Can J Diabetes xxx (2020) 1e6

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Canadian Journal of Diabetes

journal homepage:
www.canadianjournalofdiabetes.com

Original Research

Association Between Severity of Diabetic Ketoacidosis at Diagnosis and

Multiple Autoimmunity in Children With Type 1 Diabetes Mellitus: A
Study From a Greek Tertiary Centre
Kostas Kakleas MD, MRCPH, MSc, PhD a, *; Emre Basatemur MRCPCH, PhD b;
Kyriaki Karavanaki MD, PhD c
a
Paediatric Department, Leicester Royal Infirmary, Leicester, United Kingdom
b
Population, Policy and Practice Programme, Institute of Child Health, University College of London, London, United Kingdom
c
Diabetic Clinic, Second Department of Pediatrics, University of Athens, “P&A Kyriakou” Children’s Hospital, Athens, Greece

Key Messages

Many children with newly diagnosed type 1 diabetes present with ketoacidosis.

Metabolic acidosis promotes inflammation, infection and autoimmunity through its effect on different parts of the immune system.

The concept of disease “endotype” has recently arisen due to the differences in diabetes phenotypes.

Severity of acidosis at diabetes diagnosis could predict children at higher risk of multiple autoimmunity, who may benefit from
closer monitoring.

This association could be attributed either due to effect of acidosis on immune system or to the presence of a more aggressive
endotype.

a r t i c l e i n f o a b s t r a c t

Article history:
Objectives: Type 1 diabetes mellitus is a chronic disorder associated with development of autoimmunity.
Received 28 June 2019
In this work, we studied the relationship between severity of acidosis at diagnosis and future risk for
Received in revised form
5 May 2020 autoimmunity development in children with type 1 diabetes.
Accepted 6 May 2020 Methods: We investigated the presence of associated autoimmunity in 144 children with type 1 diabetes
(mean  standard deviation: age, 12.444.76 years; diabetes duration, 4.413.70 years). We identified
Keywords: the presence of thyroid disease, celiac disease, autoimmune gastritis and adrenal autoimmunity, and
associated autoimmunity retrospectively reviewed the files for presence of diabetic ketoacidosis at diagnosis.
diabetes endotypes Results: Autoimmunity prevalence was 16.7% for thyroid autoimmunity, 9.5% for celiac disease, 5% for
diabetic ketoacidosis gastric autoimmunity and 8.0% for multiple autoimmunities. There were strong associations between
type 1 diabetes mellitus severe acidosis at diabetes diagnosis (pH<7.10) and development of thyroid autoimmunity (odds ratio
[OR], 5.34; 95% confidence interval [CI], 1.90‒15.1; p<0.001), celiac disease (OR, 5.83; 95% CI, 1.19‒28.6;
p¼0.013), gastric autoimmunity (OR, 13.1; 95% CI, 1.22‒140; p¼0.006) and multiple autoimmunity (OR,
26.7; 95% CI, 2.36‒301; p<0.01). The associations persisted after adjustment for sex, age at diabetes
diagnosis, age at assessment, time since diabetes diagnosis and antiglutamic acid decarboxylase auto-
antibody status.
Conclusions: The severity of acidosis at diagnosis is strongly associated with the development of asso-
ciated autoimmune diseases in children with type 1 diabetes and could act as a predictive factor for
multiple autoimmunity development. This association can be either due to effect of acidosis on immune
system or to the presence of a more aggressive diabetes endotype.
Ó 2020 Canadian Diabetes Association.

* Address for correspondence: Kostas Kakleas MD, MRCPH, MSc, PhD, Paediatric Department, Leicester Royal Infirmary, Infirmary Square, Leicester LE1 5WW, United
Kingdom.
E-mail address: koskakl2@yahoo.gr

1499-2671/Ó 2020 Canadian Diabetes Association.

