Medical Hypotheses 163 (2022) 110845

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Medical Hypotheses
journal homepage: www.elsevier.com/locate/mehy

Hypothesis: Mutual dependency of ascorbate and calcidiol for optimal

performance of the immune system
Simon P.J. Albracht
Biochemist, retired from the Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, The Netherlands

A R T I C L E I N F O A B S T R A C T

Keywords: For a proper functioning, immune cells require sufficient levels of ascorbate and calcidiol in the blood. Upon
Immune system activation, immune cells increase their internal conversion of calcidiol to calcitriol. In general, calcitriol can
Ascorbate affect the expression of ca. 3% of the genes in the human genome. Among the gene products are a number of Fe
Calcidiol
(II)- and 2-oxoglutarate-dependent dioxygenases functioning as demethylases of histones, DNA, RNA and ribo­
Iron-dependent hydroxylases
Neutrophilia
somes in epigenetic gene regulation. These dioxygenases require millimolar concentrations of ascorbate for
Lymphocytes activity. Immune cells contain 1–4 mM ascorbate. It is hypothesized here that there is a mutual dependency of
ascorbate and calcidiol for optimal performance of the human immune system. Early signs for this thesis are in
literature, but were not yet recognized. Daily intake of both compounds to keep the plasma levels to ca. 75 µM for
ascorbate and to at least 75 nM for calcidiol, will reduce the risk of serious disease after infections. This applies in
particular to infected individuals with prolonged elevated levels of neutrophils. Reports state that such subjects
are often deficient in either ascorbate (vitamin C) or calcidiol (vitamin D3), but none of the studies considered the
levels of both at the same time.

Introduction primates, some fruit-eating bats and guinea pigs, a mutation occurred
during evolution, that inactivated the last enzyme in the ascorbate
It is widely accepted that the human immune system is suboptimal in biosynthetic pathway (L-gulono-1,4-lactone:oxygen 3-oxidoreductae,
individuals with a low plasma level of calcidiol (25(OH)D3, vitamin D3) EC 1.1.3.8). Hence, humans need a daily intake of nearly 0.5 g ascor­
[1,2]. This is usually caused by insufficient exposure to daylight and/or bic acid to correct for this mutation. Ascorbate is a substrate (providing
lack of supplemental calcidiol intake. Also patients with a variety of reducing equivalents) for two human copper enzymes. Dopamine
diseases, e.g. cancer, are often deficient in calcidiol. Likewise, the β-mono-oxygenase (dopamine,ascorbate:oxygen oxidoreductase (β-hy­
plasma level of ascorbate (vitamin C) seems to be important for a good droxylating), EC 1.14.17.1) converts dopamine into noradrenaline
immune system, although the clinical evidence is less clear [3,4]. As (norepinephrine) in adrenal glands and in the brain. Peptidylglycine
discussed in this paper, humans need up to 0.5 g L-ascorbic acid per day mono-oxygenase ([peptide]-glycine,ascorbate:oxygen oxidoreductase
which means that ascorbic acid is not really a vitamin but rather an (2-hydroxylating), EC 1.14.17.3) is involved in the amidation of bio­
essential foodstuff. I will present evidence for the hypothesis that the logically active peptides. Each enzyme contains two Cu atoms with
effects of ascorbate and calcidiol on the immune system are mutually different roles during catalysis. As will be discussed further on, ascorbic
dependent. acid has many other vital functions in our body.

Ascorbic acid

Nearly all mammals synthesize ascorbate in the liver [5]. In

Abbreviations: 2OG, 2-oxoglutarate; 3D, three dimensional; CYP, cytochrome P450; DNMT, DNA methyltransferase; EPR, electron paramagnetic resonance;
Fe2OGD, Fe(II)- and 2-oxoglutarate-dependent dioxygenase; FIH, factor-inhibiting HIF1α hydroxylase; JMJDs, Jumonji-C domain containing demethylases; GC,
group-specific component; HIF, hypoxia inducible factor; HRE, hypoxia responsive element; KDMs, lysine demethylases; MS, multiple sclerosis; NETs, neutrophil
extracellular traps; PBMCs, peripheral blood mononuclear cells; PHD, prolyl hydroxylase; SARS-CoV, severe acute respiratory syndrome coronavirus; SVCT, sodium-
dependent vitamin C transporter; TET, ten-eleven translocase; VDR, vitamin D receptor; VDRE, vitamin D responsive element.
E-mail address: [email protected].

https://doi.org/10.1016/j.mehy.2022.110845
Received 18 November 2021; Received in revised form 9 March 2022; Accepted 11 April 2022
Available online 14 April 2022
0306-9877/© 2022 The Author. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
S.P.J. Albracht Medical Hypotheses 163 (2022) 110845

