Novel oxolane derivative DMTD mitigates high glucose-induced erythrocyte apoptosis by regulating oxidative stress

Article abstract of DOI:10.1016/j.taap.2017.09.008

Chronic hyperglycemia is one of the characteristic conditions associated with Diabetes Mellitus (DM), which often exerts deleterious effects on erythrocyte morphology and hemodynamic properties leading to anemia and diabetes-associated vascular complications. High glucose-induced over production of reactive oxygen species (ROS) can alter the blood cell metabolism and biochemical functions subsequently causing eryptosis (red blood cell death), yet another complication of concern in DM. Therefore, blocking high glucose-induced oxidative damage and subsequent eryptosis is of high importance in the better management of DM and associated vascular complications. In this study, we synthesized an oxolane derivative 1-(2,2-dimethyltetrahydrofuro[2,3][1,3]dioxol-5-yl)ethane-1,2-diol (DMTD), and demonstrated its efficacy to mitigate hyperglycemia-induced ROS generation and subsequent eryptosis. We showed that DMTD effectively inhibits high glucose-induced ROS generation, intracellular calcium levels, phosphaditylserine (PS) scrambling, calpain and band 3 activation, LDH leakage, protein glycation and lipid peroxidation, meanwhile enhances the antioxidant indices, osmotic fragility and Na⁺/K⁺-ATPase activity in erythrocytes. DMTD dose dependently decreased the glycated hemoglobin level and enhances the glucose utilization by erythrocytes in vitro. Further, DMTD alleviated the increase in ROS production, intracellular Ca^{2 +} level and PS externalization in the erythrocytes of human diabetic subjects and enhanced the Na⁺/K⁺-ATPase activity. Taken together, the synthesized oxolane derivative DMTD could be a novel synthetic inhibitor of high glucose-induced oxidative stress and eryptosis. Considering the present results DMTD could be a potential therapeutic to treat DM and associated complications and open new avenues in developing synthetic therapeutic targeting of DM-associated complications.

Full text of DOI:10.1016/j.taap.2017.09.008

Accepted Manuscript
Novel oxolane derivative DMTD mitigates high glucose induced
erythrocytes apoptosis by regulating oxidative stress
Swamy Jagadish, Mahadevappa Hemshekhar, Somanathapura K.
NaveenKumar, Kothanahally S. Sharath Kumar, Mahalingam S.
Sundaram, Basappa, Kesturu S. Girish, Kanchugarakoppal S.
Rangappa
PII:
S0041-008X(17)30380-0
doi: 10.1016/j.taap.2017.09.008
YTAAP 14049
DOI:
Reference:
To appear in:
Toxicology and Applied Pharmacology
Received date:
Revised date:
Accepted date:
2 July 2017
5 September 2017
9 September 2017
Please cite this article as: Swamy Jagadish, Mahadevappa Hemshekhar, Somanathapura
K. NaveenKumar, Kothanahally S. Sharath Kumar, Mahalingam S. Sundaram, Basappa,
Kesturu S. Girish, Kanchugarakoppal S. Rangappa , Novel oxolane derivative DMTD
mitigates high glucose induced erythrocytes apoptosis by regulating oxidative stress. The
address for the corresponding author was captured as affiliation for all authors. Please
check if appropriate. Ytaap(2017), doi: 10.1016/j.taap.2017.09.008
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ACCEPTED MANUSCRIPT
Novel Oxolane Derivative DMTD Mitigates High Glucose Induced Erythrocytes Apoptosis by
Regulating Oxidative Stress
a, b
a
a
Swamy Jagadish , Mahadevappa Hemshekhar , Somanathapura K. NaveenKumar
b
a #
c
a,d
Kothanahally S. Sharath Kumar , Mahalingam S. Sundaram , Basappa , Kesturu S. Girish *
b, e
Kanchugarakoppal S. Rangappa
*
a
Department of Studies in Biochemistry, University of Mysore, Manasagangotri, Mysuru-570 006,
India
b
Department of Studies in Chemistry, University of Mysore, Manasagangotri, Mysuru-570 006,
India
c
Department of Studies in Organic Chemistry, University of Mysore, Manasagangotri, Mysuru-570
0
06, India
d
Department of Studies and Research in Biochemistry, Tumkur University, Tumakuru-572 103,
India
e
Institute of Excellence, Vijnana Bhavan, University of Mysore, Manasagangotri, Mysuru-570 006,
India
#
Present address: Section of Molecular Biology, Division of Biological Sciences, University of
California, San Diego, 9500 Gilman Drive, UC San Diego, La Jolla, CA 92093-0322, USA
*Corresponding authors
Prof. Kanchugarakoppal S. Rangappa
DOS in Chemistry, University of Mysore, Mysuru-570 006, India
E-mail: rangappaks@gmail.com
Prof. Kesturu S. Girish
DOS in Biochemistry, University of Mysore, Mysuru-570 006, India

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DOSR in Biochemistry, Tumkur University
Tumakuru-572 103, India
E-mail: ksgbaboo@gmail.com
Abstract:
Chronic hyperglycemia is one of the characteristic conditions associated with Diabetes
Mellitus (DM), which often exerts deleterious effects on erythrocyte morphology and hemodynamic
properties leading to anemia and diabetes-associated vascular complications. High glucose-induced
over production of reactive oxygen species (ROS) can alter the blood cell metabolism and
biochemical functions subsequently causing eryptosis (red blood cell death), yet another
complication of concern in DM. Therefore, blocking high glucose-induced oxidative damage and
subsequent eryptosis is of high importance in the better management of DM and associated vascular
complications.
In
this
study,
we
synthesized
an
oxolane
derivative
1-(2,2-
dimethyltetrahydrofuro[2,3][1,3]dioxol-5-yl)ethane-1,2-diol (DMTD), and demonstrated its efficacy
to mitigate hyperglycemia-induced ROS generation and subsequent eryptosis. We showed that
DMTD effectively inhibits high glucose-induced ROS generation, intracellular calcium levels, PS
scrambling, calpain and band 3 activation, LDH leakage, protein glycation and lipid peroxidation,
+
+
and enhances antioxidant indices, osmotic fragility and Na /K -ATPase activity in erythrocytes.
DMTD dose dependently decreased the glycated hemoglobin level and enhances the glucose
utilization by erythrocytes in vitro. Further, DMTD alleviated the increase in ROS production,
2
+
intracellular Ca level and PS externalization in the erythrocytes of human diabetic subjects, and
+
+
enhanced the Na / K -ATPase activity. Taken together, the synthesized oxolane derivative DMTD
could be a novel synthetic inhibitor of high glucose-induced oxidative stress and eryptosis.
Considering the present results DMTD could be a potential therapeutic to treat DM and associated

