Background: The enzyme paraoxonase (PON1) is present in blood plasma, bound to high-density lipoprotein (HDL) particles, and has an antioxidative role. Its physiological substrates are not clearly known. PON1’s plasma activity and the gene PON1's polymorphisms have been investigated in several studies in connection with Alzheimer's dementia (AD), AD's precursor stage, known as mild cognitive impairment (MCI), and in Parkinson's disease (PD). PON1 is also present in cerebrospinal fluid (CSF), yet PON1's properties in CSF and its clinical importance are presently very poorly known. So far, little attention has been paid to human PON1's enzyme-kinetic properties, i.e. the Michaelis-Menten parameters Km, Vmax and kcat, and their potential clinical significance.
Aims and hypotheses: Within our PhD thesis, we wanted to investigate whether the status of PON1 differs a) between PB patients with cognitive impairment and those without it and b) between AD patients and MCI patients. We also wanted to check c) whether PON1’s status in CSF differs from PON1 in blood plasma. At the same time, we wanted to develop an improved method for determining the parameters Km and Vmax based on time-concentration progress curves; such a method could then be used for measurements of the abovementioned samples. We set three hypotheses: 1) In AD and MCI patients, PON1 activity in cerebrospinal fluid is associated with PON1 activity in serum and is lower in AD patients than in MCI patients. 2) In Parkinson’s disease patients, PON1 activity in serum is lower in the group with cognitive decline than in the group without cognitive decline. 3) The measured kinetic parameters which represent the phenotype of PON1 are a better marker of cognitive decline in MCI, AD, and PD than PON1 genotype.
Methods: We enrolled 161 patients with suspected AD/MCI, out of which 88 had AD or MCI confirmed, and 231 PD patients, out of which 207 had a known cognitive status. For all patients from both groups, we determined genotype for four common SNPs on the PON1 gene and the kinetic parameters Km and Vmax for the substrate dihydrocoumarin (DHC), using a new method based on time-concentration progress curves, with blood plasma. For all patients with suspected AD/MCI, we additionally determined Km and Vmax for the substrate phenylacetate (PA) both with blood plasma and with CSF, as well as the rate of hydrolysis for the substrate paraoxon (PX) with blood plasma. For patients with confirmed AD or MCI, we additionally determined PON1 concentration with ELISA.
Results: We developed a method for kinetic parameter determination which is based on removing unneccesary data points from a progess curve the script in question has been named “iFIT” and put online for public use. We have shown that iFIT is comparable or better than existing methods. We have shown which SNPs on the PON1 gene have an effect on kinetic parameters for a particular substrate or on enzyme concentration and for which SNPs this apparent influence is probably only a consequence of linkage. The presence of PON1 in CSF has been demonstrated for all the investigated samples. It turned out that there is no correlation between the status of PON1 in blood plasma and in CSF; p-values for the correlations between PON1 concentration, PA Km, Vmax and Vmax/Km in CSF and the equivalent parameters in blood plasma were all greater than 0.05. Additionally, none of the investigated SNPs correlates with any of the parameters determined for PON1 in CSF, although these same SNPs correlate very significantly with some of the equivalent parameters for PON1 in plasma. No correlation was detected between genotype, PON1 concentration or kinetic parameters on one hand and the cognitive status of patients from either group on the other hand.
Conclusion: We did not confirm any of our hypotheses, all of which referred to either the connection between PON1 and cognitive decline or between PON1 in blood plasma and in CSF. The absence of a correlation between CSF PON1 and either genotype, plasma PON1, or cognitive status, is a surprising result which opens a great question, namely which are the factors which influence the status of PON1 in CSF. The absence of a connection between PON1 status and cognitive decline suggests that PON1 does not show perspective as a biomarker for the cognitive status of PD or AD patients; such biomarkers have to be searched for elsewhere. A useful result of our project is the iFIT program, the usefulness of which we have shown both with recombinant PON1 and biological samples.
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