Ascorbate can rapidly normalize the number of white blood cells in enzyme from chick embryos [18] (procollagen-L-proline,2-oxoglutarate:
infected patients oxygen oxidoreductase (4-hydroxylating), EC 1.14.11.2). The enzyme
catalyzes the reaction: 2-oxoglutarate + O2 + Pro-R => succinate +
A 1938 study [6] already noted that leukocyte counts in human CO2 + 4-OH-Pro-R. This enzyme is a tetrameric α2β2 protein [19], in
patients with too high (leukaemia, pneumonia) or too low (normal flu) contrast to the human enzymes PHD2 (monomer [9]) and FIH1
levels, rapidly normalized (within 30–60 min) after administration of a (homodimer [20]). As shown in Fig. 1, the continuous activity of the
single dose of 300 mg ascorbate, by oral or parenteral (bypassing the chicken enzyme is dependent on the presence of ascorbate although the
digestive tract) means. However, leukocyte counts returned to the initial activity is not. With electron paramagnetic resonance (EPR), I
original abnormal values after ca. 3 h. Also long-term effects (up to 6 could show that after a number of turnovers in the absence of ascorbate
days) were described. The conclusion was that ascorbate normalizes (situation like in trace a in Fig. 1, red arrow), the iron atom in the
leukocyte counts (and heals a number of the underlying diseases and enzyme oxidized to the ferric state (clear EPR signal). When ascorbate
infections) more rapidly than with the (at that time) conventional was added (conditions of Fig. 1, trace a, green arrow), the EPR signal
treatments. It is now known that the majority of white blood cells in decreased again. This corroborated the initial proposal [21] that
humans is made up of neutrophils (45–75%), lymphocytes (20–50%) ascorbate keeps iron in this enzyme in the ferrous state (situation in
and monocytes (3–10%). trace b in Fig. 1), so that di-oxygen can bind; ferric iron (Fe3+) does not
bind O2. This principle is still valid for a wide variety of human
Ascorbate mitigates the effects of hypoxia on cells Fe2OGDs, which need millimolar concentrations of ascorbate for their
continuous activity.
In 1924 Warburg described the effects of hypoxia in normal and in
cancer tissue on the metabolic phenotype of cells. Hypoxia shifted the
Epigenetic modifications control the interaction of HIF1αβ with its target
energy metabolism from a glucose-oxidative type (bulk of ATP produced
genes
by mitochondria, ’Grana’ as Warburg called them) to a glycolysis type
(increased ATP production in the cytosol) [7,8]. Thus, hypoxia changed
HIF1αβ moves into the nucleus and binds to a RCGTG motif in
the bioenergetic phenotype of cells.
hypoxia responsive elements (HREs) in DNA. This can lead to a change
In the last two decades the effects of hypoxia on cells have been
(stimulation or repression) in gene transcription. The human genome
intensively studied and the results confirmed the changes of the bio­
contains over one million HRE motifs, but the number of accessible
energetic phenotype of cells described by Warburg. Respiratory prob­
lems in individuals often lead to tissue hypoxia with O2 concentrations
below the normal level of 10–30 µM. In turn, this lowers the activity of a
set of iron-dependent hydroxylases that target the free hypoxia inducible
factor 1 subunit alpha (HIF1α) in cells. Human HIF1α can be hydrox­
ylated at three positions, at P402 and P564 by the prolyl hydroxylases
PHD1-3 (hypoxia-inducible factor-L-proline,2-oxoglutarate:oxygen
oxidoreductase (4-hydroxylating), EC 1.14.11.29) and at N803 by the
factor-inhibiting HIF1α hydroxylase FIH1 (hypoxia-inducible factor-L-
asparagine,2-oxoglutarate:oxygen oxidoreductase (4-hydroxylating),
EC 1.14.11.30) [9,10]. In vitro, these enzymes have Km values for O2
several fold higher (3- to 8-fold) than the normal tissue O2 concentra­
tions, so their activity in cells directly depends on the O2 concentration
[10]. Ascorbate partly counteracts the hypoxia-induced decrease of ac­
tivity by keeping the residual activity optimal, as discovered for cancer
cells in 2003 [11].
Hydroxylation marks the HIF1α subunit for rapid destruction by the
proteosomal degradation system in the cell. Under hypoxic conditions,
the HIF-hydroxylases become less active (first the PHDs and at still lower
O2 levels also FIH1 [12]) and without ascorbate their hydroxylation
activity stops altogether. In these cases, HIF1α dimerizes with HIF1β to
form the HIF1αβ heterodimer, a transcriptional regulator for hundreds
of genes. In addition to HIF1 there are two more HIF proteins, HIF2 and
HIF3 [13,14], but these are not discussed here.
The basis for the change of the bioenergetic phenotype of cells in­
cludes alterations like the upregulation of genes encoding for pyruvate
dehydrogenase kinase (PDK) [15,16] and for proteins and enzymes Fig. 1. Oxygen consumption at 37 ◦ C by prolyl 4-hydroxylase from chick em­
involved in glucose uptake, glycolysis, erythropoiesis, angiogenesis, and bryos in the absence and presence of ascorbate. The final reaction mixture (1.5
in autophagy of mitochondria [17]. Mitochondrial biogenesis is down­ mL) contained 3 mg bovine serum albumin, 25 µM FeSO4, 0.29 µM enzyme,
regulated [10,13]. 400 µM 2-oxoglutarate, 320 µM (Pro-Pro-Gly)10⋅9H2O and 1 mM ascorbate. All
additions were made in air-saturated (200 µM O2) 50 mM Tris-HCl buffer (pH
Ascorbate is essential for the activity of Fe(II)- and 2-oxoglutarate- 7.7). Oxygen consumption was measured polarograpically. The initial reaction
dependent dioxygenases (Fe2OGDs) rates were 1.1 µmol⋅min− 1 per mg enzyme (trace a) and 2.4 µmol⋅min− 1 per mg
enzyme (trace b). Using a molecular mass of 240 kDa, this gives turnover
numbers of 4.4 s− 1 and 9.6 s− 1, respectively. In trace (a) the reaction rate
PHD1-3 and FIH1 belong to the large family of Fe(II)- and 2-oxoglu­
dropped sharply after ca. 20 s (red arrow), i.e. after ca. 90 turnovers. Others
tarate-dependent dioxygenases (EC 1.14.11). I will use the abbreviation reported an activity drop after 15–30 turnovers at 30 ◦ C (activity 2.9 s− 1) [21].
Fe2OGDs to emphasize the fact that the active site in the functional The residual rate is presumably partly due to the non-enzymatic reaction re­
enzymes contains Fe2+. ported by [64] as studied in detail by [65]. Abbreviations: Fe, FeSO4; E,
I have been involved in a study on the effects of ascorbate on an enzyme; PPG, (Pro-Pro-Gly)10⋅9H2O; 2 og, 2-oxoglutarate; asc, ascorbate.
archetype Fe2OGD, namely the purified procollagen hydroxylating The figure was adapted from [18]

