Anemia, hematinic deficiencies, and hyperhomocysteinemia in gastric parietal cell antibody-positive and -negative recurrent aphthous stomatitis patients

Abstract

Background/purpose

Our previous study found the serum gastric parietal cell antibody (GPCA) positivity in 12.8% of 430 recurrent aphthous stomatitis (RAS) patients. This study assessed whether GPCA-positive RAS (GPCA⁺RAS) patients had significantly higher frequencies of macrocytosis, anemia, hematinic deficiencies, and hyperhomocysteinemia than healthy control subjects or GPCA-negative RAS (GPCA⁻RAS) patients.

Materials and methods

The mean corpuscular volume, blood hemoglobin (Hb), and serum iron, vitamin B12, folic acid, homocysteine, and GPCA levels were measured and compared between any two of three groups of 55 GPCA⁺RAS patients, 375 GPCA⁻RAS patients, and 430 healthy control subjects.

Results

We found that 55 GPCA⁺RAS patients had significantly higher frequencies of macrocytosis, blood Hb and serum iron and vitamin B12 deficiencies, and hyperhomocysteinemia than 430 healthy control subjects (all P-values <0.001) and significantly higher frequencies of macrocytosis, serum vitamin B12 deficiency, and hyperhomocysteinemia than 375 GPCA⁻RAS patients (all P-values <0.05). Moreover, 375 GPCA⁻RAS patients had significantly higher frequencies of macrocytosis, blood Hb and serum iron, vitamin B12, and folic acid deficiencies, and hyperhomocysteinemia than 430 healthy control subjects (all P-values <0.005). Normocytic anemia (53.3%) and pernicious anemia (26.7%) were the two most common types of anemia in 15 anemic GPCA⁺RAS patients. Moreover, normocytic anemia (50.5%), iron deficiency anemia (26.9%), and thalassemia trait-induced anemia (10.8%) were the three most common types of anemia in 93 anemic GPCA⁻RAS patients.

Conclusion

GPCA⁺ RAS patients have significantly higher frequencies of macrocytosis, anemia, serum iron and vitamin B12 deficiencies, and hyperhomocysteinemia than healthy control subjects.

Introduction

Recurrent aphthous stomatitis (RAS) is a prevalent disorder of the oral mucosa that presents as repeated episodes of painful ulceration, primarily involving the nonkeratinized and freely movable oral tissues. According to its clinical appearance, RAS is generally categorized into minor, major, and herpetiform variants. The labial and buccal mucosa and the tongue are among the sites most commonly affected. Current evidence suggests that RAS has a complex, multifactorial pathogenesis in which T cell-mediated immune disturbances play a central role.1–6

Patients with RAS, atrophic glossitis, burning mouth syndrome, oral lichen planus, and oral submucous fibrosis constitute a substantial proportion of the cases managed in our oral mucosal disease clinic.1–4,7–52 In individuals with any of these five disorders, laboratory investigations commonly include a complete blood count and measurements of serum iron, vitamin B12, folic acid, homocysteine, gastric parietal cell antibody (GPCA), thyroglobulin antibody, and thyroid microsomal antibody (also known as antithyroid peroxidase or anti-TPO antibody). These examinations are useful for identifying anemia, hematinic deficiencies, hyperhomocysteinemia, and the presence of GPCA, thyroglobulin antibody, or thyroid microsomal antibody. 1–4,7–52 The resulting laboratory data provide clinically relevant information that can guide the management of these frequently encountered oral mucosal disorders.

For the present investigation, the previously reported cohort of 430 RAS patients was stratified according to serum GPCA status into 55 GPCA-positive RAS (GPCA⁺RAS) patients and 375 GPCA-negative RAS (GPCA⁻RAS) patients.4 We examined whether the 55 GPCA⁺RAS patients exhibited higher prevalences of macrocytosis, blood hemoglobin deficiency, serum iron deficiency, vitamin B12 deficiency, folic acid deficiency, and hyperhomocysteinemia than either the 430 healthy control subjects or the 375 GPCA⁻RAS patients. We also determined whether the 375 GPCA⁻RAS patients continued to show increased frequencies of these hematological and biochemical abnormalities compared with the 430 healthy control subjects.

