A dysregulation in the metabolism of lipids may be an early marker of autoimmunity in Type 1 Diabetes (T1D). It would be of general importance to identify metabolic patterns that would predict the risk for T1D later in life. The aim of this study was to perform a prospective evaluation of glutamine and phospholipids levels in Brazilian first degree relatives (FDR) of patients with T1D in a mean interval of 5 years

A. Milech

B. Barone

D.B. Araujo de Pina Cabral

F. Carvalho

F. Vaziri-Sani

H. Skärstrand

J.E. P. Oliveira

J.R. Dantas

L. Zajdenverg

M. Rodacki

R. Tortora

Diabetology & Metabolic Syndrome

PubMed

Araujo de Pina Cabral, D B

Dantas, J R

Skärstrand, Hanna

Barone, B

Carvalho, F

Tortora, R

Milech, A

Vaziri Sani, Fariba

Oliveira, J E P

Zajdenverg, L

Rodacki, M

Lund University Publications

Prospective evaluation of glutamine and phospholipids levels in first degree relatives of patients with Type 1 Diabetes from a multiethnic population.

Springer - Publisher Connector

Prospective evaluation of glutamine and phospholipids levels in first degree relatives of patients with Type 1 Diabetes from a multiethnic population

METABOLIC SYNDROME
DIABETOLOGY & 
Araujo de Pina Cabral et al. Diabetology & Metabolic Syndrome  (2015) 7:52 
DOI 10.1186/s13098-015-0048-xSHORT REPORT Open AccessProspective evaluation of glutamine and
phospholipids levels in first degree relatives
of patients with Type 1 Diabetes from a
multiethnic population
D.B. Araujo de Pina Cabral1*, J.R. Dantas1, H. Skärstrand2, B. Barone1, F. Carvalho3, R. Tortora3, A. Milech1,
F. Vaziri-Sani2, J.E. P. Oliveira1, L. Zajdenverg1 and M. Rodacki1Abstract
Background: A dysregulation in the metabolism of lipids may be an early marker of autoimmunity in Type 1
Diabetes (T1D). It would be of general importance to identify metabolic patterns that would predict the risk for T1D
later in life. The aim of this study was to perform a prospective evaluation of glutamine and phospholipids levels in
Brazilian first degree relatives (FDR) of patients with T1D in a mean interval of 5 years.
Findings: Brazilian FDR (n = 30) of patients with T1D were evaluated and blood was sampled to measure the levels of
glutamine and phospholipids in the fasting serum by quantitative colorimetric method. The tests were repeated after a
mean interval of 5 years and compared to a control group (n = 20). The FDR presented lower levels of phospholipids
than controls (p = 0.028), but not of glutamine (p = 0.075). Phospholipids levels decreased over time (p = 0.028) in FDR
and were associated with Glutamic acid decarboxylase autoantibody (GADA) titers (p = 0.045), autoantibody positivity
(p = 0.008) and PTPN22 polymorphisms (p = 0.014).
Conclusions: In this Brazilian multiethnic population, there was a significant decrease in phospholipids levels in FDR in
patients with T1D during a 5-year prospective follow-up, as well as a significant association between these metabolite,
GADA and PTPN22 polymorphisms. For Glutamine no difference was found. These findings suggest that a dysregulation
in the metabolism of lipids may precede the onset of the autoimmunity in T1D.
Keywords: Metabolic, Diabetes, Non-whites, Antibodies, MultiethnicIntroduction
The autoimmune process in Type 1 Diabetes (T1D) may
be a late response to a dysregulation in the metabolism
of lipids and amino acids [1–4]. The DIPP (Diabetes
Prediction and Prevention Project) and BABYDIAB (a
German prospective study from birth on the natural
history of type 1 diabetes) studies found reduced levels of
methionine and glutamine before islet autoanibody
seroconversion in individuals that developed T1D later in
life. Oresic et al. also demonstrated decreased levels of* Correspondence: dbatistaaraujo@gmail.com
1Department of Nutrology, Federal University of Rio de Janeiro, Avenida
Epitácio Pessoa, número 2.990, apto 806, Lagoa, Rio de Janeiro, Brazil
Full list of author information is available at the end of the article
© 2015 Araujo de Pina Cabral et al. This is an
Attribution License (http://creativecommons.o
reproduction in any medium, provided the or
Dedication waiver (http://creativecommons.or
unless otherwise stated.phospholipids and triglycerides in the pre-clinical period
of T1D [2, 3].
The amino acids and lipids play an important role in
regulating β cells insulin secretion. Glutamine, the most
abundant amino acid in blood, presents antioxidant
properties, protecting β cells from oxidative insults.
Besides, phospholipids may also be a key modulator of
insulin release. Therefore, it would be of general importance
to identify metabolic patterns that would predict the risk
for T1D later in life [4–6].
As most investigations in this field have included only
Caucasians, very little is known about the natural history
of T1D in other ethnic groups, such as the Brazilian
population. It is possible that different populations present