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S.P.J. Albracht Medical Hypotheses 163 (2022) 110845

HREs is estimated at 500–1000 [14] and can greatly vary between

different cells types. It is thought that epigenetic modifications of his­
tones and DNA limit the accessibility of HREs. For DNA, the best studied
modification is the methylation of the cytosine ring at the C-5 position in
a cytosine-phosphate-guanidine (CpG) dinucleotide (5′ -CpG-3′
sequence). This involves the DNA methyltransferases DNMT1, DNMT3A
and DNMT3B (S-adenosyl-L-methionine:DNA (cytosine-5-)-methyl­
transferase; EC 2.1.1.37). Ca. 75% of the CpGs in the human genome are
methylated. An extra –CH3 group can create a small change in the local
three-dimensional (3D) shape of proteins and nucleic acids. This is why
the presence (or absence) of a methyl group can interfere with the
binding of transcriptional regulators and so with gene expression. It is
the main reason for the lack of HIF1αβ binding to HREs in methylated
CpG at the heart of the HRE motif RCGTG (as shown in vitro and in vivo
[14]).

Fe2OGDs can function as demethylases Fig. 2. Active site of human methylcytosine dioxygenase TET2 (EC 1.14.11.-).
In this stick representation, iron is in green and the other atoms are in CPK
In the last decade, a wealth of information on newly-discovered colours. Data are from PDB entry: 5DEU [66]. The Fe ion (Fe3+ in this case) is
Fe2OGDs became available. Below, I will mention some of the find­ octahedrally co-ordinated by one nitrogen atom from two histidine residues
ings that are relevant to my hypothesis. In mammals, Fe2OGDs are not each (H1382 and H1881), one oxygen atom from an aspartate residue (D1384),
only involved in procollagen and HIF1α hydroxylation or the Cu en­ two oxygen atoms from an N-oxalylglycine molecule (Oga, a mimic of 2-oxoglu­
zymes mentioned above, but also in a wide variety of other reactions, e. tarate) and one oxygen atom from water. In active enzyme, with Fe2+ and 2-
oxoglutarate, the water molecule is displaced by di-oxygen. Used CPK colours
g. in carnithine biosynthesis, as demethylases of histone proteins via
(elements colours): C, grey; N, blue and O, red.
hydroxylation of methyl groups on lysine residues (Jumonji-C domain
containing demethylases (JMJDs), present names lysine demethylases
(KDMs)) and as demethylases of methyl groups on e.g. C-5 of cytosine in reversible. Instead, the formed Fe3+ could only be reduced with an
DNA by proteins encoded by genes called TETs (ten-eleven translocases). apparent E½ of − 127 mV. The hysteresis was interpreted as due to a
As already mentioned, the presence of such methyl groups can interfere small conformational change of the iron active site, presumably
with the binding of transcriptional regulators. Such methyl groups can extending to the second co-ordination sphere. Schofield and co-workers
be hydroxylated by specific Fe2OGDs as the first step in their removal. believe that the redox function of ascorbate is not its only role in
More recently, also Fe2OGDs acting on a wide variety of methylated Fe2OGDs [23,26]. Although it is not expected that ascorbate will affect
RNAs were discovered [14,22]. A number of human Fe2OGDs families the TauD enzyme, it cannot be excluded; in one of its natural habitats,
and a long list of effects of their mutations on human phenotypes can be the large intestines in mammals, E. coli has access to ascorbate. I am not
found in [12]. Many of the Fe2OGDs are already mentioned in the EC aware of any redox titration’s of human Fe2OGDs. Maybe the presence
1.14.11 list (oxidoreductases acting on paired donors, with 2-oxogluta­ of millimolar amounts of a redox-inactive structural analogue of ascor­
rate as one donor, and incorporation of one atom each of oxygen into bate, e.g. L-gulonic-γ-lactone [26] can influence the redox behaviour of
both donors). Their number is still growing because the epigenetic its iron active site by preventing a conformational change and so this
regulation of gene expression is considered to be still in its infancy [22]. may help to protect the iron from oxidation to the trivalent state. In the
chick embryo enzyme, dehydro-ascorbate can bind and is a competitive
Structure and active site of Fe2OGDs inhibitor towards Fe2+ [21].

