Identifiability from parameter bounds. Structural and numerical aspects

Some a priori and a posteriori aspects of the identifiability problem for unidentifiable models are discussed. It is argued that the nation of identifiability from parameter bounds has a minor a priori structural relevance. The parameter bounds rationale may prove a useful a posteriori numerical notion. However, its practical potentiality needs careful evaluation, as the use of point estimates automatically builds into the model some hidden structural constraints. Examples are given.     

Surfactant disaturated-phosphatidylcholine kinetics in acute respiratory distress syndrome by stable isotopes and a two compartment model

Background

In patients with acute respiratory distress syndrome (ARDS), it is well known that only part of the lungs is aerated and surfactant function is impaired, but the extent of lung damage and changes in surfactant turnover remain unclear. The objective of the study was to evaluate surfactant disaturated-phosphatidylcholine turnover in patients with ARDS using stable isotopes.

Methods

We studied 12 patients with ARDS and 7 subjects with normal lungs. After the tracheal instillation of a trace dose of ¹³C-dipalmitoyl-phosphatidylcholine, we measured the ¹³C enrichment over time of palmitate residues of disaturated-phosphatidylcholine isolated from tracheal aspirates. Data were interpreted using a model with two compartments, alveoli and lung tissue, and kinetic parameters were derived assuming that, in controls, alveolar macrophages may degrade between 5 and 50% of disaturated-phosphatidylcholine, the rest being lost from tissue. In ARDS we assumed that 5–100% of disaturated-phosphatidylcholine is degraded in the alveolar space, due to release of hydrolytic enzymes. Some of the kinetic parameters were uniquely determined, while others were identified as lower and upper bounds.

Results

In ARDS, the alveolar pool of disaturated-phosphatidylcholine was significantly lower than in controls (0.16 ± 0.04 vs. 1.31 ± 0.40 mg/kg, p < 0.05). Fluxes between tissue and alveoli and de novo synthesis of disaturated-phosphatidylcholine were also significantly lower, while mean resident time in lung tissue was significantly higher in ARDS than in controls. Recycling was 16.2 ± 3.5 in ARDS and 31.9 ± 7.3 in controls (p = 0.08).

Conclusion

In ARDS the alveolar pool of surfactant is reduced and disaturated-phosphatidylcholine turnover is altered.     

Changes in urinary dinor dihydro F(2)-isoprostane metabolite concentrations, a marker of oxidative stress, during and following asthma exacerbations

To investigate changes in oxidant stress during and following acute asthma exacerbations, this study measured 2,3-dinor-5,6-dihydro-15-F(2t)-IsoP (F(2)-IsoP-M), the major urinary metabolite of 15-F(2t)-IsoP, in eight asthmatic adults, during and following an asthma hospitalization. F(2)-IsoP-M concentrations at admission and follow-up were significantly higher than discharge (admission median: 4.12 ng/Cr mg, range 1.89-7.8; follow-up: 2.47 ng/Cr mg (1.56-6.86); discharge: 1.42 ng/Cr mg (0.7-4.44); both p<0.01), but not significantly different between admission and follow-up. F(2)-IsoP-M concentrations at follow-up were higher than a control group with stable asthma (0.68 ng/Cr mg (0.31-1.5), p=0.0008). In conclusion, asthma exacerbations requiring hospitalization are associated with 6-fold higher urinary F(2)-IsoP-M concentrations compared to stable asthmatics. F(2)-IsoP-M concentrations decreased significantly during hospitalization, but significant elevations 3 months following hospitalization suggest ongoing oxidative stress despite clinical improvement. Urinary F(2)-IsoP-M may be a clinically useful, simple non-invasive systemic measure of oxidative stress in asthmatics, providing information not captured by spirometry or symptoms.     

Impurity analysis of retinoic acid samples

The structure of an impurity contained in samples of all trans-retinoic acid was established by means of NMR and MS spectra, and confirmed by X-ray diffraction analysis. The chemical structure of the impurity 2 was found to be strictly correlated to the synthetic procedure employed for the preparation of the retinoic acid samples. Single crystal analysis allowed us to characterise the molecular conformation and the crystal structure of 2.     

Rapid quantitative characterization of protein interactions by composition gradient static light scattering

Two new applications of the recently developed technique of composition gradient static light scattering (CG-SLS) are presented. 1), The method is demonstrated to be capable of detecting and quantitatively characterizing reversible association of chymotrypsin and bovine pancreatic trypsin inhibitor in a solution mixture and simultaneously occurring reversible self-association of chymotrypsin at low pH in the same mixture. The values of equilibrium constants for both self- and heteroassociation may be determined with reasonable precision from the analysis of data obtained from a single experiment requiring <15 min and <1 mg of each protein. 2), Analysis of the results of a single CG-SLS experiment carried out on Ftsz, a protein that self-associates to form linear oligomers of indefinite size in the presence of guanosine diphosphate, yields the dependence of the equilibrium constant for monomer addition upon oligomer size.     

Implications of macromolecular crowding for protein assembly

Recent studies have led to increased appreciation of the influence of excluded volume in solutions of high total macromolecular content ('macromolecular crowding') upon the various classes of reaction that lead to the assembly of proteins and protein complexes. In general, crowding is expected to stabilize native protein structure relative to less compact non-native structures and to favor the formation of functional complexes of native proteins. Under certain pathological conditions, 'overcrowding' may enhance the formation of nonfunctional aggregates of non-native protein (e.g. amyloid and inclusion bodies).