Encyclopedia
Celiac disease (CD) is included in the group of complex or multifactorial diseases, i.e., those
caused by the interaction of genetic and environmental factors. Despite a growing understanding
of the pathophysiological mechanisms of the disease, diagnosis is still often delayed and there
are no effective biomarkers for early diagnosis. The only current treatment, a gluten-free diet
(GFD), can alleviate symptoms and restore intestinal villi, but its cellular effects remain poorly
understood. To gain a comprehensive understanding of CD’s progression, it is crucial to advance
knowledge across various scientific disciplines and explore what transpires after disease onset.
Metabolomics studies hold particular significance in unravelling the complexities of multifactorial and
multisystemic disorders, where environmental factors play a significant role in disease manifestation
and and progression. By analyzing metabolites, we can gain insights into the reasons behind CD’s
occurrence, as well as better comprehend the impact of treatment initiation on patients. In this review,
we present a collection of articles that showcase the latest breakthroughs in the field of metabolomics
in pediatric CD, with thessi aim of trying to identify CD biomarkers for both early diagnosis and
treatment monitoring. These advancements shed light on the potential of metabolomic analysis in
enhancing our understanding of the disease and improving diagnostic and therapeutic strategies.
More studies need to be designed to cover metabolic profiles in subjects at risk of developing the
disease, as well as those analyzing biomarkers for follow-up treatment with a GFD.
- celiac disease
- gluten-free diet
- metabolomics
- children
- immune
- intestinal
1. Introduction.
Celiac disease (CD) is included in the group of complex or multifactorial diseases, i.e.,
those caused by the interaction of genetic and environmental factors [1]. It is a chronic
disease whose severity and digestive and/or systemic symptoms show great variability
among patients. However, the common characteristic to all of them is an exacerbated
immune response to gluten and related proteins, so this systemic disorder is considered
an immune-mediated disease. In fact, patients are characterized by the presence of high
titers of specific antibodies and the vast majority are carriers of the DQ2 and/or DQ8
haplotypes of the major histocompatibility complex (Human Leukocyte Antigen, HLA
class II), responsible for antigen presentation by the immune system [2–4]. In addition
to gluten intake and the presence of risk alleles in HLA, the occurrence of intestinal and
extra-intestinal symptoms in CD requires the activation of both types of immune response,
innate and adaptive, and this overactivation of the immune system is observed at the
intestinal level as well as at the peripheral and systemic levels [4].
The main gap in the knowledge of the pathogenesis of CD is an explanation of why
25–35% of the world’s healthy population has these haplotypes, but only about 1–3% will
develop CD [5,6]. It is possible that other environmental and genetic factors influence
an individual’s ability to induce and control the innate response and an individual’s
susceptibility to gluten.
Even though CD is actually one of the most frequent genetic diseases, affecting 1–3%
of the world’s population, and to a greater extent women and children [7], it is clearly
underestimated and underdiagnosed. Despite advances in knowledge and the development
of serological tests, CD continues to be difficult and costly to diagnose, largely due to the
systemic nature of CD, the lack of specificity of its clinical manifestations and the existence
of silent or latent forms [8,9]. The problem is that untreated celiacs or those whose diagnosis
has been delayed, despite having no symptoms, may have an exacerbated and chronic
activation of the immune system, which is uncontrolled for a longer time, leading to a
worse prognosis, an accentuation of symptoms and the appearance of other autoimmune
diseases such as type 1 diabetes or gluten-dependent hepatitis, which are frequent in CD
patients [10,11]. Also, they may be affected by a variety of adverse consequences, some
serious such as the development of malignant tumors [12]. Therefore, at the present time,
the main challenge, like for other genetic-based diseases (such as some types of cancer
and diabetes, among others), is to study in genetically predisposed individuals, on the one
hand, which factors are involved in the development or not of CD and, on the other, to
find biomarkers for its early diagnosis and follow-up. The idea is to avoid the side effects
when the disease has already made its debut and even prevent its appearance, something
relevant since it is currently incurable.