The Canadian Diabetes Association is the registered owner of the name Diabetes Canada.
https://doi.org/10.1016/j.jcjd.2020.05.003
2 K. Kakleas et al. / Can J Diabetes xxx (2020) 1e6

Mots clés: r é s u m é
auto-immunité associée
endotypes de diabète Objectifs : Le diabète sucré de type 1 est une maladie chronique associée au développement d’une auto-
acidocétose diabétique immunité. Dans ces travaux, nous avons étudié la relation entre la gravité de l’acidose lors du diagnostic
diabète sucré de type 1
et le risque de développement futur d’une auto-immunité chez les enfants atteints de diabète de type 1.
Méthodes : Nous avons examiné la présence d’une auto-immunité associée chez 144 enfants atteints du
diabète de type 1 (moyenne  écart type : âge, 12,44  4,76 ans; durée du diabète, 4,41  3,70 ans).
Nous avons relevé la présence de la maladie de la thyroïde, la maladie cœliaque, la gastrite auto-immune
et l’auto-immunité surrénalienne. Nous avons passé en revue de façon rétrospective les dossiers pour
relever la présence d’acidocétose diabétique lors du diagnostic.
Résultats : La prévalence de l’auto-immunité était de 16,7 % pour l’auto-immunité thyroïdienne, de 9,5 %
pour la maladie cœliaque, de 5 % pour l’auto-immunité gastrique et de 8,0 % pour l’auto-immunités
multiple. De fortes associations existaient entre l’acidose grave lors du diagnostic du diabète (pH < 7,10)
et le développement de l’auto-immunité thyroïdienne (ratio d’incidence approché [RIA], 5,34; intervalle
de confiance [IC] à 95 %, 1,90‒15,1; p < 0,001), la maladie cœliaque (RIA, 5,83; IC à 95 %, 1,19‒28,6; p ¼
0,013), l’auto-immunité gastrique (RIA, 13,1; IC à 95 %, 1,22‒140; p ¼ 0,006) et l’auto-immunité multiple
(RIA, 26,7; IC à 95 %, 2,36‒301; p < 0,01). Les associations persistaient après l’ajustement selon le sexe,
l’âge lors du diagnostic du diabète, l’âge lors de l’évaluation, la durée depuis le diagnostic du diabète et le
taux d’anticorps anti-acide glutamique décarboxylase.
Conclusions : La gravité de l’acidose lors du diagnostic est fortement associée au développement de
maladies auto-immunes associées chez les enfants atteints du diabète de type 1 et pourrait être un
facteur de prédiction du développement de l’auto-immunité multiple. Cette association peut être due
soit à la répercussion de l’acidose sur le système immunitaire, soit à la présence d’un endotype plus grave
de diabète.
Ó 2020 Canadian Diabetes Association.

Introduction the Second Department of Pediatrics, University of Athens, “P&A

Type 1 diabetes mellitus (T1D) is a chronic autoimmune disease,
characterized by destruction of insulin-producing pancreatic beta
cells and the presence of autoantibodies against beta cells. This
autoimmune response is not restricted to the pancreas, but also
affects other endocrine (thyroid gland, adrenal tissue) and
nonendocrine (gastrointestinal mucosa) tissues. Associated
autoimmunity can have a major effect on glycemic control by
impairing function of the affected organ.
The most common autoimmune disease among children with
T1D is thyroid autoimmunity with a prevalence of between 15% and
30%, followed by celiac disease (5% to 10%), gastric autoimmunity
(4% to 8%) and adrenal autoimmunity (0.5%) (1). The development
of these autoimmune diseases has been associated with different
factors, such as age at diabetes onset, duration of diabetes, sex and
presence of other autoimmune disorders in the same family (1).
Many children with newly diagnosed T1D present with ketoa-
cidosis (diabetic ketotic acidosis, DKA). In the United States, the
incidence of DKA at presentation was found to be 25%, whereas, in
Europe, reports vary between 26% and 70% (2). Despite better
education of primary and secondary health-care providers with
regard to the early diagnosis and treatment of T1D, the incidence of
DKA remains high. There is also increasing evidence that metabolic
acidosis promotes inflammation, infection and autoimmunity
through its effect on neutrophils, dendritic cells, T cells and other
parts of the immune system (3,4).
In view of the effect of acidosis on immune function, we inves-
tigated whether the severity of DKA at the diagnosis of T1D could act
as a predictor for future development of multiple autoimmunities.
Methods
Study design and population
A nested case‒control study was conducted among children and
adolescents with T1D regularly followed at the Diabetes Clinic of
Kyriakou” Children’s Hospital, Athens, Greece, during the period
2005‒2007. The study protocol was approved by the hospital’s
ethics committee. Informed consent for study participation was
obtained from the legal guardians and/or patients before blood
sampling.
The criteria for the diagnosis of T1D comprised of any one of the
following: a fasting plasma glucose level exceeding 126 mg/dL
(7.0 mmol/L), a random plasma glucose of >200 mg/dL (11.1 mmol/L)
or a 2-hour plasma glucose level of >200 mg/dL (11.1 mmol/L)
during a 75-g oral glucose tolerance test. For patients presenting
with symptoms of hyperglycemia, a single plasma glucose level
meeting these criteria was sufficient, and a confirmatory repeat test
was not required before treatment was initiated. However, for
asymptomatic patients, a repeat laboratory glucose test was
required, and the diagnosis was only confirmed if this result also
met the diagnostic thresholds (5).
The definition of diabetic ketoacidosis was hyperglycemia
(blood glucose >11 mmol/L or 200 mg/dL) with a venous pH<7.30
and/or bicarbonate <15 mmol/L. DKA was categorized according to
severity as mild (venous pH<7.30, bicarbonates <15 mmol/L),
moderate (venous pH<7.20, bicarbonates <10 mmol/L) or severe
(venous pH<7.10, bicarbonates <5 mmol/L) (6).
Measurements
Venous blood was drawn from each patient for autoantibody
estimation, and sera were immediately stored at 20 C. Data
regarding the presence and severity of DKA was determined from
blood-gas analysis at the time of T1D diagnosis, derived from the
patients’ medical records.
The quantitative detection of antibodies against glutamic acid
decarboxylase (anti-GAD) was performed by using the enzyme-
linked immunoassay (ELISA) method on an ELISA analyzer
(DYNEX DSX, Dynex Technologies, Lincoln, United Kingdom). This
method utilizes the 65-kd GAD isoform as the antigen (Euroimmun
AG, Lübeck, Germany). Titres 10 IU/mL were considered positive.
 K. Kakleas et al. / Can J Diabetes xxx (2020) 1e6 3

