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OPEN The association between dietary

acrylamide intake and the risk
of type 2 diabetes incidence
in the Tehran lipid and glucose
study
Firoozeh Hosseini‑Esfahani 1, Niloofar Beheshti 1, Amene Nematollahi 2,
Glareh Koochakpoor 3, Soheil verij‑Kazemi 1, Parvin Mirmiran 1* & Fereidoon Azizi 4

This study aimed at investigating the association of acrylamide consumption with the incidence of
type 2 diabetes (T2D) in adults. The 6022 subjects of the Tehran lipid and glucose study participants
were selected. The acrylamide content of food items were summed and computed cumulatively
across follow up surveys. Multivariable Cox proportional hazard regression analyses were performed
to estimate the hazards ratio (HR) and 95% confidence interval (CI) of incident T2D. This study was
done on men and women, respectively aged 41.5 ± 14.1 and 39.2 ± 13.0 years. The mean ± SD of dietary
acrylamide intake was 57.0 ± 46.8 µg/day. Acrylamide intake was not associated with the incidence of
T2D after adjusting for confounding variables. In women, a higher acrylamide intake was positively
associated with T2D [HR (CI) for Q4: 1.13 (1.01–1.27), P trend: 0.03] after adjusting for confounding
factors. Our results demonstrated that dietary intake of acrylamide was associated with an increased
risk of T2D in women.

Acrylamide is produced during the preparation of high-carbohydrate foods such as potato chips, french-fries
and breads which contain the amino acid ­asparagine1. When these foods are prepared at high temperatures
(120 °C), sugar reacts with asparagine through Millard Reaction and acrylamide produced up to 1 mg/kg ­food2.
Furthermore, acrylamide is also part of tobacco ­smoke3. Two main sources of public exposure to acrylamide
are food and cigarette smoke.
Dietary acrylamide could be an important human health risk factor; previous studies observed neurotoxic-
ity in workers exposed to acrylamide in the ­workplace4,5. Moreover, animal studies reported the relationship
of acrylamide intake with significant carcinogenic and neurotoxic r­ isk6,7. Epidemiological studies have issued
conclusive evidence that dietary acrylamide exposure is associated with the risk of cancers in ­humans8; there are
also studies that reported a significant association between dietary acrylamide intake and risk of renal, endo-
metrial, and ovarian ­cancers9. International Agency for Research on Cancer considers acrylamide as a probable
human ­carcinogen10. The effect of acrylamide in increasing oxidative DNA damage in target organs is one of the
suggested mechanisms that explain the carcinogenicity of a­ crylamide11. Recent studies indicated that long-term
high levels of dietary acrylamide intake may induce oxidative stress and chronic ­inflammation12. Prevalence of
type 2 diabetes (T2D), a metabolic disorders characterized by increased blood glucose concentration, increase
across the ­worldwide13. The etiology of diabetes remains mainly u ­ nspecified14; however, the common pathogenic
mediators in the natural course of diabetes is oxidative s­ tress15. These findings suggest this thesis that acrylamide
intake may be actual in the onset of T2D. In the study by Wang et al., ingested acrylamide was associated with
fasting plasma glucose elevation, oxidative DNA damage and lipid peroxidation in 3270 adults; the significant
linear positive dose–response relationship was found between urinary acrylamide metabolite and fasting plasma
­glucose16. In the study by Lin et al., a 1 unit rise in Hb adducts of acrylamide was related to both reduced blood

1
Nutrition and Endocrine Research Center, Research Institute for Endocrine Sciences, Shahid Beheshti University
of Medical Sciences, Tehran, Iran. 2Department of Food Safety and Hygiene, School of Health, Fasa University of
Medical Sciences, Fasa, Iran. 3Maraghe University of Medical Sciences, Maraghe, Iran. 4Endocrine Research Center,
Research Institute for Endocrine Sciences, Shahid Beheshti University of Medical Sciences, Tehran, Iran. *email:
mirmiran@endocrine.ac.ir

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insulin and reduced insulin resistance; this association was stronger in subjects with a lower education level,
smokers, whites and in subjects with body mass index (BMI) < 25 or > 30 kg/m2 than their ­counterparts17. The
existence of such contradictory results led us to perform a cohort study aimed at investigating the association of
acrylamide consumption with the incidence of T2D in a group of Iranian adults.

