Article 433

Authors

R. Lehmann1, 2, 3, T. Friedrich4, 7, G. Krebiehl4, D. Sonntag4, H.-U. Häring3, 5, 6, A. Fritsche3, 5, 6, A. M. Hennige3, 6

Affiliations

Affiliation addresses are listed at the end of the article

Key words ▶ GDM ● ▶ glucose tolerance test ● ▶ pregnancy ● ▶ targeted metabolomics ● ▶ metabolites ● ▶ insulin resistance ●

Abstract

received 08.01.2015 first decision 30.03.2015 accepted 10.04.2015 Bibliography DOI http://dx.doi.org/ 10.1055/s-0035-1549887 Exp Clin Endocrinol Diabetes 2015; 123: 433–438 © J. A. Barth Verlag in Georg Thieme Verlag KG Stuttgart · New York ISSN 0947-7349 Correspondence Dr. Anita M. Hennige Institute for Diabetes Research and Metabolic Diseases of the Helmholtz Zentrum München at the University of Tuebingen Otfried-Mueller-Str. 10 D-72076 Tuebingen Germany Tel.:  + 49/7071/29 83193 Fax:  + 49/7071/29 5348 [email protected]



Background/Aim:  Gestational diabetes (GDM) is a complex metabolic condition associated with hyperpglycemia that is diagnosed in an oral glucose tolerance test (OGTT) during pregnancy. For a deeper understanding of the pathology of the disease, further investigations during pregnancy are required, ideally under metabolic challenging conditions. Methods:  We performed targeted metabolomics in a group of 24 well-matched women during an oral glucose tolerance test (OGTT). 231 plasma metabolites were profiled and compared to conventional clinical diagnostics.

Introduction



Gestational diabetes mellitus (GDM) is defined as glucose intolerance in screening measures during pregnancy [1]. Approximately 15 % of pregnant women display glucose concentrations that exceed high cut-offs in an oral glucose tolerance test (OGTT) [2, 3]. GDM might lead to fetal complications including macrosomia, shoulder dystocia, neonatal hypoglycemia, and increased risk for caesarean delivery [4]. Among the adverse outcomes for the mother are hypertension, eclampsia and subsequent type 2 diabetes [5]. Approaches for screening include fasting glucose, random blood glucose and/or a glucose challenge in an 1 or 2 step oral glucose tolerance test [1, 2, 6]. Currently, all of these diagnostic tools are used in parallel for the diagnosis of GDM [3], and the diagnosis of gestational diabetes is solely dependent on elevated blood glucose levels [5, 7]. However, the underlying causes and consequences of this complex metabolic condition are far beyond glucose [8]. Especially the underlying metabolic pathophysiology in women during pregnancy that potentially sustain postpartum

Results:  A pattern of 8 metabolites differed between GDM and healthy controls as early as 30 min in an OGTT (AUC 0.977 ± 0.008), and an increase in acylcarnitine C18:0, decreased concentrations of diacyl phosphatidylcholines (PC aa) C34:4, PC aa C36:4, PC aa C38:5, Lyso PC C20:4 and arachidonic acid were associated with insulin resistance. Conclusion:  Our data suggest an additional value of metabolite pattern in the diagnosis of GDM and describe altered pathways that might be subjected to a more precise diagnosis and individualized therapy.

and increase their risk for type 2 diabetes is not well characterized [9]. Of note, only a subgroup of women with gestational diabetes develops type 2 diabetes over the years. In recent years there was a growing interest in the identification of metabolic fingerprints in women with gestation diabetes. Up to now, maternal body fluids of women with GDM have been investigated by NMR- or mass spectrometric (MS)-driven metabolomics approaches [10– 16]. In women with GDM, higher levels of certain amino acids including branched chain amino acids [15], a seven-fold increase in 3-caboxy-4methyl-5-propyl-2-furanpropanoic acid (CMPF) [15, 16] and lower levels of tauro-conjugated bile acids, long-chain poly unsaturated fatty acid derivatives and glycerophospholipids [14] have been reported. All studies investigated plasma collected in the fasted state in groups of 14–50 pregnant women with GDM [11, 12, 14–16]. In the current study, differences in metabolite profiles during a metabolic challenge were analyzed in GDM and healthy controls to gain deeper insight into the underlying pathophysiological mechanisms that emerge during pregnancy.

Lehmann R et al. Metabolic Profiles during an …  Exp Clin Endocrinol Diabetes 2015; 123: 433–438

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Metabolic Profiles during an Oral Glucose Tolerance Test in Pregnant Women with and without Gestational Diabetes

434 Article

Table 1  Clinical characteristics of the 24 pregnant women (9 with gestational diabetes mellitus (GDM) and 15 healthy controls) that were profiled by targeted metabolomics. The participants were matched by age, week of gestation, body mass index (BMI) and fasting glucose. OGTT = oral glucose tolerance test. Anthropometric data and clinical chemical parameters of the 2 groups were compared with the nonparametric Wilcoxon/Kruskal-Wallis tests (rank sums).

Methods



Subjects

Pregnant caucasian woman from the southern part of Germany participated in this study (PREG Study). The individuals were considered healthy according to routine laboratory tests and physical examination. The participants have been subjected to a large number of clinical tests. All 24 women were in the 24–27 week of gestation, 9 women were diagnosed as GDM and 15 were healthy controls. The women were matched by age, week of gestation, body mass index (BMI) and fasting glucose (further details of participant characteristics are given in ●  ▶  Table 1). EDTA-Blood drawing and handling was performed according to our recently published standard operating procedure (SOP) for the prevention of preanalytical errors [17]. In brief, whole blood was immediately placed in iced water, EDTA-plasma was separated from blood cells within 60 min, and stored at  − 80 °C until analysis. The permanent venous cannula was flushed with sterile 0.9 % sodium chloride after use. Informed written consent was obtained from all participants and the protocol of the study was approved by the Institutional Review Board of the University of Tuebingen according to the Declaration of Helsinki.

