APMP-APLAC joint proficiency testing programs for elemental analysis in food with metrological reference values: Assessment of participants' performance considering measurement uncertainties.

The Asia Pacific Metrology Program (APMP) and the Asia Pacific Laboratory Accreditation Cooperation (APLAC) joint Proficiency Testing (PT) programs for toxic elements such as cadmium (Cd) and lead (Pb) or nutritional elements such as iron (Fe) and zinc (Zn) in food were organized by the Korea Research Institute of Standards and Science (KRISS) with the aim of enhancing the quality of measurement and metrological traceability in various economies of the Asia Pacific region by evaluating the performance with rigorous evaluation. Three APMP-APLAC joint PT programs for elemental analyses were carried out by KRISS sequentially, where candidate certified reference materials (CRMs) were used as the PT materials and metrologically traceable certified reference values (RVs) were used as the PT assigned values for the evaluation of participants' results, which allows reliable evaluation of participant performance. This article describes the operation of the PTs and the overall performance of the participating laboratories. The effectiveness of these joint PT programs and trends in PT performance assessment are also discussed. These PT programs confirm the significant importance of using the metrologically traceable RVs instead of the consensus values from participants as the PT assigned value for reliable assessment. The lack of understanding of the concept of coverage factor, degree of freedom, standard uncertainty, and expanded uncertainty was revealed by some participants in these PT programs. Interpreting the zeta-scores or En scores, which are derived by using measurement uncertainties, in conjunction with the z-scores is highly meaningful for assessing participants' ability in measurement capabilities and measurement uncertainty evaluation. Assessment of participants' performance considering measurement uncertainties helps the participants to check how reasonable their measurement uncertainty estimation was. The results of PTs also demonstrated that these PT programs are useful for improving the measurement capability of the laboratories, whereas more capability-building in uncertainty evaluation is required for further improvement.

Effect of buffer composition and preparation protocol on the dispersion stability and interfacial behavior of aqueous DPPC dispersions.

The effect of the buffer composition and the preparation protocol on the dynamic surface tension (DST) and vesicle sizes of aqueous dipalmitoylphosphatidylcholine (DPPC) dispersions was studied. Four isotonic buffers were used in preparing DPPC dispersions at physiological conditions for possible biological applications: (1) a standard PBS solution; (2) the above PBS with 1mM CaCl(2); (3) PBS with one tenth the previous standard phosphate salt concentrations and 2.5 mM CaCl(2); and (4) 150 mM NaCl with 2.5 mM CaCl(2) and 10mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid). Two protocols, with a new method and an old method (Bangham method), were used in preparing the DPPC dispersions. The DPPC dispersions prepared with the new method contained mostly vesicles and were quite stable at 25 or 37 degrees C. Dynamic light scattering (DLS) and spectroturbidimetry (ST) results showed that the DPPC vesicle sizes in buffer (4) were much smaller than those in the other buffers. When the DPPC dispersions were prepared with the new method, the diameter of the DPPC particles was smaller than those with the old method. The DPPC vesicles with the new method were more stable than those with the other method. The DPPC dispersions of 1000 ppm at 37 degrees C with the new method produced, at pulsating area conditions at 20 cycles per minute, low tension minima (gamma(min)), lower than 10 mN/m, in buffers (1), (2), and (4). With buffer (4) the DSTs were lower and were achieved faster than with the other buffers. A minimum concentration of 1000 or 250 ppm DPPC was needed to produce DSTs lower than 10 mN/m within 10 min or less, with buffer (2) or (4), respectively. IRRAS results suggest that DPPC in buffer (2) or (4) forms a close-packed monolayer at the interface. These results have implications for designing efficient protocols of lipid dispersion preparation and lung surfactant replacement formulations in treating respiratory disease.

Stability and state of aggregation of aqueous fibrinogen and dipalmitoylphosphatidylcholine lipid vesicles.

The stability and state of aggregation of aqueous fibrinogen (FB) and dipalmitoylphosphatidylcholine (DPPC) vesicles in water or buffer at 25 degrees C were studied with dynamic light scattering (DLS), UV-vis spectroturbidimetry (ST), and cryo-transmission electron microscopy (cryo-TEM). In water, when 1000 ppm (0.10 wt %) DPPC dispersions were prepared with a protocol including extensive sonication, they contained mostly vesicles and were quite clear, transparent, and stable for at least 30 days. FB mixtures with water (0.075 wt %) were quite unstable and biphasic. They formed large aggregates which eventually precipitated. The addition of DPPC vesicles into these unstable FB dispersions reversed FB aggregation and precipitation and produced stable translucent microdispersions. The inferred lipid/protein aggregates were limited in size, with average diameters ranging from 200 to 300 nm. In buffer, DPPC dispersions were also clear and quite stable, with average dispersed particles diameter of ca. 90 nm. FB dissolved in aqueous buffer and formed transparent and stable solutions. Adding salt to an aggregated FB dispersion in water reversed the aggregation. FB aggregated and redissolved in the presence of the citrate and after the citrate was removed. There was no effect of citrate (present in FB initially) in the FB aggregation or redissolution. FB molecules in buffer form dimers or higher aggregates. Their average aggregation number is 2, determined with Rayleigh scattering analysis of turbidity data. The average hydrodynamic diameter of FB solutions from DLS was 30 nm. Mixing a stable FB solution in buffer and a stable DPPC dispersion in buffer produced highly unstable mixtures, in which large aggregates precipitated. These results have implications in understanding the interactions of lipids and proteins in many biological applications and food processing applications.

