Lung surfactant protein A (SP-A) and B (SP-B): Biological markers for diagnosis and infection in patients after lung transplantation

Lung rejection and infection are critical events frequently seen in patients after lung transplantation. If not rapidly diagnosed and appropriately treated, rejection and infection will result in lung dysfunction and lung failure. Results obtained in patients demonstrate that after lung transplantation the pulmonary surfactant system is impaired. In addition, it is found that during both lung rejection and infection the alveolar-capillary membrane is the site of inflammation and increased permeability. Interestingly, we (unpublished data) and others have found in patients with acute and chronic lung diseases that levels of surfactant proteins A and B are elevated in plasma. Thus it is conceivable that during rejection and infection the concentration of surfactant proteins A and B is also increased. Moreover, due to differences in the onset and spread of the disease process, the increase in concentration of surfactant proteins A and B during rejection could be different from that during infection. Therefore the aim of this study is to determine 1) whether during rejection and infection the concentration of surfactant proteins A and B in plasma is increased, and 2) whether differential changes in SP-A and SP-B levels in plasma can be used to distinguish rejection from infection in patients after lung transplantation.

Summary: The aim of this study was to determine whether lung surfactant proteins could be used as markers to diagnose rejection or infection in patients after lung transplantation. Serum concentrations have been determined for surfactant proteins A and D (SP-A and SP-D). Statistical analysis of data and correlation with patient history is in progress. Introduction: Lung surfactant proteins are normally detected at low concentrations in serum. In contrast, they are found at high concentrations in acute and chronic lung disease. Thus it is possible that they could be potentially used as markers to diagnose rejection or infection in patients after lung transplantation (LuTX). We hypothesized that due to differences in the onset and spread of the disease process, the increase in concentration of SP-A and SP-B during rejection could be different from that during infection. Materials and Methods: Two independent techniques (immunoassay, high performance liquid chromatography - HPLC) were used to measure surfactant protein concentrations in serum. Serum was obtained from patients before, up to three weeks after transplantation as well as from patients during postoperative routine follow-up exams (n = 730 serum samples). Results: Immunoassay: Using an indirect ELISA purified SP-A could only be detected at concentrations above 18 ng/ml. Using the more sensitive DELFIA technique, however, the detection limit could be decreased to 2 ng/ml. Similarly, purified SP-B could be detected down to 3 ng/ml albeit at lower signal intensity. Next, concentrations of SP-A and SP-B were determined in serum. In a large series of experiments it was found that detection of both proteins was unreliable. Employing various modifications to improve performance of assays, e. g. detergents, calcium chelators, enzymes, immunoprecipitation was unsuccessful. Thus, concentrations of SP-A in serum were determined in collaboration with IRC - International Reagents Corporation (Kobe, Japan). Similarly, another collaboration has been initiated to determine concentrations of SP-B. Finally, we measured concentrations of SP-D in serum with an ELISA-kit (Yamasa, Japan). Data reduction and correlation with patient history for SP-A and SP-D is now under progress. HPLC: Due to the heterogeneity of SP-A (hydrophilic) and SP-B (hydrophobic) a variety of separation techniques was tested (reversed-phase-, size exclusion-, ion exchange- and affinity- chromatography). Regardless of the technique used progress was always hampered by problems such as solubility of the proteins and oligomerization, unspecificity of protein spectra and high absorbance of additives, missing or unspecific binding of antibodies, memory effects and low reproducibility. Although important information regarding the behavior of surfactant protein A and B at different experimental conditions was obtained no serum samples could be reliably analyzed up until now with this method. Discussion: Analysis of SP-A and SP-B was complicated by problems based on a large extent on the grossly different chemical characteristics of both proteins. Nevertheless, concentrations of SP-A and SP-D in serum of patients after LuTX have been determined.