
rare adverse effects, and reduce costs.
Access to a laboratory and affordable testing allows one to measure biochemical
variables, such as serum 25(OH)D levels (and serum and urinary calcium, PTH, etc.), to
Biomedicines 2023, 11, 1542 16 of 26

assess the overall vitamin D/calcium status. Properly utilizing these data could provide
cost-beneficial approaches for patients and maximize efficacy while minimizing adverse
effects [149]. Considering the high cost of 25(OH)D measurements and their unavailability
to most people worldwide, laboratory testing is not essential [20]. Instead of using serum
25(OH)D concentration, the vitamin D dose can be calculated via published tables on
body-weight-based calculation for the correct dose [9,20].
Vitamin D is highly economical and has virtually no adverse effects on recommended
D3 doses, frequency, and durations. Furthermore, it is widely available as an over-the-
counter nutrient globally at an affordable cost. Maintaining vitamin D sufficiency in the
population is the most cost-effective approach to controlling the spread of viral infections
such as COVID-19 [20,100] and reducing the prevalence of chronic diseases [14,41,150,151]
and healthcare costs.

4.6. Vitamin D Doses to Maintain Therapeutic Serum 25(OH)D Concentrations

For a healthy 70 kg non-obese adult, it is recommended to consume 5000 IU (125 micro-
grams)/day or 50,000 IU (1.25 mg: one capsule) every tenth day or once a week—especially
during high-risk periods [20]. For a vitamin D-deficient person, these doses could take sev-
eral months to increase serum 25(OH)D to therapeutic levels of over 50 ng/mL [20]. Even
in a vitamin D-sufficient person [i.e., based on community guidelines of maintaining serum
25(OH)D concentration of 40 ng/mL], the mentioned oral doses of vitamin D would take
a few weeks to raise serum 25(OH)D concentration above 50 ng/mL [9]. Therefore, such
doses could be insufficient (and ineffective) to achieve the desired target serum 25(OH)D
concentration in emergencies.
Even for those with severe vitamin D deficiency, delays in raising serum 25(OH)D
concentrations can be reduced to less than four days by administering higher upfront
loading doses—between 100,000 IU and 400,000 IU based on the body weight, as a single
dose [152]. It the time is not critical, preferably higher amounts should be administered in
divided doses over a few days to improve absorption and aid 25-hydroxylation in the liver—
a rate-limiting factor [20,153]. In contrast, administration of 0.5 to 1 mg calcifediol orally
(preferably 0.014 mg/kg body weight) [20] rapidly increases serum 25(OH)D concentrations.
Administration of the correct dose will boost the immune system within one day. Therefore,
the latter is the most appropriate type of vitamin D in emergencies such as COVID-19 [9].

4.7. Vitamin D Dose Recommendations

Little vitamin D is present in natural food; thus, the dietary intake is minimal and
cannot be relied upon for the majority [154,155]. Furthermore, in the absence of exposure
to daily direct sunlight, casual exposure to the sun is insufficient to raise serum 25(OH)D
concentration [14,156]. Governments and some scientific committees such as IoM, Food
and Nutrition Board (FNNB), and USPTO in the USA, and the scientific advisory committee
(SCAN) in the UK, etc. continue to recommend doses of vitamin D of between 400 and
1000 IU/day. These low doses would raise serum 25(OH)D concentration in the circulation
to less than 6 ng/mL, which is grossly insufficient in those with vitamin D deficiency
and insufficiency [157]. Therefore, such doses consistently fail to raise serum 25(OH)D
concentrations to needed therapeutic levels [157], thus unhelpful.
Governments and appointed committees recommend doses of vitamin D to prevent
rickets in children and osteomalacia in adults [14,150,151], not for other disorders. It is only
necessary to raise serum 25(OH)D concentration to 20 ng/mL to benefit the musculoskeletal
system [16,158]. However, this does not help other body systems or disease conditions.
Therefore, it is paramount to use adequate doses of vitamin D (preferably body-weight-
based doses) [9,20] to increase serum 25(OH)D to the desired concentrations, as shown
in Figure 4.
For busy healthcare workers, it is difficult to remember the different doses of vitamin
D and serum 25(OH)D concentrations needed for various diseases. Therefore, irrespective
of age and body weight, when laboratory measurements are affordable and available, it is
Biomedicines 2023, 11, 1542 17 of 26

rational to maintain serum 25(OH)D concentrations above 40 ng/mL [159,160], preferably

