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Hematinic deficiencies and anemia in gastric parietal cell antibody-positive and -negative oral submucous fibrosis patients

Background/purpose Previous studies showed that approximately 13–15% of male oral submucous fibrosis (OSF) patients are serum gastric parietal cell antibody (GPCA)-positive. This study assessed whether serum GPCA or OSF itself was a significant factor that caused hematinic deficiencies and anemia statuses in GPCA-positive or GPCA-negative OSF patients (GPCA+/OSF and GPCA−/OSF patients). Materials and methods The frequencies of macrocytosis (mean corpuscular volume or MCV ≧ 100 fL) and blood hemoglobin (Hb), iron, vitamin B12 and folic acid deficiencies were determined and compared between any two of the four groups of 149 male OSF, 23 male GPCA+/OSF, and 126 male GPCA−/OSF patients and 149 age-matched male healthy control subjects. Results All three groups of OSF patients (including 149 OSF, 23 GPCA+/OSF, and 126 GPCA−/OSF patients) had a significantly higher frequency of Hb, vitamin B12, or folic acid deficiency and of macrocytosis than 149 healthy control subjects (all P-values < 0.05). The 23 GPCA+/OSF patients did have a significantly lower mean serum vitamin B12 level and a significantly higher MCV or frequency of vitamin B12 deficiency than the 126 GPCA−/OSF patients. Two of the 23 GPCA+/OSF patients had pernicious anemia. Of the 126 GPCA−/OSF patients, 6 had macrocytic anemia, 2 had iron deficiency anemia, and 4 had thalassemia trait-induced anemia. Conclusion We conclude that OSF itself does play a significant role in causing hematinic deficiencies and anemia in OSF, GPCA+/OSF, and GPCA−/OSF patients. The serum GPCA is the major factor that causes vitamin B12 deficiency, macrocytosis, and pernicious anemia in GPCA+/OSF patients.


Introduction
Oral submucous fibrosis (OSF) is a chronic progressive scaring oral mucosal disease characterized by juxtaepithelial inflammatory cell infiltration followed by a marked deposition of collagen in the lamina propria and submucosa of the oral mucosa and superficial muscle layer. The areca nut is the main etiological factor in OSF. 1,2 Areca nut contains alkaloids, flavonoids, and copper. Alkaloids, mainly arecoline and arecaidine, are found to stimulate fibroblasts to produce collagen. Flavonoids (tannins and catechins) can inhibit collagenase, stabilize the collagen fibrils (through an increase in cross-linking in the collagen fibrils), and in turn render collagen fibrils resistant to degradation by collagenase. The high concentration of copper in areca nut has been reported to stimulate lysyl oxidase activity, an enzyme essential to the final cross-linking of collagen fibers. 1,2 Therefore, areca nut ingredients can cause increased collagen deposition in the oral tissue, leading to OSF.
The localized mucosal inflammation caused by areca nut leads to the recruitment of activated T-cells and macrophages that can secrete transforming growth factor-b (TGFb), a pivotal regulator controlling collagen production and degradation. 1,2 TGF-b can increase the collagen production by activation of procollagen genes (resulting in enhanced production of procollagen), elevation of procollagen proteinase levels (leading to conversion of procollagens to collagen fibrils), and upregulation of lysyl oxidase activity (causing an increased production of insoluble form of collagen). Furthermore, TGF-b can inhibit collagen degradation by activating the tissue inhibitor of matrix metalloproteinase (TIMP) genes (resulting in an increased production of TIMPs that inhibit the activated collagenase) and plasminogen activator inhibitor (PAI) genes (leading to an elevated production of PAI that blocks the conversion of plasminogen to plasmin and in turn block the conversion of procollagenase to active collagenases). 1,2 Thus, TGF-b can not only increase collagen production but also decrease collagen degradation, finally resulting in development of OSF.
