**2.1 Subjects and tissue**

Biopsies were taken from healthy controls (n=7, mean age 36, 4 female) and patients with ERD (n=13, mean age 48, 3 female). The biopsies were collected during endoscopy and specimens were immediately frozen in liquid nitrogen (for later western blot analysis) or snap frozen in RNA STAT-60 (Nordic Bio Site AB, Stockholm, Sweden) (for later rt-PCR analysis) and subsequently stored in liquid nitrogen or fixed in buffered 4% formaldehyde (for later histo-morphology evaluation). The biopsies were taken in the 3 o´clock position 2 cm proximal to the gastro esophageal junction, and in the altered area for the esophagitis (red streak)(8). Protein expressions of MPO, NADPH oxidase, iNOS and nitrotyrosine were assessed by western blot technique and immunohistochemistry. Expression of the IL1β and IL6 were assessed by both western blot and rtPCR.

The Los Angeles classification was used by the endoscopist to decide the degree of esophageal inflammation (1). All patients who contributed for this study were classified as LA-A.

*Ethics.* All participants had given informed consent and the study had been approved by Ethical Committee of Göteborg University and was performed in accordance with the Declaration of Helsinki.

#### **2.2 Western blot analyses**

The frozen specimens were sonicated in a PE buffer (10 mM potassium phosphate buffer, pH 6,8 and 1mM EDTA) containing 10 mM 3-[(3-cholamidopropyl) dimethylammonio]-1 propane sulphonate (CHAPS: Boehringer Mannheim, Mannheim, Germany) and protease inhibitor cocktail tablet Complete (Roche Diagnostics AB, Stockholm, Sweden). The homogenate was then centrifuged (10,000 g for 10 min at 4ºC) and the supernatant was analysed for protein content by the Bradford method and stored at -80ºC (9). Samples were diluted in SDS buffer and heated at 70°C for 10 min before they were loaded on a NuPage 10% Bis-Tris gel, and electrophoresis run using a MOPS buffer (Invitrogen AB, Lidingo, Sweden). One lane of each gel was loaded with prestained molecular weight standards (SeeBlue, NOVEX, San Diego, CA, USA). A positive control was loaded on each gel (Table 1). After the electrophoresis the proteins were transferred to a polyvinyldifluoride transfer membrane, Hybond, 0.45μm, RPN303F, (Amersham, Buckinghamshire, UK) using an iBlot (Invitrogen AB). Membranes were than incubated with polyclonal specific antibodies directed at the MPO, NADPH oxidase (p47phox-subunit), iNOS, nitrotyrosine, IL-1β and IL-6 respectively (Table 1). An alkaline phosphatase conjugated goat anti-mouse or goat antirabbit IgG antibody (Santa Cruz) and CDP-Star (Tropix, Bedford, MA, USA) were used as a

mutation, activation of proto-oncogenes and inactivation or loss tumor suppressor genes

The aim of the present study is to elucidate the presence of radical producing enzymes, represented by NADPH oxidase, MPO and iNOS and also the expression of radical formation marker for ONOO- activity as well as nitro radical end products; nitrotyrosine. The second aim is to elucidate the histological changes and number of inflammatory cells as well as the expression of inflammatory markers IL1β and IL6 in human esophageal biopsies

Biopsies were taken from healthy controls (n=7, mean age 36, 4 female) and patients with ERD (n=13, mean age 48, 3 female). The biopsies were collected during endoscopy and specimens were immediately frozen in liquid nitrogen (for later western blot analysis) or snap frozen in RNA STAT-60 (Nordic Bio Site AB, Stockholm, Sweden) (for later rt-PCR analysis) and subsequently stored in liquid nitrogen or fixed in buffered 4% formaldehyde (for later histo-morphology evaluation). The biopsies were taken in the 3 o´clock position 2 cm proximal to the gastro esophageal junction, and in the altered area for the esophagitis (red streak)(8). Protein expressions of MPO, NADPH oxidase, iNOS and nitrotyrosine were assessed by western blot technique and immunohistochemistry. Expression of the IL1β and

The Los Angeles classification was used by the endoscopist to decide the degree of esophageal inflammation (1). All patients who contributed for this study were classified as

*Ethics.* All participants had given informed consent and the study had been approved by Ethical Committee of Göteborg University and was performed in accordance with the

The frozen specimens were sonicated in a PE buffer (10 mM potassium phosphate buffer, pH 6,8 and 1mM EDTA) containing 10 mM 3-[(3-cholamidopropyl) dimethylammonio]-1 propane sulphonate (CHAPS: Boehringer Mannheim, Mannheim, Germany) and protease inhibitor cocktail tablet Complete (Roche Diagnostics AB, Stockholm, Sweden). The homogenate was then centrifuged (10,000 g for 10 min at 4ºC) and the supernatant was analysed for protein content by the Bradford method and stored at -80ºC (9). Samples were diluted in SDS buffer and heated at 70°C for 10 min before they were loaded on a NuPage 10% Bis-Tris gel, and electrophoresis run using a MOPS buffer (Invitrogen AB, Lidingo, Sweden). One lane of each gel was loaded with prestained molecular weight standards (SeeBlue, NOVEX, San Diego, CA, USA). A positive control was loaded on each gel (Table 1). After the electrophoresis the proteins were transferred to a polyvinyldifluoride transfer membrane, Hybond, 0.45μm, RPN303F, (Amersham, Buckinghamshire, UK) using an iBlot (Invitrogen AB). Membranes were than incubated with polyclonal specific antibodies directed at the MPO, NADPH oxidase (p47phox-subunit), iNOS, nitrotyrosine, IL-1β and IL-6 respectively (Table 1). An alkaline phosphatase conjugated goat anti-mouse or goat antirabbit IgG antibody (Santa Cruz) and CDP-Star (Tropix, Bedford, MA, USA) were used as a

(5, 7).