Nearly all Fe2OGDs have the same general 3D structure, a distorted The plasma pharmacokinetics of ascorbate
double-stranded β-helix (DSBH or jelly-roll) core fold [23]. The Fe atom
in the active site is usually bound to an amino-acid motif HXD/E-Xn-H. The pharmacokinetics of ascorbate have been extensively studied by
The X-ray structure of crystals of the human FIH1, grown in the presence Levine et al. in young men [27] and young women [28]. Volunteers (7
of Fe2+ and 2-oxoglutarate (2OG), showed an octahedrally co-ordinated men, age 20–26; 15 women, age 19–27) were hospitalized for months
Fe atom (resolution 2.4 Å). The ligands consisted of one N atom from and fed with an ascorbate-restricted diet. Plasma ascorbate levels
H199 and H279 each, one O atom from D201 and two O atoms from sharply decreased within days (Fig. 3-a) and eventually stabilized at a
2OG. The sixth ligand site was empty but was presumably occupied by a level of 8 µM. A subsequent daily dose of 30 mg had little effect (panel
water molecule [20]. In the structure of another Fe2OGD, obtained at a b), but with a daily dose of 60 mg the plasma level increased signifi­
higher resolution (PHD2, 1.7 Å [9]), this water molecule could be cantly (panel c). With the latter dose, a steady state level was only
resolved. A recent example of a human methylcytosine dioxygenase reached in all subjects after 63 days. Careful inspection of the original
(TET2, resolution 1.8 Å) is shown in Fig. 2. The position of the water figure 1A in [28] showed that the female subject starting with the
molecule is the site where O2 can bind. Interestingly, an Fe4+–– O (ferryl- highest serum ascorbate level (61 µM), showed the fastest increase (to
oxo) state is one of the intermediates in the reaction cycle of Fe2OGDs 60 µM) in the 60 mg dose period. This suggests that the initial depletion
[23–25]. period of 29 days was not sufficient to deplete all possible ascorbate
As mentioned above, the continuous activity of many, if not all, of stocks in the body (e.g. the brain). The subject with the lowest starting
the human Fe2OGDs is dependent on the presence of ascorbate. The serum level (10 µM) required the full 63 days in the 60 mg dose period
redox function (ene-diol) of ascorbate is essential, but the 1,2-dihyrox­ and even then had the lowest serum ascorbate level (ca. 39 µM) of the 15
yethyl side chain on C-4 of the lactone ring is not [26]. Redox experi­ subjects. The curve for one subject is shown in panel d. Panel e depicts a
ments, monitored by Fourier transform infrared (FTIR), of a well-studied plot of the steady-state plasma levels against the daily doses of ascor­
bacterial Fe2OGD, taurine dioxygenase (TauD; taurine,2-oxoglutarate: bate. The maximal concentration was 70–80 µM at an oral intake of
oxygen oxidoreductase (sulfite-forming), EC 1.14.11.17) from Escher­ 200–400 mg ascorbic acid per day. A higher intake is presumably
ichia coli, showed a large hysteresis behaviour ascribed to iron [25]. The required for the elderly and for people with ill-health. Excess ascorbate,
oxidation of Fe2+ occurred at an apparent E½ of +171 mV and was not even 10 g per day [29], is not harmful and is rapidly excreted in the

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S.P.J. Albracht Medical Hypotheses 163 (2022) 110845

Fig. 3. Ascorbate plasma concentrations in 15 women (age 20–27) as a function of dose. From the start, all volunteers (inpatients) received only ascorbate restricted
diets (<5 mg ascorbate per day). (a) It took up to 29 days to deplete their plasma to a steady level of ca. 8 µM (arrow). Note that the healthy subjects started with
ascorbate levels between 10 and 60 µM. (b) Subjects received 15 mg ascorbate twice a day until, after 55 days, all had reached a steady-state level. (c) Subsequently,
subjects received 30 mg ascorbate twice a day. All reached a steady-state level after 63 days, although some did so already within half that time. (d) Steady-state
ascorbate concentration in plasma for one subject on a dose of 60 mg daily. Note that for this subject it took 36 days to reach that level. (e) Steady-state ascorbate
concentrations in plasma for subjects as a function of all doses (30, 60, 100, 200, 400, 1000 and 2500 mg per day; mean of 6 subjects).
The figure was adapted from [28]

