Is hyperhomocysteinemia relevant in patients with celiac disease?

Abstract

AIM: To investigate whether this might be related to the presence of hyperhomocysteinemia.

METHODS: From January 1998 to December 2008, we evaluated the presence of hyperhomocysteinemia in a series of 165 adult celiac disease (CD) patients (138 females and 27 males, mean age 43 years).

RESULTS: Hyperhomocysteinemia was evident in 32 patients (19.3%), although most of them had moderate levels (mean value 25 mcg/ml; range 15-30). Only one patient had a history of myocardial infarction (heterozygosis for N5-N10-metil tetrahydrofolate reductase mutation).

CONCLUSION: The systematic assessment of hyperhomocysteinemia seems, at present, unjustified in CD patients.

Key Words: Celiac disease, Endoscopy, Histology, Hyperhomocysteinemia

INTRODUCTION

Hyperhomocysteinemia, considered as an important risk factor in venous thrombosis[1-3], has a prevalence in the general population of 5%-7%[4], and causes damage of the vascular endothelium by disrupting the release of nitric oxide, an important vasodilator factor[5], followed by platelet activation and thrombus formation[4].

Celiac disease (CD) is a gluten-sensitive enteropathy due to intolerance to dietary wheat gliadin and related proteins in genetically predisposed individuals[6]. The malabsorption of folates and vitamins (the deficiency of which may be a cause of hyperhomocysteinemia) is frequent in CD patients, either in the classic or oligosymptomatic type[7], and several cases of thrombosis have been reported in patients with CD before establishing a diagnosis of gluten-related duodenal mucosal damage[8,9]. Thus, we investigated the presence of hyperhomocysteinemia in a series of patients with CD, to see whether it might be increased and represent a marker of increased venous thrombosis in these patients.

MATERIALS AND METHODS

Patients

In the period January 1998-December 2008, 165 patients with CD (27 men, 138 women, mean age 43 years) were studied. Inclusion criteria were: positivity for anti-endomysial IgA and anti-transglutaminase IgA antibodies (Eurospital, Trieste, Italy) and duodenal histology suggestive for CD.

Histological assessment

Four samples were obtained by endoscopy forceps from the proximal and distal parts of the duodenum. The biopsies, correctly oriented on acetate cellulose filters (Bio-Optica, Milano, Italy), were fixed in 10% buffered formalin, processed and included in paraffin. After obtaining 5 μm thick sections, these were stained with Hematoxylin-Eosin; some sections were also processed for immunohistochemistry and stained with an anti-CD3 monoclonal antibody (Dako, Denmark) to identify intra-epithelial lymphocytes (IEL). IEL density was expressed as the number of IEL/100 epithelial cells, with a density value of > 25 cells considered as pathological. Histological classification was based on the Marsh-Oberhuber criteria[10] and a new, recently proposed simpler classification[11,12] (Table 1).

Table 1 The Marsh-Oberhuber classification of duodenal histological lesions in celiac disease, compared to the “simplified classification”[11,12].

Histologic type	IEL	Glandular crypts	Villi	Simplified
0	Normal (< 25/100 epithelial cells)	Normal	Normal	Normal
1	Increased	Normal	Normal	Grade A
2	Increased	Hyperplastic	Normal	Grade A
3a	Increased	Hyperplastic	Mild atrophy	Grade B1
3b	Increased	Hyperplastic	Moderate atrophy	Grade B1
3c	Increased	Hyperplastic	Severe (total) atrophy	Grade B2

IEL: Intra-epithelial lymphocytes.

Laboratory assessment

Serum homocysteinemia, vitamin B12 and folic acid levels were measured in all patients. In case of hyperhomocysteinemia, mutations in N5-N10-metil tetrahydrofolate reductase (MTHFR), cystathionine beta synthetase (CBS) and the prothrombin gene were searched for. DNA was extracted from whole blood collected in tubes containing K3-EDTA using a commercial kit (Genomic DNA Isolation kit, Puregene -Gentra System). DNA analysis for MTHFR gene mutation (C677T) was performed by a PCR-RFLP method, as previously described[13]. A fragment of 232 base pairs was then amplified by polymerase chain reaction. The fragment was digested by Hinf I restriction enzyme, and subsequent electrophoresis on ethidium bromide stained 3% agarose gel was performed.

