Biliary atresia is a devastating disease for which current diagnostic and treatment methods are inadequate. Because early diagnosis is essential for effective drainage via Kasai portoenterostomy, better non-invasive diagnostic techniques are needed. This is especially important since many neonatal diseases can be confused with BA, and diagnosis currently requires operative cholangiogram and liver biopsy (1
). Thus, although children with BA have a more severe form of liver disease than children with many other types of cholestasis, this may not be initially apparent based on clinical presentation or routinely investigated laboratory parameters as supported by data in . For this study we hypothesized that liver injury in infants with BA is distinct enough from other types of neonatal cholestasis that combinations of serum biomarkers might distinguish infants with BA from other cholestatic children. For these studies we started with serum from 38 infants, aligned more than 1100 spots across 19 large format DIGE gels, and used bioinformatics techniques to identify 11 proteins whose abundance could classify serum from cholestatic infants into BA or non-BA groups. We recognize that this work will need replication with independent serum samples. Nonetheless, these promising data suggest that non-invasive tests for BA are achievable, especially if serum biomarkers are combined with additional imaging and clinical parameters. Test methods, however, will need to be simplified before these biomarkers could be used clinically.
Before discussing the potential significance of proteins in our classifier, we note that a recent analysis demonstrated changes in serum apolipoprotein C-II and transthyretin in infants with BA at early and late stages of disease (12
). Although these proteins were among our diagnostic biomarkers, comparing their data with ours is difficult because of few control samples in their study (n = 2) and their emphasis on changes in serum proteins with disease progression in BA, versus our emphasis on differential diagnosis between BA and other cholestatic disease. In both studies, changes identified are “relative” protein abundance levels, but diagnostic tests will require alternative approaches to determine absolute serum biomarker levels. Nonetheless, their data suggest that biomarkers for BA could change as disease progresses. Our data only apply to children at initial contact with pediatric gastroenterologists (i.e., at the time of initial diagnosis).
In addition to potential diagnostic significance, identified biomarkers can be tied to prior observations about BA and cholestatic disease. While we need to avoid over-interpretation, it is worth considering potential biological significance of our findings. Both apolipoprotein C-II and E were more abundant in serum from BA than non-BA infants consistent with previously reported apolipoprotein E elevations in individuals with biliary tract obstruction (13
). These proteins are linked on chromosome 19 and their expression is regulated by two hepatic control region cis
-acting liver enhancers (16
) that are activated by the farnesoid X-activated receptor (FXR; NR1H4), a nuclear hormone receptor that induces gene expression in response to several bile acids (17
). Remarkably, expression of complement component 3, another protein that was more abundant in BA than non-BA serum is also activated by FXR (18
). Collectively, these data suggest that FXR activity is higher in liver of infants with BA than in non-BA cholestasis, but there are other possible explanations.
Elevated apolipoprotein C-II in infants with BA correlates with old observations about dyslipoproteinemia in cholestatic disease. In particular, lipoprotein-X (LP-X) was previously suggested to help distinguish BA from other types of neonatal cholestasis (19
). LP-X is an unusual lamellar particle with high free cholesterol and phospholipid levels that accumulates in serum during biliary tract obstruction (20
). LP-X is also found in individuals with deficiency in lecithin cholesterol acyl transferase (LCAT), an enzyme that produces cholesterol ester from free cholesterol and lecithin. Interestingly apolipoprotein C-II, inhibits LCAT (22
) consistent with the idea that LP-X may be elevated in children with BA compared to other neonatal cholestatic diseases because of increased apolipoprotein C-II.
The identified potential biomarkers also suggest a more significant pro-inflammatory state in BA than other neonatal cholestatic diseases consistent with prior observations (23
). For example, complement C3 and factor B are acute phase reactant proteins that were more abundant in BA versus non-BA serum. These same proteins are elevated in adults with large bile duct obstruction or viral hepatitis (26
) and in primary biliary cirrhosis (29
). In contrast, serum levels of C3 and factor B were reduced in chronic active hepatitis and cryptogenic cirrhosis suggesting that their abundance may be useful as part of a diagnostic fingerprint that distinguishes BA from other neonatal liver diseases. Note, however that although recent data suggest that complement may be activated in BA (31
), our data do not provide insight into complement activation.
Low serum levels of transthyretin (prealbumin), apolipoprotein H and alpha2-HS glycoprotein are also consistent with a pro-inflammatory state in infants with BA since these proteins are negative acute phase reactants (32
). Furthermore, although low serum transthyretin is often assumed to reflect malnutrition, transthyretin falls rapidly in rats after biliary tract obstruction (36
) and inflammation is among the most potent transthyretin regulators (35
). Interestingly, mannose binding lectin (MBL2) is a component of the innate immune system (38
) but amyloid P, an acute phase reactant in mice closely related to C-reactive protein, is not a human acute phase reactant (40
). Collectively, elevated levels of positive acute phase proteins and reduced levels of negative acute phase proteins in serum of infants with BA is consistent with a pro-inflammatory state in BA compared to other forms of neonatal cholestasis. Caution must be used interpreting these data however, since FXR mRNA is repressed in mouse liver during the acute phase response (41
), at least after treatment with some inflammatory mediators. While this might seem to contradict the hypothesis outlined above that FXR activity is elevated in liver of infants with BA, FXR mRNA levels and FXR activity need not be related since FXR is activated by bile acids that accumulate more significantly in cholestatic than inflammatory liver disease.
Developing rapid non-invasive diagnostic tests that clearly distinguish BA from other forms of neonatal cholestasis is important. Our studies suggest that although no single marker reliably separates BA from non-BA serum samples, combinations of markers could be valuable. In particular, application of modern statistical methods to complex data sets provides a new opportunity to distinguish BA from other types of liver disease. Interpretation of the biological significance of identified potential biomarkers, however, is challenging. First, since we compared serum from infants with BA to serum from non-BA cholestatic infants, these data provide no insight into how these protein levels might compare to healthy infants. In addition, these analyses provide limited insight into hepatic function, liver pathology, or the extent of hepatic injury. Comparable proteomic analyses of liver tissue might provide additional valuable information, but would likely require analysis of relatively large biopsy samples to avoid the complex issues that arise in sampling heterogeneous tissue. Finally, while it is tempting to speculate that these biomarkers reflect specific disease processes as we have done, the complexity of BA and non-BA liver disease makes it possible to generate hypotheses, but not to draw conclusions about disease etiology or pathogenesis based on our data. Nonetheless, these new data provide a rationale for additional studies to validate and extend our observations with the goal of developing reliable non-invasive diagnostic testing for BA. This work will require quantitative analysis of serum protein abundance for selected biomarkers (e.g., by ELISA) and the analysis of serum from many additional children with BA and non-BA liver disease. Definitive diagnosis may require a combination of serum biomarkers, clinical criteria, and imaging results to provide an unambiguous diagnosis without liver biopsy or cholangiogram. These results also support further investigations into the role of FXR and inflammation in BA.