The only current treatment for CD is a lifelong strict gluten-free diet (GFD) that
achieves a remission of symptoms within a few days or weeks and a restoration of intestinal
villi and immune homeostasis within a few months. We ourselves have found that, after
18 months of a strict GFD follow-up, the celiac has most of the parameters equivalent to
those of a healthy child [13–15]. This is an important finding, but, even so, the usual delay
in diagnosis (because of its difficulty), in combination with the time it takes to stabilize
the disease, is too long, and in this period they may develop complications that will result
in sequelae that range from minor (e.g., permanent short stature, dental enamel failure
and psychiatric problems) to serious, such as tumors, in the long term. The fact is that the
GFD seems to play an important role in the pathogenesis of tumor development, since
some studies have described a greater development of tumors the later the diagnosis is
made and in patients who have not followed the GFD [16]. Thus, untreated CD is associated
above all with T-cell lymphoma (EATL) [12] and small bowel adenocarcinoma [17],
although a higher incidence of non-Hodgkin’s lymphoma and colorectal cancer has also
been described [18,19].
As CD is a systemic and complex disease, it is necessary to look at it from different
perspectives. Personalized or precision medicine refers to the application of biotechnology,
genetic profiling, “omics” sciences and the incorporation of clinical and environmental
factors to evaluate individual risks and design strategies for the prevention, diagnosis,
treatment or follow-up of the disease at the right time and in the right patient, with the
minimum toxicity and maximum possible efficacy. One of the sciences that has been
booming in recent years is metabolomics, which deals with the study of chemical processes
in which small molecules, called metabolites, which can be both endogenous and xenobiotic,
are measured. These molecules give information about a metabolic process that has taken
place in the organism, and metabolomics can be considered, from this perspective, as an
approach to cellular metabolism that other “omics” sciences cannot provide [20,21]. In this
sense, metabolomics is presented as a fast and non-invasive tool that could represent a step
forward in the knowledge of many diseases through the study of the metabolic profile by
obtaining what are known as “metabolomic fingerprints or signatures” resulting from the
interaction of the genome, epigenome, transcriptome, proteome and the environment.
Metabolomics studies are especially relevant in those multifactorial and multisystemic
pathological situations where the environmental factor plays a relevant role in the
appearance and development of the disease. Technologies such as metabolomics could
define the alterations that occur in the genetically predisposed individual, as well as after
certain changes, such as the GFD, helping to better understand these complex interactions,
and thus may be useful for the diagnosis and monitoring of CD [22]. In this review, we
cover several articles highlighting the latest advancements in the field of metabolomics in
pediatric CD. Specifically, we explore studies that examine plasma and urine samples, with
a special focus on the role of the GFD.
2. Plasma Metabolomic Profile
2.1. CD’s Inherent Footprint and Role of the GFD
2.1. CD’s Inherent Footprint and Role of the GFD
Several molecules have been proposed as potential CD biomarkers. In this regard,
Auricchio et al., 2019 [102] found that the serum phospholipid profile is different in children
who develop CD compared to healthy children with similar genetic profiles (specific celiac
human HLA DQ2 or DQ8), even before the introduction of gluten to the diet at 4 months of
age. They followed a cohort of children from families with a CD case from birth to 8 years
of age, with sampling at 4 and 12 months of age (and at CD diagnosis in cases >24 months
of age), finding in lipidomic analysis based on LC coupled with MS and multiple reaction
monitoring (MRM) that the lipid profile is fairly constant in each individual, in both groups,
suggesting that it could be constitutive. In the age-grouped analysis, they found that children
who developed CD had increased lyso- and phosphatidylcholine (PC) serum levels
(PC 40:4 showed the greatest difference between the two groups), as well as alkylacylphosphatidylcholine
(PC-O). Specifically, two alkylacylphosphatidylcholipids (PC O-42:0 and
PC O-38:3), together with breastfeeding and one phosphatidylcholine (PC 34:1), were defined
as predictors of CD development. They found that phosphatidylethanolamines (PE)
PE 34:1 and PE 36:1 were decreased in celiac patients.
