| Intrinsic factor (IF) is a transport glycoprotein secreted by the parietal cells of the gastric fundic mucosa, which is necessary cofactor for the absorption of orally ingested vitaminB12 ( VitB12). IF has two binding sites, one each for Vit B12 and for the ileal receptor recognizing the IF-VitB12 complex that could not be hydrolyzed by hydrolase in the intestinal tract. It is known that IF deficiency induces malab-sorption of VitB12 which leads to subsequent megaloblastic changes as pernicious anemia in A type atrophic gastritis. Reports that reflected the correlation between the level of serum IF and gastric mucosal functional status is few. To study the relation, we separated and purified human IF, and maked the radio-immunity kits of IF. Eventually, we determined the content of serum IF by the method of radioimmunoas-say.Materials and MethodsTissue samples. Human normal gastric mucosa were gained fromCollege of Forensic Medicine of China Medical University. Two New Zealand male white rabbits with 2 kilogram. Samples of patients serum were gained from The People Hospital of Qinghai province from October, 2000 to August, 2001. These patients included 49 cases of gastric cancer ( age from 32 - 86 years) , 22 cases of stomach ulcer ( age from 22 - 67 years) , 25 case of atrophic gastritis ( age from 20 - 65 years). All of patients were diagnosised by endoscopy and/or histo-logic examination. Fasting sera from 96 patients obtained in the early morning were stored at -20℃. 40 cases normal serum samples were gained from healthy volunteers to donate blood.Chemicals. DEAE-Sephadex A50, Sephadex G-75 and CNBr activated Sephrose 4B were purchased from Pharmacia Corp. Acrylam-ide, Bis-acrylamide and SDS were purchased from Sigma Corp. 131I was a product of Institute of Atomic Energy in Beijing. Protein marker was purchased from Huamei Corp. All other chemicals were of reagent grade.Preparation of gastric mucosal extract. The mucosa that was thawed was separated by sharp dissection, drained on filter paper, weighed,, minced and homogenized in cold 0. 01M phosphate buffer, pH7.0(2ml/g tissue) , after two centrifuge, the supernatant was the extract.A column of 3cm x60cm was packed with diethylaminethvl (DE-AE)-Sephadex A50 and equilibrated with the above buffer. A-30 ml portion of mucosal extract was applied to the top of the column. The column was eluated with a linear NaCl gradient The protein peaks were pooled, concentrated, dialyzed and stored at - 20℃, respectively.Gel filtraton chromatography of partially purified IF on Sephadex G-75. 1-ml portion of fourth peak of above chromatography was appliedto the column. The column was eluted with 0. 01M phosphate buffer, pH7. 0. Protein peaks were pooled, concentrated and stored at -20℃.Affinity chromatography of partially purified IF on VitB12-Sepha-rose 4B. VitB12 were covalently coupled to CNBr-activated Sepharose 4B resulting in 1.17mg of B12 coupled per ml of Sepharose. Partially purified IF was passed over the column. A linear gradient of guanidine HC1 (0 to 4.0M) was used to separate IF.Protein determination. Protein was determined by the method of Lowry et al. with bovin serum albumin as a standard.The identification of Protein peaks and the evaluation of molecular weight of them were performed on Sodium Dodecyl Sulfate- Polyac-rylamide Gel Electrophoresis (SDS-PAGE)Preparation of the antiserum to IF and determination of valence. Rabbits were immunized by injecting the partially purified IF together with an equal volume of Freund s complete adjuvant into the back intramuscularly and subcutaneously at 2 week intervals. Valence of antibodies was determined by a double immunodiffusion method.The purified IF was labeled with I in accordance with the Iodo-gen method. A SephadexG-25 column was used to remove free 131I from 131I-IF.Serum IF level of groups was determined by the method of Radio-immunoassay.Statistical analysis The significance of differences in mean values was determined by Student's t test for unpaired... |
PDF Full Text Request