EL PRIMER TRIUNVIRATO

Pepsin (an acidic protease) is the primary proteolytic enzyme of the digestive system. It is produced by the chief cells of the stomach lining as pepsinogen and cleaved to pepsin in an acidic environment. Pepsin is not known to be produced by extra-oesophageal tissues and is therefore, first, a measure of reflux if found in the upper airway, and second, reflux aspiration if found in the lungs. Importantly, it is potentially a causative agent in extra-oesophageal injury. Animal studies have shown that BAL pepsin is a highly sensitive and specific marker of aspiration after forced gastric juice aspiration, with raised pepsin levels being detected up to 48 hours post aspiration event (Badellino et al., 1996; N. A. Metheny et al., 2004; H. B. Zhang, Van Hook, Fan, Mallory, & Elidemir, 2005). Various methods of pepsin measurement are discussed within the literature.

1.4.1.1 Enzymatic pepsin assays

The enzymatic activity of human pepsin 3b is pH dependent, having maximum activity at pH 2 becoming inactive at pH 6.5. It remains stable up to pH 7 but becomes irreversibly damaged at pH 8 (Nikki Johnston, Dettmar, Bishwokarma, Lively, & Koufman, 2007). Enzymatic assays include a sample re-acidification step (to re-activate any pH deactivated pepsin) followed by a quantification of

substrate digestion measured by protein digestion, spectroscopy, or other methods. Sample pepsin concentration is determined relative to a standard curve of substrate digestion activity for known pepsin concentrations, and then normalized to total sample protein. In samples up to pH 7, pepsin retains approximately 79% of its enzymatic activity on re-acidification (Nikki Johnston et al., 2007). Previous studies have shown correlative relationships between airway sample pepsin measurements and other clinical parameters of reflux or disease using the enzymatic assay (Sabeena Farhath et al., 2006; S. Farhath et al., 2008; U. Krishnan, Mitchell, Messina, Day, & Bohane, 2002; Potluri et al., 2003).

However, this type of assay has a number of limitations. In addition to the stomach, pepsinogen is also produced in the lung (pepsinogen C) and is found in serum (pepsinogen A and C) (Foster, Aktar, Kopf, Zhang, & Guttentag, 2004). Thus, activation of pepsin by acidification could also potentially cleave pepsinogen to pepsin, leading to false positive results. Other potential problems include interference from other proteases (such as cathepsins), freeze/thawing of samples affecting activity and highly proteinaceous samples inhibiting pepsin activity (Badellino et al., 1996; He et al., 2007; U. Krishnan et al., 2002)). Most importantly, if the sample pH is above pH 7, enzymatic activity may be reduced to a variable degree between samples upon reactivation, leading to potential false-negative results and difficulties in quantitative comparisons between samples (Nikki Johnston et al., 2007).

1.4.1.2 Immunologic pepsin assays

Enzyme Linked Immunosorbent Assays (ELISAs) and Western blotting techniques can be used to assess pepsin concentrations, but both are dependent on the quality and specificity of the pepsin antibody, many of which are known to cross- react with pepsinogen. More specific antibodies to human pepsin have been developed, and these have been shown to have a high specificity through gel electrophoresis and mass spectrometry studies (Crapko, Kerschner, Syring, & Johnston, 2007; Knight, Lively, Johnston, Dettmar, & Koufman, 2005). Both sandwich and indirect pepsin ELISA methods have been published. These techniques are cheap, more sensitive (0.2 ng/ml) than published enzymatic

primary antibody increases the ratio of antigen to non-specific protein bound to the platform. There is only one commercially available assay (USCN Life Science). However, assay documentation suggests that it is not fully validated across multiple respiratory sample types, and on inquiry, the antibody used has significant cross-reactivity with pepsinogen. Much pepsin research is therefore still dependent on ‘in-house’ kits.

