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Molecule-specific diagnosis of metabolic diseases

Lysosomal storage diseases (LSDs) are a group of rare inherited metabolic diseases that are difficult to diagnose due to their rather unspecific clinical signs. In cooperation with the company Centogene AG, Prof. Dr. Michael Przybylski from the Steinbeis Transfer Center for Biopolymer Analysis and Biomolecular Mass Spectrometry at the University of Konstanz has developed a method that involves a novel substance class and enables the parallel measurement of enzyme activity based on fluorometry and mass spectroscopy. The method, for which the inventors have recently filed a patent, forms the basis for the development of rapid and highly specific enzyme test systems that allow the reliable and unequivocal diagnosis of diseases. In the interview with BIOPRO Baden-Württemberg, Prof. Przybylski gives us some insights into his invention.

BIOPRO: Prof. Przybylski, what motivated you to develop the innovative method?

Prof. Dr. Dr. Michael Przybylski works at the Steinbeis Transfer Centre for Biopolymer Analysis and Biomolecular Mass Spectrometry at the University of Konstanz © Bettina Baumann / BioLAGO

Przybylski: Our goal is to develop new methods for the diagnosis of lysosomal storage diseases. We also want to validate their clinical benefit and use them for diagnostic and therapeutic applications worldwide. We have developed a new substance class of substrate derivatives for lysosomal enzymes as well as a method that is able to reliably diagnose around 40 of the 70 – 80 or so known lysosomal storage diseases. Some of these test systems are already in the early stages of clinical testing at different clinical centres. Over the next few years, the extension of the previously available range of diagnostic methods will make a significant contribution to the development of new therapeutic substances, including for patients and diseases that cannot currently be treated.

Lysosomal storage diseases manifest in a number of organs. Does this in any way influence their diagnosis?

The diagnosis of LSDs is often rather difficult because the clinical signs are often vague and the different LSDs can be easily confused with each other. Differential diagnosis is difficult to the extent that doctors find it difficult to definitely diagnose the type of LSD based on clinical signs, i.e. pathological organ alterations such as the enlargement of the spleen or liver. Where tests are available, the different LSDs need to be tested individually, which requires considerable effort and can also lead to delays in diagnosis. However, the success of enzyme replacement therapies requires tests to be carried out as early as possible, as therapies are unable to reverse existing damage. If there is no test and therefore no diagnosis, effective treatment is not possible.

What is the current clinical practice for diagnosing LSDs?

The current standard for diagnosing LSDs is to determine the catalytical activity of lysosomal enzymes. A blood sample is mixed with a substrate derivative, which is a molecule that is specifically metabolised by the enzyme used for the diagnosis. The resulting product is measured using fluorimetry, an analytical technique for measuring the concentration of the product, from which the activity of the enzyme can then be deduced.


As far as I know, you mainly use mass spectrometric methods. Can you tell me why?

Yes, of course. Over the last few years, mass spectrometry-based tests have been developed for the diagnosis of 6 or 8 storage diseases. These tests use specific substrates that can only be used in mass spectrometry. It is therefore rather difficult to compare these data with those obtained using conventional fluorimetric methods. However, clinical and diagnostic screenings, which need to be carried out in order to establish and validate the new methods, rely on data that are comparable. This also applies to future serial applications.

The research that you are doing in the transfer project aims to provide new perspectives for molecule-specific diagnoses and hence approaches for the development of new therapies, both in Germany and worldwide. Have your methods, for which you have recently filed patents, already led to successful outcomes?

Single blood droplets on filter paper are all that is needed for diagnosis. © Bettina Baumann / BioLAGO

We have succeeded in developing a new diagnostic test principle using a new substance class. Our substrate analogues allow the determination of enzyme activity using fluorimetric analysis as well as tandem mass spectrometry using one and the same substrate. The new substrate analogues contain a functional group that can be determined fluorimetrically after the substrates have been converted by the enzyme. We use an isotope-labelled variant of the enzymatic product as a standard in mass spectrometric as well as fluorimetric analyses. The isotope-labelled variant is chemically identical with the product, but has a slightly different mass, which is why mass spectrometry can definitely distinguish it from the product. The use of identical substrates for both mass spectrometric and fluorimetric analyses has the advantage that the clinical results become comparable, thus making the data highly reliable. 

What are your substrates exactly?

The substrate analogues are novel umbelliferyl derivatives which are linked to the enzyme’s normal substrate, a hydrocarbon like glucose for example. Umbelliferone is a derivative of the secondary plant substance coumarin, the only difference being that it has an additional hydroxyl group that lights up blue under UV light. This extra hydroxyl group is the reason why umbelliferyl derivatives can be used for fluorimetric measurements. In addition, these substrate analogues allow the development of methods for the simultaneous determination of multiplex enzymes (multiplex diagnostics) in one sample by using umbelliferyl derivatives with different substitutions.

How do such multiplex measurements work?

Multiplex diagnostics assays allow the simultaneous measurement of the activity of several enzymes, and hence the diagnosis of several LSDs, in one sample. This has previously only been possible with mass spectrometric and not fluorimetric methods. Multiplex means that several enzymatic reactions are carried out in parallel. Several substrate analogues, which are linked to different hydrocarbons, are used in a single test and each is specifically metabolised by only one of the enzymes included in the analysis. The different enzymes lead to different products, which in turn provide information about the activity of the respective enzymes.

How many enzymes can be analysed in parallel? Is there an upper limit?

The number of enzymes that can be tested simultaneously is somewhat limited as they do not convert the substrates in equal measure. We therefore use enzymes with similar substrate conversion rates in one assay. The parallel measurement of different enzyme activities is of special advantage in clinical settings and also has the potential to be used for newborn screening. We can produce kits that are able to measure 5-8 enzymes simultaneously. Our patent-pending innovation relates to the multiplex determination of 20 to 25 storage diseases simultaneously.

What are your future plans for the transfer project?

First, we plan to develop further substrate analogues for about 20 storage diseases, validate the test systems for the diagnosis of the diseases using patient samples, and for application in clinical diagnosis. We will also compare the method with existing methods. The second part of the project focuses on the development of a method based on affinity mass spectrometry that enables the direct molecule-specific determination of the enzymes. This method measures the concentration of the enzyme directly, not indirectly by way of the concentration of its product.

The method has the great advantage that it differentiates between the complete absence and presence of functionally defective enzymes, which is not possible with activity measurements. The combination of this new method with the already established enzyme activity measurement method provides information on whether an enzyme is present or defective. The development of the new affinity mass spectrometry-based method is still in its early stages. The application of the developed methods is not limited to lysosomal storage diseases and their enzymes, but can be applied to a broad range of other protein and peptide biomarkers.

Further information:
Prof. Dr. Dr. Michael Przybylski
Steinbeis Transfer Center for Biopolymer Analysis and Biomolecular Mass Spectrometry
University of Konstanz
Universitätsstraße 10
78457 Konstanz
Tel.: +49 (0)7531/ 882249
E-mail: michael.przybylski(at)uni-konstanz.de

Centogene AG
Freiburg Business Location
Center for Human Genetics
Heinrich-von-Stephan-Str. 5
79100 Freiburg
E-mail: office(at)centogene.com

Website address: https://www.gesundheitsindustrie-bw.de/en/article/news/molecule-specific-diagnosis-of-metabolic-diseases