In vivo vitamin C supplementation increases phosphoinositol transfer protein
expression in peripheral blood mononuclear cells from healthy individuals
Helen R. Griffiths1*, Rachel S. Willetts1, Melissa M. Grant1†, Nalini Mistry2, Joe Lunec2‡
and Ruth J. Bevan2
1School of Life and Health Sciences, Aston University, Aston Triangle, Birmingham B4 7ET, UK
2Genome Instability Group, Department of Cancer & Molecular Medicine, University of Leicester, Leicester LE2 7LX, UK
(Received 4 January 2008 – Revised 9 June 2008 – Accepted 6 August 2008 – First published online 24 October 2008)
Ascorbate can act as both a reducing and oxidising agent in vitro depending on its environment. It can modulate the intracellular redox environ-
ment of cells and therefore is predicted to modulate thiol-dependent cell signalling and gene expression pathways. Using proteomic analysis of
vitamin C-treated T cells in vitro, we have previously reported changes in expression of five functional protein groups associated with signalling,
carbohydrate metabolism, apoptosis, transcription and immune function. The increased expression of the signalling molecule phosphatidyl-
inositol transfer protein (PITP) was also confirmed using Western blotting. Herein, we have compared protein changes elicited by ascorbate
in vitro, with the effect of ascorbate on plasma potassium levels, on peripheral blood mononuclear cell (PBMC) apoptosis and PITP expression,
in patients supplemented with vitamin C (0–2g/d) for up to 10 weeks to investigate whether in vitro model systems are predictive of in vivo
effects. PITP varied in expression widely between subjects at all time-points analysed but was increased by supplementation with 2g ascorbate/d
after 5 and 10 weeks. No effects on plasma potassium levels were observed in supplemented subjects despite a reduction of Kþchannel proteins
in ascorbate-treated T cells in vitro. Similarly, no effect of vitamin C supplementation on PBMC apoptosis was observed, whilst ascorbate
decreased expression of caspase 3 recruitment domain protein in vitro. These data provide one of the first demonstrations that proteomics
may be valuable in developing predictive markers of nutrient effects in vivo and may identify novel pathways for studying mechanisms of
action in vivo.
Peripheral blood mononuclear cells: Proteomics: Phosphoinositol transfer protein: Ascorbic acid
Micronutrient intake at low level is essential for normal cellu-
lar metabolism; the role of vitamin C in collagen biosynthesis
has been established for 70 years. Latterly, effects of micronu-
trients have beeninvestigated
approaches(1,2). However, limiting factors to the widespread
application of proteomics in nutrition include the limited
signal:noiseratio, where variability
complicates the analysis of effects of a nutrient and highlights
the need for experimental validation of potential biomarkers of
In addition to the difficulty of inter-individual variability,
the availability of material for study ex vivo is normally
restricted to biological fluids such as plasma and cerebrosp-
inal fluid(3)or less-invasive samples of tears, urine or
saliva. Analysis of the plasma proteome is also complicated
by the large dynamic range of protein concentrations, over
ten to twelve orders of magnitude, which further compounds
the difficultyin measuring
Plasma has an essential buffering role and may accumulate
products secreted from living and dying cells, particularly
if these products are ineffectively cleared or if they are pro-
duced in excess, for example by tumours(6). Approaches to
improve the reproducibility of proteomic methods for appli-
cation to plasma and other biological fluids have recently
been investigated and strategies which reduce the variability
imposed by depletion of abundant proteins have been
Proteomic technologies have been successfully applied to
improve understanding of cellular effects of micronutrients
in vitro and have led to novel hypotheses concerning pathways
that may be regulated by micronutrients(8). Peripheral blood
mononuclear cells (PBMC) may constitute a useful surrogate
model of cellular responses in vivo in which to validate
markers of nutrient effect which have been determined
in vitro by proteomics.
In addition to its role in collagen biosynthesis, vitamin C
has variably been reported to improve immune function
and decrease cardiovascular risk, possibly by interfering
*Corresponding author: Professor Helen R. Griffiths, fax þ44 121 359 5142, email email@example.com
†Present address: Birmingham University Dental School, UK.
‡Present address: School of Health, Cranfield University, UK.