ACCEPTED MANUSCRIPT
complications and open new avenues in developing synthetic therapeutic targeting of DM-associated
complications.
Key words: Eryptosis; Oxidative stress; Diabetes mellitus; Glycated hemoglobin; DMTD; PS
externalization.
Introduction
Diabetes mellitus (DM), which is known for its abnormal glucose levels in the circulation is
often associated with significant hemorheological and biochemical malfunctions in red blood cells
(RBCs), and platelet hyper-reactivity (Borst et al., 2013; Faggio et al., 2017; Mischitelli et al.,
2
016a). Chronic hyperglycemia is one of the defining characteristics of DM and is shown to play a
crucial role in orchestrating the diabetes-associated vascular and vital organ complications, which
results in overproduction and accumulation of ROS (Brownlee, 2001; Green et al., 2004; Nishikawa
et al., 2000). Chronic hyperglycemia-induced overproduction or accumulation of ROS is shown to
propagate serious pathological conditions such as micro/macro vascular abnormalities (Baynes and
Thorpe, 1999; Stratton et al., 2000), endothelial dysfunction (Wong et al., 2010), altered DNA-
protein interactions, structural changes in membrane lipids (Hunt et al., 1988) renal polyol formation
(Kikkawa et al., 2003), accumulation of advanced glycation end products (AGEs) (Hunt et al., 1988)
etc.
RBCs are crucial players in transporting oxygen to all the tissues and elimination of carbon
dioxide (Briglia et al., 2015; Pagano and Faggio, 2015; Signoretto et al., 2016). These anuclear cells
are highly susceptible to the direct exposure of endogenous and exogenous ROS, which induces
oxidative stress (Silva-Herdade et al., 2016; Bonaccorsi et al., 2013). The RBCs are vulnerable to
lipid peroxidation and glycation due to the high iron and polyunsaturated fatty acid content (Hunt et

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+
+
al., 1988). Hyperglycemia also induces RBC membrane protein glycation and decreases Na /K -
+
+
ATPase activity defining intracellular Na and K concentration A remarkable decrease in the
+
+
Na /K -ATPase activity has been implicated in the pathology of diabetes-induced polyneuropathy
and related complications (Vague et al., 2004). The pathological changes due to chronic
hyperglycemia and elevated ROS levels are known to induce deleterious effects on the morphology
and hemodynamic features of RBCs compromising their lifespan by driving them to suicidal death, a
process known as eryptosis. It is characterized by cell shrinkage and scrambling, which are regarded
as the hallmark of apoptosis. Activation of Calpain is another crucial event in hyperglycemia-
induced eryptosis (Samanta et al., 2012). Calpain, a calcium-dependent thiol protease is involved in
fragmentation of RBC cytoskeleton proteins spectrin and band 3 causing severe hemolysis and
subsequent anemic condition (Briglia and Faggio, 2017; Faggio et al., 2015; Lang et al., 2013;
Starodubtseva et al., 2008).
The key molecular events of oxidative stress-mediated eryptosis include increased formation
2
+
of endogenous ROS, which activates Ca permeable channels resulting in elevated intra-erythrocyte
2
+
+
Ca concentration and direct activation of calcium sensitive K channels (Bratosin et al., 2001). This
+
results in hyper-polarization of RBCs with K ion leakage and thus raising the electrical driving
force for chloride ion exit (Almasry et al., 2017; Bissinger et al., 2014). Finally, the leakage of KCl
from the cells with osmotically obliged water decreases the cell volume as well as the loss of
membrane integrity, leading to cell membrane scrambling with subsequent externalization of
phosphatidylserine (PS) at the cell surface (Borst et al., 2012; Chung et al., 2007; Zwaal et al., 2005).
In diabetic patients, the eryptotic erythrocytes bind to endothelial cells via CXCL16/SR-PSO
signaling cascade (Borst et al., 2012). The PS-exposing erythrocytes further increase the risk of
thrombosis by the activation of blood clotting cascade (Andrews and Low, 1999). Finally,

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erythrophagocytosis i.e., the engulfment of these PS-exposing erythrocytes results in the clearance of
circulating blood, thus eventually leading to anemia (Lang et al., 2008).
Even though there are many existing plant-derived molecules such as xanthohumal (Qadri et
al., 2009), probucol (Shaik et al., 2013), naringin and teriflunomide (Zierle et al., 2016) that can
inhibit eryptosis and associated oxidative stress, there is a great demand for synthetic small
molecules, which can curb hyperglycemia-induced oxidative stress and eryptosis. Therefore, the
present study was aimed to synthesize a potent small molecule that can inhibit hyperglycemia-
induced premature death of RBCs. Heterocyclic compounds containing oxolane moiety have been
employed to treat variety of inflammatory conditions such as Crohn’s disease, HIV, cancer etc. (Luo
et al., 2000). There exists a patent on 1, 3 dioxolane derivative as a potent leukotriene B4 (LTB4)
biosynthesis inhibitor, which is an important proinflammatory metabolite of arachidonic acid
pathway. Bioactive molecule Odisolane, an oxolane moiety containing molecule is shown to exhibit
anti-angiogenic property by targeting VEGF, AKT and ERK signaling pathways (Dërmaku-Sopjani
et al., 2014; Lee et al., 2016; Mischitelli et al., 2016b, 2016c). Therefore, in this study we have
synthesized an oxolane derivative 1-(2,2-dimethyltetrahydrofuro[2,3][1,3]dioxol-5-yl)ethane-1,2-
diol(DMTD) and evaluated its ability to inhibit hyperglycemia-induced oxidative damage on RBCs
and subsequent cell death.
Materials and methods
Chemicals & reagents
1
All reagents and starting materials were purchased from Sigma Aldrich and SDFCL. H (400
1
3
MHz) and C (100 MHz) NMR spectra of isolated compound was recorded on Agilent 400MHz
NMR spectrometer using CDCl3 as solvent. Chemical shifts are given as δ value with reference to

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tetramethyl silane (TMS) as an internal standard. Infrared spectra were recorded on Shimadzu FT-IR
model 8300 spectrophotometer.
All flourogenic, non-flourogenic substrates were from Sigma Chemicals, USA. Polyclonal
anti-Band 3 antibody was from Sigma and monoclonal anti-Calpain antibody was from Santa Cruz
Biotechnology, Dallas, USA. Calcein-AM, antibodies against GAPDH and β-tubulin were from Cell
Signaling and Technology, Danvers, USA. O-phthalaldehyde (OPT), phenylmethylsulfonyl fluoride
(
PMSF), dichlorofluorescein diacetate (DCFDA), reduced glutathione (GSH) and oxidized
glutathione (GSSG) were obtained from Sigma Chemicals, St. Louis, USA. Lactate dehydrogenase
LDH), creatine kinase (CK), blood urea nitrogen (BUN), alanine transaminase (ALT), and aspartate
(
transaminase (AST) assay kits were purchased from Agappe Diagnostics Limited, India. Glycated
hemoglobin detection kit was purchased from Diatek Healthcare Pvt.Ltd., Kolkata India. Glucose
Test kit was purchased from Arkray Healthcare Pvt. Ltd. Surat, India. All other reagents were of
analytical grade and procured from Sisco Research Laboratories, India. The synthesis and
characterization of the DMTD is provided as the supplementary information. DMTD is a water-
soluble compound, hence stock solutions are made in respective assay buffers.
Isolation of human erythrocytes and preparation of erythrocyte ghost samples
Human blood samples were processed as previously described (Lang et al., 2015). Whole
blood samples were collected from healthy volunteers within the age group of 20-55, non-smokers
and non-alcohol consumers free of any medications, and from routine diabetic patients with plasma
glucose level >150 mg/dL. The blood drawn was instantly mixed with acid citrate dextrose (ACD)
anti-coagulant (containing 85 mM sodium citrate, 78 mM citric acid and 111 mM D-glucose) in the
ratio 6:1 (blood: ACD v/v) following centrifugation at 1,000×g at 4 °C for 15 min. Then, the plasma
and buffy coat layers were discarded. The erythrocyte layers were washed three times with 150 mM