above 50 ng/mL—a range between 50 and 90 ng/mL [9,20,157].
When 25(OH)D measurements are unavailable or unnecessary, an accepted vita-
min D dose for extra-skeletal beneficial effects of “non-obese” adults is 5000 IU per day
(125 µg/day) [161,162]. In the vast majority, this would raise serum 25(OH)D concentration
to approximately 40 ng/mL over a period. These doses exceed outdated governmental
recommendations [133,162,163]. Persons with obesity/overweight and multiple comor-
bidities, taking medications that increase 24-hydroxylase catabolic activity, and those with
gastrointestinal fat malabsorption disorders require two- to four-fold higher doses than the
above [9].

4.8. Strength and Limitations

Vitamin D is a vast field of science that encompasses all body systems. Over 120,000 peer-
reviewed scientific articles on vitamin D have been published on PubMed alone, covering
several aspects of its biology, physiology, clinical characteristics, and pathology related to
vitamin D deficiency. Therefore, neither a single article nor a book could cover all aspects. This
article focuses on physiological, clinical, and practical elements that would help practicing
clinicians and healthcare workers better manage their patients. These were strengthened by
providing pertinent references under each sub-heading for further reading and exploring
other areas of interest.

5. Conclusions
Vitamin D3 supplements are widely available, inexpensive. It is a safe micronutrients
that should be considered as long-term prophylactic agents or adjunct therapy. When
available and practical, direct and safe skin exposure to sunlight is preferable to obtain
vitamin D3 . Vitamin D deficiency is associated with many common chronic diseases, such
as rickets, osteomalacia, metabolic and immune disorders, autoimmune diseases, and
cancer. It also impacts pregnancy and children’s growth, including brain functions: the lack
of it could lead to premature death. Consequently, its deficiency significantly increases the
vulnerability and severity of metabolic diseases such as diabetes, obesity, and metabolic
syndrome and worsens infections and cancer.
In addition, hypovitaminosis D increases the risks for bacterial and viral infections,
associated complications, and deaths. Those affected are particularly vulnerable to viral res-
piratory illnesses such as seasonal flu, influenza, respiratory syncytial virus, and COVID-19.
Worldwide, sepsis is responsible for approximately seven million annual deaths: a major
contributory factor for this is vitamin D deficiency [164]; half of these premature deaths
could have been prevented by taking proactive actions to treat them aggressively with
vitamin D and calcifediol as described in this article. The goal is to rapidly raise serum
25(OH)D concentration above 50 ng/mL, allowing it to boost the immune system [20]. This
would have cost a fraction of a one-day hospital stay (less than two USDs person).
This principle applies to those infected with or PCR-positive persons for SARS-CoV-2
(during the COVID-19 pandemic and in the future) [59,116,165]. Since February 2020,
over 900 peer-reviewed scientific articles related to vitamin D and COVID-19 have been
published—the first recommendation of using high doses of vitamin D was published
on 28th February 2020 [166,167]. Based on the mentioned vast literature, vitamin D’s
importance in rectifying the impairment of immune functions to overcome COVID-19 has
recently been highlighted [168,169].
The long-term use of adequate vitamin D doses, such as 5000 to 7000 IU (125 to
175 micrograms)/day, will reduce the risks for infections and complications [50,162,170],
racial disparities in infections-related clinical outcomes, including COVID-19 [116], and
the spread of COVID-19, hospitalization and mortality [59]. Children and elders with
severe hypovitaminosis D have a high risk of developing life-threatening infections [171]—
conditions, such as Kawasaki-like disease or multi-system inflammatory syndrome [33–35],
which can be significantly reduced by proactively providing vitamin D supplements.
Biomedicines 2023, 11, 1542 18 of 26

