Journal of Endocrinological Investigation

https://doi.org/10.1007/s40618-023-02110-7

ORIGINAL ARTICLE

Glycemic variability leads to higher levels of auto‑oxidized oxysterol

species in patients with type 1 diabetes mellitus
U. Ünlütürk1,2 · A. B. Bahçecioğlu2 · A. Samadi3,4 · İ. Lay3 · M. Bayraktar1,2 · S. Dağdelen1,2

Received: 5 February 2023 / Accepted: 8 May 2023

© The Author(s), under exclusive licence to Italian Society of Endocrinology (SIE) 2023

Abstract
Purpose Hyperglycemia and glycemic variability (GV) are associated with oxidative stress in patients with diabetes mellitus
(DM). Oxysterol species, produced by the non-enzymatic oxidation of cholesterol, are potential biomarkers of oxidative
stress. This study examined the relationship between auto-oxidized oxysterols and GV in patients with type 1 DM.
Methods Thirty patients with type 1 DM using a continuous subcutaneous insulin infusion pump therapy and a healthy
control group (n = 30) were included in this prospective study. A Continuous Glucose Monitoring System device was
applied for 72 h. Blood samples were taken for oxysterols produced by non-enzymatic oxidation [7-ketocholesterol (7-KC)
and cholestane-3β, 5α, 6β-triol (Chol-Triol)] levels at 72 h. Short-term glycemic variability parameters, mean amplitude
of glycemic excursions (MAGE), the standard deviation of glucose measurements (Glucose-SD), and mean of daily differ-
ences (MODD) were calculated with continuous glucose monitoring data. HbA1c was used to evaluate glycemic control
and HbA1c-SD (the SD of HbA1c over the past year) for long-term glycemic variability.
Results 7-KC and Chol-triol levels were significantly higher in the study group than in the control group. Strong positive
correlations were found between 7-KC with MAGE(24–48 h) and Glucose-SD(24–48 h). 7-KC was positively correlated
with MAGE(0–72 h) and Glucose-SD(0–72 h). No significant correlation was found between HbA1c and HbA1c -SD with
oxysterol levels. The regression models showed that SD(24–48 h) and MAGE(24–48 h) predicted 7-KC levels while HbA1c
did not.
Conclusions Glycemic variability leads to higher levels of auto-oxidized oxysterol species in patients with type 1 DM inde-
pendent of long-term glycemic control.

Keywords Glycemic variability · Oxidative stress · Oxysterol · Mean amplitude of glycemic excursions · Mean of daily
differences · Type 1 diabetes mellitus

Introduction

HbA1c is commonly used to indicate long-term glycemic

U. Ünlütürk and A. B. Bahçecioğlu contributed equally to this work.
* U. Ünlütürk
ugurunluturk@gmail.com
1
Division of Endocrinology and Metabolism, Hacettepe
University School of Medicine, Ankara, Turkey
2
Department of Internal Medicine, Hacettepe University,
Ankara, Turkey
3
Department of Medical Biochemistry, School of Medicine,
Hacettepe University, Ankara, Turkey
4
Present Address: Joint Laboratory of Applied Ecotoxicology,
Korea Institute of Science and Technology Europe, KIST
EU), Campus 7.1, 66123 Saarbrucken, Germany
control during diabetes mellitus (DM) management [1].
The Diabetes Control and Complications Trial and UK Pro-
spective Diabetes Study have demonstrated the relationship
between HbA1c and diabetic complications [2–5]. Glycemic
variability (GV) refers to the magnitude of fluctuations in
serum glucose levels over a period of time. It is increasingly
recognized that glycemic variability is also associated with
diabetic micro- and macrovascular complications. Long-
term GV is positively associated with micro- and macro-
vascular complications and mortality, independent of HbA1c
level [6, 7]. It was also shown that short-term glycemic vari-
ability might negatively affect microvascular complications
[8].

