Nanoparticle Tracking Analysis for the Enumeration and Characterization of Mineralo-Organic Nanoparticles in Feline Urine

Urinary stone disease, particularly calcium oxalate, is common in both humans and cats. Calcifying nanoparticles (CNP) are spherical nanocrystallite material, and are composed of proteins (fetuin, albumin) and inorganic minerals. CNP are suggested to play a role in a wide array of pathologic mineralization syndromes including urolithiasis. We documented the development of a clinically relevant protocol to assess urinary CNP in 9 healthy cats consuming the same diet in a controlled environment using Nanoparticle Tracking Analysis (NTA®). NTA® is a novel method that allows for characterization of the CNP in an efficient, accurate method that can differentiate these particles from other urinary submicron particulates. The predominant nanoscale particles in feline urine are characteristic of CNP in terms of their size, their ability to spontaneously form under suitable conditions, and the presence of an outer layer that is rich in calcium and capable of binding to hydroxyapatite binders such as alendronate and osteopontin. The expansion of this particle population can be suppressed by the addition of citrate to urine samples. Further, compounds targeting exosomal surfaces do not label these particulates. As CNP have been associated with a number of significant urologic maladies, the method described herein may prove to be a useful adjunct in evaluating lithogenesis risk in mammals

Nanoparticle Tracking Analysis for the Enumeration and Characterization of Mineralo-Organic Nanoparticles in Feline Urine.

Urinary stone disease, particularly calcium oxalate, is common in both humans and cats. Calcifying nanoparticles (CNP) are spherical nanocrystallite material, and are composed of proteins (fetuin, albumin) and inorganic minerals. CNP are suggested to play a role in a wide array of pathologic mineralization syndromes including urolithiasis. We documented the development of a clinically relevant protocol to assess urinary CNP in 9 healthy cats consuming the same diet in a controlled environment using Nanoparticle Tracking Analysis (NTA®). NTA® is a novel method that allows for characterization of the CNP in an efficient, accurate method that can differentiate these particles from other urinary submicron particulates. The predominant nanoscale particles in feline urine are characteristic of CNP in terms of their size, their ability to spontaneously form under suitable conditions, and the presence of an outer layer that is rich in calcium and capable of binding to hydroxyapatite binders such as alendronate and osteopontin. The expansion of this particle population can be suppressed by the addition of citrate to urine samples. Further, compounds targeting exosomal surfaces do not label these particulates. As CNP have been associated with a number of significant urologic maladies, the method described herein may prove to be a useful adjunct in evaluating lithogenesis risk in mammals

2D-ToGo workflow: increasing feasibility and reproducibility of 2-dimensional gel electrophoresis

Two-dimensional gel electrophoresis (2-DE) is one of the most powerful methods for studying global protein profiles. However, due to the multiple manual steps involved in gel based processing it is challenging to achieve the necessary overall reproducibility for a reliable comparative analysis, especially between different laboratories. To improve the 2-DE technique for quantitative analyses we have set up a robust 2-DE workflow, called 2D-ToGo, which utilizes latest innovations concerning instrumentation, consumables and protocols. Quantitative data analyses indicate the high reproducibility between replicate gels processed at a single site (intra-laboratory variation: CV 20%). The data-sets of the inter-laboratory comparison revealed similar results displaying a variation of CV 23%. The technical improvements given by our 2-DE workflow have a positive impact on process robustness and most importantly, reproducibility. Accordingly, many of the well-known challenges for resolving and quantitating up to thousands of different protein components in a given biological sample are minimized

Representative histogram showing size distribution and cumulative percentage (left) of submicron particulate matter from urine obtained from a healthy cat.

<p>After a 4 hr incubation at 37 deg C. A representative frame from the captured video analyzed by NTA is shown as well (right).</p

Characteristic of submicron particulates in healthy cat urine (n = 9).

<p>Characteristic of submicron particulates in healthy cat urine (n = 9).</p