Results
This study was done on 2707 men and 3315 women (n = 6022), respectively aged 41.5 ± 14.1 and 39.2 ± 13.0 years.
The participants include 5563 censors and 549 incident cases of T2D during a median of 6.63 years of follow up.
The mean ± SD of dietary acrylamide intake was 57.0 ± 46.8 µg/day (median: 46.4 µg/day).
The baseline characteristics of the subjects across quartiles of acrylamide intake are shown in Table 1. We
found that those who consumed higher acrylamide intakes were younger (Q1: 43.2 ± 14.3, Q4: 37.9 ± 12.7,
P < 0.001). The percentage of active smokers was higher in the upper quartiles of acrylamide intake (Q1-Q4:
13.0, 16.7, 22.2, 28.0%). The level of physical activity was higher in the upper quartiles of acrylamide intake (Q1:
495 ± 684, Q4: 640 ± 995, P < 0.001).
Moreover, individuals in the upper quartiles of acrylamide intake had higher intake of total energy (Q1:
1879 ± 481, Q4: 2704 ± 610 kcal, P < 0.001), carbohydrate (Q1: 58.4 ± 6.00, Q4: 59.6 ± 5.66% of energy, P = 0.001)
and lower total fat (Q1: 30.3 ± 5.66, Q4: 29.3 ± 6.73% of energy, P = 0.003) and saturated fatty acid intakes (Q1:
9.97 ± 2.45, Q4: 9.45 ± 5.40% of energy, P = 0.008). The distribution of FBG, SBP, DBP and TG did not differ across
quartiles of acrylamide intake; however, the level of WC (Q1: 88.2 ± 13.5, Q4: 90.3 ± 12.8 cm, P = 0.007) was

Acrylamide intake
Q1 Q2 Q3 Q4 P
n 1504 1506 1506 1506
Acrylamide (µg/day) 23.3 ± 5.98 38.9 ± 4.30 55.8 ± 6.03 106 ± 68.3
Baseline age (years) 43.2 ± 14.3 40.8 ± 13.5 39.0 ± 13.1 37.9 ± 12.7 < 0.001
Women (%) 73.3 59.8 41.7 40 < 0.001
Smoking (%) < 0.001
Non-smokers 69.6 66.5 58.7 51.8
Passive smoker 17.4 16.3 19.1 20.1
Active smoker 13.0 16.7 22.2 28.0
Family history of diabetes (%) 14.4 12.3 14.4 14.3 0.60
Physical activity (MET/min/week) 495 ± 684 500 ± 733 587 ± 871 640 ± 995 < 0.001
Education (≥ 12 years) 20.2 25.5 29.9 32.2 < 0.001
BMI (kg/m2) 27.3 ± 4.92 27.0 ± 4.74 26.7 ± 4.70 26.9 ± 4.68 0.17
Waist circumference (cm) 88.2 ± 13.5 88.5 ± 13.0 88.8 ± 13.2 90.3 ± 12.8 0.007
Fasting blood glucose (mg/dl) 90.2 ± 9.39 90.4 ± 8.95 90.1 ± 8.91 90.7 ± 8.95 0.29
Systolic BP (mmHg) 111 ± 16.9 111 ± 16.7 112 ± 15.7 111 ± 14.8 0.23
Diastolic BP (mmHg) 73.9 ± 10.6 74.4 ± 10.9 74.63 ± 10.7 75.02 ± 10.5 0.10
Triglyceride (mg/dl) 134 ± 76.9 138 ± 79.1 138 ± 82.7 140 ± 88.0 0.27
HDL (mg/dl) 47.3 ± 11.4 45.7 ± 11.0 44.7 ± 10.7 44.7 ± 10.4 < 0.001
Energy intake (kcal/day) 1879 ± 481 2152 ± 465 2432 ± 495 2704 ± 610 < 0.001
Carbohydrate (% of energy) 58.4 ± 6.00 59.0 ± 5.37 59.1 ± 5.31 59.6 ± 5.66 0.001
Protein (% of energy) 14.6 ± 2.75 14.7 ± 2.49 14.7 ± 2.20 14.9 ± 3.24 0.37
Total fat (% of energy) 30.3 ± 5.66 29.7 ± 5.12 29.6 ± 5.09 29.3 ± 6.73 0.003
SFA (% of energy) 9.97 ± 2.45 9.62 ± 2.13 9.61 ± 3.40 9.45 ± 5.40 0.008
MUFA (% of energy) 10.2 ± 2.52 9.96 ± 2.05 9.96 ± 2.18 9.96 ± 5.25 0.14
PUFA (% of energy) 6.15 ± 1.89 6.02 ± 1.59 6.00 ± 1.54 6.05 ± 5.11 0.89
Fiber (g/1000 kcal) 10.9 ± 11.1 10.2 ± 4.01 9.75 ± 2.56 9.63 ± 2.78 < 0.001
Breads (g/day) 94.0 ± 42.0 142 ± 51.0 189 ± 73.0 256 ± 225 < 0.001
Confectionary products (g/day) 4.28 ± 3.82 6.28 ± 5.24 7.66 ± 7.73 9.19 ± 11.2 < 0.001
Roasted nuts (g/day) 4.03 ± 6.13 5.43 ± 8.12 6.92 ± 8.53 8.46 ± 13.4 < 0.001
Snacks (g/day) 5.25 ± 5.38 8.16 ± 7.66 11.6 ± 11.3 15.9 ± 28.1 < 0.001
Coffee (g/day) 1.56 ± 2.68 3.87 ± 5.43 7.87 ± 10.2 39.7 ± 65.7 < 0.001
Fast food (g/day) 9.01 ± 9.85 13.0 ± 12.6 15.7 ± 16.7 20.9 ± 28.9 < 0.001
Bakery products (g/day) 8.36 ± 8.99 13.0 ± 13.4 16.2 ± 17.1 19.3 ± 21.3 < 0.001

