Haapalahti, Mila; Kulmala, Petri; Karttunen, Tuomo J; Paajanen, Laura; Laurila, Kaija; Mäki, Markku; Mykkänen, Hannu; Kokkonen, Jorma
Department of Pediatrics, Oulu University Hospital, Oulu, Finland; Department of Pathology, University of Oulu, Oulu, Finland; Foundation for Nutrition Research; the Pediatric Research Center, Medical School, University of Tampere, Tampere, Finland; Department of Pediatrics, Tampere University Hospital, Tampere, Finland; and the Department of Clinical Nutrition, University of Kuopio, Kuopio, Finland
Received September 13, 2004; accepted December 15, 2004.
Address correspondence and reprint requests to Mila Haapalahti, University of Oulu, Department of Pediatrics, P.O. Box 5000, FIN-90014 University of Oulu, Finland. (e-mail: firstname.lastname@example.org).
This study was supported by the Juho Vainio Foundation, Finland; the Alma and K.A Snellman Foundation, Oulu, Finland; and the Pediatric Research Foundation, Helsinki, Finland.
The classic form of celiac disease (CD) in children typically presents as a malabsorption syndrome with failure to thrive. However, in the last few decades the pattern of childhood CD seems to have changed towards clinically milder features and older age groups (1-4). Currently, a typical patient may only have mild or no abdominal symptoms (5). Patients with CD may have extra-intestinal effects in the skin, nervous system and liver, and if untreated CD may be associated with an increased risk for many autoimmune diseases (6).
Adults with untreated CD have abnormalities in iron, folate and vitamin B12 status with decreased body weight, fat mass and bone mineral density (7-11). These changes in body composition have also been found in untreated child and adolescent celiac patients (12-15). Silent cases of CD have been shown to have decreased bone mineral density (16-17), but it is not known whether the untreated subjects have also iron, folate and other vitamin deficiencies.
It has been suggested that nutritional status associates with the degree of mucosal injury, as patients with severe villous atrophy have poorer nutritional status than do those with milder changes in the small bowel. Kemppainen et al. (18) found a positive correlation between serum ferritin and erythrocyte folate concentrations and grade of villous atrophy in adults with symptomatic celiac disease, but Dahele and Gosh (19) found no association between vitamin B12 concentration and the grade of villous atrophy in patients with partial/subtotal villous atrophy.
We specifically wanted to elucidate whether screen-detected CD cases have signs of nutritional abnormalities. This knowledge is important when considering the need for more active case finding of the disease. In addition we were interested in the association between nutritional status and grade of mucosal injury. The present study is a part of the recent population study on the prevalence of celiac disease in Finnish children (20).
MATERIALS AND METHODS
The study group was drawn from a cohort (n = 3654, median age, 12 years; age range, 7 to 16 years) of healthy schoolchildren living in northern Finland, collected for a Study of Risk Factors for Type 1 Diabetes in 1994 (21). Seven years later their sera were studied by immunoglobulin A and immunoglobulin G-class transglutaminase (tTG) and endomysial (EMA) antibodies, and 56 (1.5%) tTG- or EMA-positive individuals were detected (20). By 2001, 12 biopsy-proven celiac patients had already been diagnosed. The remaining 44 antibody-positive subjects who had not been diagnosed with CD were invited to undergo small-bowel biopsy and antibody testing. Thirty-six (82%) agreed to undergo biopsy, and 27 had mucosal villous atrophy with crypt hyperplasia typical of CD. After excluding one subject who had already started the gluten-free diet, the final CD group consisted of 26 patients (18 females, eight males; median age, 19 years; age range, 16 to 25 years). These subjects all had a normal gluten-containing diet and none of them used vitamin or mineral supplements.