peculiarities in the development of T1D. Therefore, it isOpen Access article distributed under the terms of the Creative Commons
rg/licenses/by/4.0), which permits unrestricted use, distribution, and
iginal work is properly credited. The Creative Commons Public Domain
g/publicdomain/zero/1.0/) applies to the data made available in this article,
Araujo de Pina Cabral et al. Diabetology & Metabolic Syndrome  (2015) 7:52 Page 2 of 4important to identify if the same metabolic alterations
occur in non-Caucasian individuals. This was the first
study that analyzed the metabolic profile of FDR of
patients with T1D in a multiethnic population.
We aimed to perform a prospective evaluation of
glutamine and phospholipids levels in Brazilian FDR
of patients with T1D in a mean interval of 5 years.Methods
Subjects
First degree relatives (FDR) of Brazilian patients with
T1D (n = 30, baseline sample) were interviewed and
blood was sampled for DNA extraction, metabolites and
autoantibody measurement. Participants were classified
as whites or non-whites (mostly Afro-descendants) based
on their phenotype and family background [7]. The results
from metabolites were compared to non-diabetic controls
(n = 20).Measurement of metabolites
The quantitative colorimetric assays for glutamine and
phospholipids measurement were performed at the
Federal University of Rio de Janeiro, Rio de Janeiro,
Brazil. The metabolites were measured in fasting serum of
FDR and in control group by quantitative colorimetric
method (EnzyChrom Assay Kit of Bioassay Systems).
Since the reference range for these tests have not been
defined, the frequency of individuals above and below the
50th percentile (p50) obtained in the healthy controls was
investigated.Measurement of autoantibodies
Antibodies against GAD, insulin and insulinoma-associated
antigen-2 (GADA, IAA and IA2A, respectively) were mea-
sured by radioimmunoassay (RIA) [8–11].
Serum samples were also analyzed by a standard radioli-
gand binding assay (RBA) for the three individual ZnT8A
variants (ZnT8RA, ZnT8WA, ZnT8QA) as well as with the
ZnT8TripleA assay, as previously reported [8, 11].Genetic analysis
The FDR were genotyped for INS gene and PTPN22
polymorphisms. SNP for INS and PTPN22 (polymorph-
ism RS620W rs2476601) were genotyped with fluorogenic
allele-discrimination chemistry [12–14].Prospective evaluation
After 5 years (a mean interval of 3.8 to 6.3 years), the
measurement of glutamine, phospholipids and autoanti-
bodies was repeated. The same technique described above
was used.Statistical analysis
Mann–Whitney U test and chi-square were used for
comparison between groups. Spearman coefficient was
used to test correlation between continuous variables, and
Fisher’s exact t-test to test correlation between categorical
variables. A p-value <0.05 was considered significant.
The software applied was SPSS for Windows, version 17.0
(SPSS Inc.)
Results
The general characteristics of the sample are summarized
in Table 1.
Glutamine levels
The mean levels of glutamine found in FDR and controls
are shown in Table 1. There was no significant variation
in the levels of this metabolite in a period of 5 years
(p = 0.075) (Fig. 1).
The majority of the FDR presented glutamine levels
below the 50th percentile in relation to the median of
the control group (23 % or 71.9 % in the baseline sample
and 26 % or 86.7 % in the prospective sample), but
the difference between these groups was not significant
(p = 0.075). There was no association between glutamine
levels and positive titers of antibodies (p = 1.000).
Phospholipids levels
The mean levels of phospholipids found in FDR and
controls are shown in Table 1. There was a significant
variation in the levels of this metabolite in a period of
5 years (p = 0.028) (Fig. 2). The majority of the FDR
showed phospholipids levels below the 50th percentile
in relation to the median of the control group (21 % or
65.6 % in the baseline sample and 24 % or 80 % in the
prospective sample), with a significant difference between
these groups (p = 0.028). There was a significant association
between low phospholipids levels, positive titers of GADA
(p = 0.045) in FDR at baseline, and between positivity
for autoantibody (GADA) (p = 0.008) in the prospective
evaluation.
Genetic polymorphisms
For PTPN22, the prevalence of homozygous was 93.3 %
and heterozygous was 6.7 %. None of the FDR pre-
sented homozygous T/T for rs24766001 polymorphism of
PTPN22.
In the prospective evaluation, there was a significant
association between PTPN22 polymorphisms and low
phospholipids levels (p = 0.014).
The prevalence of rs689 polymorphism of the insulin
gene was: homozygous at 43.3 %, heterozygous 40 % or
homozygous at 16.7 % . There were no significant differ-
ences between polymorphisms of the insulin gene and
autoantibodies or metabolites.
Table 1 General characteristics of the study sample
Basal Prospective Controls p
valueFDR FDR