Table 1.  Baseline characteristics of adult participants of the Tehran Lipid and Glucose Study across quartiles
of acrylamide intake (n = 6022). Values are mean ± SD unless otherwise listed. Q quartiles of choline intake,
BMI body mass index, BP blood pressure, HDL high density lipoprotein cholesterol, MUFA mono-unsaturated
fatty acids, PUFA poly-unsaturated fatty acids, SFA saturated fat.

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higher in the upper quartiles of acrylamide intake and the level of HDL-C (Q1: 47.3 ± 11.4, Q4: 44.7 ± 10.4 mg/
dl, P < 0.001) was lower in higher quartiles of acrylamide intake.
The main contributors of acrylamide intake were for bread food items and coffee. The contribution of acryla-
mide intake from bread food items decreased from lower to upper quartiles of acrylamide intake, but the con-
tribution of coffee increased from lower to upper quartiles of acrylamide intake (Fig. 1).
The Cox-proportional HRs for T2D based on quartiles of acrylamide intake is shown in Table 2. Acrylamide
intake was positively associated with the incidence of T2D in model 1 (P for trend = 0.009); however, our results
showed no association in model 2 after adjusting for confounding variables. Also the results of subgroup analy-
sis were shown in this table. The association of acrylamide intake with T2D was not significant in subgroups

100%

90%

80%

70%
bakery products
60% fast food
confeconary products
50% coffee
snacks
40%
roasted nuts
30% breads

20%

10%

0%
q1 q2 q3 q4

Figure 1.  Contributors of dietary acrylamide intake from food items across quartiles of acrylamide intake.

Total acrylamide intake

Median intake (µg/day)
All participants (n = 6022)
Model 1
Model 2
Smoking*
Non-exposed (n = 3714)
Passive smoker (n = 1204)
Active smoker (n = 1104)
Sex†
Men (n = 2707)
Women (n = 3315)
BMI††
< 25 kg/m2 (n = 2134)
≥ 25 kg/m2 (n = 3888)
Q1 Q2 Q3 Q4 P for trend
24.2 38.9 55.5 87.7
1 1.01 (0.94–1.08) 1.04 (0.97–1.12) 1.10 (1.02–1.18) 0.009
1 1.00 (0.93–1.08) 1.02 (0.94–1.11) 1.06 (0.98–1.16) 0.13
1 1.01 (0.92–1.11) 1.01 (0.92–1.11) 1.09 (0.98–1.22) 0.24
1 0.92 (0.77–1.10) 0.96 (0.80–1.16) 0.96 (0.79–1.16) 0.70
1 1.08 (0.90–1.21) 1.15 (0.95–1.40) 1.14 (0.94–1.38) 0.18
1 0.93 (0.83–1.07) 0.96 (0.84–1.08) 0.96 (0.84–1.11) 0.85
1 1.02 (0.93–1.12) 1.05 (0.95–1.16) 1.13 (1.01–1.27) 0.03
1 1.05 (0.92–1.19) 1.04 (0.91–1.19) 1.07 (0.93–1.24) 0.37
1 0.97 (0.88–1.06) 1.00 (0.91–1.07) 1.05 (0.94–1.16) 0.27