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NaCl and centrifuged at 1,000×g at 4°C for 10 min. The supernatants were discarded after each
centrifugation. The erythrocyte layers were re-suspended in phosphate-buffered saline (PBS) pH 7.4,
containing 1 mM NaH2PO4, 16 mM Na2HPO4 and 140 mM NaCl and washed thrice with PBS.
Finally, 0.4% and 10% hematocrit were prepared in Ringer’s solution (125 mM NaCl, 5 mM KCl, 1
mM MgSO4, and 32 mM HEPES/ NaOH, 5 mM glucose, and 1 mM CaCl2 pH 7.4) and used for the
assays. The Institutional Human Ethical Committee (IHEC-UOM No.118/Ph.D./2015-16 & IHEC-
UOM No.146/Ph.D./2016-17) University of Mysore, Mysuru, has approved the study. All subjects
have read and signed a written informed consent before their enrollment into the study.
For the preparation of erythrocyte ghost samples, after the incubation erythrocyte membranes
were isolated from the hematocrit (10% washed RBCs in PBS) in accordance with previously
described experimental protocol with slight modifications (Lang et al., 2005). After incubation for
1
2 h, the control and treated erythrocytes were subjected to centrifugation at 1000×g for 10 min and
the supernatant was discarded. The cells were then lysed with 30 volumes of ice cold lysis buffer (5
mM phosphate buffer pH 8 containing 1mM EDTA along with protease inhibitor cocktail containing
1
0 µg/mL pepstatin A, 10 µg/mL leupeptin, 5 µg/mL aprotinin and 0.1 mM PMSF) for 20 min at 4
C. The ghost membrane and cytosolic fractions were separated by centrifugation at 37,000×g for 15
min at 4 °C. Membranes were then pelleted and lysed using a lysis buffer containing 125 mM NaCl,
5 mM HEPES/ NaOH pH 7.3, 10 mM EDTA, 10 mM sodium pyrophosphate, 10 mM sodium
°
2
fluoride, 0.1% SDS, 0.5% deoxycholic acid, 1% Triton X-100, 10 µL β-mercaptoethanol. Protein
content in the membrane fractions was determined by using the Lowry’s method by using bovine
serum albumin (BSA) from sigma as standard. Finally, membrane fractions with equalized protein
content, were subjected to SDS-PAGE (10% gel) and immune blotted by western blotting.
Treating erythrocytes with Glucose In vitro

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Washed erythrocytes were suspended to a final reaction mixture of 0.4% hematocrit in PBS
containing varying concentration of glucose (5, 10, 25 and 45 mM) and incubated for 6 h at 37 °C.
In a second set of experiments, erythrocytes were treated with high glucose concentration of 45 mM
with varying time intervals (2, 6, 12, 24 and 48 h) at 37 °C according to the previously described
method (Viskupicova et al., 2015), at the end of the incubation the eryptotic markers such as
increase in ROS level, intracellular calcium and PS externalization were measured. Usually, the
cytosolic glucose concentration of human erythrocytes is maintained about 4.5-5mM by non-insulin
dependent glucose transporter-1 (GLUT-1), which is elevated under hyperglycemia, hence
incubation of erythrocytes with high glucose concentration above 45 mM is routinely used as a
model to mimic the in vivo hyperglycemic condition in diabetes and erythrocytes treated with 5 mM
glucose is considered as control samples.
Determination of endogenous ROS, intracellular Calcium and PS scrambling in erythrocytes
In order to determine the oxidative stress-induced erythrocyte death, eryptotic markers such
as ROS generation, elevated intracellular calcium level and PS externalization were evaluated in
0
.4% hematocrit, as described accordingly (Lupescu et al., 2012). For the induction of eryptosis,
erythrocytes were treated with high glucose concentration (45 mM) and for inhibition studies,
erythrocytes exposed to high glucose (45 mM) were then treated with DMTD (10 and 25 µM). The
final volume was made up to 200 µL with Ringer’s solution containing 2 mM CaCl2 and incubated
for 6 h at 37 °C with constant shaking. Further, endogenous ROS generation, intracellular calcium
and phosphatidylserine (PS) externalization was determined using fluorescent dyes such as DCFDA,
Fura2-AM and Annexin V-FITC respectively. After incubation, cell suspensions were centrifuged at
2
2
80×g for 5 min and supernatants were removed, the settled erythrocytes were then suspended in
00 µL Ringer’s solution and stained independently with DCFDA (10 µM), Fura2-AM (5 µM) and

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Annexin V-FITC (1 µg/mL) for respective incubation period at 37 °C, finally cells were washed and
analyzed by exciting the samples at wavelength of 488 nm and an emission wavelength of 530 nm
TM
2+
using FACSVerse
flow cytometer (BD Biosciences, USA) for ROS generation. The Ca -
dependent fluorescence intensity was measured with an excitation wavelength of 340 nm and 380
nm and an emission wavelength of 530 nm using a Varioskan multimode plate reader (Thermo
Scientific, USA). Similarly, for the determination of PS externalization, erythrocytes were stained
TM
using FITC conjugated Annexin V for 20 min, washed and analyzed by FACSVerse
flow
cytometer (BD Biosciences, USA).
Immunoblot
Immunoblot of erythrocytes membrane samples were carried out as described accordingly
(Pantaleo et al., 2016). Briefly, 10% hematocrit in PBS were treated with high glucose 45 mM as
agonist, for inhibition studies, erythrocytes exposed to high glucose were incubated with DMTD (10
&
25 µM) for 12 h, after incubation erythrocytes ghost samples were isolated and their protein
content was determined by Lowry’s method. Erythrocyte membrane protein were then separated by
SDS-PAGE using 10% polyacrylamide gel and subsequently transferred electrophoretically to PVDF
membrane. The membranes were then blocked with 5% skimmed milk in TBST (10 mM Tris-HCl
pH 8, containing 150 mM NaCl and 0.05% Tween-20 in double distilled water) for 1 h followed by
TBST wash and incubation overnight with primary antibodies calpain (1:3000) and Band 3 (1:5000)
at 4 ºC. The membranes were then washed with TBST and incubated with horseradish peroxidase
(HRP)-conjugated respective secondary antibodies for 1 h. The blots were then washed thrice with
TBST and incubated with a chemiluminescent substrate and visualized using chemiluminescence
imaging system (Alliance 2.1, Uvitek, UK).
Imaging of erythrocytes via Atomic force microscopy (AFM)

