The use of anti-transglutaminase and anti-gliadin antibodies to compare the effect of gluten active and gluten-free diet patients suffering from celiac disease in child and adult

Dhiaa aldin.B.Aljamrawy, Karrar A. Ali Beg, Israa K. AL-Yasiri. The use of anti-transglutaminase and anti-gliadin antibodies to compare the effect of gluten active and gluten-free diet patients suffering from celiac disease in child and adult. Car-diometry; Issue 31; May 2024; p. 75-80; DOI: 10.18137/cardi-ometry.2024.31.7580; Available from: https://www.cardiometry. net/issues/no31-may-2024/use-anti-transglutaminase-anti-gli-adin-antibodies

Celiac disease (CD) is a systemic autoimmune condition that affects genetically susceptible individuals who develop an intolerance to dietary gluten. The primary environmental trigger for CD is the consumption of particular gluten peptides (1). Gluten is a collection of ethanol-soluble proteins known as prolamins and glutelins, which are present in grains such as wheat, rye, oat, and barley. These proteins, abundant in glutamine and proline residues, are not easily broken down by human intestinal proteases and contribute to the elasticity of dough required for leavening and shaping (2). The prevalence of celiac disease (CD) can vary across different regions, but on a global scale, it has been reported to have an average prevalence ranging from 0.5% to 1% (3). Among Arabian countries, the prevalence rates of celiac disease (CD) are generally similar, and Saudi Arabia has been reported prevalence, reaching up to 3.2 percent (4) .The prevalence of celiac disease (CD) in Iraq was found to be 11.2% (5) . Celiac disease can present with classic symptoms like diarrhea, weight loss, stomach pain, bloating, malabsorption, and failure to thrive. In adults, nonclassic symptoms are more common than classic and may include less specific gastrointestinal symptoms or extraintestinal signs such as anemia, osteoporosis, elevated liver enzymes (transaminitis), and recurrent miscarriage (6). Celiac disease is commonly attributed to an autoimmune response that is triggered by T-cells. When modified gliadin-derived peptides are presented to CD4+ T helper cells via HLA molecules DQ2 and DQ8, both T-cells and B lymphocytes become activated. This activation leads to the production of antibodies against gluten and tissue transglutaminase (tTG) in the lamina propria, ultimately resulting in the activation of self-reactive T helper cells (7). The typical diagnosis of celiac disease involves a combination of clinical diagnosis, serologic tests, confirmed by a small bowel biopsy showing the characteristic histology associated with celiac disease. Additionally, serological tests can be used for monitoring the response to a gluten-free diet (8). Serology is widely recognized as a noninvasive and efficient method for identifying autoantibodies associated with celiac disease. This approach involves testing blood samples for the presence of specific antibodies targeting tissue transglutaminases, gliadin. Serological tests are considered highly sensitive and specific, making them valuable tools in the diagnosis of celiac disease (9).

MATERIALS AND METHODS:

This research was carried out between the 15 ᵗʱ of October 2023 and the 15 ᵗʱ of February 2024 involved 100 participants, divided into 60 CD patients and 40 controls, with an age range of 7-65 years who attended the Digestive Center in Marjan Medical

City/Babylon Governorate and Among the patients, 40 were classified as having active CD, and all of them were newly diagnosed. Additionally, 20 patients were a gluten-free diet, 40 apparently healthy individuals as control who had been selected to be matched with the patients regarding age and gender. Informed consent was obtained from all the study participants. Blood samples were obtained from both groups and stand at room temperature to allow clot and then separated by centrifugation at 1500 g for 10 min and stored at -20°C until use. Immunological tests as Anti-tissue Transglutaminase (tTg) and antigliadin antibody (AGA) were determined using commercially available ELISA kits (Aeskulisa, Germany). ELISA was performed according to manufacturer instructions. Serum samples presenting results >15 U/mL were considered to be positive for the anti IgA and IgG of both anti tTG and anti gliadin.

RESULTS

Table (1-1):Distribution of demographic characteristics of celiac disease patients.

Determination the concentration of the serum level of the Anti-tissue Transglutaminase (tTG) Antibodies : IgA and IgG

The mean ± SE serum anti-tTG IgA level was 96.482 ± 21.248 U/mL, 132.890 ± 18.312 U/mL and 5.040 ± 0.492 U/mL in the diet group (G1), active group (G2) and in the control group (G3), respective-

Table (1-1):Distribution of demographic characteristics of celiac disease patients.

