PATIENTS AND METHODS

Study design/patients

Between January 2000 and April 2001, patients with IDA referred to our gastroenterology department from the haematology department were studied for evaluation of their anaemia, as previously reported.^5,9,13 IDA was defined as a haemoglobin concentration <14 g/dl for males and <12 g/dl for females, in the presence of ferritin <30 μg/l and mean corpuscular volume <80 fl. As reported elsewhere,^5,13 patients presenting manifest causes of blood loss, such as active gastrointestinal haemorrhage, epistaxis, menometorrhagia or heavy menstrual loss, or any other non-gastrointestinal disease likely to cause IDA were not referred to us from the haematology department. All patients underwent colonoscopy to exclude bleeding sites. A positive colonoscopy or refusal to undergo the examination were exclusion criteria for the present study. All patients completed a detailed questionnaire regarding diet and habits, as previously published.^5,9 Also excluded from the study protocol were patients with previous gastric surgery, and those who previously received anti-H2 pylori treatment or antisecretory drugs. Alcoholism, drug addiction, anorexia, and vegetarian or iron poor diet were also considered as causes for exclusion. Withdrawal from oral iron therapy, vitamin C supplementation, or any other over the counter medications containing iron or AA for at least four weeks before the examinations was a criterion for eligibility.

According to these strict criteria, we initially enrolled 52 eligible IDA patients. These patients underwent gastroscopy with multiple biopsies, as described below. We excluded from the present study however four patients in whom duodenal histology revealed coeliac disease, one with gastric cancer and four with peptic ulcer, as these findings were likely to be the cause of their IDA. The remaining 43 patients with unexplained IDA were subsequently assigned to the following groups according to their gastric histology.

Group A: IDA positive/H pylori positive patients

This group comprised 30 patients (24 females and six males; median age 47 years, range 19–66) with a diagnosis of H pylori associated gastritis and no other likely causes of IDA, as described elsewhere.^5,9,13

Group B: IDA positive/H pylori negative controls

Thirteen (12 females and one male; median age 37 years; range 18–60) patients presenting with a normal gastric histology, with no other likely causes of IDA, were included as the first control group.

Group C: IDA negative/H pylori positive controls

During the same time, we selected among outpatients undergoing gastroscopy in our department for evaluation of dyspepsia, those negative for macroscopic lesions (ulcer, cancer) but positive at histology for H pylori gastritis. Exclusion criteria for this group included: IDA, peptic ulcer (present or past), malignancies, moderate to severe oesophagitis (endoscopic diagnosis), histological diagnosis of coeliac disease, previous gastric surgery, previous anti-H pylori treatment, and previous or current use of antisecretory drugs, iron, or AA acid preparations.¹³

Eleven patients (nine females and two males; median age 48 years, range 19–67) with H pylori positive chronic superficial gastritis were included in this second control group.

The main characteristics of the three groups are shown in table 1.

A subgroup of patients in group A was re-evaluated six months after appropriate eradication therapy. All patients and controls gave written informed consent; the local ethics committee approved the study protocol.

Methods

Gastroscopy

All patients and controls underwent gastroscopy at 9:00 am after an overnight fast. All individuals were sedated before the procedure using intravenous midazolam (2.5–10 mg). Endoscopists were unaware of the presence/absence of IDA. Three biopsy samples from the antrum (smaller and greater curve, anterior or posterior walls) and three from the midbody along the greater curve were taken using a standard biopsy forceps, as reported elsewhere.^14,25 Two duodenal biopsies (II portion) were also taken to exclude coeliac disease.

Histological procedures

All gastric biopsies were immediately fixed in Bouin’s solution for 4–8 hours at room temperature, rinsed in 0.1 M phosphate buffered saline solution, pH 7.4, and embedded in wax. Serial 5 μm thick sections of gastric mucosa, perpendicular to the mucosal surface, were stained with haematoxylin-eosin for conventional histopathological examination. H pylori infection was evaluated with Giemsa staining. Assessment of the degree of gastritis was performed according to the updated Sydney system.²⁶ The following scores were assigned to each graded variable: 0=absence, 1=mild, 2=moderate, and 3=severe degree. Atrophy of the fundic mucosa was defined as focal or complete replacement of oxyntic glands by metaplastic pyloric or intestinal glands.²⁵ The diagnosis of fundic atrophy was confirmed by fasting hypergastrinaemia and reduced pepsinogen I levels. Atrophy of the antral mucosa was defined as focal or complete replacement of antral glands by intestinal metaplastic epithelium.

