Project details


Multiple research projects - laboratory Pediatrics

mass spectrometry method development isotopes

Prof. dr. A.K. Groen

Type of project:
TTT project (year 1), Pilot project (year 2 or 3), Stage Wetenschap / Researchproject of MD/PhD programme

Nature of the research:
Van basaal tot toegepast wetenschappelijk onderzoek

Fields of study:

Background / introduction
Project 1: Investigation of deoxycholic acid 7α-hydroxylation in cultured rat hepatocytes

Background: Some cholic acid (CA) is lost during the enterohepatic circulation by spill over into the colon and bacterial formation of deoxycholic acid (DCA) due to 7α-dehydroxylation. In humans DCA is partly absorbed and taken up in an independent DCA pool with its own enterohepatic circulation. It is known that rodents are able to reconvert DCA to CA in the liver. The impact of this process on the turnover of the CA pool is subject of research in our laboratory.

Questions: to measure the actual 7α-hydroxylation of DCA to CA in cultured rat hepatocytes and to develop tools to influence the conversion rate. To measure the rate of conversion DCA labeled with a stable isotope (13C, 2H) can be added to the medium and the appearance of isotopically labeled tauro-CA determined using HPLC coupled online to tandem mass spectrometry (LC/MS/MS).

Project 2: Measurement of deoxycholic acid kinetics in small plasma samples or blood spots of rodents and humans

Background: Deoxycholic acid (DCA) is formed by bacterial 7α-dehydroxylation of cholic acid (CA) in the colon. DCA in rodents is absorbed from the colon in the free form and conjugated with taurine in the liver. Part is converted to CA (project 1), part is secreted into bile as part of a DCA pool. The DCA pool size is small. Measurement of DCA kinetics (pool size, turnover rate) has been performed using bile samples. Ideally measurements are performed in small plasma samples, but then the free DCA originating from the colon, must be removed.

Procedure: A complex procedure to separate conjugated and free bile acids have been described in the 1980’s. Thereafter new extraction and separation techniques based on solid phase extraction and TLC have been developed that could simplify the procedure. Techniques will be derived from the literature and modified to allow the separation in small plasma samples. The same procedure will be applied to human plasma samples. Here the plasma volumes can be larger.

Project 3a and 3b: Measurement of chenodeoxycholic acid kinetics in rodents and rat hepatocytes

Background: Cholic acid (CA) and chenodeoxycholic acid (CDCA) are the primary bile acids formed from cholesterol. In humans CDCA and CA are both about 40% of the total bile acid pool. In rodents CDCA is rapidly converted to α- and β-muricholic acid and their metabolites. The kinetics of CDCA formation and conversion as well as the kinetics of the muricholic acids are largely unknown. Nothing is known about potential recoversion of muricholic acids to CDCA. Recently a pilot experiment has been undertaken in which rats were infused with deuterated CDCA and bile samples were collected.

Procedures: techniques will be tested to isolate the deuterated muricholic acids from the bile samples with high efficiency. Then deuterated muricholic acids will be administered to rats and mice to study the isotope dilution kinetics leading to values for pool sizes and turnover rates. Also experiments will be performed in which cultured rat hepatocytes will be supplied with deuterated CDCA measuring the conversion to the muricholic acids or with deuterated muricholic acids to measure potential reconversion to CDCA.

Project 4: Further development of the yeast based conversion of glucose to CO2 for measurement of 13C abundances

Background: Measurement of 13C abundance of glucose is important for medical and nutritional application as well as food adulteration. This involves release of glucose from a biological matrix or carbohydrate structure (starch, disaccharides, glycoproteins) and conversion of glucose to CO2. This conversion is possible applying complex GC/C/IRMS or LC/C/IRMS equipment. Recently our laboratory developed a method to convert plasma glucose selectively into CO2 applying baker’s yeast in the fermentation mode in breath collection tubes without prior treatment of the plasma. 13C abundance could be directly measured by a simple breath 13C analyzer (GC/IRMS). So far, we showed the proof of principle. Further development is necessary. The procedure must be optimized to reduce the required plasma volume. The application in full blood should also be tested. Large scale validation against GC/C/IRMS is planned. The application for glycogen turnover studies should be investigated. Furthermore, the application to food matrices and complex carbohydrates needs to be studied.

Project 5: In vivo validation in humans of a colon delivery device using stable isotope labeled compounds and Isotope Ratio Mass Spectrometry

Background: For the treatment of diseases of the large intestine (inflammatory diseases and colon cancer) and for the study of fermentation processes a colon delivery device is required. The UMCG hospital pharmacy in cooperation with the department of pharmacy of the RUG, has recently developed a coated capsule device which releases its material at a pH>7.0. At this moment in-vivo validation studies are in progress. The first data suggest that the capsule opens near the cecal valve. It is unsure whether it happens at the ileal site or the colon site. Further investigations are necessary in this respect.

Procedure: Coated capsules filled with stable isotope (13C) labeled urea and iron will be administered to healthy subjects. The release of material will be monitored by measurement of 13C enrichments in blood urea and in breath CO2 as well as measurement of iron release by clinical MRI instrumentation. MRI results should indicate whether the material has been released in the terminal ileum or the proximal colon.

This project is still under discussion, to make sure that MRI is clearly able to differentiate between ileum and colon.
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