LA-A.

Declaration of Helsinki.

**2.2 Western blot analyses** 

from healthy volunteers and patients with ERD.

IL6 were assessed by both western blot and rtPCR.

**2. Material and methods 2.1 Subjects and tissue** 

substrate to identify immunoreactive proteins by means of chemiluminescense. Images were captured by a Chemidox XRS cooled CCD camera, and analyzed with Quantity One software (BioRad laboratories, Hercules, CA, USA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (Imgenex, San Diego, CA, USA) was used as control for equal loading, and for each tested sample the ratio of primarily antibody/GAPDH was used.


MPO; myeloperoxidase, iNOS; inducible nitric oxide synthase, NADPH-oxidase; nicotinamide adenine dinucleotide phosphate oxidase, GAPDH; Glyceraldehyde-3-phosphate dehydrogenase, IL; interleukin

Table 1. Antibodies and controls used in Western Blot analyses and immunohistochemistry

#### **2.3 Immunohistochemistry**

The mucosal specimens were fixed in buffered 4% formaldehyde and embedded in paraffin. Sections for immunohistochemistry (3µm) were deparaffinized and then boiled for 15 min in 10mM citrate buffer (pH 6.0) for antigen retrieval. The Immunocruz TM Staining System (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used for the immunohistochemistry protocol. After inhibition of endogenous peroxidase activity, the slides were pre-incubated with serum block and then incubated with primary antibodies against MPO, NADPH oxidase iNOS and nitrotyrosine (see table 1) over night in dilutions of 1:200, 1:50, 1:1000 and

Expression of Reactive Oxygen Species in Reflux Disease 83

group of observation was identified using Wilcoxon´s signed rank test. Nonparamitric correlation analysis was performed by Spearman´s rank correlation test. A p-value ≤0.05

**60kDa 47kDa**

**66kDa 31kDa**

**130kDa 21.5kDa**

was considered to be of statistical significance.

**A D**

**B E**

**HI-60 HI-60**

**12-354 Serum**

**RAW 264.7 Serum**

Fig. 1. Typical western blot for MPO (Panel A), NADPH oxidas (Panel B), iNOS (Panel C), nitrotyrosine (Panel D), IL-1β (Panel E) and IL-6 (Panel F) with a band at 60 kDa, 66 kDa, 130 kDa, 47 kDa, 31 kDa and 21.5 kDa respectively, in the positive control cell lysate HL-60, cell lysate 12-354, mouse macrophage cell line RAW 264.7 and serum, and in human

A typical western blot for MPO, NADPH oxidase, iNOS, nitrotyrosine, IL-1β and IL-6 are shown in figure 1A-F. A significant increase of MPO protein expression (ERD, p=0.0001, red streak, p=0.005) as well as a significant increase of nitrotyrosine expression (ERD, p=0.05, red streak, p=0.05) was detected in ERD-patients compared healthy controls using western blotting (figure 2A and D). No significant differences in expression of NADPH oxidase, iNOS (figure 2B and C), IL1β or IL6 were detected (data not shown). However at gene expression level using rt-PCR technique, IL6 was significantly increased in ERD-patients (p=0.01), whereas there was no difference in IL1β between the groups at gene level (data not

Immunohistochemistry performed on endoscopically retrieved mucosal biopsies revealed a distinct staining for MPO in epithelial lymphocytes and also weak staining in the squamous epithelial cells (figure 3 A). Immunostaining for NADPH oxidase was detected in basal layer of the epithelium and papillae (figure 3B), whereas iNOS was localized mainly to the upper and mid-zone layer in the epithelium (figure 3C). Nitrotyrosin immunoreactivity was

No signs of active inflammation, defined as presence of lymphocytes, amount of eosinophiles granulocyts or plasmacells, were detected in the esophageal mucosa (table 3).

detected in the epithelium in both the basal and in the upper layer (figure 3D).

**C F**

esophageal mucosal biopsy retrieved during endoscopy

**4.1 Biochemical signs of inflammation** 

**4.2 Histological signs of inflammation** 

**4. Results** 

shown).

1:500 respectively. Control sections were incubated with normal rabbit or mouse IgG 0.4µg/µL instead of the primary antibody. After being washed, the slides were incubated with biotinylated secondary antibody and the complex was detected using horseradish peroxidase (HRP)-streptavidin. The colour was developed using 3,3'-diaminobenzidine.