Abbreviations: PITP, phosphoinositol transfer protein; PBMC, peripheral blood mononuclear cell; Tris, 2-amino-2-hydroxymethyl-1,3-propanediol; TTBS, Tween
British Journal of Nutrition (2009), 101, 1432–1439
q The Authors 2008
British Journal of Nutrition
with reactive oxygen species signalling(9–11). Up-regulation
of collagenmatrix production
SHSY5 cells(12)by proteomics, and using T cells exposed
to vitamin C in vitro, we have previously demonstrated that
vitamin C increases the expression of phosphatidylinositol
transfer protein (PITP) within 5min of ascorbic acid
treatment, an observation which was confirmed by Western
blotting(13). Proteins which showed a decrease in expression
in this model included the caspase 3 recruitment domain,
which if observed in vivo, may contribute to enhanced
cell survival following vitamin C supplementation, and the
Kþchannel, which may affect physiological potassium
In order to evaluate whether a reductionist approach
using in vitro proteomics can identify possible markers of
nutrient effects in vivo, we have isolated PBMC from
healthy volunteers taking part in a vitamin C supplemen-
tation study. The effect of vitamin C supplementation on
PBMC apoptosis, plasma Kþ
expression was investigated pre-, during and post-supplemen-
tation. If it is possible to use proteomics to interrogate
in vitro models we suggest that this may provide an efficient
process to identify novel biomarkers of nutrient functional
effect in vivo which can be further validated in intervention
levels and PBMC PITP
The study of T cell protein expression was undertaken in a
random subset of subjects (n 55) taking part in a larger trial
(n 209)(14). The subject recruitment protocol and study
design for the larger trial is described later.
Intervention study design
The vitamin C intervention study was carried out according
to strict protocols approved by Leicestershire, Rutland and
Northamptonshire Ethics Committee, Leicestershire Partner-
ship Trust Research Offices and University Hospitals of
Leicester NHS Trust.
Subjects (209) were recruited on to the trial from the Lei-
cestershire area following response to local advertisements.
Eligibility of each subject was determined by completion
of a confidential questionnaire outlining the inclusion and
exclusion criteria of the study. The principal inclusion criteria
were healthy individuals of 18 years or over who were
not supplementing their diet with any mineral or vitamin
and had no history of gastrointestinal irritation, thalassaemia
or haemochromatosis. Exclusion criteria were set to include
subjects who may have been at increased risk of adverse
side-effects following supplementation with vitamin C,
smokers who are known to have altered vitamin C status(15)
and women who were or thought they might be pregnant.
If subjects were found to be eligible for inclusion on to the
trial, written and informed consent was obtained.
The intervention study was designed as a double-blind
randomised controlled trial. Upon recruitment, each subject
was randomly assigned to one of four study groups compris-
ing group A (100mg vitamin C/d), group B (500mg vitamin
C/d), group C (2000mg vitamin C/d) and group D (placebo).
At week 0, subjects were given sufficient supply of tablets
for the remainder of the trial and received the appropriate
dose of vitamin C by taking two tablets per day, one in
the morning and one in the evening. Both vitamin C and
placebo tablets were supplied by DHP pharma (Crickhowell,
Powys, UK) and placebo was composed of microcrystalline
At week 0, subjects were required to attend the phlebotomy
clinic at the Clinical Research Unit, Leicester Royal Infirmary
NHS Trust in order to provide a fasting (12h overnight) blood
sample (50ml). Subjects returned at intervals of 1, 5 and 10
weeks to provide further blood samples.
Processing of blood samples
Each blood sample was collected into lithium/heparin vacutai-
ners (Sarstedt, Leicester, UK) and an EDTA vacutainer and
was stored on ice for a maximum of 2h before separation of
leucocytes or plasma, as described later.
Isolation of peripheral blood mononuclear cells from
PBMC were isolated from whole blood by density gradient
centrifugation, which produced a high yield of mononuclear
1·5ml Histopaque 1077 (Sigma-Aldrich, Poole, UK) was
aliquotted into a 15ml centrifuge tube and 10ml of whole
blood (diluted 1:1 with PBS) was carefully layered on top.
Following centrifugation at 400g for 30min at 188C,
blood components were separated into four layers; the
uppermost layer containing plasma was removed for vitamin
C analysis and the second layer containing PBMC was
collected. PBMC were subsequently washed twice with
PBS and centrifuged at 700g for 30min at 188C prior to
storage at 2808C.
