Sampling Requirements: The dynamic nature of the RAS with very short angiotensin metabolite half-lives down to seconds as well as rapid on-going angiotensin formation via renin are well known sources of contradicting angiotensin data in the literature. It is essential to thoroughly control the whole process from sampling down to signal detection, assuring the integrity and reproducibility of angiotensin data. This requires sampling in the presence of a special optimized angiotensin protease inhibitor cocktail (Circulating Angiotensin Levels) or adhering to sophisticated sample analysis protocols (Equilibrium Analysis, Tissue Analysis).

Angiotensin Sequence Similarity vs. Antibody Specificity: Different angiotensin metabolites share sequences with corresponding sections in their upstream metabolites including highly abundant angiotensinogen. Therefore, antibody cross-reactivity is a common issue for immunoassay based angiotensin quantification methods. Poor specificity and the incompatibility of these methods with internal standard technology is one of the main reasons why angiotensin data are often contradicting among different studies so far.

With the RAS-Fingerprint™, we provide a premium quality and full service solution to these issues, including consulting support for your study. Our expertise in RAS sample collection and processing in combination with high-end LC-MS/MS technology guarantee the highest analytic performance and quality standard for each individual sample. Angiotensin metabolite panels (RAS-Fingerprint™) depict additional information by visualizing the enzymatic connections between individual angiotensin metabolites. The graphs provide an attractive and unique way of displaying your data, thus facilitating the transfer of your findings to your targeted audience. Different functional RAS-Fingerprint™ panels are available for circulating plasma angiotensin levels, endogenous tissue angiotensin levels and plasma equilibrium levels.

RAS equilibrium analysis is a versatile diagnostic tool to perform a comprehensive biochemical characterization of the soluble RAS in plasma using standard Li-Heparin or serum samples. Angiotensin product/substrate ratios for a certain enzymatic cleavage directly reflect the activity of corresponding enzymes. Absolute equilibrium levels further provide information on the state of soluble RAS activity making equilibrium analysis a perfect screening tool for the soluble RAS in patient samples.

Circulating angiotensin levels are the levels of angiotensin metabolites that are present in the blood vessel, which is punctured for the collection, and might differ among different blood vessels. The quantification of circulating angiotensin levels represents a major challenge for analytic methods. The sampling process itself is very critical as angiotensin metabolite levels are extremely dynamic and rapidly change upon sampling, if not all angiotensin-metabolizing enzymes are immediately and efficiently blocked. Beside an effective protease inhibitor cocktail, which we can provide on request, the inhibitor cocktail needs to be rapidly added to the blood samples during the sampling procedure followed by cooled centrifugation and immediate freezing of the samples until analysis.

The origin of tissue angiotensin levels is still controversially discussed in literature. Plenty of factors affect local angiotensin levels in different tissues. While some tissues are supposed to contain renin and angiotensinogen, supporting local angiotensin formation, others rely on the internalization of angiotensins present in the vasculature. The angiotensin metabolite pattern in tissues is affected by local endothelial RAS components as well as circulating RAS components that regulate the abundance of various effector peptides in individual tissues.

Attoquant Diagnostics is the unique provider of mass spectrometry based angiotensin quantification.

We use mass spectrometry for all our assays due to its unchallenged accuracy to discriminate multiple peptides and its high sensitivity to detect hormones in the low picomolar molecular range of biological samples.
The combination of ultra-pressure-liquid chromatography-tandem mass spectrometry (LC-MS/MS) and stable-isotope-labeled internal standards provides unchallenged specificity and sensitivity in all our assays and is the backbone of our service.

In order to assay Neprilysin, for instance, we determine the turnover of Ang I to Ang 1-7 in presence and absence of a specific Neprilysin inhibitor.

The measurement of protease activities in tissue and plasma samples is a challenging task as the importance of assuring signal specificity is widely underestimated. Common strategies aiming at determining the activity of proteolytic enzymes in complex samples employ the cleavage of artificial substrates.  Colored or fluorescent products that are released from a quenched artificial substrate upon protease cleavage are measured and enzyme activities are quantified using a standard curve of recombinant enzyme that is usually prepared in matrix free buffers. Importantly, biological samples contain dozens of proteases that can cleave such artificial substrates, which usually contain very few amino acids that aim at mediating the selectivity for a certain enzyme. As a consequence, especially low abundance enzymes cannot be accurately quantified in complex samples using commercially available colorimetric or fluorimetric kits.

Our LC-MS/MS based approaches combine the conversion of natural substrates into the natural product of a certain enzymatic reaction with the highly sensitive and direct quantification of the enzyme specific product. The additionally employed specific inhibitors guarantee the selectivity of the assay. Standard curves of recombinant enzymes are routinely prepared in the original sample matrix.