Materials and methods

Subjects

A total of 430 RAS patients who had previously been reported were included in this study and were categorized according to their serum GPCA status into 55 GPCA⁺RAS patients (16 men and 39 women; age range, 24–90 years; mean age, 61.7 ± 12.5 years) and 375 GPCA⁻RAS patients (120 men and 255 women; age range, 18–91 years; mean age, 51.3 ± 16.6 years).4 For each RAS patient, one healthy individual matched for sex and age (within ±2 years) was selected, yielding a control group of 430 healthy control subjects (136 men and 294 women; age range, 18–91 years; mean age, 52.8 ± 16.5 years).4 All participants were consecutively recruited from the Department of Dentistry, National Taiwan University Hospital (NTUH) between July 2007 and June 2023. A diagnosis of RAS was established when patients had experienced at least one episode of oral ulceration per month over the preceding years.1–4 The detailed exclusion criteria for the RAS patients and the inclusion and exclusion criteria applied to the healthy control group have been reported previously.1–4

Venous blood samples were obtained from all study participants, including the 55 GPCA⁺RAS patients, 375 GPCA⁻RAS patients, and 430 healthy control subjects. Laboratory analyses included complete blood count and measurements of serum iron, vitamin B12, folic acid, homocysteine, and GPCA. Written informed consent was obtained from every participant before enrollment. The study protocol was approved by the Institutional Review Board of National Taiwan University Hospital (IRB No. 202402086RINC).

Determination of blood hemoglobin, iron, vitamin B12, folic acid, and homocysteine levels

The complete blood count, as well as serum iron, vitamin B12, folic acid, and homocysteine levels, were measured using routine tests conducted in the Department of Laboratory Medicine at the NTUH.1–4,7–52

Determination of serum gastric parietal cell antibody level

The serum GPCA level was measured using the indirect immunofluorescence technique, with the rat stomach serving as the substrate, as previously described.11–13 Sera were considered positive if they exhibited fluorescence at a dilution of 1:10 or more.

Statistical analysis

The mean corpuscular volume (MCV) and mean blood hemoglobin (Hb) and serum iron, vitamin B12, folic acid, and homocysteine levels were compared between any two of three groups of 55 GPCA⁺RAS patients, 375 GPCA⁻RAS patients, and 430 healthy control subjects by Student’s t-test. The differences in frequencies of microcytosis (defined as MCV <80 fL),28–30 macrocytosis (defined as MCV ≥100 fL),31–33 blood Hb and serum iron, vitamin B12, and folic acid deficiencies, hyperhomocysteinemia, and serum GPCA positivity were compared between any two of three groups of 55 GPCA⁺RAS patients, 375 GPCA⁻RAS patients, and 430 healthy control subjects by chi-square test. Moreover, comparisons of frequencies of macrocytic, normocytic or microcytic anemia between 55 GPCA⁺RAS patients and 375 GPCA⁻RAS patients were also performed by chi-square test. The result was considered to be significant if the P-value was less than 0.05.

Results

Comparisons of MCV and mean blood Hb and serum iron, vitamin B12, folic acid, and homocysteine levels between any two of three groups of 55 GPCA⁺RAS patients, 375 GPCA⁻RAS patients, and 430 healthy control subjects are shown in Table 1. Because men tended to have higher blood Hb and serum iron levels than women, these two mean levels were calculated separately for men and women. We found significantly lower mean blood Hb (for men and women) and serum iron (for women only), and vitamin B12 levels as well as significantly higher mean serum homocysteine level in 55 GPCA⁺RAS patients than in 430 healthy control subjects (all P-values <0.05, Table 1). Moreover, we also discovered significantly higher MCV, mean serum folic acid level, and mean serum homocysteine level in 55 GPCA⁺RAS patients than in 375 GPCA⁻RAS patients (all P-values <0.001, Table 1). Furthermore, 375 GPCA−RAS patients also had significantly lower MCV, mean blood Hb (for men and women) and serum iron (for men and women), vitamin B12, and folic acid levels than 430 healthy control subjects (all P-values <0.05, Table 1).

Table 1.

Comparisons of mean corpuscular volume (MCV) and mean blood concentrations of hemoglobin (Hb), iron, vitamin B12, folic acid, and homocysteine between any two of three groups of 55 gastric parietal cell antibody (GPCA)-positive recurrent aphthous stomatitis (RAS) patients (GPCA⁺RAS patients), 375 GPCA-negative RAS patients (GPCA⁻RAS patients), and 430 healthy control subjects.