The control subjects were selected from the same cohort as the CD subjects (21) via screening with a questionnaire sent to 1010 subjects aged 16 to 21 years. In all, 149 of the 829 responders (82%) fulfilled the criteria for healthy control subjects reporting no gastrointestinal symptoms and having no diagnosed diseases, and 29 (16 females, 13 males; median age, 18 years; age range, 16 to 21 years) were willing to participate in the investigations. In 1994 their tTG and EMA were negative, and in 2003 tTG was also negative (EMA was not evaluated in 2003).
All celiac patients underwent upper gastrointestinal endoscopy, gave blood samples for measurement of tTG, EMA and the nutritional status and completed a questionnaire on gastrointestinal symptoms. At the control visit within 4 to 6 months of the diagnosis, the presence of tTG or EMA and nutritional status by the previously described serum tests was checked. Dietary compliance and symptoms were also interviewed by a physician and dietitian. The control subjects had the same tests for nutritional status than the CD subjects.
Upper gastrointestinal endoscopy was performed with an Olympus GIF-IT140 endoscope (Olympus, Tokyo, Japan). Biopsy samples were taken from the duodenal bulb and mid-duodenum, two biopsies from each site. Formalin-fixed hematoxylin and eosin-stained biopsy specimens were examined under light microscopy using morphometric techniques by a pathologist blinded to any clinical data.
Celiac Disease Diagnosis
The celiac disease diagnosis was confirmed according to the ESPGHAN criteria (22). A ratio of villous height to crypt depth less than 2 (i.e., villous atrophy with crypt hyperplasia) was considered be indicative of celiac disease. Serum endomysial antibodies were detected using an indirect immunofluorescence method with human umbilical cord as antigen. Typical fluorescence pattern in sera diluted in 1 to 5 or more were considered positive. Tissue transglutaminase antibodies were performed with the Celikey assay (Pharmacia Diagnostics, Uppsala, Sweden) in accordance with the manufacturer's instructions. The cut-off for positivity was 5 U/mL. Frozen small-intestinal biopsy samples were stained for HLA-DR expression and γδ+ T-cell receptor-bearing intraepithelial lymphocytes; their density was counted as described previously (24).
The nutritional status was evaluated by serum ferritin and transferrin receptor, whole blood folate, serum pre-albumin and serum vitamin B12-vitamin concentrations. In control subjects serum transferrin receptor and vitamin B12 were analyzed from plasma samples, but all other analyses were made from serum samples. Anthropometrical status was evaluated by body weight, height and body mass index (BMI). According to the reference values of Oulu University Hospital, serum ferritin <5 μg/L in females and <20 μg/L in males, whole blood folate <85 nmol/L, serum pre-albumin <0.23 g/L in females and <0.24 g/L in males and serum vitamin B12-vitamin <180 pmol/L were considered as low concentrations. Serum transferrin receptor was considered to be elevated if >2.3 mg/L and serum transferrin receptor-ferritin index (TfR-F Index) was considered to be abnormal at values >1.8 (25). Subjects aged ≤19 years were considered underweight if their body weight was ≤−15% of the average body weight of the Finnish growth charts for children and adolescents (the Pediatric Research Foundation, Helsinki, Finland). The subjects ≥19 years were considered underweight if their BMI was <18.5 kg/m2. The cut-off BMI for overweight was 25 kg/m2 in girls ≥16 years and in boys ≥17 years, and the cut-off BMI of the 16 year-old boys was 24 kg/m2 (26).
The data was analyzed by SPSS 11.5 for Windows (SPSS, Chicago, IL). Differences in the nutritional parameters between the CD and control groups were analyzed by independent samples Student's t-test or Mann-Whitney U-test. Comparisons between bulbus and mid-duodenal biopsies were made by the paired samples Student's t-test and associations between the nutritional parameters by Pearson or Spearman correlation tests. The changes in the nutritional status on the gluten-free diet were analyzed by the paired samples Student's t-test or Wilcoxon's signed rank test.