Diagnosis of celiac disease was based on determination of
antitissue transglutaminase IgA autoantibodies, or, in the case
of patients with immunoglobulin A deficiency, determination of
antigliadin immunoglobulin G antibodies. In all cases, diagnosis
was confirmed by typical findings on jejunal biopsy, including
lymphocytic infiltration and hypertrophy of the crypts and villous
atrophy (Marsh II classification). Antitissue transglutaminase
immunoglobulin A class antibodies were detected using the ELISA
method (DYNEX DSX).
The presence of antithyroglobulin or antithyroid peroxidase
antibodies were considered to be indicative of thyroid autoimmu-
nity. Antithyroglobulin and antithyroid peroxidase antibodies were
determined using the luminescence method (ILMA, Nichols,
Germany) with an Advantage (Nichols) analyzer.
The presence of parietal cell antibodies and antiadrenal anti-
bodies was considered indicative of gastric and adrenal autoim-
munity, respectively. The semiquantitive determination of parietal
cell antibodies and antiadrenal antibodies was performed using an
indirect immunofluorescent assay on a fluorescence microscope
(Model DM LB2, Leica, Wetzlar, Germany). Titres 1:40 for parietal
cell antibodies were considered positive.
Multiple autoimmunity was defined as the presence of 2 or
more of the following associated autoimmune diseases: celiac
disease, thyroid autoimmunity, gastric autoimmunity and adrenal
autoimmunity.
Analysis
Data were summarized using mean and standard deviation if
approximately normally distributed, or median and interquartile
range if not normally distributed. Crude associations between
severity of acidosis at the time of diabetes diagnosis and subse-
quent autoimmunity were assessed using odds ratios and chi-
square tests. Multivariable logistic regression was used to deter-
mine the association between severity of acidosis at diagnosis and
subsequent autoimmunity, while adjusting for sex, age at diabetes
diagnosis, age at assessment and diabetes duration. A second model
development of thyroid autoimmunity, celiac disease, gastric
autoimmunity and multiple autoimmunity (Table 2). However, the
measures of effect were imprecise, with wide confidence intervals.
Unadjusted analyses, with pH grouped into 3 categories, are shown
in Table 3. There was evidence of a biologic gradient in the rela-
tionship between degree of acidosis and risk of thyroid autoim-
munity, but analysis was limited by the study’s sample size.
The associations between severe acidosis at T1D and all of the
study outcomes persisted after adjustment for sex, age at diabetes
diagnosis, age at assessment and diabetes duration (model A,
Table 2). The associations between acidosis and thyroid autoim-
munity, celiac disease and multiple autoimmunity also persisted
after additional control for anti-GAD status (model B, Table 2). The
anti-GAD status could not be included in the multivariable model
with gastric autoimmunity as the outcome, in view of problems
related to perfect prediction resulting from the study size limitation
(all children with gastric autoimmunity had positive anti-GAD
status). Crude and adjusted associations between all exposure
variables included in the models and the outcomes of interest are
shown in Supplementary Tables 1, 2, 3 and 4.
Discussion
Our study is the first in the literature to report a significant
relationship between low pH at diabetes diagnosis and the future
development of thyroid, gastric, celiac and multiple-organ auto-
immunity (2 autoantibodies). The association remained signifi-
cant after adjusting for sex, diabetes duration, anti-GAD status and
age at diabetes diagnosis. Crude analysis demonstrated evidence of
a biologic gradient in the relationship between the degree of
acidosis and risk of thyroid autoimmunity, but it was not possible to
examine for a biologic gradient in multivariable analysis due to the
sample size limitation.
Table 1
Descriptive characteristics of the study population