Group	MCV (fL)	Hb (g/dL)		Iron (μg/dL)		Vitamin B12 (pg/mL)	Folic acid (ng/mL)	Homocysteine (μM)
		Men	Women	Men	Women
GPCA+RAS patients (n = 55)	91.8 ± 7.5	13.9 ± 1.8 (n = 16)	13.1 ± 1.2 (n = 39)	96.4 ± 43.2 (n = 16)	85.2 ± 33.1 (n = 39)	596.0 ± 310.0	15.9 ± 6.4	13.9 ± 15.5
aP-value	0.105	<0.001	<0.001	0.386	<0.001	0.021	0.065	<0.001
bP-value	<0.001	0.644	0.072	0.643	0.740	0.252	<0.001	<0.001
GPCA−RAS patients (n = 375)	87.1 ± 8.5	14.1 ± 1.6 (n = 120)	12.7 ± 1.3 (n = 255)	92.1 ± 33.6 (n = 120)	83.2 ± 35.3 (n = 255)	640.4 ± 261.6	12.6 ± 6.6	8.3 ± 6.5
aP-value	<0.001	<0.001	<0.001	0.004	<0.001	0.034	<0.001	0.364
cHealthy control subjects (n = 430)	90.8 ± 3.7	15.2 ± 0.7 (n = 136)	13.6 ± 0.7 (n = 294)	102.9 ± 26.1 (n = 136)	99.8 ± 28.7 (n = 294)	677.9 ± 238.0	14.3 ± 6.0	8.6 ± 2.0

^aComparisons of means of parameters between 55 GPCA⁺RAS or 375 GPCA⁻RAS patients and 430 healthy control subjects by Student’s t-test.

^bComparisons of means of parameters between 55 GPCA⁺RAS and 375 GPCA⁻RAS patients by Student’s t-test.

^cThe hematological data of 430 healthy control subjects were retrieved from our previous study.4

According to the World Health Organization (WHO) criteria, microcytosis of erythrocyte was defined as having MCV <80 fL,28–30 macrocytosis of erythrocyte was defined as having MCV ≥100 fL,31–33 and men with Hb < 13 g/dL and women with Hb < 12 g/dL were defined as having Hb deficiency or anemia.53 Furthermore, patients with the serum iron level <60 μg/dL,54 the serum vitamin B12 level <200 pg/mL,55 or the serum folic acid level <4 ng/mL56 were defined as having serum iron, vitamin B12 or folic acid deficiency, respectively. In addition, patients with the blood homocysteine level >12.6 μM (which was the mean serum homocysteine level of healthy control subjects plus two standard deviations) were defined as having hyperhomocysteinemis. By the above-mentioned definitions, 5.5%, 9.1%, 27.3%, 20.0%, 21.8%, 0.0%, and 27.3% of 55 GPCA⁺RAS patients and 13.9%, 2.4%, 24.8%, 24.0%, 4.8%, 2.9%, and 7.2% of 375 GPCA⁻RAS patients were diagnosed as having microcytosis, macrocytosis, blood Hb and serum iron, vitamin B12, and folic acid deficiencies, and hyperhomocysteinemia, respectively. We found that 55 GPCA⁺RAS patients had significantly higher frequencies of microcytosis, macrocytosis, blood Hb and serum iron and vitamin B12 deficiencies, and hyperhomocysteinemia than 430 healthy control subjects (all Pvalues< 0.001, Table 2) and significantly higher frequencies of macrocytosis, serum vitamin B12 deficiency, and hyperhomocysteinemia than 375 GPCA⁻RAS patients (all P-values <0.05, Table 2). Moreover, 375 GPCA⁻RAS patients had significantly higher frequencies of microcytosis, macrocytosis, blood Hb and serum iron, vitamin B12, and folic acid deficiencies, and hyperhomocysteinemia than 430 healthy control subjects (all P-values <0.005, Table 2).

Table 2.