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AFM imaging of erythrocyte samples were carried out as described accordingly (Shi et al.,
013). Briefly, 1% hematocrit in PBS were treated with glucose 5 mM as control and high glucose 45
2
mM as agonist respectively; for inhibition studies, RBCs pretreated with high glucose were incubated
with DMTD (25 µM) for 6 h. Following incubation, erythrocytes were imaged according to the
method described previously, using cantilever: ACTA - 40N/m with tip radius of curvature 10-20 nm,
along with probe made of silicon pyramidal shape were used to image erythrocytes in non-contact
mode with Park systems NX-10 AFM. Suwon, Korea.
Determination of erythrocytes viability via calcein fluorescence and LDH release assays
To assess the viability of erythrocytes in 1% hematocrit and PBS, the cells were treated with
high glucose (45 mM), and for inhibition studies, high glucose-exposed erythrocytes were incubated
with DMTD (0-25 µM) for 6 h at 37°C, followed by staining with calcein AM (10 µM) and
incubated for 30 min at 37 °C. Further, cells were washed and measured by exciting the samples at
4
88 nm, and emission was detected at 585 nm (NaveenKumar et al., 2015). LDH release in
erythrocytes was determined by using commercially available LDH kit, according to the
manufacturer’s protocol. The assay was performed in a time course of decrease in NADH
absorbance at 340 nm for 3 min.
Determination of Lipid peroxidation (LPO) and Protein carbonyl content (PCC)
LPO status was assessed in erythrocyte membrane samples by estimating thiobarbituric acid
reactive substances (TBARS) as described accordingly (Sharma et al., 2015). Briefly, 10%
hematocrit in PBS were treated with high glucose 45 mM as agonist, for inhibition studies,
erythrocytes exposed to high glucose were incubated with DMTD (0-25 µM) for 6 h, erythrocytes
ghost samples were isolated and their protein content was determined by Lowry’s method.
Erythrocyte ghost samples (1 mg protein) were added to tubes containing 1.5 mL acetic acid (20%

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v/v, pH 3.5), SDS (8% w/v, 0.2 mL) and thiobarbituric acid (0.8% w/v, 1.5 mL). The mixture was
heated on a boiling water bath for 45 min. Adducts formed were extracted into 1-butanol (3 mL) and
the TBARS formed was read at 532 nm and quantified using TMP as the standard. Values are
expressed in terms of malondialdehyde (MDA) equivalents as nmol MDA formed per mg protein.
PCC in erythrocyte membranes was measured as described accordingly [48] using
dinitrophenylhydrazine (DNPH). To 0.2 mL of erythrocyte membrane samples (1 mg protein), an
equal volume of 10 mM DNPH in 2 N HCl was added and incubated for 1 h, shaking intermittently
at room temperature. Corresponding blank was carried out by adding only 2 N HCl to the sample.
After incubation, the mixture was precipitated with 20% TCA and centrifuged. The precipitate was
washed twice with acetone and dissolved in 1 mL of Tris-buffer (20 mM, pH 7.4 containing 140 mM
NaCl, 2% SDS-w/v) and the absorbance was read at 360 nm. The difference in absorbance is
determined and expressed as nmol of carbonyl groups per mg protein, using an extinction coefficient
-
1
-1
of 22 mM cm
Determination Glucose 6 phosphate dehydrogenase (G6PDH) activity and estimation of
GSH/GSSG levels
G6PDH activity was estimated by monitoring the increase in absorbance at 340 nm for 3 min
due to NADP-dependent glucose 6-phosphate transformation (Sundaram et al., 2014). Briefly, for
both G6PDH activity and GSH/GSSG level estimation, washed erythrocytes (5% hematocrit in PBS)
were treated with high glucose (45 mM) as agonist, and for inhibition studies, high glucose-exposed
erythrocytes were incubated with DMTD (0-25 µM) for 6 h at 37 °C. After incubation, erythrocytes
were suspended and lysed in distilled water. The lysates were used to determine G6PDH activity in
the reaction mixture (1 mL) containing Tris-HCl buffer (50 µM, pH 7.5 containing 3.8 mM NADP,

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3
.3 mM glucose-6-phosphate, and 6.3 mM MgCl2). The activity was expressed as nmol NADPH
formed/min/mg protein.
GSH and GSSG levels were determined as described previously with slight modifications
Sundaram et al., 2014). In brief, after incubation, erythrocytes samples were treated with o-
(
phthalaldehyde (1 mg/mL) at room temperature for 15 min to estimate GSH level and for GSSG
level, erythrocytes were treated with N-ethylmaleimide (10 µM) prior to o-phthalaldehyde addition,
and the resulting fluorescence was measured at 430 nm by exciting the samples at 365 nm. The
concentration of GSH and GSSG were determined from the standard curve.
Determination of stress marker enzyme activities
Superoxide dismutase (SOD) and Catalase (CAT) activities
SOD and CAT activities in erythrocyte lysates were assessed as described accordingly
(Sundaram et al., 2015). In brief, 5 % hematocrit in PBS were treated with glucose 5 mM as control
and high glucose 45 mM as agonist respectively, for inhibition studies, erythrocytes exposed to high
glucose were incubated with DMTD (5 - 25 µM) for 6 h, after incubation erythrocytes were lysed in
distilled water and were used for the determination of enzyme activities such as SOD, CAT, GRd,
and GPx. To determine SOD activity, 0.1 mL of erythrocyte lysate (0.05 mg protein) was added to
the reaction mixture (1 mL) consisting of phosphate buffer (16 mM, pH 7.8) containing TEMED–
EDTA (8 mM/0.08 mM) mixture and quercetin (0.15% w/v). The decrease in absorbance was
monitored for 1 min at 406 nm. The amount of protein that inhibits quercetin autoxidation by 50% is
defined as one unit. Similarly, The CAT activity in erythrocyte lysate was determined by measuring
the hydrolysis rate of H2O2 at 240 nm. To the reaction mixture (1 mL) containing sodium phosphate
buffer (100 mM, pH 7.4), H O (8.8 mM), erythrocyte samples (0.05 mg protein) was added. The
2
2

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decrease in absorbance was monitored for 3 min at 240 nm and the activity was expressed as mmol
-
1
-1
H2O2 decomposed per min per mg protein (Ɛ=43.6 mM cm ).
Glutathione reductase (GRd) and Glutathione peroxidase (GPx) activity activities
The glutathione reductase (GRd) activity was measured in erythrocyte lysates as described
accordingly (Sundaram et al., 2015). Briefly, 0.1mL of erythrocyte lysate (0.05 mg protein) was
added to the reaction mixture (1 mL) containing phosphate buffer [100 mM, pH 7.0 containing 2
mM EDTA, 20 mM GSSG and 2 mM NADPH (in 0.1% NaHCO (w/v)]. The decrease in absorbance
3
was monitored at 340 nm for 3 min and the activity was expressed as µmol GSSG reduced per min
per mg protein.
GPx activity was measured with a coupled enzyme assay as described accordingly (Tung et
al., 2017). The assay mixture (1 mL) contained 770 µL of 50 mM sodium phosphate (pH 7.0), 100
µL of 10 mM GSH, 100 µL of 2 mM NADPH, 10 µL of 1.125 M sodium azide, 10 µL 100 U/mL
glutathione reductase and 10 µL erythrocyte lysates. The mixture was allowed to equilibrate for 10
min. The reaction was started by adding 50 µL of 5 mM H2O2 to the mixture and NADPH oxidation
was measured during 5 min at 340 nm. One unit of glutathione peroxidase is defined as the amount
+
of enzyme able to produce 1.0 µmol NADP from NADPH per min. GPx activity was determined
-
1
-1
using the molar extinction coefficient 6.22 M cm for NADPH at 340 nm and expressed as µmol
NADPH oxidized per min per mg protein.
Measurement of Glycated hemoglobin/HbA1C and glucose utilization in erythrocytes
The levels of glycated hemoglobin or HbA1c, an amadori product of protein glycation in
hemoglobin was detected by using Glycohemoglobin detection kit. The assay was carried by ion
exchange resin method according to manufacturer’s protocol. Briefly, 10% hematocrit in PBS were