also controlled for anti-GAD autoantibody status. Variable Value

In view of the sample size limitation and small numbers of Sex* (n¼144)
children with some of the outcomes, pH was categorized into a Male 75 (52.1%)
binary variable representing the presence or absence of severe Female 69 (47.9%)
acidosis (<7.10 or 7.10) for the main analysis. Supplementary Age at T1D diagnosis, years y (n¼143) 8.0 (3.5)
Age at assessment, years y (n¼143) 12.5 (5.0)
analysis was performed with acidosis grouped into 3 categories
Time since T1D diagnosis, years z (n¼143) 3.1 (1.6‒7.2)
(<7.10, 7.10x<7.20, 7.20) to test for a biologic gradient (dose‒ pH at T1D diagnosis * (n¼115)
response relationship) in the unadjusted relationship between 7.30 26 (22.6%)
severity of acidosis and risk of autoimmunity. However, this anal- 7.20x<7.30 24 (20.9%)
ysis was limited by the small number of cases in each exposure 7.10x<7.20 34 (29.6%)
<7.10 31 (27.0%)
category. Analyses were performed using Stata version 14.2 (Sta- Thyroid autoimmunity * (n¼144)
taCorp, College Station, Texas, United States). Yes 24 (16.7%)
No 120 (83.3%)
Results Celiac disease * (n¼105)
Yes 10 (9.5%)
No 95 (90.5%)
Characteristics of the study population, which included 144 Gastric autoimmunity * (n¼101)
children and adolescents (75 boys and 69 girls) with type 1 dia- Yes 5 (5.0%)
betes, are shown in Table 1. The age (mean  standard deviation) of No 96 (95.1%)
patients at assessment was 12.55.0 years, with a mean age at T1D Adrenal autoimmunity * (n¼143)
Yes 0 (0%)
diagnosis of 8.03.5 years, whereas median diabetes duration was No 143 (100%)
3.1 (interquartile range, 1.6e7.2) years. Data regarding pH at Multiple autoimmunity * (n¼100)
diabetes diagnosis were available for 115 children (79.9%). Twenty- Yes 8 (8%)
four children had evidence of thyroid autoimmunity at assessment No 92 (92%)
GAD antibody status (anti-GAD) * (n¼128)
(16.7%), 10 had celiac disease (9.5%) and 5 had evidence of gastric
Positive 68 (53.1%)
autoimmunity (5.0%). None of the children were positive for anti- Negative 60 (46.9%)
adrenal antibodies, whereas 8 (8.0%) had evidence of multiple
GAD, glutamic acid decarboxylase; T1D, type 1 diabetes.
autoimmunity. * Data expressed as number (%).
In the unadjusted analyses, there were strong crude associations y
Data expressed as mean (standard deviation).
z
between severe acidosis at T1D diagnosis and subsequent Data expressed as median (interquartile range).
4 K. Kakleas et al. / Can J Diabetes xxx (2020) 1e6

Table 2
Associations between severe acidosis (pH<7.10) at T1D diagnosis and subsequent development of autoimmunity: Crude and multivariable analyses

Outcome pH at diabetes diagnosis Crude analyses Multivariable analyses

y z x
<7.10, n (%) 7.10, n (%) Crude OR * (95% CI) p Value Model A Model B
{ {
Adjusted OR * (95% CI) p Value Adjusted OR * (95% CI) p Value