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Oxysterols are compounds formed by the oxidation of
cholesterol by enzymatic and non-enzymatic pathways [9].
While oxysterols have a role in many biological activities
such as inflammation, immunosuppression, cytokine pro-
duction, and platelet activation, there are also data showing
that they are involved in the pathogenesis of diseases such
as DM, atherosclerosis, age-related macular degeneration,
Alzheimer's disease, colon cancer and osteoporosis [10,
11]. 7-ketocholesterol (7-KC) and cholestan-3β,5α,6β-triol
(Chol-triol) are oxysterols produced by free radical-mediated
non-enzymatic oxidation of cholesterol. Concentrations of
these oxysterols are accepted as indicators of free radical-
mediated auto-oxidation and oxidative stress [12].
Reactive oxygen species and oxidative stress are thought
to be involved in the pathogenesis of the relationship
between glycemic variability and diabetic complications
[13–16]. Recent studies showed that these auto-oxidized
oxysterols are higher in individuals with type 1 and 2 DM
than in the healthy control groups [17]. However, no study
has examined the relationship of auto-oxidized oxysterols
with glycemic variability in DM.
In this prospective study, we aimed to investigate the rela-
tionship between auto-oxidized oxysterols (7-KC and Chol-
triol levels) and both short-term and long-term glycemic
variability in patients with type 1 DM.
Methods
Subjects and setting
Consecutive 30 patients with type 1 DM using a continu-
ous subcutaneous insulin infusion (CSII) pump therapy
who applied to the tertiary endocrinology outpatient clinic
and age- and sex-matched healthy controls were included
in the study. Inclusion criteria included being older than
18 years of age, using an appropriate CSII pump for type
1 DM, agreeing to use a continuous glucose monitoring
system (CGMS) for 72 h, and providing blood samples to
measure oxysterol levels at 72 h. Exclusion criteria for the
study included pregnancy or lactation, using statin or oral
antidiabetic drugs[18], presence of chronic inflammatory
disease, hypertension, atherosclerotic heart disease, hepatic
disease, malignancies, and inherited metabolic disorders.
The study protocol adhered to the Declaration of Helsinki
Guidelines and was approved by the Ethics Committee of
Hacettepe University (GO 16969557-271). Informed consent
was obtained from each study participant.
Samples
Blood samples were collected in ethylenediaminetetraacetic
acid dipotassium (EDTA-K2)-containing tubes for oxysterol
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Journal of Endocrinological Investigation
and HbA1c measurements and serum separation tubes for
the other biochemistry parameters (Becton, Dickinson and
Company, Franklin Lakes, NJ, USA). The samples were cen-
trifuged immediately for oxysterol analysis, and plasma was
stored at − 80 °C.
Study protocol
The blood tests of patients with type 1 DM, including
HbA1c, lipid profile, and renal and hepatic function tests,
were performed. A Medtronic iPro™2 with Enlite Sensor
was applied to the patients as a CGMS device for 72 h.
Blood samples were taken from the patients for oxys-
terol levels [7-ketocholesterol (7-KC), cholestane-3β, 5α,
6β-triol (cholestanetriol, Chol-triol)] at 72 h and CGM
device was removed at the end of 72 h. MAGE is defined
as “the mean amplitude of glycemic excursions from nadir
to peak BG level and vice versa, which exceed one SD of
glucose variation” [19]. MAGE and standard deviation of
glucose measurements (Glucose-SD) were calculated using
72-h CGM data as short-term within-day glycemic variabil-
ity for 24–48 h and 0–72 h. MODD is the mean of daily
differences, which 24 h mean absolute differences between
two values measured at the same time point [20]. MODD
was calculated for short-term between-days glycemic vari-
ability for three consecutive days using 72-h CGM data. The
HbA1c level of the admission day and the previous HbA1c
level in the past year were used to calculate HbA1c-SD as a
long-term variability parameter.
Biochemical characteristics
The biochemical profiles of the patients were measured after
12 h of fasting in the morning. A Beckman Coulter AU680
analyzer (Beckman Coulter, Fullerton, CA, USA) was used
to measure routine biochemical tests of the patients with spe-
cial kits and reagents used for each biochemical test. HPLC
(DGU-20A-3R; Shimadzu Corp., Kyoto, Japan) was used
to measure HbA1c.
Oxysterol analysis
The liquid chromatography coupled with tandem mass
spectrometry (LC–MS/MS) method was used to analyze
the oxysterols based on the previously reported method 21
performed in a triple quadrupole system (Shimadzu 8040-
Japon). Plasma 7-KC and Chol-triol were derived from N,
N-dimethylglycine esters to increase ionization and cleavage
of 7-KC and Chol-triol for mass determination of oxysterol
species in human plasma. Only free and unesterified oxys-
terol species were quantified, and sample saponification was
unnecessary. As internal standards, 3β, 5α, trihydroxycho-
lastane D7 (Toronto Research Chemicals, Toronto, Canada)
Journal of Endocrinological Investigation