The concentration of total homocysteine in plasma (K3-EDTA tubes) was determined by high performance liquid chromatography, as previously described[14]. Basal hyperhomocysteinemia (normal value 5-15 μmol/L) was classified as moderate (16-30 μmol/L), intermediate (31-100 μmol/L) and severe (> 100 μmol/L) according to Hankey et al[15]. In all patients, the presence of any thrombotic episode was also evaluated.

The study was approved by the Institutional Review Board of the Desio Hospital.

RESULTS

Histological findings

Most CD patients (24/32, 75.0%) showed mild to severe villous atrophy, with the latter being present in 41.0% of patients (Table 2).

Table 2 Demographic, histological findings and associated diseases of 32 celiac disease patients with hyperhomocysteinemia.

No.	Sex/age (yr)	Histology (Marsh/simplified classification)	Associated diseases
1	F/37	Marsh 2 (Grade A)	IgA deficit
2	F/38	Marsh 3a (Grade B1)
3	M/34	Marsh 3a (Grade B1)
4	F/32	Marsh 3c (Grade B2)
5	F/20	Marsh 3c (Grade B2)
6	F/47	Marsh 3b (Grade B1)
7	F/64	Marsh 1 (Grade A)
8	F/41	Marsh 3c (Grade B2)	Epilepsy
9	F/22	Marsh 2 (Grade A)	IgA deficit
10	F/61	Marsh 3a (Grade B1)
11	M/44	Marsh 3c (Grade B2)	NASH
12	F/35	Marsh 3c (Grade B2)	type 1 diabetes
13	F/39	Marsh 3c (Grade B2)
14	F/40	Marsh 3c (Grade B2)
15	F/56	Marsh 3c (Grade B2)	PBC
16	F/35	Marsh 3c (Grade B2)
17	F/42	Marsh 2 (Grade A)
18	M/33	Marsh 3b (Grade B1)	Sarcoidosis
19	F/31	Marsh 3a (Grade B1)
20	F/42	Marsh 3a (Grade B1)
21	M/28	Marsh 3a (Grade B1)
22	F/41	Marsh 3c (Grade B2)
23	F/45	Marsh 3c (Grade B2)
24	F/21	Marsh 3a (Grade B1)
25	F/29	Marsh 3b (Grade B1)
26	F/55	Marsh 3a (Grade B1)	Osteoporosis
27	F/78	Marsh 3c (Grade B2)
28	M/47	Marsh 3c (Grade B2)
29	M/63	Marsh 3b (Grade B1)	Psoriasis, myocardial infarction
30	M/18	Marsh 3a (Grade B1)
31	M/40	Marsh 1 (Grade A)
32	F/32	Marsh 3a (Grade B1)	Sarcoidosis

IgA: Immunoglobulin A; PBC: Primary biliary cirrhosis.

Laboratory findings

Overall, hyperhomocysteinemia was detected in 32 (19.4%) CD patients (24 women, 8 men); average symptoms’ onset was 7 (range 1-40) years. Table 3 shows the serologic findings of these patients. Most patients (29/32, 91.0%) had moderate hyperhomocysteinemia, two (6.0%) intermediate and one (3.0%) severe increase of this value. Mutation of MTHFR was found in 13 (41.0%) patients, 7 homozygotes and 6 heterozygotes; one patient displayed heterozygotic mutation of the prothrombin gene. No CBS mutations were found.

Table 3 Serologic findings of 32 celiac disease patients with hyperhomocysteinemia.

No.	Serum homocysteine (NV 5-15 μmol/L)	Serum B12 vitamin (NV 190-66 pg/mL)	Serum folic acid (NV 2-14 ng/mL)	Genetic mutation
1	14	365	10
2	13.5	267	4.7
3	15	369	6
4	20	356	1
5	13	174	5	MTHFR (het)
6	21	311	1.5
7	15	354	12
8	44	293	5.2	MTHFR (hom)
9	15	493	6	MTHFR (hom)
10	17	333	1	MTHFR (het)
11	19	383	3
12	17	198	2
13	15	400	3	MTHFR (het)
14	27	720	4	MTHFR (hom)
15	13	699	3	MTHFR (hom)
16	21	246	2.4
17	18	265	5.1
18	19	282	1	Prothr (het)
19	20	457	3	MTHFR (hom)
20	14	555	0.5
21	13	291	2
22	23	280	6
23	14	329	1	MTHFR (het)
24	17	684	10
25	19	566	5.6
26	20	188	2
27	20.5	154	3
28	20.5	216	2	MTHFR(het)
29	16	164	13	MTHFR (het)
30	25	555	8
31	149	150	2	MTHFR (hom)
32	31	385	2	MTHFR (hom)

het: Heterozygosis; hom: Homozygosis; MTHFR: N5-N10-metil tetrahydrofolate reductase.