		Groups						P. value
		Patients with Celiac (Diet group)		Patients with Celiac (Active Group)		Control
Age	Mean± SD (Range)	26.3±15.6 (10-65)		20.6±10.6 (7-45)		23.1±9.9 (8-52)		0.201
Gender	Male (%)	6	30.0%	14	35.0%	10	25.0%	0.621
	Female (%)	14	70.0%	26	65.0%	30	75.0%
BMI (kg/m2)	Mean± SD (Range)	22.66±5.11 (15.12-32.05)		21.11±6.05 (8.67-35.16)		23.17±4.99 (16.46-31.02)		0.166
History of family	Yes	5	25.0%	7	17.5%	-	-	0.007
	No	15	75.0%	33	82.5%	40	100.0%
Compare between percentages using Pearson Chi-square test (χ2-test) at 0.05 level.
Compare between means using ANOVA at 0.05 level.

ly. The results revealed a highly significant difference between the patients and control groups regarding the anti-tTG IgA at P.value (P <0.001). Further comparisons using the least significant difference (LSD) showed no significant differences between the patient groups (G1 Vs. G2) (P <0.120), but higher significant differences were observed between (G1 Vs. G3), as well as between (G2 Vs. G3) (P-value < 0.001). As shown in Table 1.2.

While, the mean ± SE serum anti-tTG IgG level was 148.119 ± 32.072 U/mL, 233.230 ± 28.118 U/ mL and 4.957 ± 0.419 U/mL in the diet group (G1), in the active group (G2) and in the control group (G3), respectively. The results show a significant difference between the patient and control groups in terms of the anti-tTG IgG at (P <0.001). Further comparisons using the least significant difference (LSD) revealed differences between the patient groups (G1 Vs. G2) (P <0.018), with higher significant differences observed between (G1 Vs. G3), as well as between (G2 Vs. G3) (P-value < 0.001). As shown in Table 1.3

Determination the concentration of the serum level of the Anti-Gliadin Antibodies : IgA and IgG

The mean ± SE serum GLIA-IgA level was 10.8405 ± 1.468 U/mL, 43.6720 ± 8.133 U/mL and 4.7400 ± 0.485 U/mL in the diet group (G1), the active group (G2) and the control group (G3), respectively. The study result revealed a significant difference between the patient and control groups in terms of the anti-gliadin IgA at (P <0.001). Further comparisons using the least significant difference (LSD) show highly significant between (G1 Vs. G2) at (P <0.001), while there was no significant differences observed between (G1 Vs. G3) (P-value = 0.499). But, this study show highly significant difference between (G2 Vs. G3) at (P <0.001) As shown in table 1.4.

While, The mean ± SE serum GLIA-IgG level was 19.7465 ± 2.921 U/mL, 52.9375 ± 7.276 U/mL and 6.5850 ± 0.442 U/mL in the diet group (G1), the active group (G2) and the control group (G3), respectively. The study result revealed a significant difference be-

Table (1-2): A comparison the serum levels of anti TTG-IgA among the studies groups.

Parameters		Mean	SD	SE	F (P. value)
TTG-A	Patients with Celiac (Diet group) G1	96.482 a	95.022	21.248	23.657 (P <0.001)
	Patients with Celiac (Active Group) G2	132.890 a	115.817	18.312
	Control G3	5.040 b	3.112	0.492
	Least Significant Difference (LSD)	Comparisons		P. value (between groups)
	37.57	(G1 Vs. G2)		0.120 NS
		(G1 Vs. G3)		<0.001 HS
		(G2 Vs. G3)		<0.001 HS

One- Way ANOVA; LSD as Post hoc test for equal variances ; ns : no significant at 0.01;Hs: highly significant at 0.001; G1: Diet group; G2: Active Group; G3:control.

Table (1-3): A comparison the serum levels of anti TTG-IgG among the studies groups

Parameters		Mean	SD	SE	F (P. value)
TTG-G	Patients with Celiac (Diet group) G1	148.119 a	143.429	32.072	31.517 (P <0.001)
	Patients with Celiac (Active Group) G2	233.230 b	177.834	28.118
	Control G3	4.957 c	2.656	0.419
	Least Significant Difference (LSD)	Comparisons		P. value (between groups)
	57.32	(G1 Vs. G2)		0.018 S
		(G1 Vs. G3)		<0.001 HS
		(G2 Vs. G3)		<0.001 HS

One- Way ANOVA; LSD as Post hoc test for equal variances ; s :significant at 0.01;Hs: highly significant at 0.001; G1: Diet group; G2: Active Group; G3:control.

Table (1-4): A comparison the serum levels of anti GLIA-IgA among the studies groups.