Gastric juice pH determination and storage

At gastroscopy, immediately after passing the endoscope into the stomach, a sterile Teflon catheter was passed through the biopsy channel, and starting from the midbody along the greater curve approximately 5 ml of gastric juice were aspirated and collected in a sterile tube containing EDTA. Gastric juice pH values were immediately measured with a glass electrode pH meter (HI9321; Hanna Instruments, Padova, Italy), as described elsewhere.¹³ Two aliquots (0.5 ml) of gastric juice were then stored at −80°C, each with 25 μg of hypoxanthine (reference standard for high performance liquid chromatography) and 1.5 ml of 2% metaphosphoric acid.

Plasma collection and storage

Immediately prior to the time of gastroscopy, 10 ml of plasma were obtained from each patient and collected in a sterile tube with EDTA. Two aliquots of 0.5 ml were treated and stored as reported above for gastric juice.

Ascorbic acid determination

AA was measured in plasma and gastric juice samples, treated as described above, by a Shimadzu liquid chromatograph on an analytical Supelcosil LC-18-DB column (24 cm×4.6 mm, 5 μm, Supelco) plus guard column, by using in line both a photodiode array detector set at 265 nm and an ESA CoulArray (oxidation potential +500 mV). All determinations were carried out within seven days of collection. Samples were centrifuged for 10 minutes and 20 μl of the supernatant injected into the high performance liquid chromatography system. AA was quantitated by comparison of areas with those of authentic standards, including the reference standard (hypoxanthine), as described elsewhere.²⁷ The mobile phase consisted of 0.02 M NaH₂PO₄, 0.06% (w/v) metaphosphoric acid, and 0.4% (v/v) CH₃CN (pH 3.0); flow 0.8 ml/min. The limit of calculation for the assay used to allow statistical analyses and ratio calculation when the compound was not detectable was 0.1 μg/ml. Normal values for plasma AA in 50 healthy individuals (31 females and 29 males, median age 32 years, range 18–55) in our laboratory were 7.5–30 μg/ml.

Statistics

Continuous data are expressed as median (range) and were evaluated by appropriate statistical tests (t test or Mann Whitney U test). Proportions were compared by means of Fisher’s exact test. Correlations were evaluated using the Spearman rank correlation test. A value of p<0.05 was considered statistically significant.

RESULTS

Extension and degree of gastritis

Thirteen (43 %) of 30 patients with H pylori infection and IDA (group A) had atrophic gastritis in the fundic mucosa, nine (69%) also had atrophic changes in the antrum, while the remaining four patients had an antral chronic superficial gastritis. All but one of the other 17 patients (94%) had a chronic superficial gastritis of the antral and corporal mucosa (pangastritis). In contrast, none of the H pylori positive controls (group C) had atrophic changes in the antral or fundic mucosa, and only five (5/11, 45%) had gastritis extended to the corporal mucosa, the others presenting with an antrum restricted gastritis (p=0.0069). The median (range) values of the sum of all Sydney score variables for the antral and body mucosa were, respectively: 3 (0–5) and 7 (2–12) in group A, and 3 (1–6) and 1 (0–4) in group C. While there were no differences between groups regarding antral scores, the gastric body score was significantly higher in group A compared with group C (p<0.0001).

Intragastric pH

Median intragastric pH values were 7 (1.9–8.2) in group A, 2 (1.2–7) in group B, and 2.1 (1.3–5) in group C (fig 1A). Group A was found to be significantly different compared with groups B and C (p<0.0001) whereas no difference was found between the two control groups (p=0.8).

Gastric juice ascorbic acid (JAA)

As shown in fig 1B, gastric juice ascorbic acid (JAA) concentration was significantly lower in group A (median 4.4 μg/ml, range 0.1–23.4) compared with group B (median 17.5 μg/ml, range 0.34–44.8; p=0.0033) and group C (median 7.06 μg/ml, range 4.2–28; p=0.029). The difference between the two control groups was not significant (p=0.09).

Plasma ascorbic acid (PAA)

Median plasma ascorbic acid (PAA) concentrations were: 11 μg/ml (range 5.9–21.4), 19 μg/ml (range 5.3–49), and 8.28 μg/ml (range 2.69–21.4) in groups A, B, and C, respectively. Patients with IDA and H pylori (group A) had significantly lower PAA concentrations compared with group B (p=0.002). Group C controls also had lower, yet not significantly different (p=0.09), concentrations compared with healthy controls. Smoking status did not significantly affect PAA concentrations. In fact, the median PAA concentration was 14.5 μg/ml (range 5.5–32.4) in smokers and 10.5 μg/ml (range 5.7–75) in non-smokers (p=0.9).