urine, provided that the kidneys are functioning normally [30]. Interestingly, in the ascorbate depletion studies of Levine et al. [27]
it was noted that at the nadir of depletion, six of the seven male vol­
Tissues, plasma and blood cells contain widely different concentrations of unteers had mild but distinct fatigue and/or irritability symptoms,
ascorbate without scurvy. These symptoms, often noticed in ascorbate deficient
individuals [34], disappeared within several days during the 30- or 60-
The levels of ascorbate can differ considerably in tissues and cells. In mg daily dose periods. As the brain and nerve cells have a high ascorbate
guinea pigs (with sufficient ascorbate in their food) they are highest in content, this may point to a functional relationship.
the brain (2–10 mM) and adrenal glands (4–10 mM) [31]. This also
holds for humans [32]. The cells in these tissues concentrate ascorbate Ascorbate can greatly reduce severe symptoms due to neutrophil
via the sodium-dependent vitamin C transporter 2 (SVCT2, Vmax 1 pmol. extracellular traps (NETs)
min− 1 per cell, Km 8–69 µM). Maximal ascorbate levels in human im­
mune cells are lower: neutrophils (1.3 mM), monocytes (3.1 mM), Neutrophils are the first (transient) responders to inflammation, a
platelets (3.6 mM) and lymphocytes (3.9 mM). They are already reached little later followed by lymphocytes and monocytes/macrophages. In
with an intake of 100 mg ascorbate per day [27,28]. These cells use healthy individuals, the normal plasma half-life of neutrophils is 8–20 h
SVCT1 (Vmax 15 pmol.min− 1 per cell, Km 65–252 µM) [31]. Red blood [35] and after their spontaneous apoptosis (natural cell death) they are
cells contain the same ascorbate concentration as plasma. They possess removed by macrophages. Thus, neutrophils are short-lived cells. Viss­
no SVCT and passively take up oxidized ascorbate (uncharged) via their ers and Wilkie [36] discovered that in mice, deficient in L-gulono-1,4-
glucose transporter. In bone marrow, the haematopoietic and multi­ lactone oxidase (GULO − /− mice), ascorbate is required for the
potent stem cells, as well as haematopoietic progenitor cells contain 2 to apoptosis of neutrophils as well as for their clearance by macrophages.
20-fold more ascorbate than differentiated cells. The required concen­ Under normoxic conditions, neutrophils from ascorbate deficient mice
trations of ascorbate for many Fe2OGDs and Cu mono-oxygenases are in contained elevated levels of HIF1α, indicating that the HIF-hydroxylases
the millimolar range. also did not function properly.
Assuming an ascorbate concentration in the human brain (volume A subsequent study showed, that when GULO − /− mice were sup­
ca. 1.2 L) of 5 mM, the brain would contain ca. 1 g ascorbate (Mass plied with sufficient ascorbate for 2–4 weeks, after which the supply was
176.1 Da). As nerve cells have a high ascorbate content, the complete discontinued, their phenotype remained unaffected for 3 weeks. How­
central nervous system may contain considerably more ascorbate. An ever, a rapid weight loss occurred in week 4 and the animals showed
estimate for the amount of ascorbate in neutrophils in blood (1.3 mM severe malaise with scurvy-like symptoms [33].
ascorbate; radius 6.5 µm; amount of cells 4.15x109 L-1; 5.5 L blood) gives A little later it was discovered [37] that septic GULO − /− mice
6 mg and for platelets ca. 8 mg. Plasma itself (5.5 L; 70 µM) holds ca. 68 showed NETs in the lungs. With sufficient ascorbate in their food this
mg. Upon systemic depletion, the brain gives up lastly its ascorbate was attenuated. A suspension of isolated human neutrophils stimulated
stocks [32]; upon replenishment the brain presumably takes up firstly by phorbolmyristate acetate also formed NETs. This was greatly reduced
the available ascorbate [33]. with neutrophils preloaded with 3 mM ascorbate. NETs are extracellular

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S.P.J. Albracht Medical Hypotheses 163 (2022) 110845