and 3β-hydroxy-5- cholesten-7-one D7 (Avanti Polar Lipids, Table 1  Clinical and biochemical characteristics of the study and the
Inc., Alabaster, AL, USA) were used for 7-KC and C-triol control groups
measurements. Eight-point calibrators (3.12–400 ng/mL) Study group Control group p
were prepared for the measurement. Plasma quality control (n = 30) (n = 30)
samples (QC) were prepared by spiking the known amounts
Gender (number, F/M) 28/2 28/2 1.00
of 7-KC and C-triol standards to obtain endogenously 40/40
Age (years) 32.4 ± 10.1 36.8 ± 11.2 0.115
and 150/150 ng/mL levels, respectively. Chromatographic
Duration of DM (years) 15 ± 8 N/A N/A
separation was performed on a Symmetry C18 column
Body mass index (BMI) 24.2 ± 3.4 21.2 ± 1.7 < 0.001
(100 mm × 2.1 mm, 5 mm) using a linear water and acetoni- (kg/m2)
trile gradient (pH 3; 1 mM ammonium formate) (Thermo ALT (U/L) 16.7 ± 8.4 20.2 ± 6.2 0.071
Fisher Scientific, Waltham, MA, USA). Mass spectrometry AST (U/L) 18.5 ± 4.8 19.6 ± 4.3 0.370
analysis was performed in positive ionization mode with HbA1c (%) 8.2 ± 1.3 4.2 ± 0.1 < 0.001
electrospray ionization. LDL (U/L) 116 ± 18 93 ± 14 < 0.001
Total cholesterol (mg/dL) 192 ± 23 156 ± 23 < 0.001
Statistical analysis Triglyceride (mg/dL) 93 ± 62 126 ± 6 0.005
7 -KC (ng/mL) 42.8 ± 3.9 18.8 ± 3.8 < 0.001
Statistical analyses were performed using SPSS version Chol-triol (ng/mL) 20.3 ± 1 10.37 ± 3.8 < 0.001
22.0. For categorical variables, frequencies and percentages 7 -KC/TC 22.5 ± 3.5 12.4 ± 3.2 < 0.001
were used as descriptive statistics, and standard deviation Chol-triol/TC 10.7 ± 1.5 6.9 ± 2.9 < 0.001
and mean for parametric continuous variables. Pearson’s
Chi-square test was performed for categorical data. Kol- P values with statistical significance are shown in bold
mogorov–Smirnov and Levene’s tests were used to evaluate HbA1c glycated hemoglobin, LDL low-density lipoprotein, 7-KC
ketocholesterol, Chol-triol Cholestan-3β,5α,6β-triol, TC total choles-
the normality of continuous variables. The Student-t test terol, Data are presented as mean ± SD, n Number of cases
was performed to compare continuous variables between
the study and control groups. Pearson correlation coefficient
was used to detect the relationships between oxysterol levels retinopathy, and 16.7% (n = 5) had neuropathy. None of them
and GV parameters. Predictors of oxysterol level were deter- had a macrovascular complication. There was no comorbid-
mined by multiple linear regression analysis. A p value of ity or smokers in the control group.
less than 0.05 was considered statistically significant. MAGE, MODD, Glucose-SD, and HbA1c-SD levels of
the study group are presented in Table 2.

Results Correlation analysis between oxysterol levels

Characteristics of the study and the control groups
The clinical and biochemical characteristics of the study
(n = 30) and the control group (n = 30) are presented in
Table 1. There was no significant difference between the
groups regarding age and gender. Although the BMI of the
control group was higher than that of the study group, it was
within the normal range in both groups. HbA1c, low-density
lipoprotein (LDL), triglyceride, and total cholesterol (TC)
levels were significantly higher in the study group. 7-KC and
Chol-triol levels were also significantly higher in the study
group. Since oxysterols are products formed by the oxidation
of cholesterol, 7-KC and Chol-triol levels were normalized
to total cholesterol (TC) levels. The ratios of 7-KC/TC and
Chol-triol/TC were also used for analysis. When oxysterol
levels were corrected for TC, the proportions of 7-KC/TC
and Chol-triol/TC remained significantly different in the
study group.
6.7% (n = 2) of the patients were smokers. 13.3% (
n = 4) of the patients had nephropathy, 13.3% (n = 5) had
and parameters of glycemic variability
7-KC and 7-KC/TC levels were strongly positively cor-
related with MAGE (24–48 h) and Glucose-SD (24–48 h)
(Table 3 and Fig. 1). Moderate positive correlations were
determined between both 7-KC and 7-KC/TC with MAGE
(0–72 h) and Glucose-SD (0–72 h) (Table 3 and Fig. 2).
No significant correlation was found between HbA1c and
HbA1c -SD with oxysterol levels. There was no significant
correlation between Chol-triol and Chol-triol/TC levels with
glycemic variability parameters (Table 3).
Predictors of oxysterol levels
We performed multiple linear regression analyses to deter-
mine factors affecting the 7-KC level. Chol-triol was not
used in regression models since there was no correlation
between Chol-triol and the parameters of glycemic vari-
ability. In the correlation analysis, a robust correlation
was observed between MAGE (24–48 h) and Glucose-SD
(24–48 h) (r = 0.919, p < 0.001). Therefore, two independent

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Table 2  Results of short- and long-term glycemic variability parameters