(n = 30) (n = 30) (n = 20)
Age (years) 17.96 17.36 25.60 0.108
(mean ± SD) ±9.24 ±8.68 ±9.65
Gender [n (%)]
Female 15 (50) 15 (50) 9 (45) 0.184
Etnicity[n (%)]
Whites 15 (50) 15 (50) 8 (40) 0.272
Non-whites 15 (50) 15 (50) 12 (60)
GADA [n (%)] 2 (7) 2 (7) 2 (10) 0.887
IA2A [n (%)] 2 (7) 1 (3) 0 ——
IAA [n (%)] 0 1 (3) 0 ——
ZnT8A [n (%)] 1 (3) 1 (3) 0 0.002
Glutamine (mM) (mean ± SD) 1.45 ± 0.69 1.34 ± 0.20 1.55 ± 0.27 0.075
Phospholipids (μm) (mean ± SD) 505.20 ± 194.89 468.56 ± 219.46 640.00 ± 259.20 0.028
Basal FDR = sample of the basal period; Prospective FDR = sample of the prospective period
Controls = non-diabetic individuals; SD = standard deviation; GADA = Glutamic acid decarboxylase autoantibody; IA2A = Insulinoma-associated antigen-2
autoantibody; IAA = autoantibody against insulin; ZnT8A = Zinc transporter 8 autoantibody
Araujo de Pina Cabral et al. Diabetology & Metabolic Syndrome  (2015) 7:52 Page 3 of 4Discussion and conclusions
In this study we found a significant decrease in phos-
pholipids levels in FDR of patients with T1D during a
5-year prospective follow-up, as well as a significant
association between low levels of this metabolite,
GADA and PTPN22 polymorphisms. In this study
population, most of the FDR presented lower levels of
phospholipids in comparison to the control group, similar
to the findings in the BABYDIAB cohort. Although the
majority of FDR had low glutamine levels, there was no sig-
nificant difference in comparison with controls. A positiveFig. 1 Glutamine levels (mM) in basal sample, prospective sample
and controls. The Glutamine levels did not differ between these three
groups (p = 0.075)correlation between phospholipids levels, positivity for
autoantibody and titers of GADA was found. The altered
levels of glutamine, phospholipids, and other metabolites in
children at high risk for T1D, before the seroconversion of
pancreatic autoantibodies have been repported [2, 3]. This
dysregulation in the metabolism of lipids may be an early
marker of autoimmunity, but it still remains unclear if
these individuals with low phospholipids levels will have a
higher risk of developing T1D [4–6].
The presence of the polymorphic C allele was strongly
associated with low levels of phospholipids. PreviousFig. 2 Phospholipids levels (μm) in basal sample, prospective sample
and controls. The prospective group showed diminished levels of
phospholipids in relation to the basal samples and controls (p = 0.028)
Araujo de Pina Cabral et al. Diabetology & Metabolic Syndrome  (2015) 7:52 Page 4 of 4studies have shown that polymorphisms of this gene
increases the risk of autoimmune diseases emergence by
acting in the immunoregulation [12–20]. To our know-
ledge, the association between PTPN22 polymorphisms
and metabolites has not been reported in the literature.
This finding may represent a peculiarity of our multiethnic
population and further studies in other ethnic groups are
necessary.
To conclude, our study found reduced levels of metabo-
lites in FDR of T1D patients, and a positive association
between phospholipids, GADA and PTPN22. Some of
these results may represent uniqueness of our multiethnic
population, while others peculiarities may be common to
other populations with T1D. These findings could support
further understanding of the pathogenesis of T1D, as well
as in stratification of individuals at risk of developing T1D
and, contributing to prevention strategies.
Abbreviations
GADA: Glutamic acid decarboxylase autoantibody; IA2A: Islet antigen-2
autoantibody; INS: Insulin; FDR: First degree relatives; T1D: Type 1 Diabetes;
ZnT8RA: Arginine 325 Zinc transporter 8 autoantibody; ZnT8WA: Tryptophan
325 Zinc transporter 8 autoantibody; ZnT8QA: Glutamine 325 Zinc
transporter 8 autoantibody; ZnT8A: Zinc transporter 8 autoantibody to
one, two or all three amino acid variants.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
DB, JR and HS researched the data, analyzed and wrote the manuscript.
BB, FC and RT recruited the patients and researched the data. AM, FV-Z,
JEP, LZ and MR reviewed and edited the manuscript. All authors read and
approved the final manuscript.
Acknowledgements
We thank the participants and their families in this study as well as the
nursing and medical staff for their help.
Author details
1Department of Nutrology, Federal University of Rio de Janeiro, Avenida
Epitácio Pessoa, número 2.990, apto 806, Lagoa, Rio de Janeiro, Brazil.
2Department of Clinical Sciences, Lund University, Skåne University Hospital
(SUS), Malmö, Sweden. 3Biochemistry Laboratory, Federal University of Rio de
Janeiro, Rio de Janeiro, Brazil.
Received: 26 February 2015 Accepted: 29 May 2015
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Prospective evaluation of glutamine and phospholipids levels in first degree relatives of patients with Type 1 Diabetes from a multiethnic population

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