FARMACIA, 2009, Vol.LVII, 1 99 GLUTEN SCREENING FROM PHARMACEUTICAL PRODUCTS, AN IMPERATIVE FOR CELIAC DISEASE MANAGEMENT SIMONA OANCEA1*, MIHAELA STOIA2 University “Lucian Blaga” of Sibiu, Department of Biochemistry and Toxicology, Str. I. Raţiu 7-9, 550012 Sibiu, Romania 2 Authority of Public Health, Sibiu, Str. Gh. Bariţiu 3, Sibiu, Romania *corresponding author: e-mail: simona.oancea@ulbsibiu.ro 1 Abstract Celiac disease is a chronic intestinal disorder caused by intolerance to gluten. It is recognized that pharmaceutical products may contain traces of gluten which will cause acute illness in celiac patients. In the present paper we investigated the content of potentially hidden gluten ingredient in basic children drugs using the immunochromatographic visual test. The negative gluten results are relevant to complete the existing lists of gluten-free drugs. The visual assay proved to be an efficient rapid tool for gluten screening as an alternative to the enzyme-linked immunosorbent assay (ELISA) techniques. Rezumat Boala celiacă reprezintă o disfuncţie intestinală cronică, rezultat al intoleranţei la gluten. Astăzi este recunoscut faptul că numeroase produse farmaceutice pot conţine urme de gluten ce determină simptome acute la pacienţii cu boala celiacă. În articolul prezent, am investigat conţinutul de gluten ca potenţial ingredient în produsele farmaceutice de bază destinate copiilor, utilizând metoda vizuală imunocromatografică. Rezultatele negative obţinute sunt importante şi relevante pentru completarea listelor existente, cu privire la medicamentele lipsite de gluten. Analiza imunocromatografică s-a dovedit o metodă eficientă şi rapidă de screening al glutenului ca alternativă la metodele ELISA. Keywords: celiac disease; gluten; immunochromatographic test Introduction Data from the National Institute of Health (NIH) reported that 1% of USA population is suffering from celiac disease (CD). In Europe, 1 in 150-300 individuals may suffer from CD, which still remains underdiagnosed in many European countries [1]. CD is a food-induced disorder characterized by a permanent intolerance to wheat gluten and related proteins from rye and barley. The ethiology of CD is caused by digestive disorders of the intestinal mucosa. The pathogenesis of the disease develops under an immunological mechanism driven by human leukocyte antigens-(HLA-DQ2 or DQ8)-restricted T cells [2]. Clinically, the picture of 100 FARMACIA, 2009, Vol.LVII, 1 CD goes from typical symptoms of diarrhea, abdominal pain, weight loss and anaemia to atypical symptoms of lethargy, chronic fatigue, bruising, osteoporosis, depression, hair loss, dental defects, short stature, infertility, late puberty, early menopause, unsteady gait, muscle weakness [3-5]. At present, the only efficient treatment of CD in both symptomatic and asymptomatic patients is a strict gluten-free diet throughout their lifetime (plain meats, vegetables, fruits). Gluten is present not only in cereals (wheat, rye and barley) and the baking products, but also in some food ingredients including thickeners, stabilizers, flavourings, preservatives, condiments, yeast extracts, etc. Unfortunately, gluten is also found in inactive ingredients of pharmaceutical products [6]. Complete avoidance of foods and medicines potentially contaminated with gluten is not an easy task and needs a multidisciplinary approach from patients, doctors, dieticians and pharmacists. Despite that few pharmaceuticals are labelled as gluten-free, the great majority of drug manufacturers do not specify on the product label information regarding the gluten content [7]. A great concern comes from the generic drugs, which may not contain the same excipients as the brand product. Detection of gluten in products is based on modern bioanalytical methods e.g. enzyme-linked immunosorbent assay (ELISA) and immunochromatographic techniques [8-9]. In the present paper, we investigated the content of potentially hidden gluten ingredient in basic pediatric drugs using the immunochromatographic visual test. This assay is a qualitative and semiquantitative sensitive method for the detection of gluten acting in good correlation with the ELISA assay [10]. Materials and methods Sample extraction and preparation Ten pharmaceutical products (pediatric syrups) purchased from the national pharmacy market were used in the present investigation, as follows: analgesics and anti-inflammatories (Panadol®, Paracetamol®), digestive system drugs (Debridat®, Metoclopramid®, Duphalac®), antihistaminic (Peritol®, Aerius®) and respiratory system drugs (Eurespal®, Rofedex®, Fluidol®). The samples were extracted in ethanol 60%. After 30 minutes incubation, the samples were filtered and diluted 1:10 with phosphate-buffer saline (PBS) containing 0.09% sodium azide, pH 7.4. FARMACIA, 2009, Vol.LVII, 1 101 Gluten detection The immunochromatographic visual assay was used for qualitative determination of gluten in pharmaceuticals. Immunochromatographic