EXPERIMENTAL DESIGN

Firstly, surgically resected human stomach tissue samples obtained from early gastric cancer patients (two cases; a 56 year old man and a 52 year old woman) were subjected to immunohistochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR) to check for the presence of leptin in the gastric epithelium.

Secondly, 201 H pylori positive patients with chronic gastritis (121 men and 80 women, mean age 41.5 years) were chosen from subjects who visited multiphasic health testing services held by the Second Department of Internal Medicine, Fukui Medical University. The services included upper gastrointestinal endoscopy or radiography to screen for gastric cancer. Chronic gastritis was diagnosed using these examinations.20 ,21 Patients who had a history of peptic ulcers, reflux oesophagitis, malignant diseases, gall stones, or surgery, as well as those receiving non-steroidal anti-inflammatory drugs or antibiotics were excluded from the study.H pylori infection was diagnosed using the¹³C urea breath test (UBT). Subjects fasted overnight prior to the UBT. Breath samples were collected before and 15 minutes after intake of a ¹³C urea solution (100 mg ¹³C urea in 100 ml water) and analysed using isotope ratio mass spectrometry. A rise in the delta value of ¹³CO₂ of more than 2.5/ml over baseline values was considered positive forH pylori infection. A standardised questionnaire was used to assess abdominal symptoms. This questionnaire addressed seven persistent or recurrent symptoms for more than three months which served as the primary target variables (upper abdominal pain, heart burn, regurgitation, nausea, vomiting, upper abdominal fullness, loss of appetite). The intensity of the respective symptoms was scored as 0 (absent), 1 (low intensity), 2 (moderate intensity), or 3 (severe). A total of 121 of 201 patients (60.2%) had dyspepsia symptoms. All 201 patients underwent eradication therapy forH pylori infection with omeprazole (20 mg twice daily), amoxicillin (750 mg twice daily), and clarithromycin (200 mg twice daily) for one week for gastric cancer prophylaxis. Cure ofH pylori infection was evaluated using the UBT three months after treatment. Body weights and symptom scores of the patients were measured before and three months after eradication therapy. The change in body mass index (BMI kg/m²) after eradication therapy was compared between the eradicated and non-eradicated groups. Serum samples were obtained before and three months after eradication therapy in the morning after an overnight fast, and stored at −20°C until analysed.

Thirdly, biopsy specimens from the gastric fundic mucosa were obtained before and three months after eradication therapy from 40 of 201 patients (33 patients were successfully eradicated: 20 men and 13 women, mean age 42.2 years; and seven patients were not eradicated: four men and three women, mean age 41.6 years). These samples were subjected to quantitative RT-PCR to examine the effect of eradication therapy on leptin expression in the gastric fundic mucosa. Biopsy specimens of the gastric fundic mucosa obtained from 20H pylori negative normal controls (13 men and seven women, mean age 40.8 years) were also subjected to quantitative RT-PCR to examine gastric leptin expression in controls.

These studies were performed according to the principles of the Declaration of Helsinki, and consent was obtained from each individual after a full description of the nature and protocol of the study.

IMMUNOHISTOCHEMISTRY FOR LEPTIN

The surgically resected tissue samples were fixed in Bouin's solution and embedded in paraffin. Immunohistochemical staining was performed using the avidin-biotin-peroxidase complex technique (Vectastain ABC kit; Vector, Burlingame, California, USA) described by Hsu and colleagues.22 All incubations were done at room temperature. Each section (4 μm thick) was immersed in 0.3% H₂O₂ in methanol for 30 minutes to destroy endogenous peroxidase activity, washed for 10 minutes with 0.05 M Tris buffered saline, pH 7.5, and then incubated for 30 minutes with normal goat serum to block non-specific Ig binding sites. The goat serum was removed and the slides were incubated overnight with a 1:100 dilution of the antihuman leptin rabbit antibody 18545S (IBR, Gumma, Japan). This was followed by a 30 minute wash in Tris buffered saline, 30 minute incubation with biotinylated goat antirabbit IgG, repeat wash in Tris buffered saline, and a 30 minute incubation with avidin DH biotinylated horseradish peroxidase. After a final wash in Tris buffered saline, the slides were incubated for two minutes in a solution containing 0.05% diaminobenzidine and 0.015% H₂O₂. Nuclear staining was carried out with haematoxylin. The specificity of the immunohistochemical reaction was first established by showing that non-immune rabbit serum did not reveal positive staining. In addition, the specificity of the antibody was evaluated by absorption controls. The diluted antibody was incubated with human leptin in concentrations of up to 10 nmol/ml for 24 hours at 4°C and then applied to the tissue section.

LEPTIN ASSAY

Serum leptin concentrations were determined using radioimmunoassay kits (Linco Research, St Charles, Missouri, USA) according to the manufacturer's protocol.23

QUANTITATIVE RT-PCR

Sections of surgically resected tissue samples of the gastric fundic, antral mucosa, and gastrocolic omentum were frozen and stored in liquid nitrogen. mRNA was purified from tissues using Micro FastTrack kits (Invitrogen, Carlsbad, California, USA) according to the manufacturer's protocol.24 RT-PCR for leptin was done using a novel real time quantitative RT-PCR method using an ABI 7700 sequence detector system (PE Applied Biosystems, Foster City, California, USA).25 The sense primer for leptin was 5′-CACCAAAACCCTCA TCAAGACA-3′ and the antisense primer was 5′-CTTTCTGTTTGGAGGAGACTGACT-3′. The reaction mixture was prepared according to the manufacturer's protocol using TaqMan Gold RT-PCR Kits (PE Applied Biosystems). The reactions also contained the target hybridisation leptin probe (5′-CACCA GGATCAATGACATTTCACACACG-3′). The probe was labelled with a reporter fluorescent dye 6-carboxyfluorescein at the 5′ end. The thermal cycling conditions included 48°C for 30 minutes and 95°C for 10 minutes, followed by 50 cycles of amplification at 95°C for 15 seconds and 60°C for one minute. The PCR products were also examined by 2% agarose gel electrophoresis. The gels were stained with ethidium bromide to confirm successful amplification of the expected sequences. mRNA was also purified from biopsy specimens and subjected to real time quantitative RT-PCR for measurement of leptin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as controls using TaqMan GAPDH control reagents (PE Applied Biosystems). For relative quantification of leptin expression, a calibration curve was constructed using the mRNA obtained from the gastrocolic omentum. RNA for GAPDH was used as an endogenous control. The amount of leptin RNA was normalised to the level of GAPDH.

STATISTICAL ANALYSIS

Results are expressed as mean (SD). The significance of differences between groups for mean age, BMI, and change in BMI was evaluated using an unpaired Student's ttest. Comparison of differences in BMI changes of more than 0.5 kg/m² was performed using a χ² test with Fisher's exact probability test. Differences in serum leptin concentrations and relative RNA quantification of leptin in the interval before and after eradication therapy were analysed with a paired Student's t test. Symptom scores among groups were compared by Wilcoxon's signed rank test. The relationship between the change in BMI and the change in symptom scores or the change in relative RNA quantification of leptin was evaluated by Spearman's rank correlation. Differences with a p value of less than 0.05 were considered significant.