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treated with glucose (5-45mM), for inhibition studies, erythrocytes exposed to high glucose (45 mM)
were incubated with DMTD (10-50 µM) for 6 h. After incubation the erythrocyte samples were
lysed using hemolysis reagent and incubated with latex reagent at 37 °C for 5 min. The absorbance
was measured at 415 nm. The levels of HbA1c were calculated from a standard curve using HbA1c
and expressed as % HbA1c.
The glucose utilization levels were measured using a commercially available glucose test kit.
Briefly, 10% hematocrit in PBS were treated with glucose (5-45 mM), for inhibition studies,
erythrocytes exposed to high glucose (45 mM) were incubated with DMTD (10 & 25 µM) for 24 h.
The concentrations of glucose were measured before and after the 24 h incubation by glucose
oxidase reagent according to manufacturer’s protocol. The absorbance was measured at 505 nm. The
levels of glucose utilization were calculated by subtracting glucose levels at 24 h from glucose levels
at 0 h using the equation below. The result was expressed as m mol/L.
Glucose utilization (mmol/L) = Glucose levels at 0h - Glucose levels at 24h.
+
+
Measurement of erythrocytes osmotic fragility and Na /K -ATPase activity
The erythrocyte osmotic fragility was measured as the percentage of hemolysis induced by
hypotonic solution. An osmolarity (which produces 50% of hemolysis) was calculated as described
accordingly (Jain, 1989). Briefly packed erythrocytes (50 µL) were added to the tubes containing
0
.55% NaCl with increasing doses of glucose (5-45 mM), similarly in another set of tubes along with
packed erythrocytes, 0.55% NaCl, glucose (5-45 mM) DMTD is added dose dependently ranging
from 5-50 µM. All the tubes were then incubated at room temperature for 30 min, the samples were
then subjected to centrifugation at 450×g for 10 min, supernatants were collected and hemolysis was
then estimated by measuring the absorption of hemoglobin released at 540 nm, using a US/Vis
Beckman spectrophotometer.

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+
+
The Na /K -ATPase activity was measured accordingly with minor modifications (Sompong
et al., 2015). Briefly, 10% hematocrit in PBS were treated with high glucose 45 mM as agonist, for
inhibition studies, erythrocytes exposed to high glucose were incubated with DMTD (10 & 25 µM)
for 12 h, after incubation erythrocytes ghost samples were isolated and their protein content was
determined by Lowry’s method. The erythrocyte membranes were incubated with reaction buffer A
containing 4 mM MgCl2, 3 mM ATP-Na2, and 50 mM Tris-HCl, pH 7.4, and buffer B containing
1
20 mM NaCl, 20 mM KCl, 4 mM MgCl2, 3 mM ATP-Na2, and 50 mM Tris-HCl, pH 7.4 at 37 °C
for 1 h. After the incubation, the levels of phosphate (Pi) released from ATP-Na , were measured
2
according with minor modifications (Sompong et al., 2015). The reaction mixture was incubated
with ammonium molybdate (2.5% w/v) at room temperature for 10 min. Then, ascorbic acid (2%
w/v) was added and kept at room temperature for 20 min for color development. The absorbance
was measured at 725 nm. The levels of Pi release were calculated from a standard curve using
+
+
KH2PO4. Na /K -ATPase activity was calculated using the equation below. The results were
expressed as nmole Pi/mg protein/h.
+
+
Na /K -ATPase activity= Pi in the reaction buffer B - Pi in the reaction buffer A
Recruitment of Diabetes mellitus (DM) subjects
DM subjects (n=20) from Government Ayurvedic Medical College (GAMC), Mysuru were
recruited for the study. DM subjects were recruited purely on basis of plasma glucose level
(reference range: more than 150-200 mg /dL of blood). Viral and cirrhotic infected patients were
excluded from the study. All the experiments were in accordance and approved by the Institutional
Human Ethical Committee, (IHEC-UOM No.146/Ph.D./2016-17) University of Mysore, Mysuru.
Venous blood was drawn from DM subjects and non-smoking/drug-free healthy subjects (HS, n =
2
0) by trained professionals from GAMC, Mysuru. The draw blood was immediately mixed with

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ACD in the ratio of 6:1 (blood: ACD, v/v). About 100 µL aliquots of anti-coagulated blood was used
to determine cell count in an automated hemo-analyser (Sysmex KX-21, Japan). Further, the
remaining anti-coagulated blood was then centrifuged to separate plasma. The packed RBCs were
further washed thrice with PBS and finally suspended to 0.4 % hematocrit for further fluorescence
assays.
+
+
Measurement of eryptosis markers and Na /K -ATPase activity in DM patient’s erythrocyte
samples
+
+
Eryptosis markers like elevation in ROS, intracellular calcium, PS scrambling and Na /K -
ATPase activity in erythrocytes from DM patients were determined as described previously using
Varioskan multimode plate reader (Thermo Scientifics, USA) (Lupescu et al., 2012; Sompong et al.,
2
015). Briefly, for inhibition studies isolated and washed erythrocytes from DM patients were
treated with DMTD (50 μM) after incubation for 6 h, the samples were treated with respective
fluorescent probes. Finally, cells were collected by centrifugation and fluorescence was measured.
Determination of in vivo toxicity of DMTD
Mice model was used to assess the in vivo acute toxicity of DMTD. Swiss albino mice (8
weeks old) obtained from Central Animal Facility (University of Mysore, Mysuru) were used in the
present study. The animal experiment was reviewed and authorized by Institutional Animal Ethical
Committee (UOM/IAEC/26/2016; Dated 30-07-2016) University of Mysore, Mysuru. Animal
handling and experiments were carried out in accordance with the guidelines of the Committee for
the Purpose of Control and Supervision of Experiments on Animals (CPCSEA). The obtained
animals were acclimatized for one week prior to start of the experiment. The animals were grouped
into 3 groups consisting of 6 animals per group (n=6). Group I-Saline control mice; Group II and III-
Mice treated with DMTD 50 mg/kg and 100 mg/kg body weight respectively. Aqueous solution of

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DMTD (in saline) was administered orally to the respective experimental groups using intragastric
tube (gavage) once in a day in the morning for 8 consecutive days. Body weight was checked every
day throughout the experimental period. On completion of experiment on day 8, animals were
starved overnight and were euthanized, and blood was obtained via cardiac puncture. The obtained
whole blood was allowed to clot at 4 °C and serum was obtained by centrifuging at 1200× g for 10
min and stored at -20 °C until further use. A portion of blood was mixed with anticoagulant and
subjected to complete blood count namely total leukocyte count (WBC), erythrocyte count (RBC),
platelets (PLT), hemoglobin content (Hb) and hematocrit (HCT) using an automated hematology
analyzer (Sysmex KX-21, Japan). Immediately after euthanizing the animals, vital organs such as
liver, kidney and spleen were carefully dissected out and blotted free of blood in ice cold saline,
weighed and photographed to assess the gross appearance. Furthermore, lungs, stomach, colon and
small intestine were also dissected out and washed in ice cold saline. The harvested liver, kidney,
spleen, lungs, stomach, colon and small intestine were fixed in 10% buffered formalin for overnight.
The tissue samples were subjected to dehydration by processing with different grades of alcohol and
chloroform mixture. The processed tissues were embedded in paraffin wax, and sections of 5 µm
thickness were prepared, stained with hematoxylin-eosin dye (H &E) and observed under an Axio
imager.A2 microscope (Oberkochen, Germany) and photographed. From the serum, various
biochemical parameters such as levels of lactate dehydrogenase (LDH), creatine kinase (CK), blood
urea nitrogen (BUN), alanine transaminase (ALT), aspartate transaminase (AST) were assessed
using commercial kits (Agappe) as per manufacturer’s protocol.
Protein estimation
Protein estimation was carried out according to the method of Lowry et al. (Lowry et al.,
1
951), using BSA as standard.