Comparisons of frequencies of microcytosis (mean corpuscular volume or MCV <80 fL), macrocytosis (MCV ≥100 fL), blood hemoglobin and seerum iron, vitamin B12, and folic acid deficiencies, and hyperhomocysteinemia between any two of three groups of 55 gastric parietal cell antibody (GPCA)-positive recurrent aphthous stomatitis (RAS) patients (GPCA⁺RAS patients), 375 GPCA-negative RAS patients (GPCA⁻RAS patients), and 430 healthy control subjects.

Group	Patient number (%)
	Microcytosis (MCV <80 fL)	Macrocytosis (MCV ≥100 fL)	Hemoglobin deficiency (men <13 g/dL, women <12 g/dL)	Iron deficiency (<60 μg/dL)	Vitamin B12 deficiency (<200 pg/mL)	Folic acid deficiency (<4 ng/mL)	Hyperhomocysteinemia (>12.6 μM)
GPCA+RAS patients (n = 55)	3 (5.5)	5 (9.1)	15 (27.3)	11 (20.0)	12 (21.8)	0 (0.0)	15 (27.3)
aP-value	<0.001	<0.001	<0.001	<0.001	<0.001	NA	<0.001
bP-value	0.126	0.028	0.819	0.629	<0.001	0.407	<0.001
GPCA−RAS patients (n = 375)	52 (13.9)	9 (2.4)	93 (24.8)	90 (24.0)	18 (4.8)	11 (2.9)	27 (7.2)
aP-value	<0.001	0.004	<0.001	<0.001	<0.001	0.001	<0.001
cHealthy control subjects (n = 430)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	8 (1.9)

NA = not assessed.

^aComparisons of frequencies of parameters between 55 GPCA⁺RAS or 375 GPCA⁻ RAS patients and 430 healthy control subjects by chisquare test.

^bComparisons of frequencies of parameters between 55 GPCA⁺RAS and 375 GPCA⁻RAS patients by chi-square test.

^cThe hematological data of 430 healthy control subjects were retrieved from our previous study.4

We also found that 15 GPCA⁺RAS and 93 GPCA⁻RAS patients had anemia (defined as having an Hb concentration <13 g/dL for men and <12 g/dL for women).53 Of the 15 anemic GPCA ⁺ RAS patients, 4 (26.7%) had PA (define as having anemia, an MCV ≥100 fL, a serum vitamin B12 level <200 pg/mL, and the presence of serum GPCA positivity), 31–33,57,58 8 (53.3%) had normocytic anemia (define as having anemia and an MCV between 80.0 fL and 99.9 fL),34–36,59–62 2 (13.3%) had iron deficiency anemia (IDA, defined as having anemia, an MCV <80 fL, and a serum iron level <60 μg/dL),28–30,53,54 and one (6.7%) had microcytic anemia (define as having anemia and an MCV <80 fL) other than IDA and thalassemia trait-induced anemia (defined as having anemia, a RBC count >5.0 M/μL, an MCV <74 fL, and a Mentzer index (MCV/RBC) < 13) (Table 3).63

Table 3.

Anemia types of 15 anemic gastric parietal cell antibody (GPCA)-positive recurrent aphthous stomatitis (RAS) patients (GPCA⁺RAS patients) and of 93 anemic GPCA-negative RAS patients (GPCA⁻RAS patients).

Anemia type	Anemic patient number (%)
	Patient number (%)	Mean corpuscular volume (fL)	Iron deficiency (<60 μg/dL)	Vitamin B12 deficiency (<200 pg/mL)	Folic acid deficiency (<4 ng/mL)	Hyperhomocysteinemia (>12.6 μM)
GPCA+RAS patients (n = 55)
Pernicious anemia (PA)	4 (26.7)	≥100	0 (0.0)	4 (100.0)	0 (0.0)	4 (100.0)
Normocytic anemia	8 (53.3)	80–99.9	5 (62.5)	2 (25.0)	0 (0.0)	4 (50.0)
Iron deficiency anemia	2 (13.3)	<80	2 (100.0)	1 (50.0)	0 (0.0)	0 (0.0)
Other microcytic anemia	1 (6.7)	<80	0 (0.0)	1 (100.0)	0 (0.0)	1 (100.0)
Total anemic patients	15 (100.0)	7 (46.7)	8 (53.3)	0 (0.0)	9 (60.0)
GPCA−RAS patients (n = 375)
Macrocytic anemia other than PA	9 (9.7)	≥100	1 (11.1)	7 (77.8)	0 (0.0)	7 (77.8)
Normocytic anemia	47 (50.5)	80–99.9	20 (42.6)	2 (4.3)	2 (4.3)	6 (12.8)
Iron deficiency anemia	25 (26.9)	<80	25 (100.0)	1 (4.0)	2 (8.0)	1 (4.0)
Thalassemia trait-induced anemia	10 (10.8)	<74	0 (0.0)	0 (0.0)	0 (0.0)	1 (10.0)
Other microcytic anemia	2 (2.2)	<80	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)
Total anemic patients	93 (100.0)	46 (49.5)	10 (10.8)	4 (4.3)	15 (16.1)