Measurement of intracellular and plasma vitamin C
Intracellular vitamin C was determined in PBMC isolated
from whole blood (4·5ml) collected into EDTA. Vitamin C
was stabilised by addition of an equal volume of 10% meta-
phosphoric acid (Sigma-Aldrich) prior to storage at 2808C.
HPLC analysis was undertaken using a Perkin Elmer
isocratic LC pump (model 250) with 15mmol/l phosphate
buffer containing 9% methanol, pH 6·0, ESA model 542 auto-
sampler (ESA Analytical Ltd, Aylesbury, UK) and a Dionex
UV detector, model UVD340U (Dionex Ltd, Camberley,
UK). Samples were separated using a Luna 5mm C18 (2)
HPLC column (150 £ 4·60mm; Phenomenex, Macclesfield,
UK) and collected using Chromeleone software, version
6.0 (Dionex Ltd). Linearity of standards (.99·9% purity;
Sigma-Aldrich) was achieved for concentrations up to
100mmol/l vitamin C with sample intra- and inter-assay CV
of ,10%. Plasma and intracellular vitamin C concentration
was calculated from a known standard curve; intracellular
ascorbate concentrations were calculated relative to sample
protein content (mmol/mg protein) as a direct reflection of
Ascorbic acid affects cellular proteomes in vivo 1433
British Journal of Nutrition
Phosphoinositol transfer protein expression analysis in
peripheral blood mononuclear cells
For SDS–PAGE and Western blotting, randomised samples
were selected for pooling according to supplementation
dose and duration. Three independent pools of ten volunteers
were collected for each dose and time-point (i.e. from 120
2-amino-2-hydroxymethyl-1,3-propanediol (Tris) hydrochlo-
ride (100ml; 40mmol/l) homogenised and incubated (at
room temperature with rotation, 30min) in the presence of
pan-protease inhibitors (1:100 Focus-Protease Arrest; Merck,
Lysates were centrifuged at 13000g for 5min to remove
cellular debris prior to analysis of supernatant for protein
content. Equal amounts (2mg) of each of ten PBMC lysates
were pooled and three pooled samples were electrophoresed
and blotted as described later.
Western blotting for phosphoinositol transfer protein
Protein samples (20mg) were separated by one-dimensional
SDS–PAGE (12·5% gel) and electroblotted on to polyvinyli-
dene fluoride membrane(13). After electroblotting at 20mA for
16h for PITP, membranes were blocked in Tween 20 (1%)
Tris-buffered saline (TTBS) containing 3% bovine serum
albumin for 2h. Membranes were incubated with either a
mouse monoclonal antibody against PITP raised against
native recombinant human PITP with no reported cross-reac-
tivity (sc-13569; Santa Cruz Biotechnology, Santa Cruz,
CA, USA) or mouse monoclonal antibody against actin
which recognises b and g actin in human samples (ab1081;
Abcam, Cambridge, UK) overnight. The membranes were
washed with TTBS (6 £ 15min) before incubation with a
peroxidase conjugated secondary antibody (Sigma Aldrich)
for 2h (15:100000, diluted in TTBS containing 0·3%
bovine serum albumin). Membranes were rinsed in Tris-buf-
fered saline (6 £ 15min) and then a chemiluminescent sub-
strate (ECL Plus, Amersham Biosciences, Little Chalfont,
UK) was used to visualise detected proteins. The images
were recorded using a GS 710 calibrated imaging densi-
tometer (Biorad, Hercules, CA, USA). Bands were analysed
and quantified using Scion software (National Insitutes of
Health, Bethesda, MD, USA).
Caspase 3 activity
the subset of volunteers randomly selected for PITP expression
analysis. For this analysis, leucocytes were thawed into lysis
buffer comprising 1% Triton and 10mmol/l dithiothreitol with
0·5mmol/l phenylmethylsulphonyl fluoride. Protein concen-
tration wasagain determinedforeachsampleusing thebicincho-
ninic acid assay(16)and lysates were analysed for caspase 3-like
Fig. 1. Protein recovery from snap-frozen peripheral blood mononuclear cells isolated from fifty-five healthy subjects at four time-points during vitamin C sup-
plementation (A, placebo; B, 100mg/d; C, 500mg/d; D, 2g/d). Cells were subsequently thawed into hypotonic lysis buffer in the presence of specific protease
inhibitors and protein was determined by the bicinchoninic acid assay. Values are medians and inter-quartile ranges with complete data ranges depicted by vertical
bars. There was no significant effect of vitamin C dose on protein yield at any time-point for any of the doses studied.