In our oral mucosal disease clinic or dental clinic, patients with atrophic glossitis, burning mouth syndrome, oral lichen planus, and recurrent aphthous stomatitis are frequently encountered and patients with OSF or specific jaw bone lesions are sometimes seen. 3e21 For these particular groups of patients, complete blood count as well as serum iron, vitamin B12, folic acid, gastric parietal cell antibody (GPCA), thyroglobulin antibody (TGA), and thyroid microsomal antibody (TMA, also known as thyroid peroxidase antibody, TPO) levels were frequently examined to assess whether these patients have anemia, hematinic deficiencies, and serum GPCA, TGA, or TMA positivity. 3e21 Because the majority of OSF patients can not tolerate spicy food, suffer from burning sensation of oral mucosa, and have a certain degree of mouth opening limitation, these functional impairments may affect normal food intake and lead to nutritional deficiencies. 22,23 Our previous study demonstrated high frequencies of vitamin B12 and folic acid deficiencies and GPCA positivity in OSF patients. 22,23 In this study, 149 male OSF patients were retrospectively collected from our oral mucosal disease clinic. The mean corpuscular volume (MCV) and blood hemoglobin (Hb), iron, vitamin B12, folic acid, and GPCA levels were examined and compared with the corresponding data in 149 age-matched male healthy control subjects. We tried to assess whether serum GPCA or OSF itself was a significant factor that caused hematinic deficiencies and anemia statuses in GPCA-positive or GPCA-negative OSF patients (GPCA þ /OSF and GPCA À /OSF patients).

Subjects
This study included 149 male OSF patients (mean age 39.2 AE 12.3 years, range 18e62 years) and 149 age-matched (AE2 years of each patient's age, mean age 40.1 AE 12.6 years, range 19e63 years) male healthy control subjects. All the OSF patients and healthy control subjects were seen consecutively, diagnosed, treated, and selected in oral mucosal disease clinic and dental clinic of National Taiwan University Hospital (NTUH) from July 2007 to July 2016. Clinical diagnosis of OSF was made when patients showed characteristic features of OSF, including intolerance to spicy foods, blanching and stiffness of the oral mucosa, fibrous bands in the buccal mucosa, and progressive inability to open the mouth. 22,23 Some OSF patients might also have burning sensation of the oral mucosa, xerostomia, the presence of vesicles or ulcers on the oral mucosa, depapillation of the tongue, and impaired tongue mobility. 22,23 Incisional biopsy of buccal mucosa was taken from 43 of 149 OSF patients. Histological diagnosis was made based on the examination of hematoxylin and eosinstained tissue sections. The histological criteria for a diagnosis of OSF were: (1) atrophic epithelium with either parakeratosis or hyperkeratosis, and (2) moderate to marked fibrosis or hyalinization in the lamina propria, submucosa, and superficial muscle layer. 22,23 The oral mucosal sites (soft palate, retromolar area, buccal mucosa, labial mucosa, floor of the mouth, and tongue) of involvement and the maximum mouth opening (MMO) of OSF patients were recorded. The severity of OSF was determined according to the sites of involvement or MMO; the more the sites involved and the less the MMO, the more severe the OSF. No mild OSF patient was included in the study, because all our OSF patients had at least three sites of involvement. OSF patients with severe systemic diseases, autoimmune diseases (such as systemic lupus erythematosus, rheumatoid arthritis, Sjogren's syndrome, pemphigus vulgaris, and cicatricial pemphigoid), inflammatory diseases, malignancy, or recent surgery were excluded. Healthy control subjects had dental caries, pulpal disease, malocclusion, or missing of teeth but did not have any oral mucosal or systemic diseases. None of our OSF patients had taken any prescription medication at least 3 months before entering the study.
All the 149 OSF patients had areca quid chewing habit; they chewed 1e150 (mean, 28) quids per day for 1e39 (mean, 14.2) years. One hundred and two (68.5%) OSF patients claimed that they swallowed some of the "juice" of areca quid during the chewing process. One hundred and thirty-five (90.6%) of the 149 OSF patients were smokers; they smoked 6e60 (mean, 23) cigarettes per day for 2e39 (mean, 18.3) years. Ninety-nine (66.4%) of the 149 OSF patients were drinkers; they drank 46e6528 (mean, 942) g of pure alcohol per week for 1e33 (mean, 13.6) years. Of the 149 healthy control subjects, none were areca quid chewers (one quid or more daily for at least 1 year), 39 (26.2%) were smokers (one cigarettes or more per day for at least 1 year), and 29 (19.5%) were drinkers (drinking more than 3 days a week).
The blood samples were drawn from all OSF patients and healthy control subjects for measurement of complete blood count as well as serum iron, vitamin B12, and folic acid concentrations. In addition, the presence of GPCA in sera of OSF patients and healthy control subjects was also checked. All OSF patients and healthy control subjects signed the informed consents before entering the study. This study was reviewed and approved by the Institutional Review Board at the NTUH.