Gastric juice/plasma AA ratio

The distribution of the juice/plasma AA ratio values of the three groups is shown in fig 2. The median juice/plasma AA ratio was 0.38 (0.001–1.09) in group A, being significantly lower compared with group B (median 0.6, range 0.06–3.25; p=0.026) and group C (median 0.6, range 0.17–4.6; p=0.029). Median ratio values did not differ between the two control groups. Moreover, only one of 30 patients in group A (3.3%) had a ratio >1 (fig 2) compared with six of 13 patients in group B (46%; p=0,0017) and four of 11 in group C (36%; p=0.0138).

Relationship between severity and degree of gastritis, intragastric pH, and gastric juice AA

Gastric juice AA concentration was negatively correlated (Spearman r=−0.40, 95% confidence interval −0.62 to −0.11; p=0.0059) with intragastric pH. In patients with gastritis (groups A and C) there was a significant negative correlation between the sum of the Sydney score variables in the corporal mucosa and JAA (Spearman r=−0.53; p=0.0039). In contrast, a correlation was not found between the sum of Sydney score variables in the antrum and JAA concentrations (p=0.44).

Among group A patients, 13 with atrophic changes in the fundic mucosa differed significantly from those without in terms of intragastric pH (median 7 v 4.8; p=0.0317), JAA concentrations (median 0.38 μg/ml v 5.6; p=0.028), and JAA/PAA ratio (median 0.043 v 0.49; p=0.004). The 17 patients in group A with no atrophic changes in the fundic mucosa differed significantly from the H pylori positive controls in terms of intragastric pH (median 4.8 v 2.1; p=0.0028) but not in terms of JAA concentrations (median 5.6 μg/ml v 7.06; p=0.18). No relationship was observed between age of the patient and gastric juice (p=0.79) or plasma (p=0.69) AA levels.

Effect of eradication in IDA patients

Seven of 11 (63%) treated group A patients were successfully cured six months after therapy. The effect of therapy on pH and JAA values was related to the presence of fundic atrophy. In fact, irrespective of the outcome of therapy, a further decrease in JAA concentrations (median from 3.6 to 0.78 μg/ml) was observed in six patients who had fundic atrophy whereas treatment had no effect on intragastric pH or JAA/PAA ratio. In contrast, in the subgroup of five patients with H pylori infection and IDA without fundic atrophy, therapy led to a decrease in pH values and to an increase in JAA levels (median from 9.02 to 14.48 μg/ml) and in the JAA/PAA ratio, as detailed in table 2. However, statistical evaluation of these data did not appear to be meaningful on account of the small sample size.

DISCUSSION

The main finding of our study was that patients with H pylori gastritis, associated with otherwise unexplained IDA, presented with a concomitant increase in intragastric pH and a decrease in gastric juice AA.

Patients with IDA associated with H pylori gastritis showed an increase in intragastric pH, presenting with a median value of pH >3, a value known to be critical in the process of iron absorption.²⁸ Moreover, patients with H pylori gastritis and IDA had significant lowering of JAA concentrations compared with both healthy and H pylori positive controls. These findings suggest that these two physiological mechanisms, which are necessary for alimentary iron to be absorbed in the duodenal mucosa, are impaired in patients with H pylori gastritis and IDA.

It is well known that H pylori infection when affecting the gastric body, irrespective of the presence of fundic atrophy, induces gastric acid hyposecretion.²⁹ It has also been clearly demonstrated that H pylori gastritis induces lowering of intragastric AA concentrations and that H pylori eradication can reverse this negative effect.^{22–24,30,31} However, all of these previous studies focused on features different from ours, and indeed the possibility that such alterations in intragastric pH and AA might interfere with the process of iron absorption has not been considered until now.

Intragastric acidity is a key factor in the process of iron absorption^17–19,28 but AA is also essential for alimentary iron absorption. It is worthwhile stressing that this has given rise to the intriguing hypothesis that iron, like vitamin B12, has a gastric intrinsic factor (that is, AA) that aids absorption.¹⁹ In fact, AA not only converts ferric iron to the ferrous form, which maintains solubility at the alkaline pH of the duodenum, but also forms chelates with ferric chloride which is also stable at a pH >3.²⁸

The possibility that an increased intragastric pH impairs alimentary iron absorption and determines IDA has been considered in the course of antisecretory therapy,³² and the investigation of Zollinger-Ellison syndrome patients treated with proton pump inhibitors for long periods failed to reveal any significant changes in iron homeostasis.³³ However, it is worthwhile noting that the model of hypochlorhydria induced by antisecretory drugs is different from that due to fundic gastritis. In fact, intragastric pH was still <3 for more than one third of the day during a 24 hour monitoring period in patients treated with antisecretory drugs at standard doses.³⁴ It has also been demonstrated that pharmacologically induced hypochlorhydria results in depletion of gastric juice vitamin C concentration in H pylori positive, but not H pylori negative, subjects.³⁵

Previous studies have suggested that otherwise unexplained IDA can be caused by hypo/ achlorhydria due to atrophic body gastritis.^36,37 However, the possibility that similar mechanisms lead to IDA in some H pylori infected subjects without atrophic gastritis is far less commonly considered.