plugs, composed of DNA and a variety of cell-degrading proteins from its mono-anionic form in blood (pH 7.4). This charged molecule cannot
neutrophils, that can trap and destroy pathogenic intruders. It is an anti- permeate through membranes.
microbial mechanism that neutrophils can use in addition to phagocy­
tosis of pathogens. As discussed later on, NETs are the main cause of Plasma calcidiol
organ failure in patients with neutrophilia.
Recently it was reported that neutrophils isolated from twenty sepsis Plasma calcidiol has skeletal and non-skeletal functions
patients in hospital IC units, who appeared to be highly deficient in
plasma ascorbate (5.8 ± 11.8 µM), showed spontaneous NETosis when The role of plasma calcidiol (vitamin D) in bone metastasis has been
stimulated by phorbolmyristate acetate. This was 18-fold higher than known for a long time. In humans, calciol (pre-vitamin D3) is formed in
with neutrophils from healthy subjects (with 44.2 ± 21.9 µM plasma the skin from 7-dehydrocholesterol by UV light (270–300 nm) and this
ascorbate), where it was barely detectable. When neutrophils from the apolar compound is for 90% bound to the group-specific component
patients were incubated with 1 mM ascorbate for 2 h, the induced protein (GC; often called DBP, vitamin D binding protein) [42]) in blood.
NETosis was back to the same level as that in neutrophils from 20 Calciol is then hydroxylated to calcidiol (often indicated as 25(OH)D3)
healthy subjects incubated under the same conditions [38]. by liver 25-hydroxylase, the mono-oxygenase CYP2R1 (calciol,NADPH-
hemoprotein reductase:oxygen oxidoreductase (25 hydroxylating); EC
Lack of ascorbate can be the primary cause for death of patients with 1.14.14.24; CYP stands for cytochrome P450). Calcidiol (healthy serum
neutrophilia levels > 75 nM; plasma half-life ca. 2 weeks) is inactive and needs to be
activated by a second hydroxylase, mainly in kidneys. This 1-α-hy­
The occurrence of massive amounts of blood neutrophils (neutro­ droxylase (CYP27B1; calcidiol,adrenodoxin:oxygen oxidoreductase (1-
philia) has been noted in critically-ill patients infected with the envelope hydroxylating); EC 1.14.15.18) then converts calcidiol into the active
viruses severe acute respiratory syndrome coronavirus-1 (SARS-CoV-1), form calcitriol (1,25(OH)2D3; serum level ca. 0.1 nM; plasma half-life
Middle East respiratory syndrome coronavirus (MERS-CoV), SARS-CoV- 4–6 h). The activity of the kidney CYP27B1 is regulated by Ca2+,
2 and severe influenza (see e.g. [39]). A recent paper of Schurink et al. phosphate and their regulating hormones (e.g. parathyroid hormone
[40] reported on autopsy and extensive histopathologic analyses of all (PTH) and calcitonin). Note that both CYP enzymes are iron-containing
organs from twenty one IC patients deceased from SARS-CoV-2. Virus- hydroxylases with Fe2+ bound in a heme group. As with Fe2OGDs, also
infected cells were found in at least one organ, but the longer the disease in these enzymes an Fe4+– – O (ferryl-oxo) state is an intermediate in the
lasted, the less virus was detected. No virus was found in skeletal mus­ reaction cycle.
cles, the genital tract or the brain. All bodies had neutrophils and Calcitriol binds tightly (Kd 0.1 pM) to the vitamin D receptor protein
neutrophilic plugs in the interstitial spaces in the respiratory tract, as (VDR). VDR is expressed in nearly all human cell types (200–25,000
well as in heart, liver and brain. Brain hypoxic changes occurred in all copies per cell) [43]. It is a 50 kDa protein and there are a few poly­
patients, with extensive inflammatory response in all white and grey morphic forms. It can also bind calcidiol but with a 10- to 100-fold lower
matter, irrespective of the disease course. Most affected (n = 9) were the affinity than calcitriol. Once loaded with calcitriol, VDR binds to the
cranial medulla oblongata (centre of respiratory rhythm generation) and retinoid X receptor (RXR). The VDR-RXR heterodimer, a transcriptional
the olfactory bulbs (smell-taste centre). Among others, a massive acti­ regulator, binds to the vitamin D responsive elements (VDREs). This
vation and clustering of microglia with formation of nodules was results in the activation/repression of hundreds of target genes. Bouillon
detected (not described until then). Also the bone marrow showed clear et al. [43] estimated that calcitriol is involved in the regulation of
deviations. The lethality of the disease was caused by neutrophilic plugs expression of ca. 3% of the genes in the human genome. This number
involved in coagulopathy and by prolonged activation of neutrophils. was confirmed by a recent group meeting of world experts on vitamin D
The plugs were either NETs or NETs aggregated with platelets. The [1].
authors proposed that the lethality of the infection was not caused by the
virus itself, but “Instead a maladaptive immune stimulation could occur, Calcidiol and ascorbate show a mutual dependency for optimal
resulting in continuous neutrophil activation and organ damage.”. performance of human cells; basis for the hypothesis
Based on the information above, my conclusion is that the alveolar
and other organ damage observed in deceased SARS-CoV-2 patients [40] The immune system cannot properly function without sufficient
may have been largely caused by a lack of ascorbate. Indeed, the very calcidiol in the blood [43]. Recently [1], the calcidiol levels in plasma
first report on plasma ascorbate levels in SARS-CoV-2 patients (n = 18) have been redefined as follows: severe deficient, <30 nM; deficient,
with acute respiratory distress syndrome (ARDS) [41] showed that all <50 nM; sufficient, >75 nM. In this context it is worthwhile to mention
patients were highly deficient in ascorbate; 17 had levels below the two studies from Munger et al. on serum calcidiol levels and the risk of
detection limit of the used method (<8.5 µM) and one had 13.6 µM. multiple sclerosis (MS) [44] and type 1 diabetes [45] among 7–8 million
Although all patients survived, the long-term effects of the disease US military personnel. In 148 subjects with diagnosed MS, the risk
remain to be awaited. decreased with increasing calcidiol levels. This inverse relation was
An extensive medical overview [4] on human ascorbate plasma strongest for subjects who had the lowest levels during their adolescence
levels and medically advised daily intake of ascorbic acid over the years, (especially the ages 16–19 years). On average, the risk decreased 41%
mentioned that in the UK 4% of the population over the age of 65 and with every 50 nM calcidiol increase. Compared to subjects with < 75 nM
40% of the care-home residents over 65 years had plasma ascorbate plasma calcidiol, the risk was 51% reduced in subjects with > 100 nM
levels of 11 µM or less, i.e. they were highly deficient in ascorbate. [44]. The study on subjects with diagnosed type 1 diabetes (n = 180)
Ascorbic acid is auto-oxidizable, although strictly speaking O2 (S = 1, showed a similar pattern. Compared with subject with < 75 nM plasma
a triplet state) cannot react with ascorbate (S = 0, a singlet state), calcidiol, the risk was 40–54% lower with levels of 75–100 nM, and
because it is a spin-forbidden reaction. However, with trace amounts of 44–63% lower with levels > 100 nM [45].
transition-metal ions (Fe, Cu) a speedy auto-oxidation occurs at neutral In 2010 an instructive review from Baeke et al. on the specific effects
pH (but not at acidic pH). Dehydro-ascorbate is highly unstable and of calcidiol on immune cells appeared [46]. Most white blood cells
rapidly disintegrates. This is presumably the main reason for the short (macrophages, dendritic cells, B and T lymphocytes) require more than
plasma half-life of ascorbate in human blood (2–3 h after oral intake). In 50 nM plasma calcidiol for optimal performance. When activated by
cells, any formed dehydro-ascorbate is reduced to ascorbate by dehydro- immune signals, these cells increase the internal expression of CYP27B1
ascorbate reductase (glutathione:dehydroascorbate oxidoreductase, EC (allowing local production of calcitriol) as well as of VDR. This is sum­
1.8.5.1). Keep in mind that ascorbic acid (pKa values 4.2 and 11.6) is in marized in Fig. 4.

