Intestinal parameters of oxidative imbalance in celiac adults with extraintestinal manifestations

Table 1 Characteristics of the study groups

Groups of patients	Age (yr, mean ± SD)	n (%)
Total	70.74 ± 14.22	85
Female		71 (83.5)
Male		14 (16.5)
Active CD
Newly diagnosed CD	34.7 ± 14.9	7
Female		5 (71.4)
Male		2 (28.6)
Nontreated CD	38.2 ± 10.7	22
Female		18 (81.8)
Male		4 (18.2)
Treated CD	45.7 ± 16.1	31
Female		28 (90.3)
Male		3 (9.7)
Control	38.5 ± 13.2	25
Female		20 (80)
Male		5 (20)

Active CD: Celiac patients with active disease; Newly diagnosed CD: Celiac patients at diagnosis of CD; Nontreated CD: Celiac patients not adhering to a gluten-free diet; Treated CD: Celiac patients on a gluten-free diet.

Table 2 Clinical characteristic of study groups n (%)

Groups of patients	Iron deficiency/anemia	Chronic abdominal pain	Osteopenia/osteoporosis	Menstrual disorders	Abnormal liver tests	Others
Active CD (n = 29)	11 (37.9)	7 (24.1)	4 (13.8)	1 (3.4)	5 (17.2)	1 (3.4)
Treated CD (n = 31)	15 (48.4)	4 (12.9)	5 (16.1)	-	5 (16.1)	2 (6.5)

Active CD: Celiac patients with active disease; Treated CD: Celiac patients on a gluten-free diet.

Table 3 Human oligonucleotide primers for detection of mRNA by RT-PCR

Gene	Primer sequence	t	PCR product
IL-1β	Forward 5’-ACA TCA GCA CCT CTC AAG -3’,	60 °C	141 bp
	Reverse 5’-AGT CCA CAT TCA GCA CAG -3’
TNF-α	Forward 5’-GCC CAG GCA GTC AGA TCA TCT TC -3’,	58 °C	181 bp
	Reverse 5-TGA GGT ACA GGC CCT CTG ATG G-3’
IL-10	Forward 5’-AGC TAT CCC AGA GCC CCA GAT CCG ATT TTG G-3’,	60 °C	328 bp
	Reverse 5’-AAG CTG AGA ACC AAG ACC CAG ACA TCA AGG CG-3’
HSP-70	Forward: 5’-GCC CCA ACA GAT TGT TGT CTT-3’,	59.5 °C	111 bp
	Reverse: 5’-CCA CCA AGC AGA CGC AGA T-3’
HIF-1α	Forward 5’-GGT TCT CAC AGA TGA TGG TG-3’,	60 °C	239 bp
	Reverse 5’-TTC TTC CTC GGC TAG TTA GG-3’
SOD	Forward: 5’-GAA GGT GGG AAG CAT TA-3’,	57 °C	300 bp
	Reverse: 5’-ACC TTT GCC CAA GTC ATC TG-3’
BAX	Forward 5’-CGT CCA ACC CAC CCT GGT CT-3’,	55 °C	195 bp
	Reverse 5’-TGG CAG CTG ACA TGT TTT CTG AC-3’
β-actin	Forward 5’-GGG TAC ATG GTG GTG CCG-3’,	54 °C	307 bp
	Reverse 5’-AGC GGG AAA TCG TGC GTG-3’

t: Annealing temperature; IL-1β: Interleukin 1β; TNF-α: Tumor necrosis factor alfa; IL-10: Interleukin 10; HSP-70: Heat-shock protein 70; HIF-1α: Hypoxia-inducible factor 1α; SOD: Superoxide dismutase.

Table 4 Blood test results in the study groups

	Controls(n = 25)	Active CD(n = 29)	Treated CD(n = 31)
WBC (103/μL)	5.69 ± 1.55	5.81 ± 1.58	5.36 ± 1.36
RBC (106 cells/μL)	4.7 ± 0.37	4.49 ± 0.39a	4.45 ± 0.42a
Hemoglobin (g/dL)	13.4 ± 1.4	12.6 ± 1.8ac	13.3 ± 1.1
Hematocrit (%)	41.6 ± 9.6	37.6 ± 4.4ac	39.5 ± 3.2
PLT (103/μL)	248.52 ± 47.63	267.18 ± 96.8	252.52 ± 64.38
Vitamin D (ng/mL)	29.7 ± 5.1	19.4 ± 9.0bc	25.2 ± 11.2a
Total protein (g/L)	73.2 ± 6.1	71.2 ± 7.9	71.6 ± 3.3
AST (U/L)	18.0 ± 8.5	28.0 ± 19.3a	23.2 ± 6.5a
ALT (U/L)	22.0 ± 4.3	26.7 ± 20.1	24.2 ± 12.5
AP (U/L)	59.6 ± 19.7	62.2 ± 29.1	59.2 ± 27.3
GGTP (U/L)	24.4 ± 14.6	21.3 ± 13.6	21.3 ± 22.62
bilirubin (μmol/L)	10.5 ± 4.1	8.2 ± 5.0a	8.1 ± 3.3a
Uric acid (μmol/L)	190.8 ± 23.0	260.2 ± 53.7b	261.7 ± 51.8b

Data are given as mean ± SD.

^aP < 0.05,

^bP < 0.001 vs controls;

^cP < 0.05 vs Treated CD. Active CD: Patients with active CD; Treated CD: Celiac patients on a gluten-free diet; WBC: White blood cell count; RBC: Red blood cell count; PLT: Platelet count; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; AP: Alkaline phosphatase; GGTP: γ-glutamyltransferase.

Table 5 Clinical characteristics of the study groups: serum levels of celiac antibodies and the degree of intestinal mucosal damage n (%)

	Control (n = 25)	Treated CD (n = 31)	Nontreated CD (n = 22)	Newly diagnosed CD (n = 7)
Antibody titer
0	25	30 (96.7)	0	0
1	0	1 (3.3)	9 (40.9)	1 (14.3)
2	0	0	3 (13.6)	2 (28.6)
3	0	0	10 (45.5)	4 (57.1)
Antibody titer	0	0.03 ± 0.2	2.0 ± 0.9bd	2.4 ± 0.8bd
Degree of intestinal mucosal damage1
Normal mucosa 0	24 (96)	13 (41.3)	2 (9)	1 (14.3)
Marsh 1 1	1 (4)	5 (16.1)	7 (31.8)	0
Marsh 2 2	0	0	0	0
Marsh 3a 3	0	5 (16.1)	6 (27.3)	1 (14.3)
Marsh 3b 4	0	7 (22.6)	5 (22.7)	3 (42.9)
Marsh 3c 5	0	1 (3.2)	2 (9)	2 (28.6)
Intestinal mucosal damage1	0.04 ± 0.2	1.7 ± 1.8b	2.5 ± 1.6bc	3.7 ± 1.4bc

¹Intestinal mucosal damage was classified according to Marsh parameters, and each stage was given a score from 0 (normal mucosa) to 5 (total villous atrophy).

^bP < 0.001 vs controls;

^cP < 0.05,

^dP < 0.001 vs treated CD. Data are frequency counts (percentage of total) or the mean ± SD. Treated CD: Celiac patients on a gluten-free diet; Nontreated CD: Celiac patients not adhering to a gluten-free diet; Newly diagnosed CD: Celiac patients at diagnosis of CD; antibody titer 0: Negative; 1: Low [TGA <3x the upper limit of normal (ULN); EmA (+)]; 2: High [3xULN<TGA<10x ULN ; EmA (++)], 3: Very high [TGA>10x ULN; EmA (+++)].