Short-term glycemic variability

MAGE (mg/dl), (24–48 h) 112.3 ± 57.6

MAGE (mg/dl), (0–72 h) 103.4 ± 34.7
MODD (mg/dl) 51.4 ± 30.9
Glucose-SD (mg/dl), (24–48 h) 52.1 ± 21.9
Glucose-SD (mg/dl), (0–72 h) 54.9 ± 16.5
Long-term glycemic variability

HbA1c-SD (%) 0.7 ± 0.7

Data are presented as mean ± SD

MAGE mean amplitude of glycemic excursions, MODD mean of daily differences, Glucose-SD standard deviation of glucose measurements,
HbA1c glycated hemoglobin

Table 3  Correlations between oxysterol levels and glycemic variabil- determined as independent variables. SD (24–48 h) pre-
ity parameters dicted a 7-KC level, while HbA1c did not. When Glucose-
7 KC 7 KC/TC Chol-triol Chol-triol/TC SD (24–48 h) increased by 10 mg/dL, 7-KC increased by
r r r r 6.7 ng/mL. Glucose-SD (24–48 h) accounted for 38.9% of
the changes in 7-KC levels.
MAGE, (24–48 h) 0.558** 0.468** 0.109 0.190 Graphical representations of significant regression coef-
MAGE, (0–72 h) 0.491** 0.396* − 0.52 0.091 ficients are shown in Fig. 3A and B.
MODD, (0-72 h) 0.37 0.106 0.043 0.090
Glucose-SD, 0.678** 0.509** 0.106 0.155
(24–48 h)
Glucose-SD, 0.445* 0.386* 0.012 0.128
Discussion
(0–72 h)
HbA1c 0.107 0.809 − -0.198 − 0.80 This is the first study to evaluate the relationship between
HbA1c -S 0.027 0.966 − 0.16 0.044 glycemic variability and auto-oxidized oxysterol levels in
DM. A strong correlation between MAGE and the plasma7-
r values with statistical significance are shown in bold KC level was shown in type 1 DM patients with insulin
7-KC 7-ketocholesterol, TC total cholesterol, Chol-triol pumps.
cholestane-3β, 5α, 6β-triol, MAGE mean amplitude of glycemic
excursions, MODD mean of daily differences, Glucose-SD standard
The relationship between different oxidative stress mark-
deviation of glucose measurements, HbA1c glycated hemoglobin, ers and glycemic variability has been evaluated in various
r = Pearson correlation coefficient studies to date. In the first study [13], urinary 8-iso PGF2α
*p < 0.05 excretion rate, which is used as an oxidative stress marker,
**p < 0.01 was found to be higher in the patient group than in the con-
trol group, and it was found to be significantly correlated
with MAGE. Subsequently, Ceriello et al. showed that glyce-
models were designed to eliminate the multicollinearity mic variability has more detrimental effects than consistent
problem: model 1 included MAGE (24–48 h), and model 2 high glucose on endothelial function and oxidative stress by
included Glucose-SD (24–48 h). using the markers 3-nitrotyrosine and 24 h urinary excre-
Table 4 exhibits the results of the linear regression tion rate of 8-iso-prostaglandin F2α (8-iso-PGF2α) [21].
model 1, analyzing the association of age, duration of DM, Thiobarbituric acid reactive substance, 8-hydroxydeoxy-
smoking, LDL, total cholesterol HbA1c levels, and MAGE guanosine, and high-sensitive C-reactive protein (hs-CRP)
(24–48 h) with 7-KC level. MAGE (24–48 h) was associated are other oxidative stress markers associated with short-term
with 7-KC level, whereas HbA1c did not significantly pre- glycemic variability in the literature [22].
dict 7-KC level. An increase of 10 mg/dL in MAGE 24–48 h Although plasma or urine 8-iso-PGF2α and hs-CRP are
resulted in a rise of 6.1 ng/mL of 7-KC. MAGE (24–48 h) widely used in assessing oxidative stress, oxysterol species
explained 26.6% of the changes in 7-KC levels. are less well-known markers. Data show that oxysterols are
The results of linear regression model 2 are shown in markers of oxidative stress and are involved in the patho-
Table 5. Age, duration of DM, smoking, LDL, total cho- genesis of atherosclerosis [23, 24]. In a study of carotid
lesterol, HbA1c levels, and Glucose-SD (24–48 h) was atherosclerosis in Finnish men with hypercholesterolemia,

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Fig. 1  A Positive correlation between 7-ketocholesterol and MAGE (24–48 h) B Positive correlation between 7-ketocholesterol and Glucose-SD
(24–48 h). r = Pearson correlation coefficient