Determination of complete blood count as well as serum iron, vitamin B12, and folic acid concentrations The complete blood count as well as serum iron, vitamin B12, and folic acid concentrations were determined by the routine tests performed in the Department of Laboratory Medicine of NTUH. 3e15, 17,18 Determination of serum gastric parietal cell antibody level The serum GPCA level was detected by the indirect immunofluorescence technique with rat stomach as a substrate as described previously. 6e8 Sera were scored as positive when they produced fluorescence at a dilution of 20-fold or more. 6e8

Statistical analysis
Comparisons of the MCV and mean blood levels of Hb, iron, vitamin B12, and folic acid between 149 OSF, 23 GPCA þ / OSF, or 126 GPCA À /OSF patients and 149 age-matched healthy control subjects as well as between 23 GPCA þ / OSF and 126 GPCA À /OSF patients were performed by Student's t-test. The differences in frequency of blood Hb, iron, vitamin B12 or folic acid deficiency or of macrocytosis (MCV S 100 fL) between any two of the four groups of 149 OSF, 23 GPCA þ /OSF, and 126 GPCA À /OSF patients and 149 healthy control subjects were compared by chi-square test. The result was considered to be significant if the P-value was less than 0.05.

Results
The mean MMO of 149 OSF patients were 30.1 AE 8.5 mm; 75 had MMO & 31 mm and 74 had MMO between 31 mm and 42 mm. The soft palate, retromolar area, and buccal mucosa were the three sites where were involved by OSF in Table 1 Comparisons of the mean corpuscular volume (MCV) and mean blood concentrations of hemoglobin (Hb), iron, vitamin B12, and folic acid between any two of the four groups of 149 oral submucous fibrosis (OSF) patients, 23 gastric parietal cell antibody (GPCA)-positive OSF patients (GPCA þ /OSF patients), 126 GPCA-negative OSF patients (GPCA À /OSF patients), and 149 healthy control subjects. There were 23 GPCA þ /OSF patients and 126 GPCA À /OSF patients in this study. Comparisons of the MCV and mean blood concentrations of Hb, iron, vitamin B12, and folic acid between any two of the four groups of 149 OSF, 23 GPCA þ /OSF, and 126 GPCA À /OSF patients, and 149 healthy control subjects are shown in Table 1. We found that both 149 OSF and 126 GPCA À /OSF patients had a significantly lower mean serum vitamin B12 (P < 0.001) or folic acid level (P < 0.001) than healthy control subjects (Table 1). Moreover, the 23 GPCA þ /OSF had a significantly lower mean blood Hb, iron, vitamin B12, or folic acid level and a significantly higher mean MCV level (all P-values < 0.001) than healthy control subjects. In addition, the 23 GPCA þ / OSF also had a significantly lower mean serum vitamin B12 level (P Z 0.001) and a significantly higher mean MCV level (P Z 0.029) than healthy control subjects (Table 1).

Group
Patient number (%)  33 In this study, the 23 GPCA þ /OSF patients did have a significantly lower mean serum vitamin B12 level and a significantly higher MCV or frequency of vitamin B12 deficiency than the 126 GPCA À /OSF patients. Because GPCA þ / OSF patients may have partial or total destruction of their gastric parietal cells which can secrete intrinsic factors to help absorb vitamin B12 from the ileum, 27,28 our findings indicate that the serum GPCA-positivity can explain why 23 GPCA þ /OSF patients have a significantly lower mean serum vitamin B12 level and a significantly higher MCV or frequency of vitamin B12 deficiency than 126 GPCA À /OSF patients.
Regarding the serum iron level, Ramanathan 34 reported that 5 (63%) of the 8 OSF patients have a depressed serum iron level below the normal value. Rajendran et al. 35 found a significantly lower serum iron level in 50 OSF patients than in 50 age-and sex-matched control subjects. Anuradha and Devi 36 attributed the deficiencies of vitamin C and iron in 36 OSF patients to the increased utilization of these two substances in collagen synthesis. Tadakamadla et al. 37 also discovered a significantly lower mean serum iron level in 50 OSF patients than in 50 age-and sexmatched control subjects. In this study, there were no significant differences in the mean serum iron level and frequency of iron deficiency between 149 OSF or 126 GPCA À /OSF patients and 149 healthy control subjects. However, the 23 GPCA þ /OSF patients did have a significantly lower mean serum iron level than the 149 healthy control subjects. Because GPCA þ /OSF patients may have partial or total destruction of their gastric parietal cells which can secrete HCl to help absorb iron from the small intestine, 27,28 the lack of HCl secretion can explain why GPCA þ /OSF patients have a significantly lower mean serum iron level than healthy control subjects.