Western blot analysis (WB) provides the advantage of being able to differentiate positive signal due to pepsin, pepsinogen, or other interfering proteins because proteins are separated by molecular weight. A further advantage is that, unlike enzymatic assays, it measures protein levels, and thus clinical samples above pH 7 should not test ‘false negative’ by WB analysis. WBs for pepsin analysis have been reported, including in the analysis of laryngeal and sub-glottic biopsies (Kim, Lee, Yeo, Kim, & Cho, 2008; Knight et al., 2005; N. A. Metheny et al., 2004). The Wisconsin group have now validated this method for analysis of BAL, with a pepsin lower limit of sensitivity of 0.05 ng (Johnston et al, unpublished data). The main disadvantage of WB is that it is semi-quantitative at best and in bronchial lavage samples, there is no reliable and validated protein which can be used for normalisation.

1.4.1.3 Human studies

Pepsin has been proposed as a useful biomarker of reflux aspiration in adults and in paediatric studies. Krishnan et al. detected pepsin enzymatic activity in 31 of 37 tracheal aspirates from children with GORD compared with 0 of 26 healthy controls (A. Krishnan et al., 2007). Significantly raised BAL pepsin levels have also been found in children with chronic cough and proximal reflux (Farrell, McMaster, Gibson, Shields, & McCallion, 2006). Similarly, Starosta et al. showed raised BAL pepsin levels in 96 children with chronic lung disease, with and without GORD (Starosta et al., 2007). However, specificity was low, and there was extensive group overlap. Pepsin has also been used as a marker of aspiration in children ventilated on intensive care. However, results vary widely with reported pepsin positivity in tracheal aspirate ranging from 13.5% to 70% (Gopalareddy et al., 2008; Kathleen L. Meert, Daphtary, & Metheny, 2002), possibly due to differences in assay sensitivity. Interestingly, recent studies using very sensitive assays have found low pepsin levels in healthy paediatric control

patients (McNally et al., 2011). The authors acknowledge that reflux of gastric contents into the pharynx occurs frequently in normal, asymptomatic children, and healthy adults have been shown to aspirate nasopharyngeal secretions during sleep. Therefore, this finding is perhaps unsurprising (Gleeson, Eggli, & Maxwell, 1997; Ramaiah, Stevenson, & McCallion, 2005).

Other groups have correlated pepsin levels with respiratory pathology. pH-MII studies in lung transplant recipients have shown that acid and alkaline reflux is common (Blondeau, Dupont, et al., 2008). Pepsin levels in BAL from lung transplant patients correlate with acute rejection, implying a contribution of pepsin to allograft injury (Stovold et al., 2007; Ward et al., 2005). Farhath et al

found 92% pepsin positivity on analysis of tracheal aspirates from preterm ventilated neonates, and correlated pepsin levels with development of bronchopulmonary dysplasia (Sabeena Farhath et al., 2006; S. Farhath et al., 2008). Finally, correlations between BAL pepsin levels and airway neutrophils or inflammatory cytokines have been reported in GORD, cystic fibrosis, and post lung transplant (Blondeau, Merters, et al., 2008; McNally et al., 2011; Starosta et al., 2007).

Pepsin levels in more readily available samples such as sputum, saliva, and exhaled breath condensate have also been examined (A. Krishnan et al., 2007). Analysis of induced sputum from healthy children found pepsin was detectable in saliva from 17 of 19 children pre-sputum induction, and 19 of 19, post-sputum induction (median 14.7 ng/ml, range 8.3-25.6 ng/ml) (Ervine, McMaster, McCallion, & Shields, 2009). There is no evidence that pepsin is produced by the salivary glands; thus, these results are again consistent with the concept of normal physiological reflux in children (Ramaiah et al., 2005). In the adult literature, a variety of assays have been used to detect pepsin within sputum and saliva in patients with GERD and respiratory symptoms (Kim et al., 2008; Knight et al., 2005; Li et al., 2008; Potluri et al., 2003; Strugala, Dettmar, & Morice, 2009; Wang et al., 2010)). The variation in the frequency of pepsin detection in control subjects and the levels of pepsin reported in salivary/sputum samples is striking. In addition, pepsin levels detected in BAL

collection. When clinical specimens are analyzed by ELISA, antibody interference is always possible and particularly so when dealing with thick and proteinaceous samples, such as sputum. Therefore, rigorous assay validation is essential. Authors have called for assay standardization as a priority, so that normative values can be established for paediatric and adult age groups (Davis et al., 2010).