Thyroid autoimmunity (n¼115) 5.34 (1.90‒15.1) <0.001 3.98 (1.32‒11.8) 0.013 3.26 (1.02‒10.4) 0.048
Yes 13 (41.9%) 10 (11.9%)
No 18 (58.1%) 74 (88.1%)
Celiac disease (n¼89) 5.83 (1.19‒28.6) 0.014 6.25 (1.15‒34.1) 0.033 5.89 (1.08‒32.3) 0.039
Yes 5 (21.7%) 3 (4.6%)
No 18 (78.3%) 63 (95.5%)
k k
Gastric autoimmunity (n¼86) 13.1 (1.22‒140) 0.006 15.3 (1.43‒164) 0.009 — —
Yes 4 (17.4%) 1 (1.6%)
No 19 (82.6%) 62 (98.4%)
Multiple autoimmunity (n¼85) 26.7 (2.36‒301) <0.001 27.6 (2.84‒267) <0.001 21.7 (2.20‒214) 0.001
Yes 7 (30.4%) 1 (1.6%)
No 16 (69.6%) 61 (98.4%)

CI, confidence interval; OR, odds ratio; T1D, type 1 diabetes.

* ORs comparing outcomes in children with severe acidosis at diabetes diagnosis (pH<7.10) vs children without severe acidosis at diabetes diagnosis (pH7.10).
y
Crude p values obtained using chi-square test.
z
Results from multivariable logistic regression models adjusting for sex, age at diabetes diagnosis, age at assessment and time since diabetes diagnosis at assessment.
x
Results from multivariable logistic regression models adjusting for sex, age at diabetes diagnosis, age at assessment and time since diabetes diagnosis at assessment, and
also controlling for antiglutamic acid decarboxylase (anti-GAD) autoantibody status.
{
Adjusted p values for the effect of pH category, obtained from likelihood-ratio tests comparing nested models with and without pH category.
k
Anti-GAD status could not be included in the model with gastric autoimmunity as the outcome, due to perfect prediction (all children with the outcome were anti‒GAD-
positive).

One possible explanation for the observed associations may be
the effect of pH on protein structure and function. Acidic pH has
been shown to contribute to the pathogenesis of cystic fibrosis and
the development of neoplastic conditions and multiple sclerosis
(7e10). The pathophysiologic mechanism could be explained by the
fact that pH changes the shape and the conformation of proteins
(11). Low pH promotes protein unfolding and causes easier heat
denaturation (12). The unfolding of proteins has been implicated in
the development of amyloidosis (13). In addition, change in the pH
can result in impaired enzymatic activity and deregulation of
normal cell metabolic pathways. This has been demonstrated in
studies where the presence of metabolic acidosis was associated
with reduced free T3 and T4 production and suppressed insulin-
mediated glucose metabolism (14). Based on the previous
hypothesis, metabolic acidosis could result in unfolding of the
protein located on the surface of the thyroid gland, intestine and/or
stomach; change its conformation; and reveal amino acid
sequences (antigens) that are recognized as foreign by the
organism, thus causing the production of antibodies against them.
This process may take a few years, but eventually may lead to the
development of other autoimmune diseases.
The association between DKA severity and multiple autoim-
munity could also be attributed to the effect of pH on the
function of the immune system (15). Acidosis promotes syn-
thesis and antigen-binding ability of antibodies, as well as sur-
vival, and phagocytosis of neutrophils (16,17). It also enhances
activation of the complement system and increases the pro-
duction of proinflammatory cytokines, nitric oxide and tumor
necrosis factor (18,19). Furthermore, the presence of extracellular
acidosis was found to increase the production of interleukin-6, a
proinflammatory cytokine, and reduce the production of
interleukin-10, a cytokine with anti-inflammatory proprieties
(20,21). An acidic extracellular environment has also been
shown to alter T-cell metabolism via regulation by the T-regu-
latory cell transcription factor FOXP3, and increase T-cell motility
(22e24).