Of the 93 anemic GPCA−RAS patients, 9 (9.7%) had macrocytic anemia (define as having anemia and an MCV ≥100 fL) other than PA,31–33 47 (50.5%) had normocytic anemia,34–36,59–62 25 (26.9%) had IDA,14–17,53,54 10 (10.8%) had thalassemia trait-induced anemia,63 and 2 (2.2%) had microcytic anemia other than IDA and thalassemia traitinduced anemia (Table 3).

Comparisons of frequencies of macrocytic, normocytic or microcytic anemia between 55 GPCA⁺RAS and 375 GPCA⁻RAS patients are shown in Table 4. By the statistical analyses, there were no significant differences in frequencies of overall anemia and macrocytic, normocytic or microcytic anemia between 55 GPCA⁺RAS and 375 GPCA⁻ RAS patients (all P-values >0.05, Table 4).

Table 4.

Comparisons of frequencies of macrocytic, normocytic or microcytic anemia between 55 gastric parietal cell antibody (GPCA)-positive recurrent aphthous stomatitis (RAS) patients (GPCA⁺RAS patients) and 375 GPCA-negative RAS patients (GPCA⁻RAS patients).

Anemia type	Anemic patient number (%)		*P-value
	GPCA+RAS patients (n = 55)	GPCA−RAS patients (n = 375)
Macrocytic anemia	4 (7.3)	9 (2.4)	0.121
Normocytic anemia	8 (14.6)	47 (12.5)	0.841
Microcytic anemia	3 (5.5)	37 (9.9)	0.422
Total anemic patients	15 (27.3)	93 (24.8)	0.819

^*Comparison of frequencies of macrocytic, normocytic or microcytic anemia between 55 GPCA+RAS patients and 375 GPCA−RAS patients by chi-square test.

Discussion

The present study investigated the hematological characteristics of RAS patients with serum GPCA positivity and demonstrated several important findings. First, compared with healthy control subjects, the 55 GPCA⁺RAS patients had significantly lower blood Hb (for both men and women), lower serum iron (for women only), and lower serum vitamin B12 levels, together with significantly higher serum homocysteine levels. Second, compared with the 375 GPCA⁻RAS patients, the 55 GPCA⁺RAS patients exhibited significantly higher MCV, serum folic acid, and homocysteine levels. Third, the 55 GPCA⁺RAS patients had significantly higher frequencies of macrocytosis, vitamin B12 deficiency, and hyperhomocysteinemia than 375 GPCA⁻RAS patients, and significantly higher frequencies of microcytosis, macrocytosis, anemia, iron deficiency, vitamin B12 deficiency, and hyperhomocysteinemia than 430 healthy control subjects. Finally, although all 55 GPCA⁺RAS patients were positive for serum GPCA, only 21.8%, 27.3%, 9.1%, 27.3%, and 20.0% developed vitamin B12 deficiency, anemia, macrocytosis, hyperhomocysteinemia, and iron deficiency, respectively. These findings indicate that GPCA positivity is an important but not the sole determinant of hematological abnormalities in RAS patients.22–24 Similarly, our previous studies also found vitamin B12 deficiency, anemia, macrocytosis, hyperhomocysteinemia, and iron deficiency in 10.9%, 22.2%, 8.1%, 18.0%, and 19.7% of 284 GPCA⁺ atrophic glossitis patients, in 16.5%, 30.3%, 19.3%, 29.4%, and 16.5% of 109 GPCA⁺ burning mouth syndrome patients, and in 28.8%, 29.5%, 20.9%, 54.7%, and 23.7% of 139 GPCA⁺ oral lichen planus patients, respectively.22–24