Table 2.  Hazard ratios (95% CI) for the association of acrylamide intake and type 2 diabetes incidence in
all participants and subgroups. Model 1: adjusted for age and sex, Model 2: Model 1 plus education, physical
activity, family history of type 2 diabetes, body mass index, energy intake, triglyceride/high density lipoprotein
cholesterol, smoking. *Adjusted for age, sex, education, physical activity, history of type 2 diabetes, body
mass index, energy intake, triglyceride/high density lipoprotein cholesterol. † Adjusted for age, education,
physical activity, family history of type 2 diabetes, body mass index, energy intake, Triglyceride/High density
lipoprotein cholesterol. †† Adjusted for age, education, physical activity, family history of type 2 diabetes, energy
intake, triglyceride/high density lipoprotein cholesterol.

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of smoking categories. Our results indicated that after adjusting for confounding factors, a higher acrylamide
intake was positively associated with T2D [HR (CI) for Q4: 1.13 (1.01–1.27), P for trend: 0.03] in women. In
two subgroups of normal and overweight or obese individuals, there were no association between acrylamide
intake and T2D.

Discussion
The present study examined the association of acrylamide intake with T2D incidence in a group of adults in the
TLGS after 6.63 years of follow up. Our findings showed that the contribution of acrylamide intake from bread
food items was higher than other food groups including bakery products, coffee, roasted nuts, snacks, confec-
tionary products and fast foods. Also acrylamide intake was positively associated with T2D incidence in total
population; however, this association was not remained after adjusting for confounding variables. In this study
we observed that a higher acrylamide intake was positively associated with the incidence of T2D in women,
independent of other risk factors of T2D. There were no associations of acrylamide intake with T2D incidence
in subgroups of smoking status, normal or overweight and obese individuals.
There is a large variation in estimation of dietary acrylamide intake in different countries. The median of
dietary acrylamide intake ranged from 5.9 µg/day in 45–74 year old in Japan to 45 µg/day in a Swedish study
among participants aged 45–73 years. A review study reported that European countries had higher dietary
acrylamide estimation than other countries in the world especially in Asia, which might be due to differences
in study designs, methods and dietary habits; however, the estimation of dietary acrylamide intake in our study
was slightly higher than the Swedish participants, a European country. It might be due to eating foods in a way
that results in higher acrylamide f­ ormation18.
In our study, the main food items responsible for acrylamide intake were respectively breads (52–62%), cof-
fee (6–28%), snacks (7–10%), bakery products (5–7%), fast foods (4–6%) and roasted nuts (1–2%) in general
population; while unlike our study, the expert committee on food additives reported that the principal food
items accountable for acrylamide intake were respectively potato crisps (6–46%), potato chips (16–30%), coffee
(13–39%), pastries and sweet biscuits (10–20%) and breads (10–30%)19. In our study, most participants con-
sumed a high carbohydrate diet (~ 60% of energy intake) especially from refined sources (e.g. white bread)20.
Also these results demonstrated that dietary acrylamide intake might be driven from staple foods such as breads
that consumed in ­breakfast18. So food industry operators can apply methods to reduce acrylamide formation in
bakery products such as using natural antioxidants obtained from spices and herb extracts in bakery p ­ roducts21.
Furthermore, it was demonstrated that the more important source of acrylamide exposure is smoking com-
pared to dietary ­intakes22. Moreover acrylamide toxicity has a cumulative nature, so the chronic exposure at low
doses via different routes is important. Also acrylamide has been proven to disrupt the endocrine ­system23,24, so
low dose chronic of acrylamide exposure may create important health problems and it may be a contributing
factor to induce diabetes. Acrylamide exposure at both high and low doses can change oxidative stress parameters
and inflammation that can lead to intensive lipid peroxidation and ­atherosclerosis25.
To our knowledge, this is the first population-based study to report an association of dietary acrylamide