Fig. 2  A Positive correlation between 7-ketocholesterol/ Total cholesterol and MAGE (0–72 h). B Positive correlation between 7-ketocholes-
terol/ Total cholesterol and Glucose-SD (0–72 h). r = Pearson correlation coefficient

oxysterol 7β-hydroxycholesterol, which can be converted to
7-KC, was shown to be the strongest predictor of the pro-
gression of carotid atherosclerosis [25]. Similarly, in another
study, accumulation of 7-KC and the other non-enzymatic
oxysterols, 7alpha-hydroxycholesterol and 7beta-hydroxy-
cholesterol (7betaOH), was shown to play a role in the devel-
opment of inflammation and macrophage foam cell death as
well as atherosclerosis [26] We also previously demonstrated
that 7-KC and Chol-triol levels, the main biomarkers among
oxysterols to measure oxidative stress, were significantly
higher in patients with type 1 DM [17]. Similarly, Abo et al.
observed that plasma and erythrocyte 7-KC levels were
higher in patients with type 2 DM than in control subjects
[27]. Furthermore, in similar studies involving patients with
both type 1 and type 2 DM, oxysterol levels, including 7-KC
and Chol-triol, were higher in both groups than in the control
group [18, 28]. These studies concluded that the increase
in oxysterol levels was associated with hyperglycemia and
showed that their levels were decreased with insulin, oral
hypoglycemic agents, and statins. Although the increase in
oxysterols was attributed to hyperglycemia in these studies,
it is impossible to make a clear interpretation since there is
no glycemic variability data in these studies.
A limited number of studies suggested that oxysterol lev-
els are affected by glycemic control and HbA1c, but these
studies have not considered glycemic variability [17, 18].
HbA1c represents the average serum glucose levels over the
previous 2–3 months, reflecting glycemic control. But it does
not provide information about actual glucose fluctuations
[29]. Glycemic variability, on the other hand, is a concept
that gives more precise data about these fluctuations and is
directly related to oxidative stress and, therefore, diabetic

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Table 4  Multiple linear regression analysis − Model 1 with MAGE growth factor-binding protein-3 more than stable hypergly-
(24-48 h) cemic states [31]. In a study conducted with patients with
Standard- p 95% CI type 1 DM, an increase in inflammation and oxidative stress
ized ß markers (sICAM-1, 8-iso-PGF2α, nitrotyrosine, and IL-6)
(regression Lower Upper
was found after hypoglycemia [32]. These markers also were
coefficient)
higher in patients who recovered after hypoglycemia with
Age − 0.415 0.082 − 0.345 0.022 hyperglycemia than those who recovered with normogly-
Duration of DM (years) 0.106 0.592 − 0.141 0.242 cemia [32].
Smoking 0.043 0.804 − 4.896 6.249 Diabetes mellitus-related complications and oxidative
LDL 0.198 0.443 − 0.071 0.157 stress in chronic hyperglycemia are known to be associ-
Total cholesterol 0.126 0.661 − 0.077 0.120 ated with the polyol pathway, activation of the hexosamine
HbA1c 0.169 0.309 − 0.523 1.575 pathway and protein kinase C, and formation of advanced
MAGE (24–48 h) 0.610 0.002 0.017 0.066 glycation end-products (AGE) [33]. In addition to these
well-known pathways, glycemic variability triggers unusual
The p-value with statistical significance is shown in bold
biochemical pathways that result in oxidative stress and may
­ 2 = 0.266, F (7, 22) = 2500, p = 0.047
Model: adjusted R
contribute to DM complications [34]. Studies have shown
LDL low-density lipoprotein, HbA1c glycated hemoglobin, MAGE
that rising glycemic variability increases nitric oxide (NO)
mean amplitude of glycemic excursions, Glucose-SD standard devia-
tion of glucose measurements and superoxide levels [13, 35]. Reacting NO with super-
oxide anion (­ O2−) may result in highly reactive peroxyni-
trite (ONOO.-), increasing lipid peroxidation and protein
Table 5  Multiple linear regression analysis—Model 2 with Glucose- nitration and leading to LDL oxidation [36]. Oxysterol for-
SD (24–48 h) mation due to glycemic variability may occur through this
Standard- p 95% CI pathway. Also, in a cell culture study, authors showed that
ized ß AGE in macrophages induces intracellular accumulation of
(regression Lower Upper
7-KC and 7betaOH [37]. This finding suggests that oxys-
coefficient)
terol may play a role in the pathways leading to diabetes
Age − 0.327 0.123 − 0.292 0.037 complications.
Duration of DM 0.113 0.532 − 0.121 0.228 In our study, besides a significant correlation between
Smoking 0.026 0.870 − 4.659 5.470 MAGE and oxysterol levels, there was no relationship with
LDL 0.202 0.387 − 0.059 0.147 HbA1c-SD, a long-term glycemic variability parameter.
Total Cholesterol 0.054 0.831 − 0.078 0.096 Therefore, the findings of this study support the idea that
HbA1c 0.051 0.736 − 0.800 1.116 glycemic variability may contribute to the development of
Glucose-SD (24–48 h) 0.675 < 0.001 0.065 0.179 complications by causing oxidative stress by mechanisms
other than chronic hyperglycemia. Biochemical studies have
The p-value with statistical significance is shown in bold
shown that the half-lives of oxysterols are limited to hours—
Model: adjusted ­R2 = 0.389, F (7, 22) = 3640, p = 0.009
a few days [38–40]. The half-life of 7-KC was calculated as
LDL low-density lipoprotein, HbA1c glycated hemoglobin, Glucose-
1.5 h by Larrson et al. [41]. The result that oxysterol levels
SD: standard deviation of glucose measurements
were not associated with long-term glycemic variability
may be due to their short half-lives. While both 7-KC and
complications independent of HbA1c [8, 13]. CGM systems the Chol-triol levels were significantly higher in the study
have been frequently used in recent years to assess glyce- group than in the control group, we did not find a correlation
mic variability, and they have improved limitations asso- between glycemic variability and Chol-triol level, despite
ciated with HbA1c and self-monitoring of blood glucose the existence of 7-KC. This result may be related to the fact
[30]. Several studies have shown that glycemic fluctuations that 7-KC is the primary product of non-enzymatic choles-
might lead to oxidative stress by mechanisms independ- terol oxidation due to oxidative stress [12]. In addition, the
ent of chronic hyperglycemia [13, 31, 32]. Monniere et al. difference in metabolism and half-lives of 7-KC and Chol-
reported that urinary excretion of 8-iso-PGF2α was strongly triol may also have contributed to this result.
correlated with acute glucose fluctuations, whereas there The present study had several limitations. One of them
was no correlation between urinary 8-iso PGF2α excretion is that the study group comprised a limited number of
rates and HbA1c, mean daily glucose concentrations [13]. participants. In addition, no correlation was found between
A kidney cell culture study showed that varying glucose MODD and oxysterol levels. This result may be related
concentrations increased the production of inflammatory to the fact methods of glycemic variability measurements
cytokines, transforming growth factor-β and insulin-like were based on only 3-day CGMS data. Studies of glycemic