In the present study, we linked for the first time alterations in gastric acidity and AA in H pylori gastritis with the presence of IDA, demonstrating that patients with unexplained IDA associated with H pylori gastritis are characterised by a concomitant increase in intragastric pH and a lowering of intragastric concentrations of AA. These alterations are consequent to the peculiar pattern of gastritis observed in these patients. In fact, a significant percentage (43%) of our H pylori positive IDA patients presented atrophic changes in the gastric body, and the remaining had a chronic superficial gastritis extended to the fundic mucosa, in contrast with H pylori positive controls.

Moreover, in our study, we observed an inverse correlation between intragastric pH and JAA levels (fig 2) and, while we failed to find any correlation between JAA and the degree of gastritis in the antral mucosa, as reported by others,³⁰ JAA concentrations were lower in patients with more severe damage in the gastric body. This finding is in agreement with those of others^23,24 and suggests that increased intragastric pH may be more important than the polymorphonuclear leucocyte oxidative burst caused by the inflammatory activity of H pylori gastritis³⁰ in determining JAA depletion. However, depletion of JAA in patients with H pylori gastritis may also be due to its consumption by scavenging reactive oxygen species produced by mucosal inflammation and by preventing the formation of N-nitrose compounds.³⁸

Another interesting result obtained in the present study was the finding of lower plasma AA concentrations in all groups of patients with gastritis compared with controls with a healthy stomach. This observation is in agreement with a recent study carried out on a large number of individuals with/without H pylori infection that suggested impaired systemic bioavailability of AA in patients with H pylori, independent of diet.³⁹ Alternatively, the lower plasma AA concentrations in patients with gastritis may be the consequence of increased active secretion of AA from plasma to the gastric juice in the attempt to restore the physiological ratio between the two compartments.

If H pylori gastritis affects gastric acid secretion and AA stability, eradication should lead both to reversal of these alterations and to recovery from IDA. Unfortunately, in the present study, evaluations were not repeated in all patients after treatment to establish the effect of possible recovery of acid secretion and AA on IDA. However, we evaluated the effect of H pylori cure on intragastric pH and JAA concentrations in a subgroup of patients with IDA and H pylori gastritis. The effect of the cure in these patients was related to the presence of atrophic body gastritis before therapy. In fact, after treatment, the subgroup of patients with chronic superficial H pylori gastritis showed a decrease in intragastric pH and increase in JAA as well as in the juice/plasma ratio. In contrast, as antimicrobial therapy had no effect on gastric atrophy, as reported elsewhere,⁴⁰ we observed no changes in intragastric pH or JAA after treatment in the subgroup of patients with atrophic body gastritis. We also observed that while the subgroup of patients with atrophic body gastritis had significantly lower levels of JAA compared with H pylori positive controls, this difference was not significant for the subgroup of patients with chronic superficial gastritis. This unexpected result may have two possible explanations: firstly, it is possible that in patients with a chronic superficial gastritis the observed alteration of intragastric acidity plays a major role in affecting iron availability, even in the presence of JAA concentrations similar to those of H pylori positive controls; and secondly, it is possible that the difference between chronic superficial gastritis patients and controls in terms of JAA concentrations is small and therefore a larger number of patients are needed for it to be detectable.

It is also possible to speculate that looking at the association between IDA and gastritis from another viewpoint, gastric damage could be a consequence of IDA, as occurs in mouth and tongue epithelium and as suggested in the very early literature,⁴¹ and not therefore the cause of the anaemia. However, we consider this unlikely as the association reported by others^1–4 and us¹³ is not between IDA and non-specific gastritis, eventually caused by nutritional deficiency, but between IDA and H pylori infection and the related gastritis, as also suggested by reversal of IDA after H pylori cure.^5,6

A possible limitation of the present study is the poor repeatability of the gastric juice pH measurements performed during gastroscopy. However, while a formal evaluation both of basal and pentagastrin stimulated gastric acid secretion may have been more correct to assess the amount of gastric acid secreted, the gastric pH may per se indicates whether the gastric juice affects the stability of ferric iron, as suggested by in vitro studies.²⁸

In conclusion, our findings indicate that patients with IDA associated with H pylori gastritis present with alterations in the intragastric milieu, such as increased pH and decreased AA. The concomitant occurrence of these two alterations may plausibly account for impaired iron absorption and consequent IDA in these patients.