787 STOMACH Gut: first published as 10.1136/gut.51.6.787 on 1 December 2002. Downloaded from Human transforming growth factor α (TGF-α) is digested to a smaller (1–43), less biologically active, form in acidic gastric juice T Marchbank, R Boulton, H Hansen, R J Playford ............................................................................................................................. Gut 2002;51:787–792 Background: Transforming growth factor α (TGF-α) is a 50 amino acid peptide with potent prolifera- tive and cytoprotective activity present in gastric mucosa and juice. Aims: To determine the forms and biological activity of natural and recombinant TGF-α following incu- bation with acid pepsin. Patients: Human gastric juice was obtained under basal conditions from patients taking acid suppres- sants and from volunteers undergoing intragastric neutralisation. See end of article for Methods: Samples were analysed using mass spectroscopy and/or high pressure liquid chromatogra- authors’ affiliations phy with radioimmunoassay. Biological activity was determined using thymidine incorporation into rat ....................... hepatocytes and an indomethacin/restraint induced gastric damage rat model. α α Correspondence to: Results: TGF- 1–50 is cleaved to TGF- 1–43 by acid pepsin and this is the predominant form in normal Professor R J Playford, gastric juice. However, intragastric neutralisation or taking acid suppressants caused the predominant Gastroenterology Section, α α 3 form to be TGF- 1–50.TGF- 1–43 had only half of the ability to maximally stimulate [ H]thymidine incorpo- Imperial College School of α ration into primary rat hepatocytes (28 177 (1130) DPM/well for 2.16 nM TGF- 1–43 v 63 184 (3536) Medicine, Hammersmith α Hospital Campus, Du Cane DPM/well for TGF- 1–50; p<0.001). A similar reduced potency was seen when used in an indomethacin α Rd, London W12 0NN, induced rat gastric damage model (0.18 µmol/kg/h of TGF- 1–43 reduced ulcer area by 19% whereas UK; [email protected] α TGF- 1–50 reduced area by 62%; p<0.001). Conclusions: α α Accepted for publication TGF- 1–50 is cleaved to the TGF- 1–43 form by acid pepsin, causing 2–5-fold loss of bio- 30 April 2002 logical activity. Such changes may have relevance to the actions of acid suppressants and the import- ....................... ance of this peptide in both normal and abnormal growth. http://gut.bmj.com/ ransforming growth factor α (TGF-α) is a 50 amino acid Methods of analyses of samples to determine the forms peptide present in gastric juice and throughout the of TGF-α Tgastrointestinal tract.1 It is a potent stimulant of Preparation of gastric juice samples prior to high pressure proliferation of several gastrointestinal cell lines in vitro2–5 and liquid chromatography (HPLC) in the rat intestine when given systemically.6 In addition, Samples of gastric juice which were to have HPLC performed TGF-α protects against injury in a variety of gastric damaging were first concentrated using octadecylsilane (C18) cartridge on September 23, 2021 by guest. Protected copyright. 10 models such as those induced by ethanol or stress,78 and chromatography, as described by Elson and colleagues. Eluates were then dried on a centrifugal evaporator (Savant; human gastric juice concentrations of TGF-α increase in Farmingdale, New York, USA) and resuspended in 0.1% response to administration of noxious agents such as trifluoroacetic acid (TFA) prior to injection into the HPLC loop. indomethacin.9 Taken together, these studies suggest TGF-α plays a role in growth, differentiation, and maintenance of Reverse phase HPLC mucosal integrity of the bowel. Studies involving reverse phase HPLC used a Hewlett Packard As TGF-α is present in human gastric juice, it is constantly 1100 (Stockport, UK) consisting of a quaternary pump deliv- bathed in acid and pepsin. There has been virtually no work ery system, Rheodyne-7725 sample injector, anda1mlinjec- however examining whether it is adversely affected by being tion loop. The column used was an analytical “Jupiter” C5, 300 present in such a harsh environment. We therefore examined A, 5 µm (4.6×150 mm) column (Phenomenex UK Ltd). After the effect of acid and pepsin on the form and biological activ- application of the sample, the column was eluted isocratically ity of TGF-α in vitro and in vivo and determined the forms of (15% acetonitrile (AcN), 0.1% TFA) for 10 minutes before a TGF-α present in gastric juice obtained from subjects under gradient from 15–40% AcN, 0.1% TFA was run over 30 varying acidic conditions. minutes. Samples were collected on a Foxy-Jr fraction collec- tor and the system was controlled by a HP pentium PC with HP chemstation software. Samples were dried on a centrifugal evaporator and resuspended in water or Tris buffer prior to mass spectroscopy or radioimmunoassay, respectively. MATERIAL