Table 3
Crude associations between pH at T1D diagnosis and subsequent development of autoimmunity, with pH grouped into 3 categories
y
pH at diabetes diagnosis Outcome Crude OR (95% CI) P Value * P Value for linear trend

Yes, n (%) No, n (%)

Thyroid autoimmunity ` (n¼115) <0.001

7.20 4 (8.0%) 46 (92.0%) Baseline Baseline
7.10x<7.20 6 (17.7%) 28 (82.4%) 2.46 (0.62‒9.72) 0.183
<7.10 13 (41.9%) 18 (58.1%) 8.31 (2.11‒32.7) <0.001
Celiac disease (n¼89) 0.052
7.20 2 (5.4%) 35 (94.6%) Baseline Baseline
7.10x<7.20 1 (3.5%) 28 (96.6%) 0.63 (0.05‒7.41) 0.707
<7.10 5 (21.7%) 18 (78.3%) 4.86 (0.80‒29.6) 0.057
Gastric autoimmunity (n¼86) 0.008
7.20 0 (0%) 34 (100%) Baseline Baseline
7.10x<7.20 1 (3.5%) 28 (96.6%) —z —z
<7.10 4 (17.4%) 19 (82.6%) —z —z
Multiple autoimmunity (n¼85) 0.001
7.20 0 (0%) 33 (100%) Baseline Baseline
7.10x<7.20 1 (3.5%) 28 (96.6%) —z —z
<7.10 7 (30.4%) 16 (69.6%) —z —z

CI, confidence interval; OR, odds ratio; T1D, type 1 diabetes.

* p Value obtained using chi-square test.
y
p Value obtained using chi-square test for linear trend.
z
ORs could not be derived for gastric autoimmunity and multiple autoimmunity, as there were no positive cases in the baseline pH category (7.20).
 K. Kakleas et al. / Can J Diabetes xxx (2020) 1e6
Low pH interacts with the immune system through the presence
of acid-sensing ion channels on immune system cells. Acid-sensing
ion channels are expressed by dendritic cells, transport Naþ
through the cell membrane, are very sensitive to tissue acidosis and
are activated by low pH (25e27). Furthermore, extracellular
acidosis promotes the maturation of dendritic cells, increases their
antigen-presenting capacity and enhances the production of
interleukin-12, thus favouring the development of T-helper 1 cell
immunity (28,29). This deregulation of the immune system pro-
moted by acidic pH could eventually result in predisposition for the
future development of multiple autoimmunity.
On the other hand, the presence of multiple autoimmunity in
children with severe DKA at diabetes diagnosis could simply reflect
a more aggressive autoimmune endotype that manifests as both
multiple autoimmunity and as extent of beta-cell destruction (30).
The concept of disease “endotype” arose from the heterogeneity in
the genetic susceptibility to T1D, the different age at presentation
and disease progression rate, the presence of different autoanti-
bodies and variety of complications and the response to the treat-
ment (31). DKA at T1D diagnosis has been shown to be associated
with a more aggressive disease subtype (32). In a study of adoles-
cents with T1D who received hematopoietic stem cell trans-
plantation, those without DKA at T1D diagnosis required lower
doses of insulin and had a reduced rate of remaining b-cell
destruction posttreatment, whereas those with DKA at diagnosis
responded slower to transplantation and required a higher insulin
dose at 1 year post-transplantation (32,33).
The association between severe DKA and presence of multiple
autoimmunity persisted after adjusting for sex, age at diabetes
diagnosis, age at assessment, and T1D duration, which could act as
confounding factors. This is important as other studies have
revealed that the development of autoimmunity is associated with
female sex, increased diabetes duration and younger age at dia-
betes diagnosis (34,35). In addition, younger age at diabetes diag-
nosis is associated with lower number of insulin-containing islets,
higher frequency of DKA (particularly among children <2 years
old), higher overall number of autoantibodies and increased fre-
quency of complications and mortality (36,37). Sex, however, is not
associated with presence of DKA at diagnosis (38).
In our study, the association between severity of DKA and
multiple autoimmunity remained after controlling for the anti-GAD
status. The presence of anti-GAD has been associated with gastric
and thyroid autoimmunity, and has been proposed as a predictor
for the development of additional autoimmunity in adolescents
with T1D (39). To our knowledge, no study in the literature has
reported an association between anti-GAD positivity and DKA at
diagnosis; however, a recent study has identified presence of IA-2A
antibodies as a risk factor for development of DKA (40).
Several studies in the literature have identified that specific
haplotypes with high-risk human leukocyte antigen-DR and -DQ
genes are associated with a more severe presentation and rapid
progression of T1D (41,42). On the other hand, the TCF7L2 poly-
morphism was associated with a milder immunologic phenotype
(43). The effect of genetics in the development of DKA has been
shown to vary between studies and both high-risk and protective
haplotypes have been identified (44,45). In our study, we did not
perform genetic analysis, which is one of its limitations, as the
presence of a specific genotype could have predisposed our
patients to a more severe presentation at diagnosis and rapid
progression to multiple autoimmunity.
Our study has some additional limitations. There was only 1
pancreatic autoantibody available for study (anti-GAD). The sample
size and small number of children with the outcomes of interest
limits the power of the study. As a result, the measures of effect are
imprecise, with wide confidence intervals. The sample size limita-
tions also prevented pH from being fully explored as an ordinal
5
rather than binary variable. Thus, it was not possible to examine for
a biologic gradient between degree of acidosis and risk of auto-
immunity in the adjusted multivariable analyses. Our findings may
be incidental and do not represent a true association between
severe ketoacidosis and future development of autoimmunity. The
role of pH on the function and regulation of the immune system is
still under investigation and the exact mechanisms have not been
fully clarified. In addition, the newly introduced concept of T1D
endotypes raises the question of whether future development of
multiple autoimmunity in children with severe ketoacidosis
reflects their increased genetic susceptibility to develop more
severe autoimmune disease, rather than the predictive role of pH in
the development of autoimmunity.
If there is a true causal association between the severity of
acidosis at diabetes diagnosis and future development of multiple
autoimmunity, then early diagnosis of T1D before the development
of severe DKA could potentially reduce the risk of development of
multiple autoimmunity. It could also help to identify children at
higher risk of multiple autoimmunity, who may benefit from closer
monitoring.
Conclusions
Our study is the first study in the literature to show a positive
association between severity of ketoacidosis at diabetes diagnosis
and future development of multiple autoimmunity in children and
adolescents with T1D. In view of the limited sample size and
imprecise effect estimates, the results of this study should be
considered to be hypothesis-generating, and larger studies will be
needed to confirm the observed associations. Future research
should also address the potential role of pH in immune system
function, as well as characterize the specific T1D endotypes with
rapid and more severe disease progression that are associated with
severe acidosis and multiple autoimmunity.
Supplementary Material
To access the supplementary material accompanying this article,
visit the online version of the Canadian Journal of Diabetes at www.
canadianjournalofdiabetes.com.
Author Disclosures
Conflicts of interest: None.
Author Contributions
K.Kak. conceived and designed the study, drafted the manu-
script and completed the corrections. E.B. performed the statistical
analysis and interpretation of the data and reviewed the manu-
script. K.Kar. critically reviewed the manuscript. All authors
participated in final approval of the submitted version.
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Supplementary Table 1
Associations between all study covariates and thyroid autoimmunity