sticks (purchased from OPERON S.A., Spain) which contain monoclonal antibodies specific for gluten of wheat, barley and rye, were dipped into solution samples diluted 1:10. Results were read after 5 min. of chromatographic running. Development of a red band in addition to the control blue band on the reaction strips is considered a positive result. Results and discussion Celiac patients can manage their condition only by a strict gluten-free diet. Because of less obvious sources of gluten and lack of specification of wheat in the ingredient listing, celiac patients must check carefully the ingredients of food products and pharmaceuticals. It is known that small amounts of gluten cause intestinal damages in these individuals. Despite few common pediatric gluten-free drugs that are known, certain generic drugs may not be gluten-free. In the study, we have screened the presence of gluten in common pediatric medications (analgesics and anti-inflammatories, digestive system medications, antihistaminic, respiratory system medications) with unknown gluten status. These samples are not recognized as devoid of gluten by the celiac disease patients. Samples were extracted with ethanol 60% and diluted 1:10 in PBS. Qualitative determination of gluten was performed using the sensitive immunochromatographic test. The gluten reaction strips consist of four zones: the inferior adsorbent zone that is submerged in the sample, the red microsphere zone, the reaction zone and the superior adsorbent zone. The gluten positive samples react first with the red particles from the second zone of the strip and then migrate in the reaction zone of the strip in order to react with the anti-gliadin specific antibodies, developing a red band. A control band (blue) appears in both positive and negative samples. The sensitivity of the test is 10 ng/ml of gliadins equivalent to 2 ppm of gluten in the sample. The obtained results are given in table I. As it isshown in table I, we obtained a negative gluten test for all the samples. Consequently, the tested products do not contain gluten in concentration above 2 ppm, the sensitive limit of the test. These informations are important for completion of the existing lists of gluten-free medications (Wheaton Gluten Free Support Group). The immunochromatographic test used in the present gluten detection proved to 102 FARMACIA, 2009, Vol.LVII, 1 be a sensitive, simple and reliable test for rapid determination of gluten in pharmaceutical products. Table I Gluten content of selected pediatric pharmaceutical products determined by immunocromatographic assay Product name Active ingredient Form Manufacturer Gluten Panadol baby® Acetaminophen susp. Glaxo Smith Klein Paracetamol® Acetaminophen oral sol. Terapia Debridat® Trimebutine granules for Pfizer oral susp. Metoclopramid Metoclopramide oral sol. Pharmaplant ® Biogalenica Duphalac® Lactulose oral liq. Solvay Pharma Peritol® Ciproheptadine syrup Egis Pharmaceuticals LTD. Aerius® Desloratidine syrup Schering Plough Eurespal® Fenspiride syrup Servier hydrochloride Rofedex® Dextromethorphan syrup Biofarm Fluidol® Carbocysteine oral sol. TIS Farmaceutic Note: "-" means negative gluten test Conclusions Celiac disease (CD) is a chronic intestinal disorder of public health concern as untreated celiac patients develop disease complications and suffer from the negative impact on their quality of life. The disease results from ingestion of gluten from wheat, barley and rye. Gluten can be found not only in food products, but also as normal component of drugs. Detection of gluten in pharmaceuticals is a very important task required for displaying the gluten status of products, as the absence of gluten remains the only treatment for celiac patients. In this paper, we investigated the presence of gluten in ten common pediatric medications using a sensitive procedure, the immunochromatographic assay. The results provide information about the absence of gluten in the selected products contributing to the completion of the existing lists of gluten-free pharmaceuticals. It is known that, for celiac patients, obtaining accurate information about the gluten content of a particular pharmaceutical is a difficult and time-consuming process. FARMACIA, 2009, Vol.LVII, 1 103 References McLoughlin R, Sebastian SS, Qasim A, McNamara D, O’Connor HJ, Buckley M, O’Morain C, Celiac disease in Europe, Aliment. Pharmacol. Ther. 2003, 18 (Suppl. 3), 45– 48 2. Sollid LM, Markussen G, Ek J, Gjerde H, Vartdal F, Thorsby E, Evidence for a primary association of celiac disease to a particular HLA-DQ alpha/beta heterodimer, J. Exp. Med. 1989, 169, 345-350 3. Wieser H, Cereal protein chemistry, in Gastrointestinal immunology and gluten-sensitive disease, Feighery C, O'Farelly C (eds.), Oak Tree Press, Dublin, 1994, 191-202 4. Lo W, Sano K, Lebwohl B, Diamond B, Green PH, Changing presentation of adult celiac disease, Dig. Dis. Sci. 2003, 48, 395-398 5. 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