2. Perfil masma Metabolómico del plasma

2.1. Huella inherente de CD y papel de la GFD

2.2. Influencia genética (HLA)

3. omic Perfil mofiletabolómico de la orina

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].

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 [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.