RESUMEN
Celiac disease (CD) is a chronic autoimmune disorder induced in genetically susceptible individuals by the ingestion of gluten from wheat, rye, barley, or certain varieties of oats. A careful diet follow-up is necessary to avoid health complications associated with long-term gluten intake by the celiac patients. Small peptides (GIP, gluten immunogenic peptides) derived from gluten digestion, which are excreted in the urine and feces, have emerged as promising biomarkers to monitor gluten intake. We have implemented a simple and sensitive label-free point-of-care (POC) device based on surface plasmon resonance for the direct detection of these biomarkers in urine. The assay employs specific monoclonal antibodies and has been optimized for the detection of the 33-mer α2-gliadin, known as the main immunogenic peptide of wheat gluten, and for the detection of GIP. Direct detection in undiluted urine has been accomplished by using biosensing chips containing a robust and stable biorecognition layer, obtained after carefully optimizing the biofunctionalization protocol. Excellent limits of detection have been reached (1.6-4.0 ng mL-1 using mAb G12 and A1, respectively), which ensures the detection of gluten peptides even when the gluten intake is around the maximum tolerable amount in the digestive tract (< 50 mg) for celiac individuals. No sample pretreatment, extraction, or dilution is required, and the analysis takes less than 15 min. The assays have excellent reproducibility' as demonstrated by measuring spiked urine samples containing the same target concentration using different biofunctionalized chips prepared and stored at different periods of time (i.e., CV% of 3.58% and 11.30%, for G12- and A1-based assays, respectively). The assay has been validated with real samples. These features pave the way towards an end-user easy-to-handle biosensor device for the rapid monitoring of gluten-free diet (GFD) and follow-up of the health status in celiac patients.
Asunto(s)
Enfermedad Celíaca/orina , Dieta Sin Gluten , Gliadina/orina , Fragmentos de Péptidos/orina , Resonancia por Plasmón de Superficie/instrumentación , Anticuerpos Inmovilizados/química , Anticuerpos Monoclonales/química , Enfermedad Celíaca/dietoterapia , Diseño de Equipo , Humanos , Límite de Detección , Resonancia por Plasmón de Superficie/economía , Factores de TiempoRESUMEN
BACKGROUND: A major deficit in understanding and improving treatment in coeliac disease (CD) is the lack of empiric data on real world gluten exposure. AIMS: To estimate gluten exposure on a gluten-free diet (GFD) using immunoassays for gluten immunogenic peptides (GIP) and to examine relationships among GIP detection, symptoms and suspected gluten exposures METHODS: Adults with biopsy-confirmed CD on a GFD for 24 months were recruited from a population-based inception cohort. Participants kept a diary and collected urine samples for 10 days and stools on days 4-10. 'Doggie bags' containing » portions of foods consumed were saved during the first 7 days. Gluten in food, stool and urine was quantified using A1/G12 ELISA. RESULTS: Eighteen participants with CD (12 female; age 21-70 years) and three participants on a gluten-containing diet enrolled and completed the study. Twelve out of 18 CD participants had a median 2.1 mg gluten per exposure (range 0.2 to >80 mg). Most exposures were asymptomatic and unsuspected. There was high intra-individual variability in the interval between gluten ingestion and excretion. Participants were generally unable to identify the food. CONCLUSIONS: Gluten exposure on a GFD is common, intermittent, and usually silent. Excretion kinetics are highly variable among individuals. The amount of gluten varied widely, but was typically in the milligram range, which was 10-100 times less than consumed by those on an unrestricted diet. These findings suggest that a strict GFD is difficult to attain, and specific exposures are difficult to detect due to variable time course of excretion.
Asunto(s)
Enfermedad Celíaca/metabolismo , Dieta Sin Gluten , Exposición Dietética/análisis , Glútenes/farmacocinética , Adulto , Anciano , Enfermedad Celíaca/orina , Ingestión de Alimentos , Heces/química , Femenino , Contaminación de Alimentos/análisis , Glútenes/análisis , Glútenes/orina , Humanos , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
Background: Celiac disease (CD) patients adhering to a gluten-free diet (GFD) are exposed frequently to low levels of gluten that contribute to symptoms and persistent intestinal histologic damage. Objective: We analyzed prior clinical data to determine how much gluten is accidentally consumed while on a GFD. The aim was to understand the range of gluten consumption for a wide distribution of CD patients. Design: A meta-analysis was conducted on data from 2 different clinical programs: 1) measurements of gluten in stool and urine in CD and non-CD populations; and 2) analysis of data from trials for the investigational therapeutic latiglutenase. The stool and urine studies included controlled gluten challenges. A calibration factor was applied that allowed normal ingestion of gluten to be computed from the urine and stool measurements. From the latiglutenase trial data, a determination of gluten consumption was made by estimating how much gluten was eliminated from patients' diets due to a trial effect that led to improved histology even in the placebo group. Results: The average inadvertent exposure to gluten by CD individuals on a GFD was estimated to be â¼150-400 (mean) and â¼100-150 (median) mg/d using the stool test and â¼300-400 (mean) and â¼150 (median) mg/d using the urine test. The analyses of the latiglutenase data for CD individuals with moderate to severe symptoms indicate that patients ingested significantly >200 mg/d of gluten. Conclusions: These surrogate biomarkers of gluten ingestion indicate that many individuals following a GFD regularly consume sufficient gluten to trigger symptoms and perpetuate intestinal histologic damage.