H. R. Griffiths et al.1434
British Journal of Nutrition
activity against the synthetic substrate aspartate-glutamate-
Merck). After 1h in the dark, DEVD-aminomethylcoumarin
release was determined as fluorescence which was read at
460nm following excitation at 380nm(17).
Blood potassium measurement
Analysis was carried out on plasma samples obtained from
whole blood collected into Li/H and was undertaken at the
Department of Chemical Pathology, Guy’s and St Thomas’
Hospital NHS Foundation Trust. All samples were assayed
using a Roche Modular Analyser with the ISE1800 and
P800 sections of the module.
Potassium levels, PBMC protein and caspase 3 data are pre-
sented as the median value with 25th and 75th percentiles
and sample range with statistical analysis using GraphPad
Prism version 3.00 for Windows (GraphPad Software, San
Diego, CA, USA).
Analysis was performed using a Friedman’s test with post
hoc analysis using the Dunn post test. Groups of data were
evaluated statistically by paired comparison analysis, where
differences were considered significant when P,0·05.
Correlations were calculated with a Spearman correlation
coefficient (two-tailed test of significance).
Fig. 1 confirms that there was no significant difference in pro-
tein yield from the PBMC isolated from subjects receiving
dietary vitamin C according to the dose of supplement
given. However, there was a trend towards reduced protein
yield after 1 week, which was seen in all samples irrespective
of supplementation group and including placebo. After PBMC
harvest, the cells were split into equal aliquots according to
cell volume recovered rather than cell number. The lower pro-
tein yield suggests that the total PBMC recovery was reduced
following the first sampling. In addition, we noted that protein
yield from primary PBMC was significantly lower compared
to cultured HSB-CCRF-2 cells.
Figs. 2 and 3 illustrate that endogenous caspase 3 activity in
PBMC isolated from the peripheral blood of subjects receiving
vitamin C supplements is highly variable between individuals
and that supplementation does not affect caspase 3 activity
significantly at any dose or at any time-point during the inter-
vention. There was a trend for increased caspase 3 activity
after 1 week of supplementation with 2g vitamin C/d and
for a decrease in caspase 3 activity with 2g ascorbate/d
after 10 weeks.
To further investigate whether intracellular ascorbate status
was related to caspase 3 activity in vivo, the two parameters
were correlated using Spearman’s rank method. From these
data no relationship was found between cellular ascorbate
and caspase 3 activity in the cell lysates (Fig. 4). The levels
of measurable ascorbate were very tightly skewed towards
the limit of detection for the vitamin C assay, whereas the cas-
pase 3 activity levels that were recorded showed a closer dis-
tribution to normality.
As a decrease in the Kþtransporter was observed in T cells
exposed to vitamin C in vitro, the effect of supplementation
in vivo on plasma Kþlevels was investigated. No significant
effect of vitamin C dose or supplement duration was observed
on blood Kþlevels (Fig. 5).
To confirm whether the in vitro CCRF model of ascorbate
exposure was a model for the effects seen on the PBMC
proteome in vivo, PITP was analysed in three different pools
of ten subject PBMC proteomes under each intervention
regimen and matched across all time-points, as described in
the Experimental methods, by Western blotting. Fig. 6 (A)
shows representative Western blots from one subject pool for
Fig. 2. Lysates from peripheral blood mononuclear cells do not show
increased caspase 3 activity following differing doses of vitamin C sup-
plementation in healthy subjects (n 55) for 1 (A), 5 (B) and 10 (C) weeks.
Cellular debris was removed by centrifugation and the supernatant incubated
with aspartate-glutamate-valine-aspartate (DEVD)-aminomethylcoumarin as
a synthetic caspase 3 substrate. Released fluorescence, indicative of cas-
pase 3 activity, was measured at 460nm following excitation at 380nm and
corrected for protein content. Values are medians and inter-quartile ranges
with complete data ranges depicted by vertical bars. There was no significant
effect of ascorbate dose on caspase 3 activity as analysed by one-way
ANOVA followed by Dunnett’s multiple comparison test.