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Statistical analysis
Results were expressed as mean ± SEM of five independent experiments. Statistical significance
among groups were determined by one-way analysis of variance (ANOVA) followed by Tukey’s
#
##
###
test for comparison of means [n = 5, p */ < 0.05, p **/ <0.01, p ***/ <0.001;* significant
compared to untreated erythrocytes. # significant compared to high glucose-treated erythrocytes].
Results
Synthesis of 1-(2, 2-dimethyltetrahydrofuro [2, 3] [1, 3] dioxol-5-yl) ethane-1, 2-diol (DMTD)
DMTD was synthesized according to the previously reported method (Barton and
McCombie, 1975) with minor modifications (Fig. 1A). Synthesis of the target molecule starts with
deoxygenation of the hydroxyl group on the C-3 carbon of the compound 1 via Barton, McCombie’s
reaction (Barton and McCombie, 1975), which produces the compound 2. In order to get the DMDT
compound, selective deprotection of the terminal acetonide of compound 2 was achieved with 70%
aqueous solution of acetic acid.
-
1
Spectral characterization data of DMTD White solid; m.p. 64-66°C; IR (KBr, Cm ) 3600, 2900,
1
1
4
1
100; H NMR (400 MHz, CDCl3) δ : 5.70-5.69 (d, J = 3.6 Hz, 1H), 4.65-4.63 (t, J = 4.0 Hz, 1H),
.09-4.04 (m, 1H), 3.84 (br s, 2H), 3.75-3.73 (t, J = 3.0 Hz, 1H), 3.59-3.55 (dd, J = 2.8 & 3.2 Hz,
H), 3.44-3.39 (q, J = 6.8 Hz, 1H), 1.97-1.93 (m, 1H), 1.77-1.70 (m, 1H), 1.39 (s, 3H), 1.21 (s, 3H);
C NMR (100 MHz, CDCl3) δ 111.1, 105.1, 80.4, 78.3, 72.2, 63.5, 33.6, 26.6, 26.0.
1
3
Effect of Glucose on erythrocytes in vitro
Incubation of erythrocytes under conditions of high glucose concentrations is usually used as
a model to mimic the in vivo condition of hyperglycemia in diabetes. Exposure of erythrocytes to
high glucose significantly increased ROS and cytosolic calcium level with PS translocation to outer

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cell surface in a dose-dependent manner when compared to control (Fig. 1B). In the next set of
experiments, erythrocytes were incubated with 45 mM glucose for a different time interval starting
from 0-48 h, which resulted in significant ROS generation, increased cytosolic calcium and PS
externalization after 6 h when compared to control samples (Fig. 1B). Therefore, for further
experiments a dose of 45 mM glucose exposed for 6 h was considered as high glucose level, which
mimics the pathological diabetic complication in RBCs inducing oxidative stress. RBCs exposed to
5
mM glucose was employed as control as normal glucose level in individuals (non-diabetic
humans) prior to meal is 4 to 5.8 mM.
DMTD alleviates high glucose-induced ROS generation, intracellular calcium levels & PS
scrambling
The effect of DMTD on hyperglycemia-induced ROS production, intracellular calcium
concentration and PS scrambling in erythrocytes were measured further. As illustrated (Fig. 1C), 45
mM glucose-induced significant increase in DCF fluorescence suggesting the elevated generation of
ROS. However, glucose-induced DCF fluorescence was significantly diminished by 2 folds in
presence of 25 µM DMTD respectively, demonstrating its ability to alleviate hyperglycemia-induced
2
+
ROS production. Similarly, fluorometric analysis measuring intracellular Ca levels demonstrated
2
+
that DMTD (25 µM) effectively ameliorated high glucose-induced Ca levels in erythrocytes (Fig.
D).
1
2
+
A rise in intra-erythrocyte Ca level further triggers cell membrane scrambling with PS
translocation signaling event in erythrocytes to undergo erythrophagocytosis, which can be identified
by Annexin V-FITC binding to apoptotic erythrocytes. As illustrated in Fig. 1E, Annexin V binding
of erythrocytes was indeed enhanced by high glucose exposure. However, presence of DMTD

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significantly inhibited high glucose-induced PS externalization by 2 folds when compared to high
glucose treated cells and abrogated the annexin V binding to erythrocytes.
DMTD down-regulates the levels of membrane proteins, Calpain and Band 3
An increase in intracellular calcium level is further accompanied by activation of calcium
sensitive cysteine protease calpain, and Band 3, a membrane glycoprotein, which drives erythrocytes
towards apoptosis. High glucose pre-treated erythrocytes showed ten folds increase in the protein
levels of both calpain and band 3 when compare to control, however DMTD (25 µM) treatment
substantially decreased the expression of both calpain and band 3 by five folds when compared to
high glucose treated cells, which is evident from immunoblots (Fig. 2A and 2B).
DMTD restores high glucose-induced erythrocyte shape change
High glucose exposure in erythrocytes is followed by the structural and pathophysiological
alterations in morphology of erythrocytes, which could be observed using AFM topography. High
glucose (45 mM) induced significant alterations in their structure, leading to the loss of their normal
biconcave shape when compared to control, however treatment of affected cells with DMTD (25
µM), remarkably restored back the normal shape of erythrocytes (Fig. 2C) (Table. 1).
DMTD maintains erythrocyte viability upon high glucose stress
To further evaluate the hyperglycemia-induced erythrocyte toxicity and its protective
potential of the DMTD, calcein fluorescence assay and LDH activity were carried out. Erythrocytes
treated with 45 mM glucose exhibited decreased calcein fluorescence and this was significantly
reversed by the presence of DMTD (Fig. 3A). Further, LDH leakage is yet another important marker
used to evaluate the erythrocyte death. Treatment of erythrocytes with high glucose, induced the
release of LDH into the medium whereas, the presence of DMTD was able to significantly inhibit
LDH release owing its cytoprotective efficacy (Fig. 3B).