Effect of sonication and freezing-thawing on the aggregate size and dynamic surface tension of aqueous DPPC dispersions.

The effect of sonication and freezing-thawing on the aggregate size and dynamic surface tension of aqueous dipalmitoylphosphatidylcholine (DPPC) dispersions was studied by cryogenic-transmission electron microscopy (cryo-TEM), dynamic light scattering (DLS), UV-vis spectroturbidimetry, and surface tensiometry. When 1000 ppm (0.1 wt%) DPPC dispersions were prepared with a certain protocol, including extensive sonication, they contained mostly frozen vesicles and were quite clear, transparent, and stable for at least 30 days. The average dispersed vesicles diameter was 80 nm in water and 90 nm in standard phosphate saline buffer. After a freeze-thaw cycle, this dispersion became turbid, and precipitates of coagulated vesicles were observed with large particles of average size of 1.5x10(3) nm. The vesicle coagulation is due to the local salt concentration increase during the freezing of water. This dispersion has much higher equilibrium and dynamic surface tension than those before freezing. When this freeze-thawed dispersion was subjected to a resonication at 55 degrees C, smaller vesicles with sizes of ca. 70 nm were produced, and a lower surface tension behavior was restored as before freezing. Similar behavior was observed at 30 ppm DPPC. These results indicate that the freeze-thaw cycle causes substantial aggregation and precipitation of the vesicles. These results have implications for designing efficient protocols of lipid dispersion preparation and lung surfactant replacement formulations in treating respiratory disease and for effective administration.

Competitive adsorption of fibrinogen and dipalmitoylphosphatidylcholine at the air/aqueous interface.

The competitive adsorption of fibrinogen (FB) and DPPC at the air/aqueous interface, in phosphate buffer saline at 25 degrees C, was studied with tensiometry, infrared reflection absorption spectroscopy (IRRAS), and ellipsometry. For FB/DPPC mixtures with 750 ppm (0.075 wt%) FB and 1000 ppm (0.10 wt%) DPPC, the tension behavior was found to be similar to that of FB when alone, even with DPPC and FB being at the interface. Thus, FB interferes with adsorption of DPPC and inhibits its surface tension lowering ability. When FB protein is introduced in the solution after a DPPC monolayer has formed, the adsorption of FB is inhibited by the DPPC monolayer. When a DPPC monolayer is spread onto a solution with a preadsorbed FB layer, the DPPC monolayer excludes FB from the surface and controls the tension behavior with little inhibition by FB. When a DPPC dispersion is introduced with the Trurnit method, or sprayed dropwise, onto an aqueous FB/DPPC surfaces, the DPPC layer formed on the surface prevents the adsorption of FB and dominates the surface tension behavior. These results have implications in controlling the inhibition of lung surfactant tension behavior by serum proteins, when they leak at the alveolar lining layer, and in developing surfactant replacement therapies for alveolar respiratory diseases.

New protocols for preparing dipalmitoylphosphatidylcholine dispersions and controlling surface tension and competitive adsorption with albumin at the air/aqueous interface.

The adsorption behavior of dipalmitoylphosphatidylcholine (DPPC), which is the major component of lung surfactant, at the air/aqueous interface and the competitive adsorption with bovine serum albumin (BSA) were studied with tensiometry, infrared reflection absorption spectroscopy (IRRAS), and ellipsometry. Dynamic surface tensions lower than 1 mN/m were observed for DPPC dispersions, with mostly vesicles, prepared with new protocols, involving extensive sonication above 50 degrees C. The lipid adsorbs faster and more extensively for DPPC dispersions with vesicles than with liposomes. For DPPC dispersions by a certain preparation procedure at T>T(c), when lipid particles were observed on the surface, dynamic surface tensions as low as 1 mN/m were measured. Moreover, IRRAS intensities and ellipsometric deltaDelta values were found to be much higher than the values for other DPPC dispersions or spread DPPC monolayers, suggesting that a larger amount of liposomes or vesicles adsorb on the surface. For DPPC/BSA mixtures, the tension behavior is controlled primarily by BSA, which prevents the formation of a dense DPPC monolayer. When BSA is injected into the subphase with a spread DPPC monolayer or into a DPPC dispersion with preadsorbed layers, little or no BSA adsorbs and the DPPC layer remains on the surface. When a DPPC monolayer is spread on a BSA solution at 0.1 wt% at 25 degrees C, then DPPC lipid can displace the adsorbed BSA molecules. The lack of BSA adsorption, and the expulsion of BSA by DPPC monolayer is probably due to the strong hydrophilicity of the lipid polar headgroup. When a DPPC dispersion is introduced with Trurnit's method or when dispersion drops are sprayed onto the surface of a DPPC/BSA mixture, the surface tension becomes lower and is controlled by DPPC, which can prevent the adsorption of BSA. The results may be important in understanding inhibition of lung surfactants by serum proteins and in designing efficient protocols of surfactant preparation and administration.