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Journal of Endocrinological Investigation
Fig. 3  A Regression model-1 included MAGE (24–48 h). B Regression model-2 included Glucose-SD (24-48 h)
variability with subjects using the CGMS for extended
periods will provide more detailed information.
In conclusion, the present study showed that short-term
glycemic variability in type 1 DM correlates with levels of
7-KC, an oxysterol associated with oxidative stress, inde-
pendent of the HbA1c level. These results suggest that
glycemic variability may cause oxidative stress through
auto-oxidized oxysterols with mechanisms independent
of long-term glycemic control. In future studies, longer
follow-ups with a more extensive study group will more
clearly reveal the relationship between glycemic variabil-
ity and oxysterol.
Author contributions UÜ and ABB: conception and design of the
work, analysis, and interpretation of data, drafting the manuscript. AS
and İL: performing biochemical analyses in the laboratory and revis-
ing the manuscript. MB and SD: conception and design of the work,
analysis and interpretation of data, and revising the manuscript.
Funding This research did not receive any specific grant from funding
agencies in the public, commercial, or not-for-profit sectors.
Availability of data and materials The data are available on request
from the authors.
Declarations
Conflict of interest None of the authors have any potential conflicts of
interest associated with this research.
Ethical approval This research involves human participants. Ethical
approval has been obtained from the ethical committee of Hacettepe
University (Project number: 16969557-271).
Informed consent Written informed consent was obtained from all the
patients before inclusion.
References
1. Nathan DM, Turgeon H, Regan S (2007) Relationship between
glycated haemoglobin levels and mean glucose levels over time.
Diabetologia 50(11):2239–2244
2. (1994) Effect of intensive diabetes treatment on the develop-
ment and progression of long-term complications in adolescents
with insulin-dependent diabetes mellitus: Diabetes Control and
Complications Trial Diabetes Control and Complications Trial
Research Group. J Pediatr. 125(2):177–88.
3. (1995) The relationship of glycemic exposure (HbA1c) to the
risk of development and progression of retinopathy in the dia-
betes control and complications trial. Diabetes 44(8):968–83.
4. Lachin JM, Genuth S, Nathan DM, Zinman B, Rutledge BN
(2008) Effect of glycemic exposure on the risk of microvascular
complications in the diabetes control and complications trial–
revisited. Diabetes 57(4):995–1001
5. (1998) Intensive blood-glucose control with sulphonylureas
or insulin compared with conventional treatment and risk of
complications in patients with type 2 diabetes (UKPDS 33).
UK Prospective Diabetes Study (UKPDS) Group. Lancet.
352(9131):837–53.
6. Gorst C, Kwok CS, Aslam S, Buchan I, Kontopantelis E, Myint
PK et al (2015) Long-term glycemic variability and risk of
adverse outcomes: a systematic review and meta-analysis. Dia-
betes Care 38(12):2354–2369
7. Hirakawa Y, Arima H, Zoungas S, Ninomiya T, Cooper M,
Hamet P et al (2014) Impact of visit-to-visit glycemic vari-
ability on the risks of macrovascular and microvascular events
and all-cause mortality in type 2 diabetes: the ADVANCE trial.
Diabetes Care 37(8):2359–2365
8. Akaza M, Akaza I, Kanouchi T, Sasano T, Sumi Y, Yokota
T (2018) Nerve conduction study of the association between
glycemic variability and diabetes neuropathy. Diabetol Metab
Syndr 10:69
9. Vaya J, Szuchman A, Tavori H, Aluf Y (2011) Oxysterols for-
mation as a reflection of biochemical pathways: summary of
in vitro and in vivo studies. Chem Phys Lipids 164(6):438–442
13