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DMTD prevents high glucose-induced oxidative damage and restores endogenous antioxidant
enzyme activities
An abrupt increase in the intracellular oxidative stress markers defines the severity of the
oxidative damage in a cell. Erythrocytes treated with high glucose exhibited a severe elevation in the
levels of oxidative stress markers such as LPO and PCC to an extent of 80% and 73.5% respectively,
and decrease in G6PDH activity and the ratio of reduced/oxidized glutathione to an extent of 65%
and 51% respectively when compared to control (Fig. 3C-F). The erythrocytes treated with DMTD
(25 µM) lowered the LPO (Fig. 3C) and PCC (Fig. 3D) content effectively by 75% and 72%
respectively when compared to high glucose alone treated erythrocytes. In addition, treatment of
DMTD also restored G6PDH (Fig. 3E) activity and GSH/GSSG (Fig. 3F) ratio very effectively up
to 91% and 93% respectively aiding endogenous GSH replenishment.
Furthermore, endogenous antioxidant enzymes play a crucial role in fighting any form of
oxidative stress, hence to evaluate the impact of DMTD on hyperglycemia-induced redox imbalance
we assessed the activity of key endogenous antioxidant enzymes such as superoxide dismutase
(SOD), catalase (CAT), glutathione reductase (GRd) and glutathione peroxidase (GPx), in high
glucose-treated erythrocytes in presence and absence of DMTD (Fig. 4A and 4B). Severe changes in
the homeostasis of all the antioxidant enzymes were evident in high glucose-treated erythrocytes. A
drastic decline in the SOD and CAT activities upto 85% and 64% respectively were observed in
hyperglycemic erythrocytes, which was revoked effectively by DMTD (25 µM) to the extent of 80%
and 75% respectively. Similarly, activities of glutathione replenishing enzymes like GRd and GPx
were reduced by 85% and 70% respectively upon treatment of erythrocytes with high glucose when

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compared to control cells. Nevertheless, treatment of cells with DMTD (25 µM) restored the altered
GRd and GPx activity up to 90% and 92% respectively when compared to hyperglycemic cells.
DMTD reduces protein glycation (Glycated hemoglobin/HbA1C) and enhances glucose
utilization
The dose-dependent effect of DMTD on high glucose treated amdori product formation in
erythrocytes were shown in Fig. 5A. Erythrocytes treated with high glucose (25 and 45 mM) showed
significant increase in HbA1C levels when compared to control cells, however incubation of high
glucose exposed erythrocytes with DMTD (10-50µM) resulted in decline of HbA1C level in a dose-
dependent fashion. Further, effect of DMTD on glucose utilization in erythrocytes treated with
glucose for 24 h was determined. We observed a significant enhancement in glucose utilization by
RBCs with 45mM glucose. DMTD (10 and 25 µM) incubated along with 45 mM glucose induced a
gradual increase in glucose utilization and a more efficient utilization was found at 25 µM
concentration (Fig. 5B).
Effect of DMTD on osmotic fragility of erythrocytes
Osmotic fragility refers to susceptibility of erythrocytes to hemolysis under oxidative stress
conditions by varying the osmolarity of NaCl from 0.25 to 0.9%, as illustrated in (Fig. 5C). Osmotic
fragility of RBCs increases with high glucose level (25 and 45 mM), however there was a significant
recovery in the percentage of RBCs fragility in DMTD-treated erythrocytes when compared to high
glucose-treated RBCs in 0.55% NaCl concentration. There was a dose-dependent inhibition of
hemolysis in DMTD-treated erythrocytes.
+
+
Effect of DMTD Na /K -ATPase activity
+
+
The effects of DMTD on Na / K -ATPase activity in erythrocytes treated with high glucose
+
+
are shown in (Fig. 5D). A remarkable reduction in Na /K -ATPase activity was observed with 45

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mM glucose when compared to control. However, incubation of erythrocytes with DMTD reversed
+
+
the inhibitory effect of high glucose on Na /K -ATPase activity dose dependently as compared to 45
mM glucose-treated erythrocytes.
Effect of DMTD on erythrocyte of Diabetic patients
About 20 diabetic patient samples with plasma glucose level more than 150 mg/dL were
evaluated for their erythrocytes ROS level, intracellular calcium concentration, PS scrambling and
+
+
Na /K -ATPase activity. As illustrated in Fig. 6, there is a significant increase in ROS levels (Fig.
A), intracellular calcium concentration (Fig. 6B), PS scrambling (Fig. 6C) in erythrocytes of
6
+
+
diabetic patients. In contrast, Na /K -ATPase activity (Fig. 6D) was reduced significantly in
diabetic patients when compared to normal individuals; however, treatment of erythrocyte samples
2
+
with 50 µM DMTD reduced the increased levels of ROS, intracellular Ca , PS scrambling and
+
+
recovered back the Na /K -ATPase activity.
DMTD is non-toxic in vivo as tested in a mice model
In order to assess the acute toxicity of DMTD, mice were orally treated with two dosages of
DMTD for 8 days concurrently and monitored for the changes in body weight, toxic symptoms and
mortality rate. Fig. S1 represents the relative body weight in percentage from day 1 to day 9 (Fig.
S1 A) and percentage change in body weight on day 9 alone of experimental animals (Fig. S1 B).
Results clearly indicate that there was no significant change in the body weight of mice treated with
DMTD throughout the experimental period with respect to saline. Water and feed intake were also
monitored in experimental mice, DMTD treated mice exhibited normal water and feed intake as
compared to saline control. Furthermore, no mortality and toxic symptoms were observed in DMTD
treated mice confirming the non-toxic nature of DMTD. (All the graphs and data related to in vivo
studies were submitted as supplementary figures)

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Anti-coagulated blood isolated from mice through heart puncture was analyzed for total
blood cell count to assess the possible effect of DMTD on blood components. Supplementary table
S1 represents the total cell count including WBC, RBC, PLT, Hb and HCT of mice treated with
different dosages of DMTD (Table. S1). Results clearly indicate that there was no statistical
difference between the saline- and DMTD- treated groups. LDH, a cytoplasmic biomarker enzyme,
is widely used in toxicology and in various pathologies to assess the cell, tissue and organ damage.
Likewise, CK, a central controller of cellular energy homeostasis, plays particularly important role in
tissues with large and fluctuating energy demands. Both serum LDH (Fig. S1 C) and CK (Fig. S1 D)
activities were estimated in mice treated with DMTD. The results suggested that there was no
change in the levels of these enzymes in comparison with saline control.
Liver toxicity was assessed by estimating the serum levels of ALT, AST along with the gross
appearance and histological analysis. Liver from DMTD-treated groups showed no significant
difference in weight (Fig. S2 A) in comparison with saline control group. Serum ALT and AST
levels of DMTD-treated mice showed no difference statistically in comparison to saline control (Fig.
S2 B & C). The histological analysis of DMTD-treated liver tissue sections revealed no signs of
congestion, vacuolization, hepatocyte necrosis and infiltration of inflammatory cells. In addition,
DMTD-treated liver sections exhibited normal hepatic architecture with respect to saline control
group (Fig. S2 D). Furthermore, kidney toxicity was assessed by estimating the levels of BUN along
with the gross appearance and histological analysis. DMTD treated mice showed no significant
change in kidney weight (Fig. S3 A) and BUN levels (Fig. S3 B). Macroscopic and microscopic
analysis also revealed that DMTD-treated mice kidneys showed normal morphology and devoid of
Bowmans’s capsule damage, urinary and vesicle pole damage, necrosis, constriction of distal tubules
and interstitial nephritis, and had normal renal architecture in comparison with saline control group