Exposure variable Crude OR for thyroid autoimmunity (95% CI) p Value * Adjusted OR for thyroid autoimmunity y (95% CI) p Value z

pH at T1D diagnosis <0.001 0.048

7.10 Baseline Baseline
<7.10 5.34 (1.90e15.1) 3.26 (1.02e10.4)
Sex 0.824 0.419
Male Baseline Baseline
Female 1.11 (0.46e2.66) 1.60 (0.51e5.08)
Age at T1D diagnosis, per-year increase 0.97 (0.85e1.10) 0.605 0.61 (0.34e1.11) 0.107
Age at assessment, per-year increase 1.12 (1.03e1.23) 0.010 1.60 (0.90e2.87) 0.111
Time since T1D diagnosis, per-year increase 1.12 (1.01e1.25) 0.030 0.71 (0.39e1.28) 0.251
Anti-GAD antibody status 0.280 0.218
Negative Baseline Baseline
Positive 1.68 (0.65e4.39) 2.14 (0.64e7.18)

CI, confidence interval; GAD, glutamic acid decarboxylase; OR, odds ratio; T1D, type 1 diabetes.
* p Values obtained from chi-square tests for categorical exposure variables, and from logistic regression for continuous exposure variables.
y
Results from multivariable logistic regression model adjusting for listed exposure variables (N¼103).
z
p Values obtained from likelihood-ratio tests comparing the model with vs without the variable of interest.