The working group of Sen et al., 2019 [103] also applied lipidomics in the study of a
cohort of Finnish children in the context of the Type 1 Diabetes Prediction and Prevention
study. Based on MS and comparing plasma samples from children who developed CD
with plasma samples from healthy controls, matched for HLA risk, sex and age, they found
that CD progressors (children who developed CD) had increased triacylgycerols (TGs) of
low carbon number, double-bond count plasma levels and decreased phosphatidylcholines
and cholesterol esters levels at 3 months of age compared to controls. These differences
were not apparent at birth (cord blood) and exacerbated with age. It is proposed that
the increase in TGs of low carbon number and double-bond count is due to de novo
lipogenesis compensating for lipid malabsorption, which would occur at a very young age;
this increase in TGs has been linked in adults to increased liver fat in non-alcoholic fatty
liver disease [104]. In addition, they found decreased total essential TG levels in the plasma
of the CD progressors after gluten intake, reversing this trend, but not significantly, after
the onset of GFD, and there was an inverse relationship with the tissue transglutaminase
IgA titer (tTGA). There was also an increase in cholesterol levels after the start of GFD in the
CD progressors. On the other hand, the endogenous TGs plasma levels were decreased in
CD progressors independently of gluten intake. PCs were elevated in both CD progressors
and controls after the start of gluten intake. A difference in sphingomyelin plasma levels
was observed in CD progressors at a later age, after the introduction of GFD.
These findings suggest that a dysregulation in lipid metabolism may be associated
with the development of CD, and that it occurs in the first months of life, even before
the introduction of gluten to the diet. This could help predict the development of CD in
infants at genetic risk, even years before the appearance of specific antibodies or clinical
symptoms/signs.
However, a previous study (2016) based on the PreventCD project suggested that the
metabolic profile at 4 months (before the introduction of gluten to the diet) did not predict
the development of CD, but that metabolic pathways are affected later in life [105]. In this
study, which studied serum samples from infants at genetic risk for CD who developed CD
compared to those who did not develop the disease at 8 years of age, a trend of decreased
phospholipids levels was found in children who subsequently developed CD, although
not significantly, with a greater decrease in the subsample of children exclusively breastfed
until 4 months of age. They conclude that metabolomic studies should focus on children
who have already had gluten introduced to their diet. However, this study focused the
analysis on phospholipids and acylcarnitines, and TGs and cholesterol esters were not
measured.
Following the lipidomics approach, in a pilot study conducted by ourselves [106],
plasma lipid profile was affected in celiac patients, despite GFD treatment. Using an
LC-MS/MS platform, there plasma from 17 celiac children under a GFD treatment and
17 healthy controls (siblings) was analyzed. Among the significant molecules, it was
found that 64% were increased and 36% decreased in CD patients. Two carboxylic acids
and derivatives were increased in CD; other molecules whose levels were affected in
patients were four fatty acyls (thromboxanes and leykotrienes involved in inflammatory
pathways), five glycerolipids, eleven glycerophospholipids, one organoxigen compound
and two sphingolipids, lipid species belonging to steroid metabolism and other molecules
involved in bilirubin metabolism. In celiac patients elevated levels of molecules involved in
cell signaling pathways (ceramides, diacylglycerides and lysophospholipids) were found.
Diacylglycerides play a central role in the control of neuronal communication, phagocytosis
and the control of immune responses, and as a second messenger they play an important
role in the regulation of mTOR, recently described as a key factor in maintaining a sustained
inflammatory response in CD [107].
Aside from the lipid profile, one-carbon metabolism alterations were also found by this
group [108] under a targeted plasma metabolomics study. They observed a down-regulation
of the trans-sulphuration pathway in CD patients, despite GFD, with decreased cysteine
and cystathionine, which, together with normal glutathione and vitamin B6, suggests a
specific defect at the level of the enzymes involved in antioxidant defense, oxygen sensing,
mitochondrial function, inflammation and second-messenger signaling. This finding,
moreover, could be explained by a S-adenosyl-L-homocysteine (SAH) hydrolase mutation
that causes typical symptoms of the disease, such as growth retardation, dental anomalies
or hypertransaminasemia. In contrast, other pathways involved in one-carbon metabolism
appeared to be preserved (choline metabolism, the methionine cycle and the folate cycle),
suggesting that adherence to a strict GFD could reverse certain metabolic changes in celiac
patients, making them resemble the profile of healthy subjects. This group notes that these
metabolomic changes are, however, minor, as only approximately 4% of the total plasma
metabolome analyzed was affected [106].