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S.P.J. Albracht Medical Hypotheses 163 (2022) 110845

Fig. 4. Roles of calcidiol/calcitriol in bone homeostasis and in immune cell functioning. Bone cells require calcitriol from blood, whereas immune cells need cal­
cidiol. Abbreviations: GC, group-specific component; CYP2R1, 25-hydroxylase (lysosomal; EC 1.14.14.24); CYP27B1, 1-α-hydroxylase (EC 1.14.15.18); PTH,
parathyroid hormone; VDR, vitamin D receptor; VDRE, vitamin D responsive element. The figure is based on the information from Baeke et al. [46].

Recent studies showed that an intake of 2,000 µg calcidiol changed expected that far more Fe2OGDs genes are under control of VDR-
the expression of 702 genes in collected peripheral blood mononuclear calcitriol. Therefore I hypothesize that for the normal optimal perfor­
cells (PBMCs, a mixture of monocytes, T and B lymphocytes) from mance of human cells, the roles of calcidiol/calcitriol and ascorbate
healthy individuals [47–49]. The immune-response threshold level for show a mutual dependency (Fig. 5). The one cannot optimally carry out
calcitriol is often (much) higher than the plasma level of 0.1 nM. Local its functions in absence of the other. This is presumably most important
production within immune cells makes it therefore independent of the in cells and tissues with millimolar concentrations of ascorbate, i.e. the
systemic level and hence the regulation is quite different from that in immune system and the central nervous system.
kidneys (Fig. 4.). In view of the millimolar concentrations of ascorbate in There is widespread clinical evidence indicating that sufficient cal­
white blood cells, it is hypothesized here that also a range of Fe2OGDs is cidiol lowers the incidence of viral and bacterial infections; plasma
involved in this regulation and thus, that both ascorbate and calcidiol calcidiol levels show that ca. one third of the world population is defi­
are required for optimal functioning. cient (<50 nM) [1]. In Europe that is 40% and in the Middle East even
In 2011 it was reported for the first time by Pereira et al. [50] that 90%. In 2014 it was reported that calcidiol deficiency in Saudi Arabia
calcitriol enhanced the expression (2- to 3-fold; RNA level) of histone had epidemic proportions [52]). Giustina et al. [1] concluded sufficient
lysine demethylase JMJD3/KDM6B (EC 1.14.11.68) in several types of calcidiol to be preventive rather than therapeutic (within the experi­
human cancer cell lines. VDR, as well as an intact iron co-ordination in mental time span). Another recent medical publication [2] urged the UK
KDM6B, were absolutely required for the observed phenotype changes. and other governments to pay attention to vitamin D3 in relation to the
In tissue samples of colon cancer patients the expression (RNA level) of immune system. It is of note, that the ascorbate background of in­
both KDM6B and VDR was downregulated in tumour tissue vs. normal dividuals in these reports were disregarded. These 2020 recommenda­
tissue in ca. 60% of the patients. The authors anticipated that VDR- tions [1,2] came a decade after the importance of calcidiol for the
calcitriol was also involved in the regulation of other lysine immune system was reviewed by Baeke et al. [46]. Apparently, Gov­
demethylases. ernments Health Departments react extremely slowly.
This was substantiated in their 2012 study [51]. Their data showed
(in cancer cell lines) that calcitriol had an unanticipated wide regulatory Extensive tissue damage can lead to organ failure and rapid loss of
action on the expression of genes encoding a number of JMJDs (which calcidiol
are Fe2OGDs), as well as on genes coding for two FAD-containing lysine
demethylase families. The GC (DBP) concentration in human plasma is 4.9–6.8 µM,
In view of the 702 genes that changed expression in PBMCs of whereas the calcidiol level is 50–150 nM. Ca. 90% of calcidiol is bound
healthy subject after intake of 2,000 µg calcidiol [47–49], it can be to GC (Ka 7.108 M− 1). Serum albumin has a much lower affinity (Ka