Ascorbic acid affects cellular proteomes in vivo1435
British Journal of Nutrition
each group, dose and time. Scanned images were quantitated
using Scion software and PITP was expressed relative to actin
regimens. Fig. 6 (B) demonstrates the linearity of actin band
intensity relative to protein loading, up to 30mg protein per
lane and validates the use of normalisation methodology to
adjust for errors in protein loading. Integration of band intensi-
ties confirmed that PITP was significantly elevated after 5 and
10 weeks of supplementation with vitamin C (2g/d) compared
with the same subjects at baseline (Fig. 6 (C)).
In the present paper we have investigated whether our
previous study of the effects of vitamin C on T cell protein
expression in vitro is a predictor for changes in protein
expression or function in vivo using plasma and PBMC iso-
lated from supplemented individuals.
Fig. 3. Lysates from peripheral blood mononuclear cells (n 55) do not show increased caspase 3 activity following vitamin C supplementation (A, placebo;
B, 100mg/d; C, 500mg/d; D, 2g/d) at any time-point up to 10 weeks of supplementation. Cellular debris was removed by centrifugation and the supernatant incu-
bated with aspartate-glutamate-valine-aspartate (DEVD)-aminomethylcoumarin as a synthetic caspase 3 substrate. Released fluorescence, indicative of caspase
3 activity, was measured at 460nm following excitation at 380nm and corrected for protein content. The data are presented as the median, inter-quartile range
and complete data range. There was no significant effect of ascorbate supplementation time on caspase 3 activity as analysed by one-way ANOVA followed by
Dunnett’s multiple comparison test.
Fig. 4. Caspase 3 activity in peripheral blood mononuclear cells isolated
from subjects (n 55) post-vitamin C supplementation does not correlate with
cellular ascorbate levels.
Fig. 5. Plasma potassium levels in healthy subjects are not affected by sup-
plementation with vitamin C (2g/d) for 10 weeks. Values are medians and
inter-quartile ranges with complete data ranges depicted by vertical bars.
H. R. Griffiths et al.1436
British Journal of Nutrition
As expected, inter-individual variability proved to be high
in spite of the population size; at the extreme, caspase
3 activity variance was four times the median recorded
value. The outliers responsible for the extended whiskers to
the box plots represent caspase activity in PBMC from indi-
vidual subjects which was not sustained over time. It is not
clear why such variation exists, although increased apoptosis
of leucocytes is reported post-infection(18).
In vitro we showed a loss of the caspase 3 recruitment
domain protein between 2 and 8h, but an increase at 24h
which we considered may result in a protective effect of
short-term exposure to ascorbic acid, but that prolonged
exposure to high doses of ascorbic acid may prime the cell
for apoptosis(13); induction of DNA damage in individuals
supplemented with high doses of vitamin C has previously
been shown by Anderson et al.(19). In vitro, caspase 3 activity
in HSB-CCRF-2 cells was significantly greater after culture
with 10mmol/l ascorbate than 1000mmol/l ascorbate after
24h(14). Others have also shown using propidium iodide stain-
ing for detection of apoptosis, that incubation of primary
PBMC ex vivo with 0·2mg/ml vitamin C for 24h caused a
39% increase in the percentage of apoptotic cells, as com-
pared to those kept at the same incubation conditions without
vitamin C(20). In the context of determining apoptotic effects
of vitamin C in vitro, there are several possible confounders
which should be considered and are reviewed by Halliwell(21).
Blood potassium levels remained unaffected by vitamin C
supplementation in vivo despite a decrease in Kþchannel
Fig. 6. (A) Phosphoinositol transfer protein (PITP) levels are increased following ascorbate supplementation (2g/d) in healthy volunteers. Proteomes from
three pools of ten independent volunteers in each of the supplementation groups were extracted and subjected to one-dimensional PAGE with Western blot-
ting for PITP or actin. (B) Representative standard curve of peripheral blood mononuclear cell lysate protein loaded and actin band intensity (r20·94). Differ-
ent amounts of lysate (0, 10, 20 or 30mg protein) were separated by SDS–PAGE, subject to Western blot for actin and the integral of the resultant bands
were used to confirm linearity of band intensity. M, marker. (C) Blots were scanned and interrogated using Scion software and PITP band intensities are
expressed relative to the actin band integral for each group at each dose. 0, 1, 5, 10 refer to weeks of intervention. Mean values were significantly different
from those of week 0: *P,0·05.