Oral glucose tolerance test (OGTT)

All of the individuals reported underwent an oral 75 g glucose tolerance test. Following an overnight fast of at least 8 h, 75 g glucose was ingested at 8:00 A.M.; plasma glucose and insulin concentrations were determined before and after 30, 60, 90, and 120  min (permanent venous cannula). Plasma glucose was measured using a glucose analyzer (Yellow Springs Instruments, Yellow Springs, OH). Insulin was analyzed using the ADVIA Centaur XP immunoassay system, clinical chemical routine parameters were analyzed using the ADVIA 1 800 analyzer, and blood cell count and haemoglobin using the ADVIA 2 120 haematology system (all analyzers from Siemens Healthcare Diagnostics, Eschborn, Germany).

Calculation of insulin resistance

In a five-point OGTT, whole body insulin sensitivity was assessed from glucose and insulin values according to Matsuda and DeFronzo [18].

Metabolite profiling

Within this study 231 metabolites from chemical classes of amino acids, biogenic amines, acylcarnitines, glycerophospholipids, sphingomyelins, hexoses and free fatty acids were profiled, as these classes of compounds have been reported before to be of relevance in the diabetes context [19–21]. Metabolite profiles were measured in EDTA-plasma of 24 pregnant women in the fasted state and the subsequent changes of the metabolite pattern at 30 min and 120 min during an OGTT were monitored. The quantification of amino acids, acylcarnitines, sphingomyelins, phosphatidylcholines, hexose (glucose), and biogenic amines was performed using a AbsoluteIDQ® p180 kit (Biocrates Life Sciences AG, Innsbruck, Austria) [22]. The assay was based

Controls n week of gestation age in years BMI [kg/m2] fasting blood glucose [mmol/l] glucose OGTT 30 min [mmol/l] glucose OGTT 60 min [mmol/l] glucose OGTT 90 min [mmol/l] glucose OGTT 120 min [mmol/l] fasting insulin [pmol/l] insulin sensitivity index ­(ISI-Matsuda) blood pressure Syst. [mmHg] blood pressure Diast. [mmHg] ASAT [U/l] ALAT [U/l] GGT [U/l] creatinine [mg/dl] leukocytes [1 000/μl] hemoglobin [g/dl] cholesterol [mg/dl] triglycerides [mg/dl] HDL cholesterol [mg/dl] LDL cholesterol [mg/dl] high sensitive C reactive ­protein [mg/dl]

GDM

p

15 25.6 ± 0.3 30 ± 1 25.6 ± 0.8 4.4 ± 0.1 7.5 ± 0.3 7.4 ± 0.4 6.4 ± 0.3 6.1 ± 0.2 60 ± 7 13.1 ± 1.3

9 26.4 ± 0.6 32 ± 1 29.8 ± 2.5 4.8 ± 0.2 9.1 ± 0.4 10.6 ± 0.3 9.1 ± 0.7 7.5 ± 0.6 133 ± 24 5.0 ± 0.7

0.3 0.2 0.1 0.2 0.005   92 mg/dl (5.1 mmol/l), 1 h > 180 mg/dl (10 mmol/l) and 2 h > 153 mg/dl (8.5 mmol/l) [7]. 15 controls and 9 women with GDM were matched for age, BMI ▶  Table 1). As and week of gestation, as well as fasting glucose ( ● expected, plasma glucose levels were significantly elevated in ▶  Fig. 1) and this was accomthe GDM group during the OGTT ( ●

panied by elevated insulin level in the fasted state. This finally translates to significantly lower measures of insulin sensitivity ▶  Table 1). While in all the other metaboin the GDM group ( ● lomics studies, fasting glucose levels were significantly higher in women with GDM [10–16], we additionally matched individuals for fasting blood glucose levels to minimize the effects of differ▶  Table 1). ent basal glucose levels on the metabolic profile ( ● Women in both groups from the PREG study were considered otherwise healthy according to physical examination and did not differ in terms of blood pressure (systolic and diastolic), liver enzymes (ASAT/ALAT), kidney function (creatinine), inflammation markers (leucocytes, high sensitive C reactive protein) and cholesterol.

Kinetics of plasma metabolite patterns during an OGTT in 24 pregnant women Differences irrespective of the individual clinical phenotype

We analyzed plasma samples drawn before and during the oral glucose tolerance test in gestational week 24–27 by targeted metabolomics to identify metabolic markers that relate to altered pathways during a glucose challenge. A profile of 231 plasma metabolites per sample of 6 groups of metabolite classes including amino acids (AA), acylcarnitines (AC), free fatty acids (FFA), sphingomyelins (SM), phosphatidylcholines (PC), and lysophospatidylcholines (LPC) was analyzed in the fasted state, i. e., directly before the start and at 2 time points during an OGTT (30, 120 min). Following the initial evaluation of these measurements, 30 metabolites had to be excluded based on low signal intensities in plasma (Table S2; supplementary information). The remaining pattern of 201 metabolites was used for further analysis. We first investigated single metabolite differences without background information in terms of clinical covariables. Among the metabolites with significantly decreased levels (p 

Metabolic profiles during an oral glucose tolerance test in pregnant women with and without gestational diabetes.

Gestational diabetes (GDM) is a complex metabolic condition associated with hyperpglycemia that is diagnosed in an oral glucose tolerance test (OGTT) ...
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