In a more recent study based on a targeted plasma metabolomics analysis, Girdhar
et al., 2023 [109] found increased levels of 2-methyl-3-ketovalric acid, taurodeoxycholic
acid (TDCA), glucono-D-lactone and isoburyryl-L-carnitine, as well as significantly low
oleic acid levels (anti-inflammatory metabolite) in CD progressors (compared to healthy
children matched for age, HLA genotype, breastfeeding duration and gluten exposure
duration). Other metabolic pathways were also affected in the CD progressors, such
as the pentose phosphate pathway, unsaturated fatty acid biosynthesis and glycolipid
and linoleic acid metabolism. Notably, TDCA levels were increased to twice the normal
values. TDCA, a metabolite derived from the gut microbiota, may play a role in small
intestinal inflammation and CD pathogenesis, as its administration to C57BL/6J mice by
supplementing their diet caused a distortion in crypt structure and total or partial villous
atrophy; increased CD4+ T cells, natural killer cells and Qa-1 and NKG2D expression on T
cells (two immunomodulatory proteins); and decreased regulatory T cells in intraepithelial
lymphocytes. Therefore, TDCA could be used as an early diagnosis biomarker, and more
importantly, targeted therapies to eliminate TDCA-producing bacteria (Clostridium XIVa
and Clostridium XI) early in life could be used as a strategy to decrease the CD development
risk. On the other hand, they found that the cytokine plasma profile and other metabolites
were altered in CD progressors, even before diagnosis (other recent work (Auricchio et al.,
2023 [110]) has also focused on the serum cytokine profile and proinflammatory genes
expression in infants at CD risk), and differences were also found in the gut microbiota
composition (studied in stool) (other authors have also studied microbiota and metabolome
alterations in infants at risk of CD, in stool [111,112]). In the CD progressors, before
diagnosis, they found significantly increased levels of three proinflammatory cytokines
(IFNA2, IL-1a and IL-17E/(IL25)) and a chemokine (MIP-1b/CCl4).
Another interesting aspect to be addressed is alternative biomarkers that allow the
disease to be monitored and can also be used in the evaluation of celiac patients’ relatives.
Plasma citrulline was assessed by an LC auto sampler (and in dried blood spots) by Lomash
et al., 2021 [113], as a potential biomarker useful in the diagnosis and monitoring
of the disease, as well as in the evaluation of celiac patients’ first-degree relatives (FDRs)
(predictive value in the distinction of seronegative CD and in the progression of potential
to overt CD). This non-essential amino acid is specifically produced by proximal small
intestine enterocyte villi, so it has been proposed as a possible marker of residual intestinal
function in pathologies such as necrotizing enterocolitis in newborns, enterophaties, small
intestine transplantation or small bowel resections [114]. This work found statistically
significant differences in the median plasma citrulline levels in celiac children (20.1 mcM(IQR,
13.35–29.15)) compared to controls (serology-negative FDRs) (37.33 mcM (IQR, 29.8–42.6)).
They also found an inverse correlation between plasma citrulline levels and anti-tTG IgA
levels throughout the establishment of GFD and, in addition, in the different Marsh grades
at diagnosis; so, citrulline could be used as a surrogate biomarker for serology in disease
monitoring and in predicting the histopathological damage degree (it was effective in
distinguishing grades 3b and above but not in distinguishing 3a or less in celiac patients
and healthy asymptomatic FDRs). In addition, in patients with inconclusive serology and
biopsy results, the median plasma citrulline reflected mucosal damage (12.26 mcM) like
in potential celiacs (median plasma citrulline levels: 23.25 mcM).