Ramanathan 34 reported that 10 (77%) of the 13 OSF patients have iron deficiency anemia. The present study showed a significantly greater frequency of anemia in 149 OSF patients (9.4%) than in 149 healthy control subjects (0%). Wahi et al. 33 discovered that 6% of the 70 male OSF patients and 11% of the 34 female OSF patients had anemia, but the frequency of anemia did not differ significantly from that of the control subjects. A South African study also found no significant difference in the prevalence of iron deficiency anemia between OSF patients and the general population. 38 In a South African study, Seedat reported that serum vitamin B12 and folate levels of OSF patients are within normal limits. 38 However, Ramanathan 34 found folic acid deficiency in 6 of the 6 OSF patients. This study also discovered that 76 (51.0%) and 61 (40.9%) of OSF patients had vitamin B12 and folic acid deficiencies, respectively. Only 17 (22.4%) of the 76 vitamin B12-deficient OSF patients were GPCA-positive. In these 17 GPCA þ /OSF patients, the vitamin B12 deficiency could be attributed to the presence of GPCA in sera, because GPCA can induce destruction of gastric parietal cells and in turn result in failure of intrinsic factor production. 27,28 Lack of intrinsic factor finally leads to failure of vitamin B12 absorption. 29,30 Furthermore, the vitamin B12 deficiency in the other 59 GPCA À /OSF patients may be due to other causes including inadequate intake or malabsorption of vitamin B12, the presence of anti-intrinsic factor antibodies, or transcobalamin II deficiency, etc. 30 Folic acid deficiency may be resulted from poor nutritional intake, malabsorption, hepatobiliary dysfunction, increased folate catabolism, and medication (e.g., methotrexate, 5-fluoro-uracil, phenytoin, etc.). 28,30 In the present study, we suggest that the folic acid deficiency is probably due to poor or insufficient intake of folic acidcontaining diet because OSF patients usually have functional impairment in food intake and chewing due to the limited mouth opening. Further studies are needed to explore the real causes resulting in vitamin B12 and folic acid deficiencies in OSF patients.  Our previous study demonstrated that 14.7% of 109 and 13.2% of 68 male OSF patients are GPCA-positive. 22,23 Similar result was found in this study that showed serum GPCA-positivity in 23 (15.4%) of 149 male OSF patients. In this study, 2 (8.7%) of 23 GPCA-positive, 2 (2.6%) of 76 vitamin B12-deficient, and 2 (16.7%) of 12 macrocytosis male OSF patients had pernicious anemia by the strict WHO definitions. 6e8 Our previous studies demonstrated that 12.9% of 124 GPCA-positive, 18.9% of 90 vitamin B12deficient, and 16.7% of 60 macrocytosis male and female patients with oral mucosal diseases had pernicious anemia by the strict WHO definitions. 6e8 The discrepancies in parts of the data may be due to the gender (male vs. both sexes), age (middle-aged OSF patients vs. elderly patients with other oral mucosal diseases), and disease (OSF vs. atrophic glossitis, burning mouth syndrome, oral lichen planus, or recurrent aphthous stomatitis) differences. 6e8 Our results demonstrated that 14 (9.4%), 32 (21.5%), 76 (51.0%), 61 (40.9%), 12 (8.1%), and 23 (15.4%) of the 149 OSF patients had blood Hb, iron, vitamin B12, and folic acid deficiencies, macrocytosis, and serum GPCA positivity, respectively. All three groups of OSF patients (including 149 OSF, 23 GPCA þ /OSF, and 126 GPCA À /OSF patients) had a significantly higher frequency of macrocytosis or blood Hb, vitamin B12, or folic acid deficiency than healthy control subjects. Furthermore, the 23 GPCA þ /OSF patients had a significantly higher frequency of vitamin B12 deficiency than the 126 GPCA À /OSF patients. We conclude that OSF itself does play a significant role in causing hematinic deficiencies and anemia in OSF, GPCA þ /OSF, and GPCA À /OSF patients. The serum GPCA is the major factor that causes vitamin B12 deficiency, macrocytosis, and pernicious anemia in GPCA þ /OSF patients.