intake and T2D. Unlike to our findings in all participants, previous epidemiological studies reported the relation
between acrylamide exposure and glucose metabolism. Wang et al. found that urinary acrylamide metabolites
were positively associated with FBG in a dose response manner in a general urban adult population. Also
they indicated that acrylamide exposure is positively associated with FBG, oxidative DNA damage and lipid
­peroxidation16. In the American adult population, acrylamide exposure was associated with the insulin resist-
ance and a decrease in insulin ­levels17. In the national health and nutrition examination survey (NHANES) the
association of T2D and acrylamide biomarkers including hemoglobin adducts of acrylamide and glycidamide
(HbAA and HbGA) was reported. HbAA was linearly and inversely associated with the risk of T2D, while HbGA/
HbAA was positively and non-linearly associated with T2D p ­ revalence26. HbAA and HbGA are considered as
long-term exposure biomarkers of acrylamide in humans. Dietary acrylamide was positively correlated with
HbAA and HbGA in previous studies; however, the results indicate that non-dietary exposure of acrylamide
including tobacco smoke and environmental tobacco smoke (passive smoking) might increase the level of HbAA
and HbGA which induce the significant result in this ­study22. People who smoke tobacco products have 3–4
times higher exposure levels of acrylamide than non-smoker ­people22,27.
There were increases in gluconeogenesis and glycogenolysis as well as reduction in glycolysis in the liver of
rats. Change in gene expression may be responsible for these processes that may be associated with hypoinsu-
linemia and impaired insulin signaling ­pathway28. In addition acrylamide can increase adipocyte differentia-
tion through upregulation of the expression of adiposity-related genes. In vitro and in vivo studies found that
acrylamide exposure in daily life can induce weight gain and promote obesity. These outcomes of acrylamide
can influence different metabolic disease like d ­ iabetes29.
In our study we found that a higher acrylamide intake increased the risk of T2D incidence in women by 13%,
independent of other risk factors of T2D; this association was not observed in men.
The evidence on rats reported sex differences in FBG and insulin levels after AA e­ xposure28. They reported
increase in FBG, decrease in insulin level and pancreatic islet injury under 30 mg/kg body weight of AA exposure
for three weeks in female rats, while similar results were observed after 50 mg/kg body weight AA exposure in
male rats. These differences may be due to higher AA absorption (3.53 fold) in female than in male ­rats28.
Environmental chemicals could affect as antagonists or agonists through estrogenic or androgenic pathways.
This pathway may disorder the number of hormone receptors and disrupt the levels of endogenous hormones.
Dietary exposure of acrylamide was found to be associated with endocrine-related cancers in mammals. Also
acrylamide intake might disorder thyroid homeostasis, altered sex hormone levels in cross-sectional s­ tudies22,23.

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Our study has several strengths. We used large scale prospective association of acrylamide intake with T2D.
Also we used a full range of high quality measurement of outcome biomarkers. The measurement of acrylamide
intake was performed in frequent surveys and cumulatively summed, so changes in the exposure was considered
in determining long time risk of T2D. The use of acrylamide intake is less expensive than acrylamide biomarkers
to be applied as an exposure in large population.
However, some limitations should be regarded when interpreting these results. Although we had adjusted
a range of potential confounders in our study, but some confounding factors including eating habits and food
preparation methods were not estimated and they could not be adjusted in Cox regression models. Acrylamide
exposure from cigarette smoking and occupation were not considered in this study since detailed occupational
histories were not available in this study.

Conclusion
The results of our study demonstrated that dietary intake of acrylamide was associated with an increased risk
of T2D in women of the TLGS cohort. People especially women should be aware of the findings of our study
considering the adverse effect of acrylamide exposure with T2D in their lives.