The significantly higher frequencies of vitamin B12 deficiency, hyperhomocysteinemia, macrocytosis, anemia, and iron deficiency in 55 GPCA⁺RAS patients can be explained by the pathogenic effects of GPCA on gastric parietal cells.22–24,57,58 GPCA is an autoimmune antibody directed against gastric parietal cells and is a characteristic serological marker of autoimmune gastritis. Autoimmune destruction of gastric parietal cells reduces the secretion of intrinsic factor and hydrochloric acid. Intrinsic factor is indispensable for vitamin B12 absorption in the terminal ileum, whereas gastric hydrochloric acid is essential for releasing vitamin B12 from dietary proteins and facilitating iron absorption in the upper gastrointestinal tract. Therefore, progressive loss of parietal cell function may simultaneously impair vitamin B12 and iron absorption, eventually resulting in vitamin B12 deficiency, iron deficiency, macrocytosis, anemia, and hyperhomocysteinemia. 18–27,57,58 Moreover, vitamin B12 deficiency interferes with DNA synthesis during erythropoiesis, leading to ineffective erythrocyte maturation and the production of enlarged erythrocytes, which explains the significantly higher MCV and frequency of macrocytosis in 55 GPCA⁺RAS patients.18–24,57,58 Because vitamin B12 serves as an essential cofactor for methionine synthase, impaired vitamin B12 metabolism also results in the accumulation of homocysteine, accounting for the significantly higher serum homocysteine levels and the higher frequency of hyperhomocysteinemia observed in the GPCA⁺ RAS patients.25–27,64–66

An interesting finding of the present study is that GPCA positivity resulted in vitamin B12 deficiency, anemia, macrocytosis, hyperhomocysteinemia, and iron deficiency in only 21.8%, 27.3%, 9.1%, 27.3%, and 20.0% of the 55 GPCA⁺RAS patients, respectively. Several explanations may account for these observations. First, the presence of serum GPCA does not necessarily indicate complete destruction of gastric parietal cells. Autoimmune gastritis is a chronic and progressive disease, and the degree of parietal cell damage varies substantially among individuals.57,58 Many GPCA-positive patients may have only mild or early-stage autoimmune gastritis with sufficient residual parietal cell function to maintain adequate intrinsic factor and hydrochloric acid secretion. Consequently, these patients can still absorb sufficient vitamin B12 and iron to maintain normal hematological status.22–24,57,58

Second, the development of vitamin B12 deficiency depends on the magnitude and duration of impaired absorption. The human liver stores approximately 2–5 mg of vitamin B12, which is sufficient for several years under normal physiological conditions. Therefore, even when intrinsic factor secretion has begun to decline, serum vitamin B12 concentrations may remain within the normal range until hepatic stores become substantially depleted. This explains why only approximately one-fifth (21.8%) of GPCA⁺RAS patients had biochemical vitamin B12 deficiency despite universal GPCA positivity.18–24,57,58

Third, hyperhomocysteinemia develops only when vitamin B12 deficiency is sufficiently severe to impair homocysteine remethylation. Patients with preserved vitamin B12 metabolism or adequate folate status may maintain normal homocysteine concentrations despite GPCA positivity. 25–27,64–66 Likewise, macrocytosis represents a relatively late hematological manifestation of vitamin B12 deficiency. Macrocytic erythrocytes become evident only after prolonged ineffective erythropoiesis and gradual replacement of normal erythrocytes by newly formed megaloblastic erythrocytes. Consequently, only 9.1% of GPCA⁺RAS patients developed macrocytosis, although a larger proportion had biochemical vitamin B12 deficiency or anemia.31–33

Fourth, anemia is a multifactorial consequence of vitamin B12 deficiency and iron deficiency rather than an inevitable result of GPCA positivity alone. Hemoglobin concentration is influenced by the severity and duration of vitamin B12 deficiency, the coexistence of iron deficiency, baseline hematological status, age, sex, nutritional condition, and compensatory erythropoietic capacity.14–21 Therefore, many GPCA-positive patients retained sufficient hematopoietic reserve to maintain normal hemoglobin concentrations despite impaired gastric function. Similarly, iron deficiency developed in only 20.0% of GPCA⁺RAS patients because gastric hydrochloric acid secretion may remain adequate in many patients with early or mild autoimmune gastritis, allowing relatively normal dietary iron absorption. In addition, dietary iron intake, menstrual blood loss, gastrointestinal bleeding, inflammatory status, and other individual factors also influence body iron stores. Thus, GPCA positivity increases the risk of iron deficiency but is not sufficient by itself to produce iron deficiency in every patient.14–17