Supplementary Table 2
Associations between all study covariates and celiac disease

Exposure variable Crude OR for celiac disease (95% CI) p Value * Adjusted OR for celiac disease y (95% CI) p Value z

pH at T1D diagnosis 0.014 0.039

7.10 Baseline Baseline
<7.10 5.83 (1.19e28.6) 5.89 (1.08e32.3)
Sex 0.925 0.485
Male Baseline Baseline
Female 1.07 (0.29e3.95) 0.54 (0.10e3.10)
Age at T1D diagnosis, per-year increase 1.13 (0.92e1.38) 0.255 0.95 (0.63e1.42) 0.787
Age at assessment, per-year increase 1.01 (0.88e1.16) 0.850 1.19 (0.86e1.65) 0.291
Time since T1D diagnosis, per-year increase 0.77 (0.57e1.05) 0.098 0.65 (0.38e1.09) 0.104
Anti-GAD antibody status 0.974 0.779
Negative Baseline Baseline
Positive 0.98 (0.26e3.63) 1.28 (0.23e7.12)

CI, confidence interval; GAD, glutamic acid decarboxylase; OR, odds ratio; T1D, type 1 diabetes.
* p Values obtained from chi-squared tests for categorical exposure variables, and from logistic regression for continuous exposure variables.
y
Results from multivariable logistic regression model adjusting for listed exposure variables (N¼86).
z
p Values obtained from likelihood-ratio tests comparing the model with vs without the variable of interest.
6.e2 K. Kakleas et al. / Can J Diabetes xxx (2020) 1e6

Supplementary Table 3
Associations between all study covariates and gastric autoimmunity

Exposure variable Crude OR for gastric autoimmunity (95% CI) p Value * Adjusted OR for gastric autoimmunity y (95% CI) p Value z

pH at T1D diagnosis 0.006 0.009

7.10 Baseline Baseline
<7.10 13.1 (1.22e139) 15.3 (1.43e164)
Sex 0.664 0.576
Male Baseline Baseline
Female 0.67 (0.11e4.22) 0.57 (0.07e4.29)
Age at T1D diagnosis, per-year increase 0.93 (0.71e1.20) 0.561 1.04 (0.60e1.79) 0.901
Age at assessment, per-year increase 1.05 (0.88e1.26) 0.599 0.88 (0.53e1.44) 0.601
Time since T1D diagnosis, per-year increase 1.23 (0.92e1.38) 0.256 1.32 (0.78e2.23) 0.302
Anti-GAD antibody status —x —x
Negative Baseline Baseline
Positive —x —x

CI, confidence interval; GAD, glutamic acid decarboxylase; OR, odds ratio; T1D, type 1 diabetes.
* p Values obtained from chi-square tests for categorical exposure variables, and from logistic regression for continuous exposure variables.
y
Results from multivariable logistic regression model adjusting for listed exposure variables (N¼86).
z
p Values obtained from likelihood-ratio tests comparing the model with vs without the variable of interest.
x
ORs could not be generated for anti-GAD status as the exposure variable due to perfect prediction (all children who had gastric autoimmunity were anti-GAD-positive).
Thus, anti-GAD status was not included in the multivariable model.

Supplementary Table 4
Associations between all study covariates and multiple autoimmunity

Exposure variable Crude OR for multiple autoimmunity (95% CI) p Value * Adjusted OR for multiple autoimmunity y (95% CI) p Value z

pH at T1D diagnosis <0.001 0.001

7.10 Baseline Baseline
<7.10 26.7 (2.36e302) 21.7 (2.20e214)
Sex 0.500 0.243
Male Baseline Baseline
Female 0.60 (0.13e2.69) 0.34 (0.05e2.18)
Age at T1D diagnosis, per-year increase 0.89 (0.72e1.10) 0.282 0.69 (0.43e1.09) 0.115
Age at assessment, per-year increase 1.03 (0.89e1.20) 0.677 1.25 (0.87e1.81) 0.227
Time since T1D diagnosis, per-year increase 1.05 (0.88e1.26) 0.602 0.84 (0.55e1.29) 0.428
Anti-GAD antibody status 0.159 0.196
Negative Baseline Baseline
Positive 3.14 (0.58e16.9) 4.90 (0.44e54.4)

CI, confidence interval; GAD, glutamic acid decarboxylase; OR, odds ratio; T1D, type 1 diabetes.
* p Values obtained from chi-square tests for categorical exposure variables, and from logistic regression for continuous exposure variables.
y
Results from multivariable logistic regression model adjusting for listed exposure variables (N¼86).
z
p Values obtained from likelihood-ratio tests comparing the model with vs without the variable of interest.