Materials and methods

Study population. Participants were selected from the Tehran lipid and glucose study (TLGS), an extensive
population-based cohort study performed to find out risk factors for non-communicable diseases in a represent-
ative sample of Tehran, the capital of I­ ran30,31. The first survey was done from 1999 to 2001 on 15,005 individuals
aged ≥ 3 years, using the composite stratified cluster random sampling technique. The follow-up surveys was
done every 3 years; 2002–2005 (survey 2), 2005–2008 (survey 3), 2008–2011 (survey 4), and 2012–2015 (survey
5) and 2015–2018 (survey 6). Of subjects participating in surveys 3 and 4, we randomly selected 8048 individuals
(≥ 18 years) with completed dietary assessment for this secondary analysis. We excluded subjects with under- or
over-reporting of energy intake (< 800 or ≥ 4200 kcal/day)32 (n = 780), then a total of 7268 adult men and women
with available anthropometric, dietary, and biochemical data were followed until survey 6 (participants entering
at surveys 3 and 4 were respectively followed three and two times). Of these individuals, we precluded pregnant
or lactating women and subjects diagnosed with T2D at baseline based on measurement of fasting blood glucose
(FBG) or self-reported taking glucose-lowering d ­ rugs33 (n = 598). Finally, after precluding individuals missing
any follow-up data (n = 498), 6022 participants remained and entered the analysis (Fig. 2).
All of the participants signed the written informed consent. The study was implemented in agreement with
the Declaration of Helsinki rules and the study protocol was approved by the ethical committee of the Research
Institute for Endocrine Sciences, Shahid Beheshti University of Medical Sciences, Tehran, Iran.

Dietary assessment. Skilled nutritionists collected dietary data using a valid and reliable semi-quantitative
food frequency questionnaire (FFQ). They questioned usual dietary intakes based on standard portion sizes dur-
ing the last year through face-to-face personal i­ nterviews34,35. The consumption frequency of each food item was
changed to daily intakes (g/day) using household measures. Due to the uncompleted Iranian food composition
table (FCT), the United States Department of Agriculture (USDA) FCT was used to break down the nutrient
composition of food items (e.g., bread, legume, nuts, white or red meat) not included in the Iranian FCT.
We used the values of acrylamide content reported in the analysis of 30 food items considering cooking
methods and food preparation. The acrylamide content of traditional and industrial breads, bakery products
(cakes and biscuits), confectionary products (pastries and chocolates), snacks (potato-based and corn-based),
coffee powder, roasted nuts (almonds, pistachios, hazelnuts, peanuts and edible seeds), and fast foods (Pizza,
hamburger and sausage) was evaluated. The acrylamide concentration of food was estimated using dispersive
liquid–liquid micro-extraction system coupled with gas-chromatography mass ­spectrometry36–38. The nutrients
and acrylamide content of food items were computed cumulatively across follow up surveys from the baseline
survey until the point of the last follow-up visit or the time that diabetes diagnosed.

Physical activity measurements. An interviewer questioned physical activity levels using Persian-trans-
lated modifiable activity questionnaire (MAQ). Previous study reported high reliability and moderate validity of
this ­questionnaire39. Time and frequency of different intensity activities were collected as stated by routine activ-
ities of daily life over the past year. These data were changed into metabolic equivalent/minutes/week (MET/
min/week)40.

Blood pressure and anthropometric measurements. Skilled staff measured bodyweight using a
digital scale (Seca 707) with a precision of 100 g. Subjects had minimum clothes and were barefoot. Height was
measured by a tape measure with an accuracy of 0.5 cm in the standing position without shoes and straightened
shoulders. Trained staff measured the waist circumference (WC) in the umbilical region after a normal exhala-
tion without pressure on the body surface in the standing position and with the most miniature clothing to the
nearest 0.1 cm.
Qualified physicians evaluated systolic and diastolic blood pressure (SBP, DBP) using a standard mercury
sphygmomanometer. They asked participants to rest for 15 min, and then the physician measured blood pressure
in the sitting position while setting the cuff on the right arm. They repeated this operation twice with an interval
of 30 s. We took the average of two measures in the analysis.

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Total population of TLGS at baseline

The third aged ≥3 years
(2005-2008) 2005–2008 (survey 3), (n=12523)
or fourth 2008–2011(survey 4), (n=12823)
(2008-2011)

Completed dietary assessment at baseline

aged ≥18 years
(n=8048)

6.6 year follow up

Excluding under or over-reporters of energy

intake (<800 or ≥4200 Kcal/day), pregnant
or lactating women (n=7268)

Free of diabetes at
baseline (n = 6627)

The sixth
(2015-
2018) Excluding loss to follow up or
survey of missing data
TLGS (n=6022)

Figure 2.  Outline of study participants’ selection.