The Ethical Committee of the Faculty of Medicine, University of Oulu, approved the original study protocol in 1994 and in 2001. The subjects were informed of the positive serological findings and were asked to undergo upper gastrointestinal endoscopy to obtain mucosal biopsies for the definitive diagnosis of CD.
The control subjects had a similar female-male ratio and anthropometry, but significantly different median age (19 versus 17 years; Mann-Whitney U test P = 0.001) than, the 149 control subjects. CD and control groups had similar female-male ratio (2.25 versus 1.2; x2 = 1.1; P = 0.284) and median age (19 versus 18 years; Mann Whitney U-test, P = 0.06).
At the time of diagnosis, 18 of the 26 CD patients (69%) reported having some gastrointestinal symptoms. Heartburn was reported by 10 (38%), abdominal pain by eight (31%), diarrhoea/loose stools by six (23%) and constipation by four (15%) subjects. The remaining eight (30%) subjects reported no gastrointestinal symptoms.
In the histologic analysis of the duodenal samples of the celiac subjects, eight (31%) had partial villous atrophy, five (19%) had subtotal villous atrophy and 13 (50%) had total villous atrophy in the mid-duodenum; the respective percentages in the duodenal bulbus were 12%, 8% and 81%. Villous height and villous height/crypt depth ratio were significantly lower in bulbus than in the mid-duodenum (Table 1); two thirds (67%) of the subjects had a lower villous-crypt ratio in bulbus than in mid-duodenum.
Nutritional Status at Time of Diagnosis
The subjects with CD had significantly lower whole blood folate, serum pre-albumin and serum ferritin, concentrations, and higher serum transferrin receptor concentration and TfR-F Index than the control subjects (Table 2). Celiac patients had greater risk to have low prealbumin (OR 4.8, 95% CI 1.6-14.9). At least one abnormality (low serum ferritin, whole blood folate or serum vitamin B12-vitamin, or high TfR-Index) was found in 12 CD subjects (46%), and in nine (31%) control subjects. Vitamin B12 (Spearman rank correlation, r = −0.6; P = 0.004) and whole folate concentrations (Pearson r = −0.4; P = 0.051) correlated with TfR-F Index.
Body mass index was not different in the females of the CD and control groups (22 versus 22 kg/m2) or in the males of the respective groups (25 versus 24 kg/m2). None of the subjects older than 19 years of age had BMI <18.5 kg/m2 and only one control subject aged 17 years had body weight <−15% as compared with the growth chart. Conversely, six CD subjects and seven control subjects aged >19 years had BMI >25 kg/m2. The females with CD were shorter (162 versus 167 cm; P = 0.018) than the control females, whereas no difference was found in the males (178 versus 177 cm; not significant) (Table 3).
Mucosal Injury and Nutrition
Villous height, crypt depth and their ratio in the duodenal bulb and mid-duodenum showed no significant association with the nutritional parameters (data not shown). Neither did the titer of tTG show any association with the villous or crypt measures. However, titer of tTG associated with whole blood folate (Spearman r = −0.5; P = 0.016) and with TfR-F Index (Spearman r = 0.4; P = 0.05).
Symptoms and Nutritional Status on the Gluten-free Diet
All except one of the 26 CD patients started to follow the gluten-free diet as advised, but three cases gave it up. Of those 22 subjects following the diet, 15 (65%) reported improvement in their gastrointestinal symptoms or general well being (less tired/disappearance of dizziness). At the control visit three of the subjects reporting initially no gastrointestinal symptoms reported positive changes in their bowel function/abdominal pain and one subject reported disappearance of dizziness.
On the gluten-free diet, significant improvements in blood folate (n = 17) and serum pre-albumin (n = 9), vitamin B12 (n = 20), TfR Index (n = 16) and ferritin (n = 19) were observed. Eight subjects had used multivitamin and one had used iron supplement during the diet.