AND METHODS Materials Recombinant and purified native human TGF-α were ob- ............................................................. tained from Calbiochem (Nottingham, UK). Porcine pepsin Abbreviations: HPLC, high pressure liquid chromatography; AcN, was obtained from Sigma (P6887, Poole, UK) in the form of a acetonitrile; TFA, trifluoroacetic acid; EGF, epidermal growth factor; lyophilised powder. All other chemicals were obtained from EGF-R, epidermal growth factor receptor; PPI, proton pump inhibitor; RIA, Sigma unless stated otherwise radioimmunoassay; TGF-α, transforming growth factor α. www.gutjnl.com 788 Marchbank, Boulton, Hansen, et al Radioimmunoassay (RIA) for TGF-α a Dynatech Multimash automatic cell harvester and solubilised TGF-α-like immunoreactivity was measured using a commer- by incubation at 37°C for one hour in 200 µl of 1 M KOH. Cell cial RIA kit (BT-350; Biomedical Technologies Inc., Stoughton, extract (50 µl) was counted in a β counter in 1 ml of scintillant Gut: first published as 10.1136/gut.51.6.787 on 1 December 2002. Downloaded from Massachusetts, USA). Briefly, tubes were incubated for 24 hours (Optiphase Safe; LKB-Pharmacia, Bromma, Sweden). at 4°C, and bound and free TGF-α were separated using 50 µl/tube of donkey antisheep antibody and 50 µl of PEG followed Assay of biological activity of different forms of TGF-α in by mixing and centrifugation at 2000 rpm for 15 minutes at 4°C. vivo α Typical results gave a maximal binding ratio (Tmax tubes) of 0.5 The ability of TGF- to prevent gastric damage by indometh- and a non-specific binding ratio (To tubes) of 0.03. Sensitivity of acin and restraint in rats was assessed using previously the assay is 0.02 ng/tube. This radioimmunoassay does not cross validated methods.14 Under light ether anaesthesia, rats (male react with epidermal growth factor (EGF). Sprague Dawley, 225–250 g) had two subcutaneous cannula inserted into the back of the neck and were then placed in Pilot study to determine the effect of acid pepsin digestion of Bullman restraint cages. Once the animals had recovered, a TGF-á on immunoreactivity of TGF-á continuous subcutaneous infusion of saline or various doses α Six aliquots of TGF- were randomised to be incubated in and forms of TGF-α was started at 1 ml/h using a either Tris buffer (pH 7) or 0.1 M HCl, each containing pepsin multi-syringe infusion pump (Harvard Apparatus, Massachu- at 1 mg/ml, for one hour at 37°C. At the end of the incubation setts, USA). Thirty minutes later, 20 mg/kg of indomethacin period all samples were neutralised using 0.1 M NaOH. were injected subcutaneously via the second cannulae. Standard curves were then performed using all of these aliq- Animals were killed by stunning and cervical dislocation three uots on a single day. Data were analysed by fitting three hours later and their stomachs removed and inflated with 4 ml parameter logistic functions of loge concentration by least of 10% formalin. The next day the stomachs were opened and squares. These showed a small but significant decrease in the placed in fresh formalin prior to assessment. The stomachs α immunoreactivity of TGF- which had been treated with acid were randomly coded and all analyses of gastric damage were pepsin. Fifty per cent of maximal binding occurred at a assessed blind. Total ulcerated area (mm2/stomach) was α concentration of 5.2 ng/ml TGF- in Tris/pepsin standards and assessed using a dissecting microscope (×10) with the aid of a α 2.0 ng/ml TGF- in standard which had been treated with acid square grid. The stomachs were then embedded in wax and pepsin (significance of difference between 50% maximum the depth of damage assessed microscopically and given a α 15 binding values p<0.001). Concentrations of TGF- in gastric microscopic ulcer score, as previously described. Using this juice samples were determined taking into account these system, each stomach was given a score of 0 to 4, with 0=no small differences in immunoreactivity of the intact and damage, 1=one small erosion (less than 0.5 mm), 2=two digested forms. small or one large erosion (greater than 0.5 mm), 3=two or more large erosions, and 4=any area of ulceration extending Mass spectroscopy to the muscularis mucosa. Molecular weight was assessed by mass spectroscopy using the technique of matrix assisted laser desorption time of flight Ethics approval with a Finnigan LaserMAT mass spectrophotometer (San Local ethics approval was obtained for studies involving Jose, California, USA). Samples were mixed with 0.5 µlof human volunteers and subjects gave informed consent. http://gut.bmj.com/ alpha-cyano-4-hydroxycinnamic acid matrix (1% in 50% AcN, β 0.1% TFA). Standards used were oxidised insulin chain Study protocols (molecular weight 3496.9) and ubiquitin (molecular weight Study 1: stability of recombinant TGF-α in acid pepsin in 8564.9).