Biochemical analysis. Blood samples were collected between 7:00 to 9:00 a.m, after 12–14 h of overnight
fasting. The samples were centrifuged 30 to 45 min after collection. The technician analyzed blood samples using
Selectra 2 auto-analyzer at the TLGS research laboratory on the day of blood collection. They quantified FBG
concentration using enzymatic colorimetric method and glucose oxidase technique (Vital Scientific, Spankeren,
the Netherlands). In follow-up times, they perform the standard 2-h post-challenge blood glucose test using oral
administration of 82.5 g glucose monohydrate solution (equivalent to 75 g anhydrous glucose) for all individuals
who were not on glucose-lowering drugs.
High-density lipoprotein cholesterol (HDL-C) concentration was evaluated after precipitation of the apolipo-
protein B-containing lipoproteins with phosphotungstic acid. Triglyceride (TG) level was assessed by enzymatic
colorimetric tests using glycerol phosphate oxidase (Pars Azmoon Inc., Tehran, Iran). For glucose, inter-and
intra-assay coefficients of variations were both 2.2%. For TG, inter-and intra-assay coefficients of variations were
1.6% and 0.6%, r­ espectively41.

Outcome definition. Incidence of T2D was determined as FBG concentrations ≥ 126 mg/dl or 2-h plasma
glucose concentrations ≥ 200 mg/dl or self-reported taking glucose-lowering drugs (oral diabetes medication or
insulin injections)33.

Statistical analysis. We performed statistical analyses using the Statistical Package for Social Sciences (ver-
sion 21.0; SPSS). A two-sided P value < 0.05 was considered statistically significant. We categorized data into
quartiles of acrylamide intake. Chi-square test for categorical variables and one-way ANOVA for continuous
variables were used to compare the mean and frequency of participants’ baseline characteristics across quartiles
of acrylamide intake. P for trend across acrylamide intake categories was performed by assigning continuous
variables in a linear regression model. The normality of variables was checked by Kolmogorov–Smirnov test.
Percentage of acrylamide from different food groups across quartiles of total acrylamide intake was computed.
Multivariable Cox proportional hazard regression analyses were performed to estimate the hazards ratio (HR)
and 95% confidence interval (CI) of incident T2D. The first quartile was assumed as the reference. The median
of each quartile was utilized as a continuous variable to estimate the P-value of trends across quartiles of acryla-
mide intake in the Cox proportional hazard regression models. The confounders were selected based on litera-

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ture; also, we apply each confounder in the univariable Cox regression model; a two-tailed P-value < 0.20 was
practiced for specifying admission in the model.
Time to event was defined as the time between baseline and the event date (for event cases) or the last follow-
up (for censored participants), whichever occurred first. The event date was defined as the mid-time between the
follow-up visit date at which T2D was detected for the first time and the most recent follow-up visit before the
diagnosis. Study participants were censored due to death, loss to follow-up, or non-occurrence of T2D before
the end of follow-up. The Cox regression models were adjusted for several potential confounders. The analyses
were performed with sex and age adjustment (model 1). Model 2 was adjusted for education levels (> 14 and
≤ 14 years), smoking (non-exposed, passive smoker, and active smoker), BMI (continuous), physical activity
(continuous), family history of type 2 diabetes (yes, no), energy intake (continuous), TG/HDL ratio, plus model
1. Also multivariable Cox proportional hazard regression analyses were performed to estimate the hazards ratio
(HR) and 95% confidence interval (CI) of incident T2D in subgroups of sex, smoking and BMI. The proportional
hazards assumption was verified using the Schoenfeld residuals test and plot of log [− log (survival)] versus log
(time) to see if they are parallel.

Data availability
The datasets generated during and/or analysed during the current study are available from the corresponding
author on reasonable request.

Received: 5 March 2023; Accepted: 18 May 2023

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Author contributions
F.H.-E., N.B., and S.V.-K. collected the data and wrote the first draft of the manuscript with contributions of
G.K. and A.N.; P.M. and F.A. also supervised the study. All authors reviewed and approved the final draft of the
manuscript.

Funding
This work was supported by the Research Institute for Endocrine Sciences, Shahid Beheshti University of Medical
Sciences (Tehran, Iran) under Grant number 29965.

Competing interests
The authors declare no competing interests.

Additional information
Correspondence and requests for materials should be addressed to P.M.
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