2.2. Genetic Influence (HLA)
In the above-mentioned work [113], plasma citrulline levels were also correlated with
HLA genotype. Significantly low plasma citrulline levels were observed in subjects with
the HLA DQ 2.5 genotype with subtypes DQA1*0501 and DQB1*0201. The HLA-DQ
genotype has already been reported to influence early intestinal microbial colonization,
thus influencing the metabolome [115].
Kirchberg et al. (2016) found that the HLA genotype did not have any influence on
the serum metabolic profile in infants who were at risk for celiac disease before introducing
gluten to their diet [105].
2.2. Genetic Influence (HLA)
In the above-mentioned work [34], plasma citrulline levels were also correlated with HLA genotype. Significantly low plasma citrulline levels were observed in subjects with the HLA DQ 2.5 genotype with subtypes DQA1*0501 and DQB1*0201. The HLA-DQ genotype has already been reported to influence early intestinal microbial colonization, thus influencing the metabolome [36]. Kirchberg et al. (2016) found that the HLA genotype did not have any influence on the serum metabolic profile in infants who were at risk for celiac disease before introducing gluten to their diet [26].3. Urine Metabolomic Profile
3. Urine Metabolomic Profile
Other studies (Table 3) have also compared the urine metabolomic profile of celiac
children with healthy controls, some of them focusing on changes in the volatile organic
compounds (VOCs) profile. An example of this is Di Cagno et al., 2011 [37][116], who, using gas
chromatography mass spectrometry/solid-phase microextraction (GC-MS/SPME) analysis,
demonstrated that VOCs and free-amino-acid levels are altered in the urine (and stool) of
celiac children with more than 2 years of GFD, relating these imbalances to qualitative and
quantitative differences in the microbiota of celiac patients compared to healthy people.
They found that the CD group had higher dimethyl trisulfide and dimethyl disulfide
urine levels. In addition, with some exceptions, they also had higher urine hydrocarbon
levels. No differences in urine aldehyde levels were found between the two groups. These
findings were confirmed by NMR, which also found that the CD group had higher lysine,
arginine, creatine and methylamine mean levels, while carnosine, glucose, glutamine and
3-methyl-2-oxobutanoic acid were the highest in healthy children. This study emphasizes
that a GFD does not completely restore the microbiota or, consequently, the metabolome
of children with CD, and that there are possible metabolic markers of CD; furthermore,
it suggests that the addition of prebiotics and probiotics to the GFD could restore the
microbiota–microbiome balance in celiac children.
In relation to this aspect, Drabinska et al., 2019 [117], studied the effect of GFD supplementation
with a prebiotic (oligofructose-enriched inulin) on VOC urine concentration in
celiac children and adolescents, using GC-MS/SPME analysis. This work is based on the
idea that changes in the VOC profile in biological fluids that occur in various gastrointestinal
diseases (studied by the recent so-called “volatolomics”) are due in part to alterations in
microbiota metabolism, especially its fermentative activity, and not so much to variations
in its composition. However, GFD supplementation with this prebiotic had no impact on
most VOC urine profiles of celiac patients; only a significant change was observed in benzaldehyde
concentrations, which decreased by 36% after 12 weeks of intervention, which
may be related to a decrease in Lactobacillus counts in the prebiotic-supplemented group, as
Lactobacillus produces an aminotransferase that converts phenylalanine to benzaldehyde.
Another study, also led by Drabinska [118], aimed to optimize the GC-MS/SPME
method for the detection of changes in VOC urine profiles in celiac children compared to
healthy children. Based on Variable Importance in the Projection (VIP) scores, several CD
biomarkers could be suggested: 1,3-di-tert-butylbenzene (only found in the urine of celiac
children) and other VOCs present in higher concentrations in the urine of healthy children
(2,3-butanedione, 2-heptanone, dimethyl disulfide and octanal, and, with lower VIP scores,
2-butanone, hexanal and 4-heptanone). Again, these differences could be explained by
alterations in the celiac patients’ gut microbiota, as many VOCs are fermentation products
of the microbiota. On the other hand, the VOC levels in biological fluids (blood, urine,
sweat . . . ) could be increased by the altered intestinal permeability present in many
gastrointestinal tract diseases.