6
S.P.J. Albracht Medical Hypotheses 163 (2022) 110845

Fig. 5. Mutual dependency of calcitriol and

ascorbate for the regulation of gene expres­
sion. The figure is a schematic representation
of the regulatory roles of calcitriol-loaded
VDR and the involvement of Fe2OGDs as
demethylases in gene expression and protein
synthesis. The yellow column marked
’Fe2OGDs (ascorbate)’ shows the specific
targets (histones to ribosomes) of the deme­
thylases with the enzyme numbers as present
in the EC 1.14.11 list (date October 30,
2021), followed by one or two of the some­
times many, widely different gene names.
Example: “Histones/27, 65–69/KDM2-6” in­
dicates demethylases acting on histone pro­
teins/EC 1.14.11.27, EC 1.14.11.65–69/gene
names KDM2-6 (KDM stands for lysine
demethylase). Note that the EC 1.14.11 list
contains only demethylases that have been
studied as proteins. TETs means (the genes
encoding) ten-eleven translocases; the
expressed proteins actually function as
demethylases and were not yet in the EC
1.14.11 list. The left-hand part of the figure
is adapted from [12]. The right-hand part
shows effects (stimulation/repression) of
VDR-calcitriol on the various demethylases
genes as demonstrated by [50,51] (solid ar­
rows). The dashed arrows indicate expected
effects discussed in the present hypothesis.

6.105 M− 1), but due to its higher concentration (650 µM), it binds ca. (ignoring ascorbate) for human health [1,42,43], for the immune system
10% of calcidiol [53]. Thus, the saturation of GC with calcidiol is only [46,49], or for SARS-CoV-2 [2]. Hopefully my remarks will help to
1–3%. GC also strongly binds actin monomers (Ka 1.109 M− 1 [53]) and a change that attitude. Nature can be best understood by a multidisci­
most important function of GC is to clear extracellular G-actin (globular plinary academic approach.
actin) released from the cytoskeleton of necrotic and/or damaged cells
[54,55]. This prevents the polymerization of these apolar monomers to Concluding remarks
F-actin (fibrous actin), which can otherwise lead to obstruction of blood
capillaries. GC in blood of healthy individuals consists of up to 20–44% Based on the information above, my advice would be that in­
(mol.mol− 1) of GC-actin [56]. The plasma half-life of uncomplexed GC dividuals, especially those with a weakened immune system and the
in human blood (healthy individuals) is 2–2.5 days. However, the half- older generation, should take sufficient supplemental ascorbic acid and
life of GC-actin is only 30–60 min. Heavy trauma can therefore result in calcidiol to keep their plasma levels to 70–80 µM ascorbate (intake
dangerously low levels of GC and organ failure, because the GC pro­ 200–500 mg per day) and 80–200 nM calcidiol (intake 50–70 µg per
duction in the liver cannot keep up with such high losses [57–59]. Thus, day). This will help their immune system to combat optimally many
tissue damage, as observed in SARS-CoV-2 patients [40], may have intruding viruses and pathogenic bacteria, as well as other aberrant cells
contributed to organ failure and will also have resulted in rapid loss of like beginning cancer cells. These recommendations may diminish the
calcidiol. severity of infections and may contribute to prevent future overloading
of hospital IC units, e.g. in case of patients with SARS-CoV (all variants).
Publications on the effects of ascorbate or calcidiol on cells or An additional cheap and powerful combat method is to combine this
the immune system approach with immunotherapy with the macrophage activating factor
(MAF) prepared by partial deglycosylation of GC (reviewed in [62]).
I noticed that many of such studies were performed in vitro with cell In retrospect, the risk groups should have optimized their immune
lines grown in RPMI 1640 media, i.e. media that did not contain system at least seven to eight weeks prior to vaccination against SARS-
ascorbate or calcidiol. Studies on the effects of one of the two were al­ CoV-2, as well as after the jab, by appropriate (high) doses of ascorbic
ways performed in the absence of the other. For in vivo studies on the acid and calcidiol. This might have significantly reduced most (long
effects of calcidiol, the plasma levels of ascorbate were never taken into term) adverse effects (and even death) of this stimulation of the immune
consideration. Also when animal results were compared with those system with non-self proteins. Recent preliminary autopsy and histo­
obtained in humans, ascorbate was ignored. The same is true for the in pathological studies on eighth individuals deceased after SARS-CoV-2
vivo effects of ascorbate; calcidiol levels were not considered. vaccination showed inflammation and lymphocytic-predominant tis­
Publications that I have consulted specifically for the topics in the sue destruction in heart and lungs [63]. As normal lymphocytes contain
present hypothesis, can be roughly grouped into three categories: (a) ca. 3.9 mM ascorbate, my suggestion is that these deadly adverse effects
Studies that did not mention any vitamins, e.g. medical ones dealing were mainly due to severe ascorbate deficiency of the individuals.
with HIF and Fe2OGDs during hypoxia [12], RNA (de)methylation [22]
or SARS-CoV-2 [40], or biochemical/biophysical ones emphasizing the
Declaration of Competing Interest
mechanism of action of Fe2OGDs (e.g. [24,25,60]); (b) Papers discus­
sing the importance of ascorbate (neglecting calcidiol) for NETosis [37],
The author declares that he has no known competing financial in­
for neutrophil function during sepsis [4,38] or for inflammation and
terests or personal relationships that could have appeared to influence
cancer [32,61]); (c) Reports pointing to the importance of calcidiol
the work reported in this paper.

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