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(
Fig. S3 C). In addition, splenic damage was assessed by gross appearance, weight and histological
analysis. DMTD-treated mice showed no significant difference in spleen weight (Fig. S4 A) and the
longitudinal section of spleen revealed normal white and red pulp architecture, and were devoid of
vacuolization of splenic histocytes, hyperplasia lesions and red pulp pigmentation (Fig. S4 B).
Furthermore, the effect of DMTD on lungs, stomach, colon and small intestine were
histomorphologically assessed. The obtained result clearly indicates that there was no significant
difference in any of these histological parameters between DMTD treated and saline control groups
(Fig. S5 & S6). The in vivo acute toxicity study clearly demonstrates that DMTD is non-toxic in
nature.
Discussion
Although RBCs lack nuclei and mitochondria, they are very sensitive and are also chief
markers of health. The RBCs with their highly dedicated membrane structure are persistently
exposed to various components in the circulatory system. Therefore, they frequently interact or react
to certain components, which might trigger the process of eryptosis. Eryptosis studies usually
involves the determination of ROS generation, increased intracellular calcium level, externalization
of PS from inner leaflet to outer leaflet of plasma membrane, activation of cytoskeleton degrading
enzymes, which ultimately lead to cell shrinkage and death of erythrocytes. Some of the pathological
conditions associated with elevated rate of eryptosis include type-2 diabetes, malaria, chronic renal
failure, hemolytic uremic syndrome, sepsis, sickle cell anemia, thalassemia etc (Pretorius et al.,
2
016). Our continuing efforts and interests in developing multicomponent reaction for the synthesis
of various organic compounds and to check their potency for different biological activities (Girish et
al., 2015; Jagadish et al., 2016; Narasimhamurthy et al., 2013), prompted us to adopt the simple
synthetic route to produce oxolane derivative (DMTD) and evaluated its protective efficacy on

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hyperglycemia-induced premature death of RBCs. In the present study, we have probed the effect of
chronic diabetes-associated high glucose concentration on RBCs, particularly the events of eryptosis
and its amelioration by DMTD. Apart from xenobiotics, oxidative stress is one of the key triggers of
eryptosis (Bartoskova et al., 2013; Faggio et al., 2016; Mischitelli et al., 2017). It was found that
hyperglycemic conditions indeed increased the ROS levels, and there was a concurrent increase in
2
+
intracellular Ca levels followed by PS scrambling, which are the major sign of eryptosis. Previous
studies show that during hyperglycemic conditions, there is an increased generation of advanced
glycation end-products (AGEs), which are known pro-oxidants, and their buildup leads to chronic
oxidative stress (Vlassara et al., 1992). When RBCs are persistently exposed these AGEs, the former
undergo changes in their morphology and damage in their membranes, resulting in abnormally high
rate of eryptosis. Thus, anemia is frequently seen in diabetic patients (Awasthi et al., 2015).
Therefore, regulating oxidative stress seems to be the key to combat anemia in diabetics. Till date,
studies have demonstrated the anti-eryptotic effects of different plant-derived/synthetic/anti-oxidant
molecules including resveratrol, naringin, xanthohumol, probucol, nitric oxide, ferulic acid,
zidovudine, blebbistatin, salidroside etc., which were tested under various in vitro conditions (Qadri
et al., 2009; Shaik et al., 2013; Zierle et al., 2016). To this end, we synthesized a potent small
molecule 1-(2,2-dimethyltetrahydrofuro[2,3][1,3]dioxol-5-yl)ethane-1,2-diol(DMTD), an oxolane
derivative, and demonstrated for the first time its anti-eryptotic effects on hyperglycemia-induced
RBCs as well on RBCs from blood samples of diabetics under in vitro conditions.
2
+
In the present study, we observed an increased production of ROS and intracellular Ca ,
externalization of PS, altered levels of oxidative stress markers such as LPO, PCC, G6PDH and
GSH/GSSG ratio, and decreased activities of key endogenous antioxidant enzymes such as SOD,
CAT, GRd and GPx in hyperglycemia-induced RBCs. Upon treatment of these RBCs with DMTD,

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there was remarkable reduction in ROS levels, stabilization of oxidative stress markers and
maintains the antioxidant enzyme activities. The protective efficacy of DMTD can be attributed to
the presence of hydroxyl group in its side chain. These hydroxyl groups might be able to quench the
free radicals generated during oxidative stress and by the autoxidation of glucose in presence of
transition metal ions. In addition, DMTD may also quench the free radicals generated by
autoxidation of glycated proteins, which are formed during hyperglycemia. DMTD further restores
+
the anti-oxidant enzymes by maintaining the osmotic fragility of erythrocytes via revoking the Na /
+
K -ATPase activity (Waczulíkova et al., 2000). Thus, we can infer that DMTD has the potential to
effectively regulate oxidative stress in RBCs under hyperglycemic conditions and maintains the
antioxidant indices. To further determine whether this inhibition of oxidative stress by DMTD could
successfully reduce the rate of eryptosis, we evaluated its effect on the eryptotic events such as
2
+
increase in cytosolic Ca , activation of calpain and Band 3 glycoprotein, PS membrane scrambling,
and morphological changes in hyperglycemia-induced RBCs. DMTD considerably averted the
above-mentioned key events of eryptosis. These findings suggest that DMTD has the potential to
treat anemia and associated micro vascular complications such as RBC aggregation and endothelial
adhesion in diabetics.
Hyperglycemic condition in diabetics is also shown to cause increased rate of non-enzymatic
glycation, the process of glucose binding to amino groups of proteins resulting in the formation and
accumulation of AGEs. This in turn leads to free radical generation via auto-oxidation of glucose
and glycated proteins. AGEs also interact with cell surface AGE receptors, the ensuing events being
oxidative stress, vascular dysfunction and inflammation, which further add to diabetic complications
(Niedowicz and Daleke, 2005). The free radicals and oxidative stress cause alterations in
erythrocytes morphology and function including shape change, membrane lipid peroxidation,

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increased membrane fragility and rigidity, and lipid fluidity, this results in decreased levels of
antioxidant enzymes (Waczulíkova et al., 2000).The results of the present study demonstrate that
DMTD significantly decreased glycated hemoglobin (HbA1C) level and increased glucose utilization
by erythrocytes. Further, DMTD was also able to revoke osmotic fragility and shape change, and
conferred cytoprotective effect on hyperglycemia-induced erythrocytes.
Conclusion
Taken together, our findings confirm that high glucose or chronic hyperglycemia associated
with type 2 DM can induce oxidative damage on erythrocytes causing eryptosis. We also
demonstrate that the synthesized small molecule DMTD effectively revoked high glucose-induced
oxidative damage and subsequent apoptosis of erythrocytes. In addition, the study also demonstrates
that DMTD significantly decreased glycated hemoglobin (HbA1C) level and increased glucose
utilization and was able to revoke high glucose-induced osmotic fragility and shape change, and
conferred cytoprotective effect on erythrocytes. The in vivo studies to evaluate the toxicity of DMTD
confirmed that the molecule is non-toxic to treated mice and its major vital organs. With this, our
study suggests that DMTD could be a potential therapeutic candidate to treat chronic hyperglycemia-
induced anemia and associated microvascular complications such as RBC aggregation and
endothelial adhesion in diabetics.
Competing interests
The authors report no conflicts of interest. The authors alone are responsible for the content
and writing of the paper.
Acknowledgements

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SJ thanks UGC-BSR for research fellowship and would like to express heartfelt gratitude to
Prof. Sathyanarayana Bhat, Principal and Mr. Dwarkesh, Lab technician, GAMC, Mysuru for
helping in recruiting HG subjects for the study. Authors also thank Central instrumentation facility,
Institution of excellence (IOE), University of Mysore, Mysuru and Indian Institute of Science (IISc)
Bengaluru, for FACS and AFM facility. SJ also thanks the members of Prof. Kemparaju’s lab,
University of Mysore, Mysuru for their kind help during the study and Dr. Thushara RM, for
language editing the manuscript. This research was supported by the funds from University grant
commission (UGC), Govt. of India.
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