In seriously ill patients (e.g., non-traumatic patients admitted to ICUs) with vitamin D
deficiency, even high doses would take up to a week to increase serum 25(OH)D concentra-
tions. For such patients and in emergencies, it is best to treat them with a single oral dose
of 0.5 and 1.0 mg calcifediol (0.014 mg/kg body weight) to rapidly raise serum 25(OH)D
concentrations above the minimum therapeutic levels of 50 ng/mL [20]. This dose can be
continued every third day until a high amount of vitamin D maintains the serum 25(OHD
concentration at the desired level. Within four hours of administration of a proper dose
of oral calcifediol, serum 25(OH)D concentrations increase above 50 ng/mL, sufficient to
boost the immune system within one day.
The administration of high doses of vitamin D and/or calcifediol causes robust im-
mune responses, allowing individuals to overcome severe inflammation and oxidative
stress, thus preventing cytokine storms and ARDS [172]. Magnesium deficiency increases
the risk and worsens cytokine storm-related complications and outcomes [173]. Therefore,
it is crucial to prevent (intracellular) magnesium deficiency and provide sufficient amounts
of other micronutrients and co-factors, allowing robust immune and metabolic functions.
Because of the multiple biological and physiological benefits, the prompt administration of
proper vitamin D dose supplements is necessary to prevent complications, ICU admissions,
and death from infections [174]. Serum 25(OH)D concentration over 50 ng/mL is also
necessary to overcome some cancers and reduce all-cause mortality.
Vitamin D supplementation prevents the development of chronic diseases [175,176],
including osteoporosis and metabolic conditions [177] and acute and chronic respiratory
infections [90,91,178]. SARS-CoV-2 infection, post-vaccinations, and other coronaviral
infections are classic examples of where proper doses of vitamin D leading to sufficiency
would prevent pulmonary and endothelial cell damage, hypoxia and ARDS, coagulation
abnormalities and micro-embolism [75]. Consequently, it reduces hospitalizations, ICU
admissions, and deaths [59,178,179]. In addition, vitamin D supplements at proper doses
reduce the incidence of seroconversion and symptomatic SARS-CoV-2 [117], and minimize
complications [171], hospitalizations [180], ICU admissions [181], and deaths [59,179].
As illustrated in Section 4.7, the doses recommended (e.g., vitamin D, 400 to 1000 IU/day)
by governments and some appointed advisory bodies were designed only to overcome the
minimum serum 25(OH)D concentrations necessary to overcome musculoskeletal disorders
like rickets in children and osteomalacia adults. Such low doses do not benefit other body sys-
tems. Despite these, none of these recommendations specify that the suggested minuscule
amounts are only applied to prevent musculoskeletal diseases—rickets and osteomalacia.
Because of this narrow focus, the suggested doses and serum 25(OH)D concentrations
are misleading and outdated. Considering the above, recommendations and broader conclu-
sions by the entities mentioned above are misguiding [158,182] and cannot be generalized.
Outdated recommendations and guidelines for vitamin D need to be urgently replaced with
new documents using recently published data-driven solid recommendations, including
those from systematic reviews and meta-analyses.
Synthetic analogs of vitamin D should not be used as prevention or replacement
therapy for deficiency, and supplements or treatment for osteoporosis and infections.
Vitamin D analogs are restricted to specific indications, such as hepatic or renal failure,
hypoparathyroidism, etc. Calcifediol is the agent of choice in emergencies, where the
rapid elevation of serum 25(OH)D concentration is necessary to reduce complications
that save lives. Calculating calcifediol and vitamin D doses on a body-weight basis is
straightforward [20]—thus, an individual’s needed vitamin D dose should not guess.
Calcifediol is administered as a single dose (in conjunction with a high amount of vitamin
D to prolong the benefits of vitamin D) or every third day during an emergency. In addition,
calcifediol is indicated when 25-hydroxylation of vitamin D is impaired, as in hepatic
failure. However, it is not recommended as a replacement therapy for osteoporosis and
renal failure. For correcting vitamin D deficiency, D3 should be used.
Furthermore, 1α-hydroxylated synthetic vitamin D analogs, including 1,25(OH)D, are
indicated when renal tubular cells fail to generate calcitriol from 25(OH)D due to a lack
Biomedicines 2023, 11, 1542 19 of 26

of 1α-hydroxylation activity, as in advance renal failure. Calcitriol is also indicated in all

types of hypoparathyroidism to maintain serum-ionized calcium. Treating deficiencies
with nutrients to overcome conditions requires understanding biology and physiology
in conjunction with practicalities and cost-effectiveness. With such an understanding,
proper dosing regimens and the right frequency of administering nutrients using the three
commercially available vitamin D metabolites (i.e., parental D3 , calcifediol, and calcitriol)
are crucial for improving clinical outcomes, avoiding adverse effects, and saving lives.

Funding: The author received no grants of funding.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest: The author has no conflict of interest. This research received no grant or aid
from funding agencies or commercial or not-for-profit sectors. He did not receive any funding or
writing assistance.

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