10. Samadi A, Sabuncuoglu S, Samadi M, Isikhan SY, Chirumbolo S,
Peana M et al (2021) A comprehensive review on oxysterols and
related diseases. Curr Med Chem 28(1):110–136
11. Zarrouk A, Vejux A, Mackrill J, O’Callaghan Y, Hammami M,
O’Brien N et al (2014) Involvement of oxysterols in age-related
diseases and ageing processes. Ageing Res Rev 18:148–162
12. Iuliano L (2011) Pathways of cholesterol oxidation via non-enzy-
matic mechanisms. Chem Phys Lipids 164(6):457–468
13. Monnier L, Mas E, Ginet C, Michel F, Villon L, Cristol JP et al
(2006) Activation of oxidative stress by acute glucose fluctuations
compared with sustained chronic hyperglycemia in patients with
type 2 diabetes. JAMA 295(14):1681–1687
14. Ceriello A, Esposito K, Piconi L, Ihnat M, Thorpe J, Testa R
et al (2008) Glucose “peak” and glucose “spike”: Impact on
endothelial function and oxidative stress. Diabetes Res Clin Pract
82(2):262–267
15. Schisano B, Tripathi G, McGee K, McTernan PG, Ceriello A
(2011) Glucose oscillations, more than constant high glucose,
induce p53 activation and a metabolic memory in human endothe-
lial cells. Diabetologia 54(5):1219–1226
16. Costantino S, Paneni F, Battista R, Castello L, Capretti G, Chi-
andotto S et al (2017) Impact of glycemic variability on chro-
matin remodeling, oxidative stress, and endothelial dysfunction
in patients with type 2 diabetes and with target HbA(1c) levels.
Diabetes 66(9):2472–2482
17. Samadi A, Gurlek A, Sendur SN, Karahan S, Akbiyik F, Lay I
(2019) Oxysterol species: reliable markers of oxidative stress in
diabetes mellitus. J Endocrinol Invest 42(1):7–17
18. Ferderbar S, Pereira EC, Apolinário E, Bertolami MC, Faludi A,
Monte O et al (2007) Cholesterol oxides as biomarkers of oxida-
tive stress in type 1 and type 2 diabetes mellitus. Diabetes Metab
Res Rev 23(1):35–42
19. Service FJ, Molnar GD, Rosevear JW, Ackerman E, Gatewood
LC, Taylor WF (1970) Mean amplitude of glycemic excursions,
a measure of diabetic instability. Diabetes 19(9):644–655
20. Molnar GD, Taylor WF, Ho MM (1972) Day-to-day variation of
continuously monitored glycaemia: a further measure of diabetic
instability. Diabetologia 8(5):342–348
21. Ceriello A, Esposito K, Piconi L, Ihnat MA, Thorpe JE, Testa R
et al (2008) Oscillating glucose is more deleterious to endothelial
function and oxidative stress than mean glucose in normal and
type 2 diabetic patients. Diabetes 57(5):1349–1354
22. Chang CM, Hsieh CJ, Huang JC, Huang IC (2012) Acute and
chronic fluctuations in blood glucose levels can increase oxida-
tive stress in type 2 diabetes mellitus. Acta Diabetol 49(Suppl
1):S171–S177
23. Brown AJ, Jessup W (1999) Oxysterols and atherosclerosis. Ath-
erosclerosis 142(1):1–28
24. Björkhem I, Andersson O, Diczfalusy U, Sevastik B, Xiu RJ, Duan
C et al (1994) Atherosclerosis and sterol 27-hydroxylase: evidence
for a role of this enzyme in elimination of cholesterol from human
macrophages. Proc Natl Acad Sci U S A 91(18):8592–8596
25. Salonen JT, Nyyssönen K, Salonen R, Porkkala-Sarataho E, Tuo-
mainen TP, Diczfalusy U et al (1997) Lipoprotein oxidation and
progression of carotid atherosclerosis. Circulation 95(4):840–845
26. Berthier A, Lemaire-Ewing S, Prunet C, Montange T, Vejux
A, Pais de Barros JP et al (2005) 7-Ketocholesterol-induced
apoptosis. Involvement of several pro-apoptotic but also anti-