Ascorbic acid affects cellular proteomes in vivo1437
British Journal of Nutrition
expression in T cells in vitro. The major organ responsible for
maintaining blood potassium is the kidney and if a decrease of
channel expression occurred in vivo on supplementation, a
change in Kþtransport into T cells is unlikely to affect overall
Our earlier in vitro proteomic studies identified PITP as a
candidate marker of ascorbate effect which was mobilised
within 5min of exposure to ascorbate and was sensitive to
ascorbate concentration. PITP are ubiquitous proteins that
transport lipids, such as phosphatidylinositol and phospha-
tidylcholine, between membranes and thus have important
roles in signalling(22). Using pooled PBMC from three sets
of ten independent donors at each dose and time-point,
increased mobilisation of the signalling protein PITP was
observed which is indicative of a vitamin C-dependent
priming effect within the PBMC population in vivo(23).
It is important to note that PITP expression is detectable
at low levels in PBMC isolated from peripheral blood
and this is consistent with a role in cell signalling and
The normalisation of PITP protein expression level in
PBMC to actin levels may be questioned owing to over-
exposure of actin blots (a facet of the low expression levels
of PITP relative to actin and the need to load greater sample
volumes on gels in order to visualise both proteins on the
same gel/blot). Over-exposed actin blots are included to illus-
trate the amount of cellular extract loaded for analysis of
PITP, however, when actin was visualised after lower
PBMC loading concentrations, PITP was below the level of
detection in re-probed blots. Moreover, a standard curve of
protein loaded against actin integral showed highly significant
correlation (P,0·0001). In the absence of normalisation to
actin, the same trend of increasing PITP with vitamin C
dose and duration of supplement is observed, however, there
is large variability between different pools.
Pooling of samples was necessary as the median protein
concentration of lysates was 2mg/ml (Fig. 1), the concen-
tration of protein required to be loaded on to SDS–PAGE
for visualisation of PITP. To avoid the potential bias intro-
duced by analysis of only those lysates of protein concen-
tration .2mg/ml, pooling was undertaken. There are clear
disadvantages of such an approach, for example, only a
subset of healthy subjects may show an increase in PITP
and individual sample analysis offers further information on
inter-individual variation as reported here for the caspase
assay. Further studies are required to evaluate phosophoinosi-
tide metabolism in individual subjects following vitamin C
changes in protein expression reported here have biological
In conclusion, the present study has demonstrated that
PITP is modulated by ascorbate in PBMC in vitro and in
vivo. In contrast, functional changes in apoptosis or Kþ
homeostasis were not observed. Elevated expression of
exposure to ascorbate and confirms the value of in vitro
modelling for identifying suitable biomarkers of effect or
exposure in vivo. It also highlights the importance of verifica-
tion of observations made in cellular systems using human
intervention studies as false positives may emerge from
in vitro studies alone.
In addition to complexity in the technology which creates
‘high dimensional’ data sets(1), the genetic variability in
human studies adds further complications to the application
of proteomics to nutritional intervention studies in vivo.
One of the key issues in considering nutrition in healthy
to maintain normal physiology are, by definition, likely to
reduce any effects of diet on the proteome. By adopting an
early biomarker search strategy using a proteomic approach
in cell lines and then examining the potential for such markers
to be affected in vivo, the analysis of human populations
may be undertaken. Future advances in bioinformatics and
multiple component analysis to enable analysis of large
numbers of gel data sets (.100) each with up to 1000
spots per gel, will provide a route to work through such
inter-individual variability and large sample sizes to define
consistent patterns of effect without the need for sample
pooling or reductionism(24).
The authors gratefully acknowledge financial support from the
Food Standards Agency, UK (TO1026). H. R. G. analysed
protein, caspase activity, actin and was principal author of
the manuscript. R. S. W. analysed PITP and measured cellular
protein content. N. M. was responsible for subject recruitment,
PBMC preparation and ascorbate analysis. M. M. G. devel-
oped the PITP WB assay and advised on methodology. J. L.
was principal applicant on T01026, was responsible for project
ethics and organised supplementation. R. J. B. was responsible
for data collection and analysis. The authors declare no
conflicts of interest.
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