Another noteworthy finding is that 375 GPCA⁻RAS patients also had significantly higher frequencies of anemia, iron deficiency, vitamin B12 deficiency, folic acid deficiency, macrocytosis, microcytosis, and hyperhomocysteinemia than 430 healthy control subjects. These results indicate that GPCA-independent mechanisms also contribute to hematinic abnormalities in RAS patients. Nutritional inadequacy, gastrointestinal malabsorption unrelated to autoimmune gastritis, chronic blood loss, inflammatory conditions, genetic susceptibility, or immune dysregulation associated with RAS may all contribute to these hematological abnormalities. Therefore, although GPCA positivity represents an important pathogenic mechanism in a subset of RAS patients, it cannot fully explain all hematinic deficiencies observed in the RAS population.14–24

The findings of this study have important therapeutic implications. Because GPCA-positive RAS patients are at increased risk of vitamin B12 deficiency, iron deficiency, anemia, macrocytosis, and hyperhomocysteinemia, routine laboratory evaluation, including complete blood count, serum iron, vitamin B12, folic acid, homocysteine, and GPCA assays, should be considered in RAS patients, particularly those with persistent, recurrent, or treatment-resistant oral ulcerations.1 Patients with confirmed vitamin B12 deficiency should receive vitamin B12 replacement therapy, preferably by intramuscular administration when autoimmune gastritis or impaired intrinsic factor production is suspected, although high-dose oral vitamin B12 supplementation may also be effective in selected patients.18–21,37 Iron supplementation should be administered to patients with concomitant iron deficiency after exclusion of chronic gastrointestinal blood loss or other underlying causes.14–17 Because autoimmune gastritis is a chronic autoimmune disease, patients with GPCA positivity should undergo long-term follow-up with periodic monitoring of hematological parameters and vitamin B12 and iron status.57,58 Early identification and correction of hematinic deficiencies may restore normal erythropoiesis, normalize homocysteine metabolism, improve oral mucosal healing, reduce the recurrence of oral ulcerations, and improve patients’ overall quality of life.1

Several limitations of this study should be acknowledged. First, this was a single-center cross-sectional study conducted at a tertiary referral hospital, and therefore the study population may not be fully representative of all RAS patients. Second, because of the cross-sectional design, causal relationships between GPCA positivity and hematological abnormalities cannot be definitively established. Third, gastric functional studies, serum intrinsic factor antibody testing, serum gastrin levels, pepsinogen assays, endoscopic examinations, and histopathological confirmation of autoimmune gastritis were not performed. These investigations would provide more direct evidence regarding the severity of gastric parietal cell destruction and its relationship with hematinic deficiencies.57,58 Finally, the therapeutic effects of vitamin B12 and iron replacement were not evaluated in this study. Prospective longitudinal studies are needed to determine whether correction of these hematinic abnormalities reduces the frequency or severity of RAS.1

In conclusion, GPCA⁺RAS patients have significantly higher frequencies of macrocytosis, vitamin B12 deficiency, and hyperhomocysteinemia than GPCA⁻RAS patients, as well as significantly higher frequencies of microcytosis, macrocytosis, anemia, iron deficiency, vitamin B12 deficiency, and hyperhomocysteinemia than healthy control subjects. Nevertheless, GPCA positivity results in vitamin B12 deficiency, anemia, macrocytosis, hyperhomocysteinemia, and iron deficiency in only a subset of GPCA⁺RAS patients because autoimmune destruction of gastric parietal cells varies considerably in severity and progression among individuals, and substantial physiological reserves of vitamin B12 and iron metabolism can delay the development of hematological abnormalities.14–21,57,58 These findings emphasize that GPCA is an important risk factor rather than a definitive predictor of hematinic deficiencies in RAS patients.22–24 Early laboratory screening, identification of GPCA positivity, appropriate vitamin B12 and iron replacement therapy, and long-term follow-up may help prevent hematological complications and improve the clinical management of RAS patients.1