The Relationship between Plasma Alpha-1-Antitrypsin Polymers and Lung or Liver Function in ZZ Alpha-1-Antitrypsin-Deficient Patients

Abstract

Alpha-1-Antitrypsin (AAT) is a protein of the SERPINA1 gene. A single amino acid mutation (Lys342Glu) results in an expression of misfolded Z-AAT protein, which has a high propensity to intra- and extra-cellular polymerization. Here, we asked whether levels of circulating Z-AAT polymers are associated with the severity of lung disease, liver disease, or both. We obtained cross sectional data from the Dutch part of the Alpha1 International Registry of 52 ZZ-AAT patients who performed a pulmonary function test and donated a blood sample on the same day. From the Alpha-1 Liver Aachen Registry, we obtained a cohort of 40 ZZ-AAT patients with available data on their liver function. The levels of plasma Z-AAT polymers were determined using a LG96 monoclonal antibody-based sandwich ELISA. In a Dutch cohort, the median plasma level of Z-AAT polymers of patients diagnosed for pulmonary disease was 947.5 µg/mL (733.6−1218 µg/mL (95% CI)), which did not correlate with airflow obstruction or gas transfer value. In the Alpha-1 liver patient cohort, the median polymer level was 1245.9 µg/mL (753−2034 µg/mL (95% CI)), which correlated with plasma gamma-glutamyl transferase (GGT, rs = 0.57, p = 0.001), glutamate dehydrogenase (GLDH, rs = 0.48, p = 0.002) and triglycerides (TG, rs = 0.48, p = 0.0046). A Wilcoxon rank test showed higher Z-AAT polymer values for the liver over the lung group (p < 0.0001). These correlations support a possible link between plasma Z-AAT polymers and the liver function.

Keywords: ELISA; SERPINA1; alpha1-antitrypsin; deficiency; liver biomarkers; lung function; polymers.

Figure 1

Pulmonary function of 52 ZZ-AATD patients was measured 15 min after the inhalation of 400 µg of salbutamol followed by spirometry and a CO diffusion gas transfer. Levels of plasma Z-AAT polymers (µg/mL) were measured by ELISA based on LG96 mAb (see materials and methods). There was no correlation between plasma Z-AAT polymer values and FEV1 % pred, or Kco % pred.

Figure 2

Z-AAT polymer levels in plasma of 2 patients homozygous Mheerlen genotype and 5 with phenotype SZ mutant of AATD. The dashed line represents the lower limit of detection of the assay. The line through the closed circles represents the median value.

Figure 3

Repeatedly determined plasma levels of Z-AAT polymers in two randomly selected ZZ-AATD patients. On 14 consecutive days, plasma levels of Z-AAT polymers remained relatively stable in both patients.

Figure 4

Z-AAT polymer analysis by Western blots using two different monoclonal antibodies. Equal amounts of plasma samples from seven ZZ-AATD individuals were electrophoretically separated using 7.5% native polyacrylamide gels followed by western blots. Purified polymeric Z-AAT isolated from pooled ZZ plasma samples was used as a positive control. The western blots were probed against mouse monoclonal anti-AAT polymer antibodies LG96 (A) and 2C1 (B)(both diluted 1:700). The image demonstrates that both antibodies recognize the Z-AAT polymers, but the profiles of recognized polymers differ. This blot is the representative from n = 2 independent repeats.

Figure 5

(A–D), Correlations between plasma levels of Z-AAT polymers and liver biomarkers and patient age: (A), the gamma-glutamyl transferase (GGT) values were log2 transformed; (B), glutamate dehydrogenase (GLDH), (C), triglycerides (TG), and (D) patient age Spearman correlation was calculated from 40 pairs of data and shows a significant correlation between plasma Z-AAT polymer levels and liver markers.