The main finding of this study is that screen-detected adolescent CD patients differ from the healthy control subjects by having lower whole blood folate and serum ferritin and prealbumin concentrations, but higher serum transferrin receptor concentration and TfR-F Index. Nearly half of the celiac patients had at least one abnormality in nutritional markers (low serum ferritin, whole blood folate or serum vitamin B12-vitamin or high TfR-F Index). The CD patients had similar weight and BMI as the controls, but females with CD were shorter than the control subjects.
Similarly to Tursi et al., we found depleted iron storages in one third of the celiac patients (27). One third of our patients also had elevated TfR-F Index, indicating a progression to anemia. In identifying mild iron deficiency, especially in inflammatory disorders, the transferrin receptor and TfR-F Index are considered to be more reliable than ferritin (28). However, true deficiencies are difficult to demonstrate because of the complex association between ferritin, folic acid and vitamin B12. Serum ferritin is able to catabolize folate and thus decrease the intracellular folate concentration (29). In addition, folate requires vitamin B12 for its activation, and low folate concentrations may attributable to vitamin B12-deficiency. It has been suggested that vitamin B12 deficiency is not common among the celiac patients because vitamin B12 is absorbed in the ileum, which is thought to be spared from injury in CD. However, Dahele and Gosh (30) reported that 41% of adults with untreated CD were vitamin B12-deficient (intrinsic factor antibodies being negative in all patients); only one third of these had concomitant folate deficiency.
The proximal small bowel is an important site for absorption of nutrients. In untreated celiac disease, there is a loss of brush border proteins and enzymes needed in absorption of folic acid. Low folate status is therefore common in these patients. Folate deficiency increases the risk of cardiovascular diseases and neural tube defects of the embryo during pregnancy. In our study, only one third of the CD patients had low folate status, suggesting that the damaged part of the bowel segment was probably short, leaving enough healthy mucosa for nutrient absorption. This is further supported by the observation that most CD patients had milder villous atrophy in the mid-duodenum than in the proximal duodenum (bulbus). Progression of mucosal injury and nutritional deficiencies in celiac disease seems to be slow, as our patients were seropositive up to 7 years before the biopsy.
However, in celiac disease mucosal damage is suspected to be more severe in the proximal than in distal small intestine (31-33) and may be patchy (34); the biopsies have been recommended to obtain distally from the duodeno-jejunal region. This diagnostic practice can lead to underestimation of the diagnosed cases, especially if specimens are obtained by a capsule technique. In agreement with the recommendation by Vogelsang et al. (35), our findings support the practice of taking biopsies with an endoscope from the duodenal bulb and from the descending duodenum. This practice will maximize the likelihood of detecting the progression of villous atrophy as early as possible and starting of the gluten-free diet. It has been shown that gluten-free diet improves the quality of life also in silent/subclinical celiac patients (36,37).
We found no association between the grade of villous atrophy and the measures of nutritional status. Biopsies serve only as a measure of abnormality at the definite point of their origin, and they do not indicate the extent of the abnormality, which may better correlate with the severity of malabsorption. Validated injury index should be created for estimation the degree of mucosal damage and for development of adequate nutritional treatment in untreated patients. Even though no associations between the villous/crypt measurements and nutritional markers were found, we observed an interesting association between the titer of tTG and the serum iron and whole blood folic acid concentrations. The role of tTG in the pathogenesis of mucosal injury and its dysfunction is not fully understood, but it has been shown that serum immunoglobulin A from celiac patients inhibits crypt epithelial cell differentiation in vitro (38), suggesting a possible connection between immunoglobulin A class autoantibodies and inhibited nutrient absorption. It has been also shown that celiac immunoglobulin A targets jejunal tTG early in disease development, even before endomysial antibodies are present in the circulation (39).
In conclusion, one third of screen-detected adolescent celiac patients have abnormal folate or iron status. To prevent the progression of nutritional deficiencies, early diagnosis and treatment by gluten-free diet is recommended for celiac patients with subclinical/silent celiac disease.
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