Serum B12 vitamin levels were low in 5 (15.6%) patients and serum folate levels were low in 6 (19.0%) patients. No correlation (Spearman’s test) was found between serum homocysteine and age (r = 0.10, P = 0.58), gender (r = 0.66, P = 0.07), onset of symptoms (r = -0.06, P = 0.75), vitamin B12 (r = -026, P = 0.14), folic acid (r = 0.05, P = 0.75), and histological grading (r = -0.01, P = 0.9). Moreover, no correlation was also found between histological grading, vitamin B12 (r = -0.10, P = 0.56) and folic acid (r = -0.2, P = 0.3) values.

Clinical findings

Concerning vascular pathology, one patient with heterozygosis for MTHFR mutation and moderate hyperhomocysteinemia had myocardial infarction, whereas the single patient with severe hyperhomocysteinemia underwent coronary angiography for atypical chest pain, but no evidence of vessel pathology was found. No patient in this series had episodes of venous or arterial thrombosis, or any stroke episodes.

DISCUSSION

Our findings show that hyperhomocysteinemia is relatively frequent in patients with CD, being present in about 20% of the patients in our series. Hyperhomocysteinemia might represent a link between undiagnosed gluten-sensitive enteropathy and some of its complications[16]. Interestingly, these results were similar to those obtained in an overlapping geographic area, which showed the presence of hyperhomocysteinemia in about 20% of newly diagnosed CD patients compared to about 6% of controls[17].

Hyperhomocysteinemia may be due to genetic factors, with CBS deficiency being considered the most common genetic cause[5,17], or from acquired folate and vitamin B12 deficiencies[18,19]. A homozygous deficiency of MTHFR, the vitamin B12 dependent enzyme for the remethylation of homocysteine to methionine, may cause hyperhomocysteinemia and it has a worse prognosis than CBS deficiency for the absence of an effective therapy[20]. Moreover, treatment with a gluten-free diet and folic acid in CD patients with MTHFR variants does not consistently improve hyperhomocysteinemia[21].

Thus, CD (in which malabsorption of folate and vitamin B12 is common[22]) might lead to increased cardiovascular risks due to an increase of secondary (acquired) hyperhomocysteinemia, further aggravated by the possible presence of genetic abnormalities responsible for hyperhomocysteinemia. However, notwithstanding the relative frequency of hyperhomocysteinemia in our CD patients, this was almost always of moderate entity, with only one patient displaying high levels. Interestingly, the only patient to have a cardiovascular event (myocardial infarction) had relatively low levels of hyperhomocysteinemia and presented heterozygous mutations of MTHFR. No CBS mutations were found in our series. Only one mutation of the prothrombin gene was found, and this is in line with the paucity of reports of such mutations in CD patients[23].

In conclusion, at present it seems unnecessary to systematically investigate CD for the presence of hyperhomocysteinemia; conversely, a serological screening for CD in patients with hyperhomocysteinemia, cardiovascular events and vitamin deficiency could be considered, especially because adult CD patients may display only a few to no intestinal symptoms[24,25], and the onset of the disease may rarely be due to a thrombotic event[26-28].

COMMENTS

Background

Venous thrombosis has been reported in patients with celiac disease (CD). Since this might be related to hyperhomocysteinemia, a risk factor for vascular disease, we investigated the prevalence of hyperhomocysteinemia in a series of adult celiac patients.

Research frontiers

An increased prevalence of hyperhomocysteinemia in CD might lead to increased cardiovascular risk.

Innovations and breakthroughs

To date, most data on this topic originates from single reports, and only one other study investigated systematically celiac patients.

Applications

It appears that, given the low prevalence of hyperhomocysteinemia in celiac patients, it is unnecessary to screen systematically patients; this is useful information in terms of sanitary expenses.

Peer review

The authors evaluated in a cohort of 165 CD patients the presence of hyperhomocysteinemia during a period of time of 10 years. They showed that seems unnecessary to investigate systematically CD for the presence of hyperhomocysteinemia. Their work could contribute to the epidemiologic information of the CD in the Italian population.