Parameters		Mean	SD	SE	F (P. value)
GLIA-A	Patients with Celiac (Diet group) G1	10.8405 a	6.567	1.468	15.415 (P <0.001)
	Patients with Celiac (Active Group) G2	43.6720 b	51.437	8.133
	Control G3	4.7400 a	3.067	0.485
	Least Significant Difference (LSD)	Comparisons		P. value (between groups)
	14.56	(G1 Vs. G2)		<0.001 HS
		(G1 Vs. G3)		0.499 NS
		(G2 Vs. G3)		<0.001 HS

tween the patient and control groups in terms of the anti-gliadin IgA at (P <0.001). Further comparisons using the least significant difference (LSD) show highly significant between (G1 Vs. G2) at (P <0.001), while there was no significant differences observed between (G1 Vs. G3) (P-value = 0.110). But, this study show highly significant difference between (G2 Vs. G3) at (P <0.001) As shown in table 1.5

DISCUSSION

The immune system produces anti-tTG antibodies in response to gluten exposure, which ultimately leads to damage to the intestinal mucosa (10). The elevated prevalence of patients exhibiting positivity to Anti-tTG IgA,IgG can be ascribed to its strong sensitivity and specificity. IgA with a sensitivity of 78% to 100% and a specificity of 90% to 100%,while the sensitivity of IgG anti-tTG ranging from 75% to 95% and the specificity from 94% to 100 (11,12). Conversely, the IgA class is associated with a reduced incidence of false negative outcomes, IgG make it advisable for utilization in individuals with IgA deficiencies. In cases where IgA antibodies are entirely absent, an IgG compensatory production may be prompted by the typical T-helper 1 response in celiac disease (13). The gluten-free diet group showed a notable decrease compared to the active celiac disease group. The current study agreement with study in Baghdad city (14), and with a cross-sectional study conducted in Pakistan (15), In both studies, the average serum levels of anti-tTG IgA and IgG were notably elevated in patients diagnosed with Celiac Disease in comparison to control subjects. A retrospective study revealed that a proportion of children with celiac disease experience a significant slow decrease in anti-tTG levels after starting the gluten-free diet, but the persistent of the these antibodies positivity observed after 24 months of the diet, a period that may persist for 4 years (16). López et al. also demonstrated that elevated levels of IgG antibodies persisted in half of the evaluated patients, even after two years of a gluten-free diet. This, combined with the considerable variability in IgG antibodies used for diagnosis, has the potential to cause confusion and challenges in follow-up (17). Persistently elevated anti-tTG antibody levels are significantly associated with abnormal duodenal histology, low ferritin, and poor adherence to the gluten-free diet (18). Nevertheless, maintaining strict adherence to a GFD is difficult, leading to reported adherence rates varying from 42% to 91%. Moreover, as many as 80% of GFD-adherent patients

Table (1-5): A comparison the serum levels of anti GLIA-IgA among the studies groups.

Parameters		Mean	SD	SE	F (P. value)
GLIA-G	Patients with Celiac (Diet group) G1	19.7465 a	13.064	2.921	25.103 (P <0.001)
	Patients with Celiac (Active Group) G2	52.9375 b	46.015	7.276
	Control G3	6.5850 a	2.796	0.442
	Least Significant Difference (LSD)	Comparisons		P. value (between groups)
	13.23	(G1 Vs. G2)		<0.001 HS
		(G1 Vs. G3)		0.110 NS
		(G2 Vs. G3)		<0.001 HS

may experience unintended gluten contamination in their diet (19).

According to the anti-Giliadin antibodies the finding of the current study show the decrease of both an-ti-GLIA-A and GLIA-G after gluten diet as compare to active celiac patient and in comparison to between patients groups to control the active group show highly significant difference while no difference between the gluten free diet and control. The results of the current study where agreement with Ali et al. (2020) in Baghdad, which demonstrated a significantly higher level of anti-gliadin IgA,IgG antibody levels compared to the control group (20). The sensitivity and specificity of for both IgA and IgG AGA (Anti-Gliadin antibodies) fall within the range of 80% to 90%. It is commonly observed that specificities are lower in adults compared to children (21). While the study by Midhagen et al., 2004, suggests that there is a rapid decrease in anti-gliadin antibodies (AGA) after one year of starting a gluten-free diet (GFD) in adult patients, which similar results reported in children,This indicates that the response to a GFD in terms of AGA reduction show no differences between different age groups (22). In contrast (23),it is stated that the use of anti-gliadin antibodies (AGA) is challenging due to their low specificity (AGA-IgG) and low sensitivity (AGA-IgA). As a result, AGA are no longer considered crucial for diagnosing celiac disease. However, they may still have value in monitoring patients who have already been diagnosed with celiac disease to assess their adherence to a gluten-free diet (GFD). This is because these antibodies can reappear even after minor dietary lapses.

CONCLUSION

In conclusion, This study highlights the importance of dietary adherence in celiac disease management. Patients on gluten-free diets had significantly lower levels of anti-TG and anti-gliadin antibodies, indicating better control of the autoimmune response. Monitoring these antibody levels can be a valuable tool in assessing the effectiveness of dietary interventions in celiac disease patients. Improving adherence to the Gluten-Free Diet (GFD) and encouraging a well-rounded diet are crucial. Education and direction from dietitian and/or pediatric gastroenterologist with experience treating children with celiac disease (CD) can help achieve this. Another important component is being vigilant about identifying unintentional gluten consumption in their diet.