apoptotic calcium-dependent transduction pathways. FEBS J
272(12):3093–3104
27. Abo K, Mio T, Sumino K (2000) Comparative analysis of plasma
and erythrocyte 7-ketocholesterol as a marker for oxidative stress
in patients with diabetes mellitus. Clin Biochem 33(7):541–547
13
Journal of Endocrinological Investigation
28. Samadi A, Isikhan SY, Tinkov AA, Lay I, Doşa MD, Skalny AV
et al (2020) Zinc, copper, and oxysterol levels in patients with type
1 and type 2 diabetes mellitus. Clin Nutr 39(6):1849–1856
29. Beck RW, Connor CG, Mullen DM, Wesley DM, Bergenstal RM
(2017) The fallacy of average: how using HbA(1c) alone to assess
glycemic control can be misleading. Diabetes Care 40(8):994–999
30. Danne T, Nimri R, Battelino T, Bergenstal RM, Close KL,
DeVries JH et al (2017) International consensus on use of con-
tinuous glucose monitoring. Diabetes Care 40(12):1631–1640
31. Jones SC, Saunders HJ, Qi W, Pollock CA (1999) Intermittent high
glucose enhances cell growth and collagen synthesis in cultured
human tubulointerstitial cells. Diabetologia 42(9):1113–1119
32. Ceriello A, Novials A, Ortega E, La Sala L, Pujadas G, Testa
R et al (2012) Evidence that hyperglycemia after recovery from
hypoglycemia worsens endothelial function and increases oxida-
tive stress and inflammation in healthy control subjects and sub-
jects with type 1 diabetes. Diabetes 61(11):2993–2997
33. Ighodaro OM (2018) Molecular pathways associated with
oxidative stress in diabetes mellitus. Biomed Pharmacother
108:656–662
34. Weber C, Schnell O (2009) The assessment of glycemic variability
and its impact on diabetes-related complications: an overview.
Diabetes Technol Ther 11(10):623–633
35. Valente T, Valente F, Lucchesi MBB, Punaro GR, Mouro MG,
Gabbay MAL et al (2021) Relationship between short and long-
term glycemic variability and oxidative stress in type 1 diabetes
mellitus under daily life insulin treatment. Arch Endocrinol Metab
65(5):570–578
36. Griendling KK, FitzGerald GA (2003) Oxidative stress and car-
diovascular injury: part I: basic mechanisms and in vivo monitor-
ing of ROS. Circulation 108(16):1912–1916
37. Iborra RT, Machado-Lima A, Castilho G, Nunes VS, Abdalla DS,
Nakandakare ER et al (2011) Advanced glycation in macrophages
induces intracellular accumulation of 7-ketocholesterol and total
sterols by decreasing the expression of ABCA-1 and ABCG-1.
Lipids Health Dis 10:172
38. Diczfalusy U, Kanebratt KP, Bredberg E, Andersson TB, Bötti-
ger Y, Bertilsson L (2009) 4beta-hydroxycholesterol as an endog-
enous marker for CYP3A4/5 activity. Stability and half-life of
elimination after induction with rifampicin. Br J Clin Pharmacol
67(1):38–43
39. Bodin K, Andersson U, Rystedt E, Ellis E, Norlin M, Pikuleva I
et al (2002) Metabolism of 4 beta -hydroxycholesterol in humans.
J Biol Chem 277(35):31534–31540
40. Björkhem I, Lütjohann D, Diczfalusy U, Ståhle L, Ahlborg
G, Wahren J (1998) Cholesterol homeostasis in human brain:
turnover of 24S-hydroxycholesterol and evidence for a cerebral
origin of most of this oxysterol in the circulation. J Lipid Res
39(8):1594–1600
41. Larsson H, Böttiger Y, Iuliano L, Diczfalusy U (2007) In vivo
interconversion of 7β-hydroxycholesterol and 7-ketocholesterol,
potential surrogate markers for oxidative stress. Free Radical Biol
Med 43(5):695–701
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